WO2005031300A2 - Device for detecting biological and chemical particles - Google Patents

Device for detecting biological and chemical particles Download PDF

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Publication number
WO2005031300A2
WO2005031300A2 PCT/US2004/020640 US2004020640W WO2005031300A2 WO 2005031300 A2 WO2005031300 A2 WO 2005031300A2 US 2004020640 W US2004020640 W US 2004020640W WO 2005031300 A2 WO2005031300 A2 WO 2005031300A2
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WO
WIPO (PCT)
Prior art keywords
cantilever
detection device
suspended
region
suspended member
Prior art date
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PCT/US2004/020640
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French (fr)
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WO2005031300A3 (en
Inventor
Rashid Bashir
Amit Kumar Gupta
Dallas Morisette
Oguz H. Elibol
Gerold W. Neudeck
John P. Denton
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Purdue Research Foundation
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Application filed by Purdue Research Foundation filed Critical Purdue Research Foundation
Priority to CA002530607A priority Critical patent/CA2530607A1/en
Priority to EP04809462A priority patent/EP1651943A2/en
Priority to AU2004276725A priority patent/AU2004276725A1/en
Publication of WO2005031300A2 publication Critical patent/WO2005031300A2/en
Publication of WO2005031300A3 publication Critical patent/WO2005031300A3/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N5/00—Analysing materials by weighing, e.g. weighing small particles separated from a gas or liquid
    • G01N5/02—Analysing materials by weighing, e.g. weighing small particles separated from a gas or liquid by absorbing or adsorbing components of a material and determining change of weight of the adsorbent, e.g. determining moisture content
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C5/00—Separating dispersed particles from liquids by electrostatic effect
    • B03C5/02—Separators
    • B03C5/022—Non-uniform field separators
    • B03C5/026—Non-uniform field separators using open-gradient differential dielectric separation, i.e. using electrodes of special shapes for non-uniform field creation, e.g. Fluid Integrated Circuit [FIC]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples

Definitions

  • This disclosure relates to devices, methods of use, and methods of fabrication of such devices, for use in selectively concentrating, capturing, and detecting the presence of species, for example, chemical molecules such as gases or proteins, biological species, such as cells (e.g., bacterial cells and/or eukaryotic cells) or virus particles, spores, molds, yeast, microorganisms, and the like.
  • species for example, chemical molecules such as gases or proteins
  • biological species such as cells (e.g., bacterial cells and/or eukaryotic cells) or virus particles, spores, molds, yeast, microorganisms, and the like.
  • the invention is disclosed in the context of arrays of micro- and nano-scale structures intended to be used, for example, as mass sensors to detect the presence and/or concentration(s) of the selected species. However, it is believed to be useful in other applications as well.
  • BACKGROUND Detection of very small quantities of chemical and biological species is important in industrial, environmental, human health, and bio-security applications, to name a few.
  • Various methods, devices, and structures for the detection of chemical and biological species utilize a variety of technologies and methods.
  • many prior methods, devices, and structures lack the ability to detect low concentrations of such species, and require long turnaround times.
  • Many such methods and apparatus also generally require chemical or biological labeling of the species to be detected. All of these characteristics can complicate the detection process.
  • methods for detection of coronaviruses include virus isolation, electron microscopy, and immunofiuresence assay with human coronavirus specific fluorescein (hereinafter sometimes FITC)- conjugated antibodies.
  • FITC coronavirus specific fluorescein
  • some molecular detection methods utilize polymerase chain reaction (hereinafter sometimes PCR) amplification for screening for viruses such as coronaviruses.
  • PCR polymerase chain reaction
  • PCR polymerase chain reaction
  • PCR polymerase chain reaction
  • PCR polymerase chain reaction
  • Viruses are traditionally quantified by their replication-induced cytopathic effects on cultured cells or by immunological means such as ELISA or immuno electron microscopy.
  • the cell culture quantification methods usually take several days to weeks and some viruses such as some human respiratory coronaviruses cannot be grown in cultured cells in vitro.
  • the ELISA method involves the capture of viruses by an antibody cross-linked to a surface, such as 96-well polystyrene microtiter plates. The captured viruses are then detected with a secondary antibody that is attached to an enzyme such as horseradish peroxidase, which catalyzes a chemical reaction that produces a color change.
  • Cell culture, ELISA and immunoelectron microscopy techniques typically are labor-intensive techniques that require experienced personnel to perform.
  • RNA and DNA viruses such as HJN-1, HIV-2, hepatitis B and C viruses, dengue virus, and cytomegalovirus (see, for example, Mackay, I. M., Arden, K. E., ⁇ itsche, A., Real-time PCR in Nirol., Nucleic Acids Res., 15: 1292-1305, 2002, incorporated herein by reference).
  • pathogenic RNA and DNA viruses such as HJN-1, HIV-2, hepatitis B and C viruses, dengue virus, and cytomegalovirus
  • cytomegalovirus see, for example, Mackay, I. M., Arden, K. E., ⁇ itsche, A., Real-time PCR in Nirol., Nucleic Acids Res., 15: 1292-1305, 2002, incorporated herein by reference.
  • These assays are highly sensitive and faster than prior methods.
  • PCR-based assays are troublingly prone to contamination, which can result in false positive detection.
  • PCR-based assays generally are not suitable for high throughput detection and discrimination of viral subtypes or antigenic variants.
  • Micro- and nano-scale technology has been increasingly used in a wide variety of chemical and biological applications, including, for example, detection and characterization of biological species in biomedical applications.
  • detection and characterization of biological species in biomedical applications For example, one area of technology that has become increasingly important but not very well developed is the handling, mampulation, and characterization of viral infectious agents, for example, the detection of very low virus particle concentrations in air and their continuous monitoring from air, using biomedical micro-electromechanical- systems technology (BioMEMS).
  • BioMEMS biomedical micro-electromechanical- systems technology
  • reducing the time-to-result to be able to perform 'point-of-use' analysis has become very important.
  • Micro- and nano-scale devices could not only yield very important scientific data but could also be used in practical diagnostic applications in the health and food industries, and in biological and chemical hazard prevention systems.
  • Cantilevers, bridges, or other micro- and nano-scale suspended structures associated with semiconductor devices have been used to selectively detect biological and chemical particles, for example, as sensors or actuators in atomic force microscopy (AMF) and scanning probe microscopy (SPM).
  • AMF atomic force microscopy
  • SPM scanning probe microscopy
  • Optical lever detection techniques are used, for example, to measure the deflection of atomic force microscopy (AFM) tips.
  • a laser beam is reflected off of the tip of a cantilever and is typically detected by two photodetectors.
  • the relative intensity of the laser signal received by the two photodetectors is related to the degree to which the cantilever is deflected.
  • this technique has several shortcomings, especially as the dimensions of cantilevers are reduced in order to detect particles having sub- 100 femtogram (fg) masses.
  • the technique requires reflecting a focused laser beam off the surface of the cantilever tip, resulting in larger lateral dimensions of the cantilever.
  • the technique generally precludes simultaneous monitoring of an array of cantilevers, which is necessary or highly desirable in a variety of detection applications.
  • a relatively large off-chip system is required, including the laser, focusing optics, and photo detectors. Therefore, the degree to which such a system can be miniaturized is limited.
  • Piezo-resistive detection methods eliminate the requirement of external optical system elements by electrically measuring the deflection-induced strain on the surface of the cantilever or the change in vibration frequency due to the particle mass.
  • the technique is analogous to macroscale strain gauges that are attached to mechanical components to measure deformation.
  • the change in resistance for a piezo-electric element in response to strain is induced by cantilever deflection.
  • This change in resistance can be detected, for example, by using a Wheatstone bridge circuit.
  • Piezo-resistor elements can be built into the surface of the cantilever by standard integrated circuit fabrication techniques. This provides a flexible, integrated, and scaleable vibration sensing technique adaptable to a variety of cantilever structures. When a cantilever is deflected, a strain gradient is induced throughout the body of the cantilever, with zero strain at the center. One surface is in tension (positive strain) while the opposite surface is in compression (negative strain).
  • piezo-resistive elements are generally placed near the surface region of the cantilever. While this may be accomplished by implantation and/or diffusion into relatively thick cantilevers, it becomes very difficult to achieve in sub- 100 nanometer (nm) thick cantilevers which are generally required for detecting sub- 100 fg masses.
  • devices, methods of use, and methods of fabrication of such devices for use in selectively concentrating, capturing, and detecting the presence of species, for example, chemical molecules such as gases or proteins, biological species, such as cells (e.g., bacterial cells and/or eukaryotic cells) or virus particles, and the like are desirable.
  • species for example, chemical molecules such as gases or proteins, biological species, such as cells (e.g., bacterial cells and/or eukaryotic cells) or virus particles, and the like are desirable.
  • an apparatus comprises a filter structure adapted to be coupled to an alternating current voltage source.
  • the filter structure includes at least one electrode having a feature which promotes establishment of at least one of a region of increased electric flux and a region of decreased electric flux relative to an adjacent region to select a first species susceptible of dielectrophoretic selection.
  • the apparatus further includes a cantilever structure upon which the first species is collected.
  • the apparatus also includes at least one device for causing the cantilever structure to oscillate, and for receiving as an output from the cantilever structure the cantilever structure's frequency of oscillation.
  • the at least one device is adapted to be coupled to means for determining from the output the mass of the first species collected on the cantilever structure.
  • an integrated micro- electromechanical analyte detection device comprises a substrate, a support member coupled with said substrate, a cantilever having a fixed end coupled to said support member and a free end, and a first electrode coupled to said substrate and positioned adjacent said free end of said cantilever such that the free end and the electrode are excitable terminals for dielectrophoresis.
  • an integrated micro- electromechanical analyte detection device comprises a substrate, a support member coupled to said substrate, a cantilever having a fixed end coupled to said support member and a free end, and a transistor having at least one region formed on at least one of said free end of said cantilever and a portion of said support member.
  • an integrated micro- electromechanical analyte detection device comprises a substrate, a support member coupled to or defined by said substrate, and a suspended member having first and second ends. At least the first end is coupled to the support member such that at least a portion of the suspended member is movable relative to the substrate.
  • the integrated micro-electromechanical analyte detection device further comprises a field effect transistor having a channel defined by said suspended member.
  • an integrated micro- electromechanical detection device comprises a substrate, a first suspended member having first and second ends, at least said first end fixed with said substrate, a dielectrophoresis filter coupled to said substrate, and a fluid channel defined by said subsfrate and communicating fluid between said first suspended member and said dielectrophoresis filter.
  • a method is provided for determimng a characteristic of an analyte interacting with an integrated detection device.
  • the device includes a subsfrate and a suspended member having first and second ends. At least the first end is coupled with the substrate.
  • the method comprises applying an electric signal to the suspended member to perform dielectrophoresis on the analyte and determining a change in a parameter of the suspended member.
  • a method is provided for determimng a characteristic of an analyte interacting with an integrated detection device.
  • the integrated detection device includes a subsfrate, an electrode and a suspended member having first and second ends. At least the first end is coupled with the subsfrate.
  • the method comprises applying an electric signal to the electrode and the suspended member to perform dielectrophoresis on the analyte.
  • a method is provided for determining a characteristic of an analyte interacting with an integrated micro- electromechanical detection device.
  • the device includes a subsfrate, a support member coupled with the subsfrate and a suspended member having first and second ends. At least the first end is coupled to the support member.
  • the method comprises determining a change in a current signal conducted by a fransistor region defined at least in part by one of the suspended member and the junction of the first end of the suspended member and the support member, upon the analyte interacting with the suspended member.
  • a method is provided for fabricating an integrated circuit on a substrate.
  • the integrated circuit includes a cantilever.
  • the method comprises providing a silicon-on-insulator (SOI) wafer having a buried oxide layer (BOX), thinning the SOI layer to less than approximately 30 nm, photolithographically patterning and etching the SOI layer to define the cantilever, tWnning the exposed portions of the BOX layer, depositing an etch stop layer to the exposed portions of the SOI and BOX layers, photolithographically patterning an etch window extending laterally approximately from the free ends of the cantilever and vertically to at least the depth of the base of the SOI layer, and etching the BOX layer below the cantilever, thereby releasing an end of the cantilever.
  • SOI silicon-on-insulator
  • BOX buried oxide layer
  • the integrated circuit includes a cantilever.
  • the method comprises patterning a seed window using an insulator mask on subsfrate, providing an oxide layer displaced vertically above the seed window and laterally overlying the seed window and at least a portion of the insulator mask, growing silicon selectively from the base of said seed window vertically to the top of the insulator mask and laterally between the insulator mask and the oxide layer, and etching at least a portion of the oxide layer and the insulator mask, thereby releasing a suspended end of the selectively grown silicon from the remaining structure.
  • a method is provided for fabricating an integrated circuit on a substrate.
  • the integrated circuit includes a cantilever and a field effect transistor.
  • the method comprises providing a silicon-on- insulator (SOI) wafer, thinning the SOI in the area where the cantilever will be formed using an anisofropic etch in order to form an abrupt step adjacent the top surface of cantilever and the junction of the remaining structure, oxidizing the silicon surface of the cantilever and the step to form transistor gate oxide, depositing polysilicon gate material conformally along the top surface of the cantilever adjacent the step, anisotropically etching the polysilicon gate material through the entire deposited thickness so that a sidewall of polysilicon having a lateral width approximately equal to the deposited thickness remains on any exposed vertical surface, implanting a source and drain region using the polysilicon gate as an implantation mask, and etching to release a suspending portion of the cantilever on the end opposite the transistor gate.
  • SOI silicon-on- insulator
  • a method for fabricating an integrated circuit on a subsfrate.
  • the integrated circuit includes a field effect fransistor (FET).
  • FET field effect fransistor
  • the method comprises providing a silicon wafer, thermally growing a first thick oxide layer for subsfrate isolation, lithographically patterning and depositing a sacrificial silicon layer on the oxide layer, depositing a second thick oxide layer using plasma enhanced chemical vapor deposition (PECND), etching via holes to define the FET source and drain regions, and etching to selectively remove the sacrificial silicon layer.
  • PECND plasma enhanced chemical vapor deposition
  • the removal defines a bridge formed by a portion of the second oxide layer suspended over the thermally grown oxide layer.
  • the method further comprises collapsing the oxide bridge to leave nano-scale via holes near the anchors of the oxide bridge, and wet etching a seed hole in the first oxide layer in the source region via hole.
  • the seed hole extends down to the silicon layer surface.
  • the method further comprises growing silicon epitaxially through the via holes, removing silicon remaining on the surface by chemical mechanical polishing (CMP), implanting the source and drain regions, and etching to remove the oxide encapsulating the epitaxially grown silicon.
  • CMP chemical mechanical polishing
  • the silicon forms suspended nano-wires forming the FET channel and a thin connecting silicon plate between the wires.
  • the method further comprises etching to remove the remaining oxide covering on the device and the thin silicon plate between the wires.
  • FIG. 1 illustrates schematically an exemplary integrated micro- electromechanical analyte detection device constructed according to the present invention
  • Fig. 2 illustrates a scanning electron micrograph of an exemplary cantilever beam integrated into a semiconductor chip according to the present invention
  • Fig. 3 illustrates frequency shift of the cantilever illustrated in Fig. 2 after the addition of a single virus particle
  • Fig. 4 illustrates a perspective view of an exemplary cantilever beam and a supporting member in dynamic mechanical vibration
  • Fig. 5 illustrates an exemplary cantilever beam and supporting member in static deflection
  • Fig. 1 illustrates schematically an exemplary integrated micro- electromechanical analyte detection device constructed according to the present invention
  • Fig. 2 illustrates a scanning electron micrograph of an exemplary cantilever beam integrated into a semiconductor chip according to the present invention
  • Fig. 3 illustrates frequency shift of the cantilever illustrated in Fig. 2 after the addition of a single virus particle
  • Fig. 4 illustrates a perspective view of an exemplary can
  • FIG. 6 illustrates graphically a desired resonant frequency and minimum detectable mass for exemplary cantilevers having widths of 1/ra ⁇ and various thicknesses;
  • Figs. 7 A-E illustrate cross-sectional views of an exemplary device in various stages of semiconductor fabrication;
  • Figs. 8 A-B illustrate top views corresponding with the device of Fig. 7A-E in various states of fabrication;
  • Fig. 9 illustrates a system including scanning laser doppler vibrometer determining the resonant frequencies of unloaded and loaded suspended members;
  • Fig. 10 illustrates graphically the resonant frequency shift measured before various loaded cantilever beams measured by the system of Fig. 9;
  • Figs. 11 and 12 illustrate an exemplary method of fabricating an ulfrathin cantilever;
  • FIG. 13 illustrates a perspective view of an exemplary system for measuring the unloaded resonant frequency of an exemplary cantilever using a laser and a detector
  • Fig. 14 graphically illustrates the frequency response for the unloaded cantilever illustrated in Fig. 13
  • Fig. 15 illustrates the measurement of a loaded cantilever illustrated in Fig. 13
  • Fig. 16 illustrates the resonant frequency shift between the cantilevers illustrated in Figs. 13 and 15
  • Figs. 17A-D illustrate mechanical characterization of an exemplary cantilever using known masses positioned at the free end
  • Figs. 18 and 19 illustrate the resonant frequency change after the additional of known masses for the exemplary cantilever illustrated in Figs. 17A-D
  • Figs. 14 graphically illustrates the frequency response for the unloaded cantilever illustrated in Fig. 13
  • Fig. 15 illustrates the measurement of a loaded cantilever illustrated in Fig. 13
  • Fig. 16 illustrates the resonant frequency shift between the
  • FIG. 20-22 illustrate scanning electron micrographs of exemplary embodiments of cantilevers
  • Fig. 23 illustrates a cross-sectional view of the components and layers of an exemplary cantilever having a sensing element at the junction of the cantilever and the support member according to the present invention
  • Fig. 24 illustrates an elevation view of the areas of maximum strain for an exemplary cantilever according to the present invention
  • Figs. 25 A-D illustrate exemplary cantilever structures within fluid channels defined in integrated semiconductor devices
  • Figs. 26A-B illustrate a cross-sectional view and a top view, respectively, of an exemplary cantilever having a transistor region adjacent the area of maximum strain of the cantilever
  • Fig. 27 graphically illustrates the carrier density for the transistor region of Figs.
  • Fig. 28 illustrates DEP motion of particles between an elecfrode and a plate
  • Fig. 29 illustrates the use of DEP to concentrate analytes at free ends of a cantilever pair according to the present invention
  • Fig. 30 illustrates a perspective view of an array of cantilever pairs using DEP to concentrate analytes at the free ends of the cantilevers
  • Fig. 31 illustrates binding of Listeria monocytogenes on various surfaces
  • Fig. 32 illustrates a perspective view of a cantilever having binding agents for a target analyte and using mechanical vibration of the cantilever to aid in the release of non-target species
  • Fig. 32 illustrates a perspective view of a cantilever having binding agents for a target analyte and using mechanical vibration of the cantilever to aid in the release of non-target species
  • Fig. 33 illustrates individual elements for selectively binding a target analyte to an exemplary cantilever
  • Fig. 34 illustrates a schematic diagram of a DEP filter for a fluid channel for a device such as that illustrated in Fig. 1
  • Fig. 35 illustrates a scanning electron micrograph of fluid channels and electrodes for an exemplary DEP filter such as for the exemplary embodiment illustrated in Fig. 1
  • Figs. 36A-C illustrate DEP manipulation and separation of live and dead cells on electrodes
  • Fig. 37A-B illustrate an exemplary DEP filter for selectively capturing a target analyte
  • Fig. 38 illustrates an optical image of a device used for the DEP manipulation illustrated in Figs. 36 A-C
  • FIG. 39A-B illustrate scanning electron micrographs of a device having suspended nano-wire members
  • Figs. 40A-G illustrate perspective views of a process for fabrication of the devices illustrated in Figs. 39 A-B
  • Figs. 41 B, C, E and G are detailed views more fully illustrating the fabrication steps of Figs. 40 B, C, E and G, respectively
  • Fig. 42 illustrates the change in conductance of an exemplary suspended member after cycling between exposure to oxygen and purging in nitrogen
  • Fig. 43 graphically illustrates the cycling illustrated in Fig. 42 with the addition of a heat cycle
  • Fig. 44 illustrates a schematic elevational view of the structure of an integrated detection device having a suspended channel
  • Fig. 45 illustrates a perspective view of an exemplary integrated device having suspended nano-wire members connected by an ultra thin silicone film according to the present invention
  • Fig. 46 illustrates the exemplary device of Fig. 45 after the removal of the silicon film connecting the suspended nano-wires
  • Fig. 47 illustrates a perspective view of an exemplary device having a suspended member with an elecfrode.
  • Corresponding reference characters indicate corresponding parts throughout the several views. The exemplification set out herein illustrates an embodiment of the apparatus and such exemplification is not to be construed as limiting the scope of this application in any manner.
  • An exemplary micro- or nano-elecfromechanical analyte detection device includes a cantilever, nano-wire, or other suspended member for detecting selected biological or chemical species based on the change in resonant frequency, surface stress, or other characteristic of the suspended member upon interaction with a selected analyte.
  • analyte is defined as any biological or chemical species, including, for example, chemical molecules, proteins, bacteria, cells, virus, spores, molds, yeast, microorganisms, and the like.
  • DEP can be utilized to concentrate a selected analyte, for example at the free end of the cantilever or other suspended member.
  • the suspended member may also include a region of a transistor, for example a channel of a field affect transistor to provide on-chip electronic detection of selected analytes.
  • Integrated devices having anays of suspended members for detecting selected analytes located within a fluid channel are also disclosed as well as methods of use and methods of fabrication of the exemplary integrated devices.
  • the recent technological advances in nanotechnology and micromachining of semi-conductor materials present new opportunities for inexpensive, small and sensitive diagnostic or other detection devices capable of rapid and highly accurate detection of minute analytes, for example, sub-100 fg analytes.
  • This disclosure relates to a device that can detect the presence of analytes by either changes in surface stress or detection of added mass through a change in resonant frequency of a suspended structure, for example, a cantilever or bridge structure.
  • a micro- cantilever-based virus detection device and technique which may yield performance characteristics exceeding the sensitivity and specificity of present detection techniques, for example, PCR amplification assays and ELISAs. Calculated limits of detection of the exemplary device are 10 "17 -10 "18 grams (gm) of mass change on the cantilever surface. This translates to the mass of single virus particles.
  • influenza viruses typically are not considered bioterrorism agents, these viruses can be pandemic with devastating casualties.
  • Human rhinoviruses and coronaviruses are among the most common causes of upper- respiratory infectious diseases, and clearly, a device to rapidly measure these agents in air samples can have profound practical and economic implications.
  • a micro-scale, robust, real-time monitoring device based on integrated micro-machined ultrathin cantilever arrays with on-board signal processing for the rapid and sensitive detection of infectious agents in field settings and in primary patient care facilities.
  • an array is specific for specific pathogens and has the sensitivity to detect a single virus or toxin molecule.
  • the exemplary embodiment includes a dielectrophoresis (hereinafter sometimes DEP)-based infectious agent trapping, separation and concentration device for the detection of an airborne virus on functionalized micro-scale cantilevers.
  • DEP dielectrophoresis
  • the exemplary device provides all-electronic detection of single entities in the zeptogram and/or attogram range, has a short turnaround time of detection, and does not require labeling of entities.
  • the device includes an ulfrathin and highly sensitive mechanical structure built on an electronic chip that detects DNA, proteins, viruses, and other analytes in very low concentrations. The device may also detect the composition of analytes. By tailoring the function of the mechanical device surface, specific adsorption of biological or chemical species can be achieved, thus permitting target-specific detection.
  • the exemplary device includes nano-mechanical suspended structures, for example, cantilever ("diving board") or bridge (for example, nano-wire) sensors.
  • An exemplary method of device fabrication produces ultra-thin, and thus, highly sensitive suspended structures (less then 500 nm thick, 130 ⁇ m long, and 20 ⁇ m wide; preferably less than 30 nm thick, 5 ⁇ m long, and 2 ⁇ m wide; more preferable less than 10 nm thick, 3 ⁇ m long, and 1 ⁇ m wide; more preferably approximately 100 angsfroms (A) thick) on a silicon or other semiconductor chip.
  • the exemplary chip provides detection of analytes, for example, DNA, proteins, viruses, and chemicals, in very low concentrations, for example, less than 10 particles per milliliter.
  • suspended structures can detect the presence or binding of analytes on them, for example, by a resulting change in resonant frequency due to the added mass or a resulting change in surface stress due to the change in surface energy.
  • the suspended member of some exemplary embodiments comprise silicon, other semiconductor, metallic conductor, or non-conductive materials may be alternatively used.
  • the suspended structures for example cantilevers, can be arrayed within a micro-fluidic channel where an air or other fluid flow can be passed over the structures.
  • alternating current (ac) DEP signal (with frequency different than the resonant frequency) is applied to electrodes associated with the suspended structures, particles having permittivities greater than the carrier fluid (gas or liquid) will be trapped near the regions of highest field gradient, for example, at the tips of the suspended structures. If the suspended structures are coated with antibodies for specific viruses, only those viruses are captured. Any non-specifically bound particles can be removed by driving the suspended structures in resonance, at an appropriate amplitude, while the specifically bound particles remain attached to the antibodies present on the suspended structures. Then the attached mass can be detected by measuring a shift in the resonance frequency of the suspended structures due to the attached mass. Mechanical vibrations or bending of suspended structures are typically detected by a laser reflection based system, which is not amenable to miniaturization.
  • Capacitive detection methods may not be practical for low capacitance applications due to the signal levels, and hence susceptibility to noise, hi order to obtain useful signal levels, the total capacitance is sometimes increased by increasing the overlap area of the capacitive regions, and by keeping a minimum distance between the sensing elecfrodes.
  • the resonating structure should be as small as possible, thus limiting the available signal for capacitive detection.
  • the electrode used to drive the device may be the same electrode that supplies an electric field for the device.
  • the elecfrode is the gate or the channel of the transistor.
  • an ultratliin suspended silicon bridge for example, a nano-wire, provides the FET channel and an active resonant sensing area.
  • the suspended structure may be grown, for example, by tunnel epitaxial growth, where the growth path of the silicon is restricted and is allowed only in one direction in a cavity.
  • an oxide layer forming a roof of the cavity can be made to collapse due to stiction and subsequent growth of silicon results in very thin single crystal silicon wires suspended between two islands or supporting structures, which may form the source and drain of the FET.
  • the nano- wires can be simply used as resistors. Resistive nano-wires may provide detection of molecules binding on the wire by measuring resistance changes across the nano-wire.
  • the current through the FET is modulated as a function of the distance between the suspended channel and the gate. Also, the current will be a function of the gate bias, as in typical FETs.
  • the piezo-resistive effects on oscillating nano-wire channels also affect the modulation of current due to stress changes that the channel experiences due to mechanical oscillation.
  • the amplitude and frequency of the oscillations can be determined, with high signal levels and high signal-to-noise ratios compared to typical capacitive sensing.
  • an advantage to having the nano-wire or other suspended structure comprise the FET channel is the combined effect on the channel current of both the physical displacement of the channel relative to the gate and the piezo- resistive change from the bending of the channel.
  • the nano-wire comprises the FET gate
  • the piezo-resisitive effect acts on the gate while the displacement effect acts on the channel. Because the oscillation frequency and amplitude can be sensed electically, and high signal-to-noise ratios are expected, very small changes in resonant frequncy, due to the added mass of particles on the nano-wires can be resolved by incorporating feedback.
  • the calculated resonant frequency is about 2.123340 MHz.
  • the minimum detectable frequency is estimated to be, 5.5 x 10 "19 g, which corresponds to the approximate mass of a single protein, therefore smaller than the mass of a single virus particle or cell.
  • the exemplary device can be operated in other modes of electrical operation and/or mass detection, for example, as resistors, transistors, or capacitors with detection of a change in surface potential or a change in mass.
  • one exemplary embodiment of the device may be an integrated micro-electromechanical analyte detection device 100 comprising integrated circuit 102 which has been fabricated to include all device components, for example, fluid channel 104, cantilever arrays 106, 108, and 110, DEP filters, concentrators, or sorters (which may be configured to filter, concentrate, or sort particles; however, herein sometimes collectively referred to as "filters") 112, 114, and 116, signal processing and control circuitry 118, and fluid intake filters 120 and exhaust filters 122.
  • Integrated device 100 is capable of concentrating, capturing, and detecting the presence of selected chemical and/or biological analytes.
  • Cantilevers 106, 108, and 110, or other suspended structure, for example, a bridge has small enough dimensions to detect analytes having sub- 100 fg masses, for example, individual bacterial cells, viruses, or chemical molecules.
  • Integrated device 100 may include structures and circuitry 118 for measuring changes in surface stress, energy, deflection, or resonant frequency of cantilevers 106-110 induced by the added mass or other characteristic of an analyte bound or otherwise interacting with cantilevers 106-110.
  • the resonant mechanical oscillation (or vibration) frequency of cantilever 106 which is shown in Fig. 2 and is supported by support member 130, is decreased by a measurable quantity by the resting of analyte 132 on cantilever 106.
  • analyte 132 is a Vaccinia virus particle.
  • unloaded cantilever beam oscillation 134 has a peak resonant frequency of 1.27 megahertz (MHz) and loaded cantilever beam oscillation 136 has a peak resonant frequency of 1.21 MHz.
  • the shift in resonant frequency is directly related to the mass and therefore the identity of analyte 132 interacting with cantilever 106.
  • individual or sets of cantilevers 106, 108, and 110 may be fiinctionalized in order to provide sensitivity to particular biological or chemical analytes.
  • cantilevers 106-110 may be coated with different antibodies, antigens, or other binding agents to promote binding of a particular analyte to the individual cantilever 106-110, or may be treated with an anti-fouling agent to prevent non-specific or undesirable specific particles from interacting with cantilevers 106-110.
  • DEP filters 112-116 may be utilized in conjunction with cantilevers 106-110 in order to selectively sort, concentrate or capture specific analytes or other particles and direct them toward or away from cantilevers 106- 110.
  • Known nanomechanical sensors can detect single individual biochemical molecules (see for example, M. L. Roukes, Sci. Am. 285, 48 (September 2001, incorporated herein by reference).
  • known microstructures can detect individual bacterial cells (see, for example, Ilic, et al, 2001).
  • free end 142 is located opposite fixed end 144, which is mechanically coupled to support member 146.
  • the way to improve the mass sensitivity is to decrease k as well as increase the resonant frequency. This can be done by decreasing the size of the cantilever beam 140 as well as decreasing thickness 148 of the cantilever beam.
  • Single-crystal materials are generally used to make sensor elements due to their high mechanical quality factor. Silicon, for example, can be used for fabricating sensor elements such as cantilever 140, due to advantages such as low stress and controlled material quality, using currently available VLSI circuit fabrication facilities, miniaturization of devices, high confrol of dimensions, and the economical advantage of batch fabrication.
  • piezo-resistive detection modes are preferred, for example, utilizing piezo-electric element 150 shown in Fig. 5, especially due to the need for arrays of cantilevers and detectors, then silicon provides the capability to realize such elements 150 to detect deflections.
  • Cantilever beams were first introduced to the nanotechnology field with their use as force sensors in atomic force microscopy (AFM) (see Binnig, G., Quate, C. F. and Gerber,. Atomic force microscope, Physical Review Letters, 56:930- 933, 1986, incorporated herein by reference). They have also been used extensively as probes in various other imaging techniques, involving different interactions between the probe and the sample, (see, for example, Wickramasinghe, H.K.
  • AFM atomic force microscopy
  • cantilever beam 140 deflections 154 can be measured in the dynamic mode allowing cantilever beam 140 to be used as a micro-mechanical oscillator sensor.
  • Fabrication of exemplary embodiments of the present device may include selective epitaxial growth (SEG), epitaxial lateral overgrowth (ELO), and chemical mechanical polishing (CMP) for the micro-fabrication of thin single-crystal silicon cantilever beams or other suspended structures.
  • SEG selective epitaxial growth
  • ELO epitaxial lateral overgrowth
  • CMP chemical mechanical polishing
  • the thin suspended structures can be integrated into silicon-based micro-systems such as flow sensors, pressure sensors, bio-chemical sensors and the like.
  • the disclosed fabrication process can produce low stress sub-lOOnrn thickness cantilevers 140 for ultra-high sensitivity chemical and biological detection.
  • the minimum detectable mass with a resonant cantilever beam will depend on the geometry and the material that the cantilever is made of, as well as the minimum detectable frequency change of the cantilever beam. Also, the location of the mass to be detected on cantilever 140 will effect the sensitivity. If the mass is placed at free end 142 of cantilever 150, the device can detect lower masses, since the effective mass will be higher closer to free end 142 of cantilever 140.
  • the resonant frequency of cantilever 140 assuming small deflection on free end 142 of cantilever 140 (the mechanical system can be modeled as a mass and spring for small deflections) can be expressed as: where m eff is the effective mass of cantilever 140, and k is the spring constant.
  • the spring constant will be dependent on the geometry of cantilever 140. For a rectangular cantilever with dimensions 1 as length 158, w as width 156, t as thickness 148, and a material modulus of E, the spring constant can be expressed as,
  • m eJ ⁇ 0.24 • m cantilever .
  • the new resonant frequency can be expressed as: ⁇ y m eff + dm where ⁇ m is the mass added at free end 142 of cantilever 140.
  • Thickness 148 of cantilever 140 largely affects the resonant frequency of the system.
  • the resonant frequency, equation (2) can be expressed in terms of the cantilever properties as, which clearly indicates that, in order to keep the same resonant frequency when scaling down length 158 of cantilever 140 (which would scale down the minimum detectable mass), thickness 148 needs to be scaled down as the square of the scaling factor. So for example to scale down the length 100 times, thickness 148 would need to be scaled down 10,000 times in order to keep the same resonant frequency for cantilever 140.
  • the value of the resonant frequency will play a role for the detection of the frequency. If the resonant frequency is too high, the deflection at fee end 142 will be very small, hence may not be detectable. Also circuitry 118 (Fig. 1) used for detection may not be feasible in the giga-hertz (GHz) range. However, keeping the resonant frequency too low is also not desirable, since this may cause interference with measurements from ambient noise. Thus the design of cantilever 140 should be made for the desired resonant frequency and minimum detectable mass, as shown graphically in Fig. 6 for exemplary cantilevers having width 156 of 1 ⁇ m and various thicknesses 148.
  • the exemplary microfabrication and application of arrays of silicon cantilever beams as nanomechanical resonant sensors according to the present invention are designed to detect the mass of individual virus particles.
  • the dimensions of the fabricated cantilever beams 140 are in the range of 4-5 ⁇ m in length 158, 1-2 ⁇ m in width 156 and 20-30 nm in thickness 148.
  • the virus particles used as an analyte for the exemplary embodiment were Vaccinia virus, which is a member of the Poxviridae family and forms the basis of the smallpox vaccine.
  • the frequency spectra of cantilever beams 140, due to thermal and ambient noise, were measured using a laser Doppler vibrometer under ambient conditions.
  • the change in resonant frequency as a function of the virus particle mass binding on the surface of cantilever beam 140 forms the basis of the detection scheme.
  • a single Vaccinia virus particle has an average mass of 9.5 femto-grams (fg).
  • the exemplary device can be very useful, for example, as a component of biosensors for the detection of air-borne virus particles or other analytes.
  • Known macroscale quartz crystal micro-balance devices for the detection of virus particles require an external power supply and detection of the detachment of virus particles were measured (see, for example, M. A. Cooper, F. N. Dultsev, T. Minson, V. P. Ostanin, C. Abell, D. Klenerman, Nature Biotechnol.
  • the below discussed exemplary embodiment according to the present disclosure includes nanomechanical devices formed on an integrated circuit, with a measurement set-up sensitive enough to measure thermal and ambient noise induced deflections and thus not requiring an external source to excite the cantilever beams (see, for example, B. Ilic, B. Uic, D. Czaplewski, M. Zalalutdinov, H. G. Craighead, P. Neuzil, C. Campagnolo, C. Batt, J. Vac. Sci. Technol. B 19, 2825, 2001, incorporated herein by reference), h order to fabricate exemplary device 200, shown in various stages of fabrication in Figs.
  • P-type (100) 4" silicon-on-insulator (SOI) wafers 202 are used as the starting material, shown in Figs 7A and 8 A, for one exemplary fabrication method.
  • the wafers have SOI layer 204 of 210 nm thickness and buried oxide (BOX) layer 206 thickness of around 390 ran.
  • Wet oxidation followed by buffered hydrofluoric (BHF) etching is performed in order to thin SOI device layer 204 down to 30 nm.
  • RLE reactive ion etching
  • etch window 212 was photolithographically patterned using BHF oxide etch, as shown in Figs. 7C and 8C.
  • vapor phase etching using xenon difluoride available from Xactix, Inc., of Pittsburgh, PA
  • oxide 206 is etched in BHF, rinsed in DI water, immersed in ethanol and dried using critical point drying (hereinafter sometimes CPD), resulting in device 200 as shown in Figs. 7E and 8E.
  • one exemplary embodiment of system 220 associated with device 200 uses microscope scanning laser Doppler vibrometer 222 (Model #MSV-300 available from Polytec PI of Auburn, MA) with a laser beam spot size of around 1-2 ⁇ m.
  • Vibrometer 222 may include, for example, monitor 224, CCD camera 226, microscope 228, scanner controller 230, vibration controller 232, oscilloscope 234, sensor head 235, laser 236, beam splitter 238, detector 240, reference signal 242, and measurement signal 244.
  • analyte for example, purified Vaccinia virus particles in DI water can be introduced over cantilever beams 208 and allowed to incubate for 30 min, following which the cantilever beams are rinsed in ethanol and dried using CPD so as to minimize stiction of the cantilever to the underlying sufaces.
  • the resonant frequency of cantilever beams 208 are then measured again in order to obtain the 'loaded' resonant frequencies of cantilever beams 208 with the analyte.
  • the added mass for the corresponding change in resonant frequency can be determined.
  • the change in mass (placed right at free end 142 of cantilever beam 140 shown in Fig. 4) in relation to a change in resonant frequency can be given as,
  • k is the spring constant of cantilever beam 208
  • f 0 is the initial resonant frequency
  • ⁇ is the resonant frequency after the mass addition.
  • Cantilever beams 208 can be calibrated by obtaining their spring constant, k, using the unloaded resonant frequency measurement f 0 , quality factor Q, and the plan dimensions (length and width) of the cantilever beam.
  • the resonant frequency and the quality factor can be obtained by fitting the vibration spectra data to the amplitude response of a simple harmonic oscillator (SHO).
  • SHO simple harmonic oscillator
  • f frequency in Hz
  • f 0 is the resonant frequency
  • Q is the quality factor
  • a d c is the cantilever amplitude at zero frequency, as described by D. A. Walters, J. P. Cleveland, N. H. Thomson, P. K. Hansma, M. A. Wendman, G. Gurley, V. Elings, Rev. Sci. Instrum. 67, 3583, 1996, incorporated herein by reference).
  • the measured spring constant of exemplary cantilever beams 208 is around 0.005-0.01 N/m.
  • Virus particles shown as 132 in Fig. 2 can be counted by observing cantilever beams 208 using a scanning electron microscope (hereinafter sometimes SEM).
  • the change in frequency upon addition of mass can be detected by a laser Doppler vibrometer 246, as shown in Fig. 9.
  • the effective mass contribution of the viruses can then be calculated based on their relative position from the fixed end of the cantilever beams (for example, fixed end 144 of cantilever 140, shown in Fig. 4).
  • the resonant frequency shift (decrease) versus the effective number of virus particles observed on the cantilever beam can be plotted as shown in Fig. 10.
  • the resonant frequencies may be obtained by fitting the amplitude response of a simple harmonic oscillator to the measured data.
  • the average dry mass for a single Vaccinia virus particle was measured to be 9.5 fg, which is in the range of the expected mass of 5-8 fg (see Bahr, G.F., Foster, W.D., Peters, D.
  • exemplary cantilever beams 208 for a 1 kHz frequency shift is 160 attograms (ag) added mass (6.3 Hz/ag).
  • Another exemplary embodiment according to the present invention integrates device 200 with on-chip antibody-based recognition and concentrators, for ulfra-sensitive detection of air-bome virus particles, for example, device 100 shown in Fig. 1.
  • Another exemplary micro-fabrication technique may be used for fabricating device 300 having ultra-thin cantilever beams 302 in single crystal silicon with no stress.
  • the exemplary process utilizes a technique called MELO (Merged Epitaxial Lateral Overgrowth) and can be regarded as an extension of selective epitaxial growth (SEG) and epitaxial lateral overgrowth (ELO) of crystalline material.
  • SEG is a form of vapor phase epitaxy (VPE), and is a variation on the conventional full wafer epitaxy process known in the art.
  • the epitaxial deposition conditions are adjusted to prevent silicon deposition on the insulator region 304, for example, Si02, while silicon epitaxial growth occurs only on the exposed silicon in the seed windows 305 (see, for example, Bashir, R., Venkatesan, S., Neudeck, G. W. and Denton, J. P. A polysilicon contacted subcollector BJT for a three-dimensional BiCMOS process. IEEE Electron Device Letters 13:392-395, 1992, incorporated herein by reference). Referring to Fig.
  • Cantilevers with thickness ranging from 0.2-0.5 ⁇ m, maximum length of around 130 ⁇ m and widths of around 20 ⁇ m and 10 ⁇ m have been fabricated using the above described method and known surface micro-machining techniques.
  • Mechanical characterization of device 300 can be perfonned by measuring the resonance frequency using thermal noise to excite unloaded cantilever 302 as shown in Figs. 13 and 14, and by adding known micro-sized particles 304 as shown in Figs. 15 and 16.
  • cantilevers 302 can be scaled to thickness of less than 100 nm and can be integrated into micro-fluidic channels (for example, channel 104 of Fig. 1 ) within the substrates for a wide variety of chemical and biological detection applications.
  • thermal mechanical noise is sufficient to oscillate cantilever beams 302 whose deflections can be detected by an AFM that employ the optical lever technique (see, for example, Meyer, G.
  • the cantilever deflection signal was extracted from a Dimension 3100 SPM (see, for example, Meyer, G. and Amer, N. M. Novel optical approach to atomic force microscopy. App. Phys.
  • the technique was also used to then determine the mechanical properties such as stiffness (or spring) constant of the cantilever beams using the added mass (or Cleveland) method (see, for example, Cleveland, J. P., Manne, S., Bocek, D. and Hansma, P. K. A nondestructive method for determining the spring constant of cantilevers for scanning force microscopy , Review of Sci. Inst., 64:403- 405, 1993, incorporated herein by reference) was used.
  • polystyrene spherical beads 308-314 of known mass shown in Figs. 17A-D, were placed at the free ends of cantilever beams 302 using, a micromanipulator.
  • Spherical beads of diameter of around 5.48 ⁇ m and 3.18 ⁇ m were used in the exemplary system.
  • the masses of the beads can be calculated to be in the range of 90.5 picograms (pg) and 17.7 pg, respectively.
  • the diameter Due to variation in the diameter of individual beads from the stated specifications of the manufacturer's values, the diameter can be measured using an optical microscope.
  • the change in resonant frequency, f ls due to addition of a single mass, M 1? as shown in Fig. 19, can be measured and used to detect and extract the mass of the bead 308.
  • Table I presents the values of the measured spring constant, effective mass and mass resolution for previously microfabricated cantilever beams 320 and 330 shown in Figs. 21 and 22, respectively. These masses were detected by measuring the vibrations of the cantilever using optical means. Cantilever 320 shown in Fig. 21 was placed in a modified AFM system and the laser reflection off the cantilever surface was detected using a quad photo-detector, thus allowing us to measure the vibration of the cantilever 320, for example, as shown for cantilever 302 in Figs. 13 and 15, however, other techniques to detect the vibrations may be used. Table 1. Planar dimensions and measured values of spring constant and effective mass.
  • cantilevers 320 and 330 are capable of detecting masses down to about lOfg using a detection resolution of 1Hz change in frequency.
  • the detection of such a small frequency change likely requires driving the cantilevers using forced excitations rather than thermal noise sources.
  • a piezoelectric film connected to an external signal generator to sweep the signal frequency and measure the resonant frequency of cantilevers 320 and 330 could be utilized.
  • the design of cantilever beams is critical to the overall system design and the ability to detect single virus particles or other minute analytes. Using equations (6) and (7), the minimum detectable mass can be calculated as a function of the cantilever geometry, for example as shown in Fig. 6.
  • the minimum detectable mass is decreased.
  • the size of a cantilever would need to be reduced to 3um wide x about lOum long.
  • the resonant frequency would be larger than 1-5 MHz if a thickness of 0.5um is used. However, if the thickness is reduced down to 20nm, them the resonant frequency is also reduced to below 100kHz, which likely would be easier signal to detect and process.
  • silicon-based cantilevers which can included integrated piezo-resistive elements to provide an electrical output.
  • SOI silicon-on-insulator
  • MLO merged epitaxial lateral overgrowth
  • CMP chemical mechanical polishing
  • a tunnel is defined using deposited films and selective growth of silicon is performed to fill the tunnel with single crystal material, forming an ultra-thin cantilever or other suspended structure.
  • the films around the silicon can then be removed, thus releasing these nano-mechanical structures.
  • Another exemplary device 400, shown in Fig. 23, according to the present invention may, include piezo-resistive element(s) or fransistor region element(s) 404 integrated into suspended member 402.
  • Element 404 may be used for the detection of the deflection, vibration, or surface stress or energy of suspended member 402.
  • Element 404 can be grown selectively at the anchored or fixed end 406 where the stress is maximized, specifically, where suspended member 402 joins support member 408 of device 400, especially at the surfaces of the junction of members 402 and 408.
  • the growth of selective elements 404 enables a device such as device 100 (Fig. 1) or the devices shown in Figs. 25A-D having an array of cantilevers or other suspended members for detecting selective analytes.
  • cantilevers or other suspended structures can be fabricated with lateral dimensions limited only by the resolution of the photolithography used, and thickness defined by thin silicon-on- insulator (SOI) layers, formed by wafer bonding, separation by implanted oxygen (SIMOX), or confined lateral selective epitaxial growth (CLSEG).
  • SOI silicon-on- insulator
  • SIMOX separation by implanted oxygen
  • CLSEG confined lateral selective epitaxial growth
  • integrated piezo- resistive or fransistor detection methods using, for example, device 400 shown in Fig. 23, eliminate the need for external components by electrically measuring the surface strain or other characteristic induced by deflection or vibration of cantilever 402.
  • exemplary device 400 having a piezo-electric element 404 the resistance of element 404 changes in response to strain, which is induced in cantilever 402 by deflection.
  • cantilever 402 is deflected, a strain gradient is induced throughout the body of the cantilever, with zero strain at the center.
  • One surface is in tension (positive strain), while the opposite surface is in compression (negative strain).
  • piezo-electric element 404 Since the response of piezo-electric element 404 to strain is approximately symmetric about zero strain, an element that penetrates through the entire cantilever would not respond to deflection. The response of the top half of the element would be equal and opposite to that of the bottom half, resulting in no net response. Therefore, to be most effective piezo-electric element 404 must be restricted to the near surface region of cantilever 402. Nvhile this is simple to accomplish by implantation and/or diffusion into relatively thick cantilevers, it can be very difficult to achieve in sub-100 nm thick cantilevers.
  • exemplary device 500 shown in Figs. 26 A-B having transistor element 510 at the junction of cantilever 502 and support member 508 offers a possible solution to this limitation.
  • device 500 may utilize ultra-thin channel 510 of field effect fransistor (FET) 512, for example a MOSFET also having gate 504, source 514, drain 516, and source/drain region 511, as a piezoelectric sensing element.
  • FET field effect fransistor
  • Conductive channel 510 of FET 512 may be confined to within a few nanometers of the surface, thus in the region of maximum strain upon deflection of cantilever 502.
  • FET field effect fransistor
  • a device such as a MOSFET an advantageous deflection sensor for ultra-thin cantilevers.
  • Fig. 27 was generated for device 500 by simulation using SCRED 2.0, a tool which self-consistently solves the Schr ⁇ dinger and Poisson equations.
  • the structure simulated is a dual gate n-channel MOSFET with a background concentration of 10 16 cm "3 , a body thickness of 10 nm, an oxide thickness of 1.5 nm, and a metal gate with a work function of 4.6 eN.
  • the back gate was held at a constant zero bias, while the top gate was swept from zero to two volts.
  • FIG. 26A-B An exemplary fabrication method for device 500 shown in Fig. 26A-B may be: 1. Begin with an SOI wafer. 2. Thin the SOI in the area where cantilever 502 will be formed using an anisotropic etch. This forms an abrupt step between support member 508 and the top surface of cantilever 502. 3. Oxidize the silicon surface to form gate oxide 518. 4. Conformally deposit polysilicon gate material 504. 5.
  • polysilicon gate material 504 Anisofropically etch polysilicon gate material 504 through the entire deposited thickness. This leaves a sidewall of polysilicon on any exposed vertical surface, with a lateral width approximately equal to the deposited thickness. 6. Implant the source 514 and drain 516 regions appropriately, using polysilicon gate 504 as an implantation mask. 7. Release cantilever 502 by wet or dry etching. Another potential advantage of using a MOSFET-based deflection sensor such as device 500 is that by externally modulating the gate bias it may be possible to directly obtain the vibrational frequency spectrum of cantilever 504, through a known technique, for example heterodyne mixing, or other known signal processing techniques.
  • Heterodyne mixing uses the fact that the product of two sinusoidal signals is the sum of two sinusoids with frequencies equal to the sum and difference of the original sinusoidal frequencies. By filtering out the sum portion of the signal, the result is a lower frequency signal with the same information content as the original signal. Heterodyne mixing is well know from radio receivers that "mix down" the incoming RF signal to an "intermediate frequency” that is more easily processed by downstream circuitry. Another example of the use of mixing is the lock-in amplifier, in which a reference signal is used to stimulate a device under test (DUT). The output of the DUT is then multiplied, or “mixed,” with the reference signal, and subsequently filtered to remove the sum component.
  • DUT device under test
  • g m o is the unperturbed fransconductance
  • ⁇ s is the linear coefficient of channel mobility strain dependence
  • S(t) is the strain in the channel 510 region. Note that the strain and therefore the fransconductance are explicitly written as a function of time, ubstituting this expression for fransconductance in drain 516 current expression yields:
  • Equation (14) is the unperturbed response of MOSFET 512 under the application of a small signal.
  • the second term is the mixing term that is of interest in this discussion.
  • fransistor element 510 is located on another suspended structure, such as a bridge, rather than cantilever 502.
  • fransistor element 510 or 511 located on cantilever 502 may be a gate which cooperates with a channel located on another portion of device 500 such that the distance between the gate and channel as cantilever 502 deflects induces changes in the channel current.
  • Dielectrophoresis may also be utilized in an exemplary embodiment of the present invention. DEP is the franslational motion of neutral particles in a non-unifonn field region (see, for example, Pohl, H. A. The motion and precipitation of suspensoids in divergent electric fields, J. Appl. Phys. 22 (1951) 869- 871, incorporated herein by reference).
  • DEP has been demonstrated to be able to capture and separate biological materials in fluid using micro-fabricated elecfrodes with ac electric fields (see, for example, Li, H. and Bashir, R. Dielectrophoretic separation of live and heat-inactivated Listeria on microchips. Sensors and Actuators, hi press, 2002a, incorporated herein by reference).
  • Neutral particles including, for example, biological cells
  • DEP forces can occur on cells when a non-uniform electrical field interacts with the field-induced electrical polarized particles, as shown in Fig. 28.
  • the time-averaged DEP force F for a dielectric sphere immersed in a medium in constant field phases in space is given as:
  • DEP may be incorporated into exemplary device 600, as shown in Fig. 29.
  • Suspended, silicon members, for example, opposed cantilever pair 602 and 604 are used for a dual purpose: (1) as electrodes for DEP in order to capture air-borne or aerosolized virus particles and (2) as a mass sensor in order to detect the virus particles.
  • Cantilevers 602 and 604 are scaled down in size by the exemplary processes described above to provide the mass sensitivity necessary for detection of individual virus particles.
  • an ac electric field or another predetermined waveform is applied to cantilevers 602 and 604, which may be electrically coupled to elecfrodes 608 and 606, respectively, biological cells 616 and 618, or other analytes, will be captured at regions 614 with the largest gradient of the field.
  • the largest gradient of the field is located at free ends 610 and 612 of cantilever beams 602 and 604, respectively, as shown in Figs. 29 and 30, which, as discussed above, is also the preferred mass location for maximum change in resonance frequency for cantilevers 602 and 604.
  • the technique of DEP and manipulation of analytes by electrical forces provides a unique means to confrol the separation dynamics of biological agents and other particles.
  • the method has numerous biological and medical applications, e.g., identification and characterization of individual cells, purification of cell subpopulations from mixture suspension, etc., especially, for example, with integrated use of DEP separation and trapping of analytes such as air-borne infectious agents, combined with the detection of the analytes using micro-cantilevers.
  • the cell membrane consists of a very thin lipid bi-layer containing many proteins and is highly insulating with a conductivity of around 10 "7 S/m, while the cell interior contains many dissolved charged molecules, leading to a conductivity as high as lS/m.
  • the cell membrane Upon death, the cell membrane becomes permeable and its conductivity can increase by a factor of 10 4 due to the cell contents exchanging freely material freely with the external medium through the small pores on the membrane.
  • This large change in the dielectric properties on cell death indicates a large change in the dielectric polarizability.
  • a large difference in DEP responses positive and negative respectively
  • a selective separation can be achieved between live and dead cells.
  • DEP is particularly useful in the manipulation and separation of microorganisms and has been employed successfully in isolation and detection of sparse cancer cells, concentration of cells from dilute suspensions, separation of cells according to specific dielectric properties, and trapping and positioning of individual cells for characterization (see, for example, Wang, X., Huang, Y., Gascoyne, P. R. C. and Becker, F. F. IEEE Transactions On Industry Applications 33:660-669, 1997; Huang Y, H ⁇ lzel R, Pethig R, Wang X-B, Differences in the ac electrodynamics of viable and nonviable yeast-cells determined through combined dielectrophoresis and electrorotation studies. Phys. Med. Biol., 37:1499-1517.
  • the first report of molecular-scale particle manipulation was that of Washizu et al,, 1990, where they reported DNA fragments of 48.5 kilobase pairs (or about 30 mega-daltons (mDa) in molecular weight, which would be ⁇ 30 nm radius if the molecule were closely packed into a sphere were trapped and stretched (approximately 16 ⁇ m long) by positive DEP.
  • Attachment of the analytes, for example, proteins, on micr -fabricated surfaces is important to the success of applications such as protein chips, and the attachment and capture of cells on micro-fabricated surfaces, such as the cantilevers of the Exemplary embodiments according to the invention.
  • the attachment of proteins on surfaces is complex when compared to the attachment of DNA to surfaces. Proteins have to be attached in such a way that their structure and functionality should be retained.
  • the attachment of antibodies and proteins has been demonstrated on micro-fabricated surfaces using functional groups such as silane (see, for example, Britland, S., Arnaud, E.
  • Patterning of ligands has also been demonstrated using alkenethiolate SAMs, which were produced on Au layers and then ligands such as biotin was printed on the SAMs using micro-contact printing (see, for example, Lahiri, J., Ostuni, E., and Whitesides, G. M., Patterning ligands on reactive SAMs by microcontact printing, Langmuir, 15, 2055, 1999b, inco ⁇ orated herein by reference). Proteins micro-arrays have been demonstrated where proteins were immobilized by covalently attaching them on glass surfaces that were treated with aldehyde-containing silane reagents (see, for example, MacBeath, G. and Schreiber, S.
  • the Avidin was adsorbed onto the microchips from phosphate buffered saline or from PBS to which ammonium sulfate had been added. Avidin was also adsorbed onto bovine serum albumin (BSA) coated surfaces of oxide and platinum. Fluorescence microscopy was used to confirm adso ⁇ tion of labeled protein, or the binding of fluorescently labeled biotin onto previously adsorbed, unlabeled Avidin. When labeled biotin in PBS is presented to Avidin adsorbed onto a BSA coated microchip, the fluorescent signal was significantly higher than for Avidin adsorbed onto the biochip alone.
  • BSA bovine serum albumin
  • biotinylated BSA which adsorbs strongly onto a C 18 modified silica surface at pH 7.2 through hydrophobic interactions, has been used for analyte specific bonding. Its activity is maintained, as indicated by the strong adso ⁇ tion of streptavidin, whiph was validated through fluorescence microscopy (see, for example, Huang, T., Gaba, A., Gomez, R., Bashir, R., Sturgis, J., Robinson, J. P., Ladisch, M.
  • the SiO 2 surfaces are first derivatized with octadecyltrichlorosilane (ODTS) to form a C 18 surface.
  • ODTS octadecyltrichlorosilane
  • BSA Biotinylated bovine serum albumin
  • streptavidin can be adsorbed onto the biotinylated BSA.
  • streptavidin The role of streptavidin is to capture biotinylated monoclonal antibody and orient it in a way that enables the antibody to contact and capture Listeria monocytogenes.
  • an exemplary embodiment according to the present disclosure addressed a sandwich scheme for the C 18 modified surface using non-specific binding of Listeria monocytogenes and Listeria monocytogenes binding to biotinylated antibody cl le9 using PBS buffer at pH 7.2 and incubated for 2 hours, the comparisons for which are shown in Fig. 31.
  • An additional exemplary embodiment according to the present disclosure includes covalent attachment techniques of Immobilizing IgG C11E9 antibodies on a silicon dioxide surface for use in a biosensor to detect capture of pathogen Listeria monocytogenes (see, for example, Gaba, A., Sturgis, J., Robinson, J.
  • Various surface derivatization approaches can be utilized to anchor coronavirus specific antibodies to silicon cantilevers. For example, utilizing several heterobifunctional cross-linkers and micro-patterning of cantilever surfaces with the coronavirus specific antibodies.
  • exemplary cantilevers according to the present disclosure include well depths of about 14-16 ⁇ m, the problem of stiction could occur and hence surface modifications can be introduced to minimize such stiction of cantilevers to subsfrate after the BHF etching of the oxide encompassing the cantilevers.
  • the exemplary embodiment utilizes a coating including hydrophobic self-assembling mono layers (SAM) films.
  • SAM self-assembling mono layers
  • 1.0 mM of octadecyltrichlorosilane (OTS) in 2,2,4-trimethylpentane (isooctane) may be used as the solvent to form the SAM coatings.
  • OTS octadecyltrichlorosilane
  • isooctane 2,2,4-trimethylpentane
  • Other anti-stiction methods and surface modifications may alternatively be utilized.
  • Another exemplary embodiment utilizes covalent attachment techniques of Immobilizing IgG C11E9 antibodies on a silicon dioxide surface for use in a biosensor to detect capture of pathogen Listeria monocytogenes (see Gaba, A., Sturgis, J., Robinson, J. P., Gomez, R, Bashir, R., Ladisch, M. R.
  • a platform for placing more selective antibodies for L. monocytogenes and other target cells may be extended to the detection of virus particle. Eliminate the non-specific adso ⁇ tion of the virus particles in crucial. Therefore, anti-fouling or blocking layers such as BSA and other bio-chemical layer may be utilized. Additionally, DEP forces for the movement and manipulation of the virus particle may be utilized, if they are non-specifically adsorbed on the antibody coated cantilevers.
  • An ac signal or other waveform may be pulsed at these elecfrodes in such a way that could effectively sweep the virus particles away from or toward the cantilevers, as desired.
  • the vibrations of the cantilever could also provide a novel method to detach the biological entities captured on the cantilever beam surfaces.
  • the non-specifically bound species could be detached first, for example, at approximately 0.1-1.0 pN when an anti-fouling agent is utilized, while the specifically bound entities will not be removed, as shown in Fig. 32.
  • the amplitude of vibrations will also increase, resulting in the release of specifically bound entities, for example, at approximately 200 pN.
  • An overall system device for example device 100 shown in Fig.
  • device 100 may utilize many of the above described exemplary embodiments, including those further described below. Additionally, as shown in Fig. 33, antigen-binding sites, antibodies, linking molecules, and blocking agents may be used in conjunction with the suspended member in order to functionalize it for a specific entity.
  • device 100 may be micro-fabricated with input port tube 124 with mechanical filter 120 that only lets particles less than 0.5 urn, for example, pass through. Once particles are in the air sfream in fluid channel 104 of integrated circuit 102, and they will contact DEP potential wells from the particle sorters 112 and 114, where they would get diverted into the appropriate fluid channel chambers having an array of cantilevers 106-110.
  • Analytes with different dielectric properties or size will pass through the first sorter 112 and can be selected by the second sorter 114. Only one sorter 112 and 114 and one cantilever chamber may also be used. Cantilevers 106-110 may be coated with antibodies for specific virus or other analytes and will therefore capture these specific analytes. Another DEP filter 116 after the cantilevers can be used to confine or concentrate the analytes in the region close to cantilevers 106-110 to maximize capture by cantilevers 106-110. Optical or electrical measurement of the resonant frequency change can detect the binding of single virus particles, for example, electrical measurement utilizing signal processor and confrol circuitry 118 implementing any of the above discussed embodiments for electromechanical sensing.
  • Mesh filters 122 at the output port of device 102 is useful to ensure that there is a mechanism to contain analytes inside device 102 if infectious agents are found in the air sample.
  • HEPA type filters and the rotary rod method for capturing viral particles that are in the range of 0.5um or less in size my be used to concentrate analytes.
  • an exemplary apparatus for validating the inventive concept may consist of a closed plastic box where known quantities of a virus can be introduced into the air, and the air then circulated over the filter or capture device. The selection of the type of blower which will push the air across the filter will be important as will the size of the plexiglass box, which may be initially fabricate to contain half a cubic foot of air.
  • Viruses are generally 0.05-0.1 microns and hence, the exemplary apparatus may use a surrogate viruses (that are not pathogenic) and focus on particle sizes in the 0.05-0.1 micron range. Calibration of the system may be done using inert particles (that are typically used for calibrating particle size instruments) in order to begin to identify optimal linear velocity through the filter/membrane, and determine the effectiveness of particle capture. Operation using surrogate virus may use standard culture methods. In addition, a labeling compound and fluorescence microscopy can be used to identify virus that are trapped in the filter or membrane. Air-spray with aerosolized virus particles may also be directly injected into device 100 at controlled rates for the pu ⁇ ose of performing proof-of-concept validation.
  • Airborne microorganisms bacterial, fungal spores, viral particles and pollen
  • Airborne microorganisms are known to cause various health effects, including infection, hypersensitivity, toxic reactions, irritations, and inflammatory response (see, for example, Agranovski, I. E., N. Agranovski, T. Reponen, K. Willeke, and S. A. Grinshpun, Development and Evaluation of a New Personal Sampler for Culturable Airborne Microorganisms, Atmospheric Environment, 1-10, 2001, inco ⁇ orated herein by reference).
  • Virus particles can range from 0.05-0.1 ⁇ m in size and are highly resistant to extreme environmental conditions. To detect a few harmful spores from the air in a regular-sized office room represents a very challenging technological endeavor.
  • the airborne particles need to be first pre-processed in order to isolate the bio-aerosol.
  • the air samples in a room need to be pre-processed in order to isolate the airborne microorganism.
  • a coarse filter can be used to filter out the larger airborne particles (see, for example, Battarbee, J. L., N. L. Rose, and X. Long, "A Continuous, High Resolution Record of Urban Airborne Particulates Suitable for Retrospective Microscopical Analysis," Atmospheric Environment, 31(2), 171-181, 1997, inco ⁇ orated herein by reference).
  • many other non-biological particles also have ⁇ m-size characteristics.
  • Cylindrical traps work by coating the inner surface of a cylinder fist with a sticky film such as cellulose. Then, the pre-processed air is feed onto the surface.
  • the viral capture efficiently is heavily influenced by the airflow velocity (see, for example, Griffiths, W. D., I. W. Stewart, S. J. Futter, S. L. Upton, and D. Mark, "The Development of Sampling Methods for the Assessment of Indoor Bioaerosols," J. Aerosol Sci., 28(3), 437-457 (1997); Maus, R., and H.
  • DEP filters or valves which are selective to any object having a dielectric constant different from the medium, for the capture of microorganisms may be utilized instead of the above mentioned approaches.
  • polystyrene beads can be separated from buffer, and cells or spores can be separated from water (see, for example, Li, H. and Bashir, R. Dielectrophoretic separation of live and heat-inactivated Listeria on microchips.
  • an exemplary device may utilize DEP filter 700 to selectively capture a particle of interest inside the integrated circuit.
  • Polystyrene beads 704 (coated with antibodies selective to target species) are flowed through the integrated circuit.
  • An array of elecfrodes 702 may be used to generate an AC electric field at a frequency on the order of 1 MHz, and a peak intensity of at least 106 V/ ⁇ m.
  • beads 704 approach electrodes 702, they will experience a DEP force which repels them away from regions where the gradient of the electric field is maximum (at the edges of electrodes 702). If this force is equal to or larger than the drag force exerted on beads 704 by the liquid flow, beads 704 remain trapped in the chamber (along with the bacterial cells they carry) while everything else in the sample flows out of the chip.
  • FIG. 37A shows 2.38 ⁇ m (diam.) beads 704 flowing freely through the chip while the electric field is off.
  • Fig. 37B shows beads 704 accumulating in the middle of electrodes 702 when the field is turned on (beads 704 cannot cross the edges of electrodes 702, where the DEP force if maximum).
  • the techniques described above can be used to concentrate bacteria, cells, viruses, D ⁇ A, or proteins, as long as their dielectric constant is different than the dielectric constant of the medium that they are suspended in. Hence, this technique can be used to separate virus particles from other particles in air, or used to concentrate particles of a particular type in a micro-chamber of interest.
  • Exemplary interdigitated microelecfrodes according to the present invention can be manufactured on silicon subsfrate using standard photolithography.
  • the electrode material can be gold, 1000 A thick, magnetron sputtered onto a 100 A thick seed layer of chromium.
  • the width of the elecfrodes and the spacing between two adjacent electrodes can be simulated so as to produce enough DEP force to stop the particle in the air-flow.
  • the electrodes can be connected to an arbitrary wavefonn generator as the AC signal source by attaching two conducting wires to the contact pads.
  • Fluorescently labeled aerosolized samples can be allowed to enter into the DEP chambers and a sinusoidal signal with varying voltage and frequency values can be applied to the electrode array.
  • the electrokinetic behaviors can then be viewed on a TV monitor through a CCD digital camera on a fluorescence microscope.
  • the filters can be designed using ANSYS or other finite element software, which will allow the modeling of the ac fields as a function of the electrode geometry and spacing.
  • the DEP forces will be calculated.
  • Another exemplary device 700 according to the present invention includes an ultra-thin suspended silicon member 702, for example, a nano-wire, providing an active resonant sensing area, for example a fransistor channel (or gate) in the case of device including a field effect transistor.
  • the suspended member 702 may be grown, for example, by tunnel epitaxial growth, where the growth path of the silicon is restricted and is allowed only in one direction in a cavity. During the formation of the cavity, an oxide layer forming a roof of the cavity can be made to collapse due to stiction and subsequent growth of silicon results in very thin single crystal silicon wires suspended between two islands, which may form the source and drain of the FET. Alternatively, by not doping the source and drain regions, the nano- wires forming suspended members 702 can be simply used as resistors. Resistive nano-wires provides detection of molecules binding on the wire by measuring resistance across the nano-wire.
  • the current through the FET specifically the FET channel formed by member(s) 702 is modulated as a function of the distance between the suspended member 702 and the gate. Also, the current will be function of the gate bias, as for typical FETs.
  • the piezo-resistive effects on oscillating nano-wire members 702 also affect the modulation of current due to stress changes that the nano-wire member experiences from mechanical oscillating.
  • the amplitude of the oscillations and the frequency can be determined, with high signal levels and sinal-to-noise ratios compared to pure capacitative sensing.
  • the oscillating frequency and amplitude is sensed electically, and high signal-to-noise ratios are expected, very small changes in resonant frequncy, due to the added mass of particles on nano-wire members 702 can be resolved by inco ⁇ orating feedback.
  • the resonant frequency is 2.123340 MHz.
  • the novel device can be operated in other modes of electrical operation and/or mass detection, for example, as resistors or transistors, with detection of a change in surface potential or a change in mass. If a resistor is fabricated, then binding of entities on the surface of the nano-wire members 702 will result in change in electrical resistance of the resistor due to change in surface properties. Members 702 can be thermally excited and the mechanical resonance can be measured electrically using capacitive technique and the bottom gate.
  • the resonance frequency will change.
  • the resonance could also be measured using the changes in resistance value of the nano-wire member.
  • nano-wire 702 can be capacitively excited using the bottom gate and the mechanical resonance can be measured electrically using the changes in resistance value of the nano-wire upon mass addition to the nano-wire member. If device 700 includes an FET, then binding of analytes on the surface of the suspended nano-wire members 702 will result in change the source/drain current due to changes in surface potential.. Once the suspended nano-wire member vibrates (thermal or capacitive excitation), the piezo-junction effect will change the source drain current and that could be electrically detected.
  • the thermal vibrations can also be detected capacitively using the bottom FET gate.
  • the suspended nano-wire member 702 can be driven into resonance using the bottom gate and the changes in source/drain current can be measured as a means to detect the vibration and thus change in resonant frequency, as discussed above for cantilever devices.
  • carbon nano-tubes and silicon nano-wires have been demonstrated as single molecule biosensors, the fabrication methods that have been used for creating those devices are typically not compatible with modem semiconductor manufacturing techniques and their large scale integration is problematic.
  • the exemplary method of fabrication of silicon nano-wires at precise locations described below overcome those limitations.
  • the exemplary method of fabrication allows for the realization of truly integrated sensors capable of production of dense arrays.
  • exemplary device 800 may be perfonned on p-type low doped silicon wafers 802.
  • a 2000 A thick oxide 804 can be grown by wet oxidation for substrate isolation.
  • a sacrificial layer 806 of amo ⁇ hous silicon with 100 A of thickness can next be deposited and defined lithographically on the silicon dioxide layer 804, as shown in Fig. 40A.
  • Another 4000 A thick oxide layer 810 can then be deposited using plasma enhanced chemical vapor deposition (PECVD).
  • PECVD plasma enhanced chemical vapor deposition
  • Via holes 808 that will subsequently be filled with silicon and act as the FET source and drain regions, can be etched using a reactive ion etch, as shown in Figs. 40 A and 41 A.
  • the sacrificial layer of polycrystalline silicon can then be removed selectively by wet etching using tefra-methyl-ammonium-hydroxide. The removal of the sacrificial layer defines a gap between the thermally grown oxide and deposited oxide.
  • Via holes 812 are then used as a mold for the epitaxial silicon to grow through which will later form the suspended nano-wire members.
  • a seed hole 816 was wet etched in thermal oxide 804 on the source side down to the silicon surface 802, as shown in Fig. 40D, in order to grow epitaxial silicon by a CLSEG with no intentional doping.
  • the exemplary fabrication process yields good quality single crystal silicon with low n-type doping ( ⁇ 10 16 cm "3 ).
  • Epitaxial silicon grows through via holes 812 at the edges of the collapsed oxide bridge 814 as well as at the interface of the collapsed region.
  • the silicon grown in the interface region between via holes 812 forms a 6-7 nm thick plate.
  • the excess silicon remaining on the surface can be removed by chemical-mechanical polishing as shown in Figs. 40E and 41E.
  • a high dose n-type blanket implant can be performed in order to form conductive source and drain regions 820 and 822, respectively.
  • a high temperature anneal can be performed to activate and drive in the implanted dopant, and to densify the PECND oxide.
  • Contact holes can next be wet etched in the oxide to access the silicon source and drain regions 820 and 822.
  • a 200 A of chromium, followed by 2500 A of gold was evaporated and patterned to define elecfrical contacts, as shown in Figs. 40E and 4 IE.
  • the final step in the exemplary fabrication process is to uncover (thus suspending) the silicon nano-wire members 824 by removing the encapsulating oxide by un-patterned wet etching in buffered hydrofluoric acid, shown in Fig. 40F.
  • the fabrication method yields a film of silicon about 7 nm thick in the collapsed regions, and 50 nm diameter suspended nano-wire members s at the edges of the film between the anchors.
  • Etching in BHF for 6 minutes removes the oxide covering on the device, leaving the plate and wires in place, with a supporting oxide layer below, as shown in Fig. 45.
  • a longer BHF etch (14 min) removes all remaining oxide as well as the thin silicon film 823 (Fig. 45) between the wires, resulting in the formation of suspended nano-wire members 824 (shown in Fig. 46, and shown in Fig. 39B as 702).
  • the samples can be soaked in methanol without drying to ensure the complete displacement of the water, and then air dried. Experiments have been performed on both the plate and wire structures in order to verify the feasibility of using the structures as field effect sensors.
  • Fig. 42 shows a decrease in the conductance of the plates upon exposure to oxygen, and recovery of the conductance after purging in nitrogen.
  • the baseline conductance of device 700 shifts for the same amount of recovery time, indicating some irreversibility of the adso ⁇ tion.
  • they were heated in vacuum at elevated temperatures (80-90°C).
  • a similar set of experiments was also performed on the nano-wire members 702. The nano-wire members 702 showed similar response to exposed oxygen, however simply purging the test chamber was not sufficient to recover the conductance of the wire device 700.
  • Device 700 conductance continued to decrease at a slower rate after the nitrogen purge was started until the chamber was evacuated. After evacuation of the chamber the conductance stabilized at a constant value, hi order to desorb the oxygen, the nano-wire device 700 was heated up to 80°C in vacuum and cooled back to room temperature. Upon this procedure device 700 conductance increased due to deso ⁇ tion of oxygen gas from the wire surface.
  • a device cycling experiment is shown in Fig. 43. The results obtained from gas phase measurements are very encouraging, and suggest that the use of a top-down nano-fabrication technique as disclosed above is capable of producing nanoscale sensors for the detection of very low concentrations of analytes.
  • exemplary device 900 includes a suspended structure, for example cantilever 902, coupled to support structure 906 and having free end 904 and elecfrode 908.
  • Electrode 908 is capable of functioning as a terminal for DEP.
  • Cantilever 902 may comprise a semiconductor, a metallic conductor, or a non-conductor, or any combination thereof.
  • Electrode 908 may be coupled with or defined by cantilever 902.
  • elecfrode 908 may comprise the entire extent of or a portion of cantilever 902.
  • elecfrode 908 may comprise a thin metal or degeratively doped semiconductor layer or otherwise defined portion of cantilever 902.
  • electrode 908 my include a conductive material extending to free end 904 of cantilever 902, forming a terminal for concentrating the selected analyte at free end 904.

Abstract

An exemplary micro or nano-electromechanical analyte detection device (100, 200, 300, 400, 500, 600, 700, 800, 900) includes a cantilever (106, 108, 110, 140, 208, 302, 320, 402, 502, 602, 604, 902), nano-wire (702, 824), or other suspended member (330) for detecting selected biological or chemical species (132, 304, 618) based on the change in resonant frequency, surface stress, or other characteristic of the suspended member upon interaction of a selected analyte. Dielectrophoresis is utilized to concentrate a selected analyte, for example at the free end (142, 306, 610, 612, 904) of the cantilever or other suspended member. The suspended member may also include a region (404, 510, 824) of a transistor (512), for example a channel of a field affect transistor to provide on chip electronic detection of selected analytes. Integrated devices having an array of suspended members for detecting selected analytes located within a fluid channel (104) are also disclosed as well as methods of use and methods of fabrication of the exemplary integrated devices.

Description

DENICE FOR DETECTING BIOLOGICAL AND CHEMICAL PARTICLES
CROSS REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 60/483,274, filed June 27, 2003, and to U.S. Provisional Application No. 60/551,131, filed March 8, 2004, the complete disclosures of which are expressly incorporated by reference herein.
FIELD OF THE INVENTION This disclosure relates to devices, methods of use, and methods of fabrication of such devices, for use in selectively concentrating, capturing, and detecting the presence of species, for example, chemical molecules such as gases or proteins, biological species, such as cells (e.g., bacterial cells and/or eukaryotic cells) or virus particles, spores, molds, yeast, microorganisms, and the like. The invention is disclosed in the context of arrays of micro- and nano-scale structures intended to be used, for example, as mass sensors to detect the presence and/or concentration(s) of the selected species. However, it is believed to be useful in other applications as well.
BACKGROUND Detection of very small quantities of chemical and biological species, for example, virus particles, bacteria, chemical molecules, or any other analyte, is important in industrial, environmental, human health, and bio-security applications, to name a few. Various methods, devices, and structures for the detection of chemical and biological species utilize a variety of technologies and methods. However, many prior methods, devices, and structures lack the ability to detect low concentrations of such species, and require long turnaround times. Many such methods and apparatus also generally require chemical or biological labeling of the species to be detected. All of these characteristics can complicate the detection process. Currently, a few reliable detection methods for some minute analytes do exist. For example, in the field of viral detection, methods for detection of coronaviruses include virus isolation, electron microscopy, and immunofiuresence assay with human coronavirus specific fluorescein (hereinafter sometimes FITC)- conjugated antibodies. Additionally, some molecular detection methods utilize polymerase chain reaction (hereinafter sometimes PCR) amplification for screening for viruses such as coronaviruses. However, for the detection of many viruses, including coronavirus, the above methods typically require dedicated equipment, facilities, and skilled persomiel to carry out the tests. Viruses are traditionally quantified by their replication-induced cytopathic effects on cultured cells or by immunological means such as ELISA or immuno electron microscopy. The cell culture quantification methods usually take several days to weeks and some viruses such as some human respiratory coronaviruses cannot be grown in cultured cells in vitro. The ELISA method involves the capture of viruses by an antibody cross-linked to a surface, such as 96-well polystyrene microtiter plates. The captured viruses are then detected with a secondary antibody that is attached to an enzyme such as horseradish peroxidase, which catalyzes a chemical reaction that produces a color change. Cell culture, ELISA and immunoelectron microscopy techniques typically are labor-intensive techniques that require experienced personnel to perform. Real-time quantitative PCR amplification assays have recently come into extensive use to identify and quantify pathogenic RNA and DNA viruses, such HJN-1, HIV-2, hepatitis B and C viruses, dengue virus, and cytomegalovirus (see, for example, Mackay, I. M., Arden, K. E., Νitsche, A., Real-time PCR in Nirol., Nucleic Acids Res., 15: 1292-1305, 2002, incorporated herein by reference). These assays are highly sensitive and faster than prior methods. However, due to the extreme sensitivity of the method, PCR-based assays are troublingly prone to contamination, which can result in false positive detection. Moreover, PCR-based assays generally are not suitable for high throughput detection and discrimination of viral subtypes or antigenic variants. Micro- and nano-scale technology has been increasingly used in a wide variety of chemical and biological applications, including, for example, detection and characterization of biological species in biomedical applications. For example, one area of technology that has become increasingly important but not very well developed is the handling, mampulation, and characterization of viral infectious agents, for example, the detection of very low virus particle concentrations in air and their continuous monitoring from air, using biomedical micro-electromechanical- systems technology (BioMEMS). In addition, reducing the time-to-result to be able to perform 'point-of-use' analysis has become very important. Micro- and nano-scale devices could not only yield very important scientific data but could also be used in practical diagnostic applications in the health and food industries, and in biological and chemical hazard prevention systems. Cantilevers, bridges, or other micro- and nano-scale suspended structures associated with semiconductor devices have been used to selectively detect biological and chemical particles, for example, as sensors or actuators in atomic force microscopy (AMF) and scanning probe microscopy (SPM). When the mass of the particle to be detected is added to the suspended structure, surface stresses, vibrational deflections, and like parameters of the structure are detectably altered, for example, by changing the resonant frequency of the structure. It has recently been demonstrated that biomolecular interactions such as DNA-DNA and protein-ligand interaction on cantilever surfaces cause bending of microcantilever beams (see, for example, Raiteri, R., Nelles, G., Butt, H.-J., Knoll, W. and Skladal, P., Sensing of biological substances based on the bending of microfabricated cantilever. Sensors Act. B. 61: 213-217, 1999; Fritz, J., Bailer, M. K., Lang, H. P., Rothuizen, H., Nettiger, P., Meyer, E., Guntherodt, H., Gerber, C, Gimzewski, J. K., Translating biomolecular recognition into nanomechanics, Science, 288: 316-318, 2001, all of which are incorporated herein by reference). The method was found to be sufficiently sensitive that it could differentiate single base-pair mismatches in a DΝA hybridization event (see for example, Hansen, K. M., Ji, H. F., Wu, G., Datar, R., Cote, R., Majumdar, A., Thundat, T., Cantilever-based optical deflection assay for discrimination of DΝA single nucleotide mismatches, Anal. Chem., 73: 1567-1571, 2001, incorporated herein by reference). Two primary methods for detecting the vibrational deflection of structures such as cantilevers and bridges are optical lever detection and piezo- resistive detection. Optical lever detection techniques are used, for example, to measure the deflection of atomic force microscopy (AFM) tips. A laser beam is reflected off of the tip of a cantilever and is typically detected by two photodetectors. The relative intensity of the laser signal received by the two photodetectors is related to the degree to which the cantilever is deflected. However, this technique has several shortcomings, especially as the dimensions of cantilevers are reduced in order to detect particles having sub- 100 femtogram (fg) masses. First, the technique requires reflecting a focused laser beam off the surface of the cantilever tip, resulting in larger lateral dimensions of the cantilever. Second, the technique generally precludes simultaneous monitoring of an array of cantilevers, which is necessary or highly desirable in a variety of detection applications. Finally, a relatively large off-chip system is required, including the laser, focusing optics, and photo detectors. Therefore, the degree to which such a system can be miniaturized is limited. Piezo-resistive detection methods eliminate the requirement of external optical system elements by electrically measuring the deflection-induced strain on the surface of the cantilever or the change in vibration frequency due to the particle mass. The technique is analogous to macroscale strain gauges that are attached to mechanical components to measure deformation. The change in resistance for a piezo-electric element in response to strain is induced by cantilever deflection. This change in resistance can be detected, for example, by using a Wheatstone bridge circuit. Piezo-resistor elements can be built into the surface of the cantilever by standard integrated circuit fabrication techniques. This provides a flexible, integrated, and scaleable vibration sensing technique adaptable to a variety of cantilever structures. When a cantilever is deflected, a strain gradient is induced throughout the body of the cantilever, with zero strain at the center. One surface is in tension (positive strain) while the opposite surface is in compression (negative strain). Since the response of a piezo-resistive element to strain is approximately symmetric about zero strain, a piezo-resistor that penetrates through the entire cantilever would not respond to the flexion. The response of the top half of the element would be equal and opposite to that of the bottom half, resulting in no net response. Therefore, to be effective, piezo-resistive elements are generally placed near the surface region of the cantilever. While this may be accomplished by implantation and/or diffusion into relatively thick cantilevers, it becomes very difficult to achieve in sub- 100 nanometer (nm) thick cantilevers which are generally required for detecting sub- 100 fg masses. Accordingly, devices, methods of use, and methods of fabrication of such devices, for use in selectively concentrating, capturing, and detecting the presence of species, for example, chemical molecules such as gases or proteins, biological species, such as cells (e.g., bacterial cells and/or eukaryotic cells) or virus particles, and the like are desirable.
SUMMARY According to one aspect of the invention, an apparatus comprises a filter structure adapted to be coupled to an alternating current voltage source. The filter structure includes at least one electrode having a feature which promotes establishment of at least one of a region of increased electric flux and a region of decreased electric flux relative to an adjacent region to select a first species susceptible of dielectrophoretic selection. The apparatus further includes a cantilever structure upon which the first species is collected. The apparatus also includes at least one device for causing the cantilever structure to oscillate, and for receiving as an output from the cantilever structure the cantilever structure's frequency of oscillation. The at least one device is adapted to be coupled to means for determining from the output the mass of the first species collected on the cantilever structure. According to another aspect of the invention, an integrated micro- electromechanical analyte detection device comprises a substrate, a support member coupled with said substrate, a cantilever having a fixed end coupled to said support member and a free end, and a first electrode coupled to said substrate and positioned adjacent said free end of said cantilever such that the free end and the electrode are excitable terminals for dielectrophoresis. According to another aspect of the invention, an integrated micro- electromechanical analyte detection device comprises a substrate, a support member coupled to said substrate, a cantilever having a fixed end coupled to said support member and a free end, and a transistor having at least one region formed on at least one of said free end of said cantilever and a portion of said support member. According to another aspect of the invention, an integrated micro- electromechanical analyte detection device comprises a substrate, a support member coupled to or defined by said substrate, and a suspended member having first and second ends. At least the first end is coupled to the support member such that at least a portion of the suspended member is movable relative to the substrate. The integrated micro-electromechanical analyte detection device further comprises a field effect transistor having a channel defined by said suspended member. According to another aspect of the invention, an integrated micro- electromechanical detection device comprises a substrate, a first suspended member having first and second ends, at least said first end fixed with said substrate, a dielectrophoresis filter coupled to said substrate, and a fluid channel defined by said subsfrate and communicating fluid between said first suspended member and said dielectrophoresis filter. According to another aspect of the invention, a method is provided for determimng a characteristic of an analyte interacting with an integrated detection device. The device includes a subsfrate and a suspended member having first and second ends. At least the first end is coupled with the substrate. The method comprises applying an electric signal to the suspended member to perform dielectrophoresis on the analyte and determining a change in a parameter of the suspended member. According to another aspect of the invention, a method is provided for determimng a characteristic of an analyte interacting with an integrated detection device. The integrated detection device includes a subsfrate, an electrode and a suspended member having first and second ends. At least the first end is coupled with the subsfrate. The method comprises applying an electric signal to the electrode and the suspended member to perform dielectrophoresis on the analyte. According to another aspect of the invention, a method is provided for determining a characteristic of an analyte interacting with an integrated micro- electromechanical detection device. The device includes a subsfrate, a support member coupled with the subsfrate and a suspended member having first and second ends. At least the first end is coupled to the support member. The method comprises determining a change in a current signal conducted by a fransistor region defined at least in part by one of the suspended member and the junction of the first end of the suspended member and the support member, upon the analyte interacting with the suspended member. According to another aspect of the invention, a method is provided for fabricating an integrated circuit on a substrate. The integrated circuit includes a cantilever. The method comprises providing a silicon-on-insulator (SOI) wafer having a buried oxide layer (BOX), thinning the SOI layer to less than approximately 30 nm, photolithographically patterning and etching the SOI layer to define the cantilever, tWnning the exposed portions of the BOX layer, depositing an etch stop layer to the exposed portions of the SOI and BOX layers, photolithographically patterning an etch window extending laterally approximately from the free ends of the cantilever and vertically to at least the depth of the base of the SOI layer, and etching the BOX layer below the cantilever, thereby releasing an end of the cantilever. According to another aspect of the invention, a method is provided for fabricating an integrated circuit on a substrate. The integrated circuit includes a cantilever. The method comprises patterning a seed window using an insulator mask on subsfrate, providing an oxide layer displaced vertically above the seed window and laterally overlying the seed window and at least a portion of the insulator mask, growing silicon selectively from the base of said seed window vertically to the top of the insulator mask and laterally between the insulator mask and the oxide layer, and etching at least a portion of the oxide layer and the insulator mask, thereby releasing a suspended end of the selectively grown silicon from the remaining structure. According to another aspect of the invention, a method is provided for fabricating an integrated circuit on a substrate. The integrated circuit includes a cantilever and a field effect transistor. The method comprises providing a silicon-on- insulator (SOI) wafer, thinning the SOI in the area where the cantilever will be formed using an anisofropic etch in order to form an abrupt step adjacent the top surface of cantilever and the junction of the remaining structure, oxidizing the silicon surface of the cantilever and the step to form transistor gate oxide, depositing polysilicon gate material conformally along the top surface of the cantilever adjacent the step, anisotropically etching the polysilicon gate material through the entire deposited thickness so that a sidewall of polysilicon having a lateral width approximately equal to the deposited thickness remains on any exposed vertical surface, implanting a source and drain region using the polysilicon gate as an implantation mask, and etching to release a suspending portion of the cantilever on the end opposite the transistor gate. According to another aspect of the invention, a method is provided for fabricating an integrated circuit on a subsfrate. The integrated circuit includes a field effect fransistor (FET). The method comprises providing a silicon wafer, thermally growing a first thick oxide layer for subsfrate isolation, lithographically patterning and depositing a sacrificial silicon layer on the oxide layer, depositing a second thick oxide layer using plasma enhanced chemical vapor deposition (PECND), etching via holes to define the FET source and drain regions, and etching to selectively remove the sacrificial silicon layer. The removal defines a bridge formed by a portion of the second oxide layer suspended over the thermally grown oxide layer. The method further comprises collapsing the oxide bridge to leave nano-scale via holes near the anchors of the oxide bridge, and wet etching a seed hole in the first oxide layer in the source region via hole. The seed hole extends down to the silicon layer surface. The method further comprises growing silicon epitaxially through the via holes, removing silicon remaining on the surface by chemical mechanical polishing (CMP), implanting the source and drain regions, and etching to remove the oxide encapsulating the epitaxially grown silicon. The silicon forms suspended nano-wires forming the FET channel and a thin connecting silicon plate between the wires. The method further comprises etching to remove the remaining oxide covering on the device and the thin silicon plate between the wires.
BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 illustrates schematically an exemplary integrated micro- electromechanical analyte detection device constructed according to the present invention; Fig. 2 illustrates a scanning electron micrograph of an exemplary cantilever beam integrated into a semiconductor chip according to the present invention; Fig. 3 illustrates frequency shift of the cantilever illustrated in Fig. 2 after the addition of a single virus particle; Fig. 4 illustrates a perspective view of an exemplary cantilever beam and a supporting member in dynamic mechanical vibration; Fig. 5 illustrates an exemplary cantilever beam and supporting member in static deflection; Fig. 6 illustrates graphically a desired resonant frequency and minimum detectable mass for exemplary cantilevers having widths of 1/raι and various thicknesses; Figs. 7 A-E illustrate cross-sectional views of an exemplary device in various stages of semiconductor fabrication; Figs. 8 A-B illustrate top views corresponding with the device of Fig. 7A-E in various states of fabrication; Fig. 9 illustrates a system including scanning laser doppler vibrometer determining the resonant frequencies of unloaded and loaded suspended members; Fig. 10 illustrates graphically the resonant frequency shift measured before various loaded cantilever beams measured by the system of Fig. 9; Figs. 11 and 12 illustrate an exemplary method of fabricating an ulfrathin cantilever; Fig. 13 illustrates a perspective view of an exemplary system for measuring the unloaded resonant frequency of an exemplary cantilever using a laser and a detector; Fig. 14 graphically illustrates the frequency response for the unloaded cantilever illustrated in Fig. 13; Fig. 15 illustrates the measurement of a loaded cantilever illustrated in Fig. 13; Fig. 16 illustrates the resonant frequency shift between the cantilevers illustrated in Figs. 13 and 15; Figs. 17A-D illustrate mechanical characterization of an exemplary cantilever using known masses positioned at the free end; Figs. 18 and 19 illustrate the resonant frequency change after the additional of known masses for the exemplary cantilever illustrated in Figs. 17A-D; Figs. 20-22 illustrate scanning electron micrographs of exemplary embodiments of cantilevers; Fig. 23 illustrates a cross-sectional view of the components and layers of an exemplary cantilever having a sensing element at the junction of the cantilever and the support member according to the present invention; Fig. 24 illustrates an elevation view of the areas of maximum strain for an exemplary cantilever according to the present invention; Figs. 25 A-D illustrate exemplary cantilever structures within fluid channels defined in integrated semiconductor devices; Figs. 26A-B illustrate a cross-sectional view and a top view, respectively, of an exemplary cantilever having a transistor region adjacent the area of maximum strain of the cantilever; Fig. 27 graphically illustrates the carrier density for the transistor region of Figs. 26 A-B for various depths from the surface of the cantilever; Fig. 28 illustrates DEP motion of particles between an elecfrode and a plate; Fig. 29 illustrates the use of DEP to concentrate analytes at free ends of a cantilever pair according to the present invention; Fig. 30 illustrates a perspective view of an array of cantilever pairs using DEP to concentrate analytes at the free ends of the cantilevers; Fig. 31 illustrates binding of Listeria monocytogenes on various surfaces; Fig. 32 illustrates a perspective view of a cantilever having binding agents for a target analyte and using mechanical vibration of the cantilever to aid in the release of non-target species; Fig. 33 illustrates individual elements for selectively binding a target analyte to an exemplary cantilever; Fig. 34 illustrates a schematic diagram of a DEP filter for a fluid channel for a device such as that illustrated in Fig. 1 ; Fig. 35 illustrates a scanning electron micrograph of fluid channels and electrodes for an exemplary DEP filter such as for the exemplary embodiment illustrated in Fig. 1 ; Figs. 36A-C illustrate DEP manipulation and separation of live and dead cells on electrodes; Fig. 37A-B illustrate an exemplary DEP filter for selectively capturing a target analyte; Fig. 38 illustrates an optical image of a device used for the DEP manipulation illustrated in Figs. 36 A-C; Figs. 39A-B illustrate scanning electron micrographs of a device having suspended nano-wire members; Figs. 40A-G illustrate perspective views of a process for fabrication of the devices illustrated in Figs. 39 A-B; Figs. 41 B, C, E and G are detailed views more fully illustrating the fabrication steps of Figs. 40 B, C, E and G, respectively; Fig. 42 illustrates the change in conductance of an exemplary suspended member after cycling between exposure to oxygen and purging in nitrogen; Fig. 43 graphically illustrates the cycling illustrated in Fig. 42 with the addition of a heat cycle; Fig. 44 illustrates a schematic elevational view of the structure of an integrated detection device having a suspended channel; Fig. 45 illustrates a perspective view of an exemplary integrated device having suspended nano-wire members connected by an ultra thin silicone film according to the present invention; Fig. 46 illustrates the exemplary device of Fig. 45 after the removal of the silicon film connecting the suspended nano-wires; and Fig. 47 illustrates a perspective view of an exemplary device having a suspended member with an elecfrode. Corresponding reference characters indicate corresponding parts throughout the several views. The exemplification set out herein illustrates an embodiment of the apparatus and such exemplification is not to be construed as limiting the scope of this application in any manner.
DETAILED DESCRIPTIONS OF ILLUSTRATIVE EMBODIMENTS An exemplary micro- or nano-elecfromechanical analyte detection device includes a cantilever, nano-wire, or other suspended member for detecting selected biological or chemical species based on the change in resonant frequency, surface stress, or other characteristic of the suspended member upon interaction with a selected analyte. For the purpose of this disclosure, analyte is defined as any biological or chemical species, including, for example, chemical molecules, proteins, bacteria, cells, virus, spores, molds, yeast, microorganisms, and the like. DEP can be utilized to concentrate a selected analyte, for example at the free end of the cantilever or other suspended member. The suspended member may also include a region of a transistor, for example a channel of a field affect transistor to provide on-chip electronic detection of selected analytes. Integrated devices having anays of suspended members for detecting selected analytes located within a fluid channel are also disclosed as well as methods of use and methods of fabrication of the exemplary integrated devices. The recent technological advances in nanotechnology and micromachining of semi-conductor materials present new opportunities for inexpensive, small and sensitive diagnostic or other detection devices capable of rapid and highly accurate detection of minute analytes, for example, sub-100 fg analytes. This disclosure relates to a device that can detect the presence of analytes by either changes in surface stress or detection of added mass through a change in resonant frequency of a suspended structure, for example, a cantilever or bridge structure. One exemplary embodiment of the present device includes a micro- cantilever-based virus detection device and technique which may yield performance characteristics exceeding the sensitivity and specificity of present detection techniques, for example, PCR amplification assays and ELISAs. Calculated limits of detection of the exemplary device are 10"17-10"18 grams (gm) of mass change on the cantilever surface. This translates to the mass of single virus particles. When this method is coupled to currently available monoclonal antibodies against viruses (see, for example, Parren, P. W., and Burton, D. R., The antiviral activity of antibodies in vitro and in vivo, Adv. Immunol., 77, 195-262, 2001, incorporated herein by reference), its specificity could surpass ELISAs since the described technique does not rely on enzymatic reaction kinetics as do the mentioned prior art techniques. The ability to detect and monitor in real-time and on a continual basis viruses and their subtypes, particularly the most contagious viruses and bioterrorism agents, can have dramatic implications in the confinement and management of the viral epidemics. One of the most contagious forms of disease spread occurs via aerosolized pathogens. Recently, considerable attention has been given the deadliest bioterrorism agents that spread through airborne particles, such as smallpox virus and anthrax toxin. Although influenza viruses typically are not considered bioterrorism agents, these viruses can be pandemic with devastating casualties. Human rhinoviruses and coronaviruses are among the most common causes of upper- respiratory infectious diseases, and clearly, a device to rapidly measure these agents in air samples can have profound practical and economic implications. Thus, there is an important need for a micro-scale, robust, real-time monitoring device, based on integrated micro-machined ultrathin cantilever arrays with on-board signal processing for the rapid and sensitive detection of infectious agents in field settings and in primary patient care facilities. According to one exemplary embodiment, an array is specific for specific pathogens and has the sensitivity to detect a single virus or toxin molecule. The exemplary embodiment includes a dielectrophoresis (hereinafter sometimes DEP)-based infectious agent trapping, separation and concentration device for the detection of an airborne virus on functionalized micro-scale cantilevers. The exemplary device provides all-electronic detection of single entities in the zeptogram and/or attogram range, has a short turnaround time of detection, and does not require labeling of entities. The device includes an ulfrathin and highly sensitive mechanical structure built on an electronic chip that detects DNA, proteins, viruses, and other analytes in very low concentrations. The device may also detect the composition of analytes. By tailoring the function of the mechanical device surface, specific adsorption of biological or chemical species can be achieved, thus permitting target-specific detection. The exemplary device includes nano-mechanical suspended structures, for example, cantilever ("diving board") or bridge (for example, nano-wire) sensors. An exemplary method of device fabrication produces ultra-thin, and thus, highly sensitive suspended structures (less then 500 nm thick, 130 μm long, and 20 μm wide; preferably less than 30 nm thick, 5 μm long, and 2 μm wide; more preferable less than 10 nm thick, 3 μm long, and 1 μm wide; more preferably approximately 100 angsfroms (A) thick) on a silicon or other semiconductor chip. The exemplary chip provides detection of analytes, for example, DNA, proteins, viruses, and chemicals, in very low concentrations, for example, less than 10 particles per milliliter. These suspended structures can detect the presence or binding of analytes on them, for example, by a resulting change in resonant frequency due to the added mass or a resulting change in surface stress due to the change in surface energy. While the suspended member of some exemplary embodiments comprise silicon, other semiconductor, metallic conductor, or non-conductive materials may be alternatively used. The suspended structures, for example cantilevers, can be arrayed within a micro-fluidic channel where an air or other fluid flow can be passed over the structures. When an alternating current (ac) DEP signal (with frequency different than the resonant frequency) is applied to electrodes associated with the suspended structures, particles having permittivities greater than the carrier fluid (gas or liquid) will be trapped near the regions of highest field gradient, for example, at the tips of the suspended structures. If the suspended structures are coated with antibodies for specific viruses, only those viruses are captured. Any non-specifically bound particles can be removed by driving the suspended structures in resonance, at an appropriate amplitude, while the specifically bound particles remain attached to the antibodies present on the suspended structures. Then the attached mass can be detected by measuring a shift in the resonance frequency of the suspended structures due to the attached mass. Mechanical vibrations or bending of suspended structures are typically detected by a laser reflection based system, which is not amenable to miniaturization. Capacitive detection methods may not be practical for low capacitance applications due to the signal levels, and hence susceptibility to noise, hi order to obtain useful signal levels, the total capacitance is sometimes increased by increasing the overlap area of the capacitive regions, and by keeping a minimum distance between the sensing elecfrodes. However, for accurate mass detection in the attogram range, the resonating structure should be as small as possible, thus limiting the available signal for capacitive detection. In one exemplary embodiment of the novel resonating channel structure, the electrode used to drive the device may be the same electrode that supplies an electric field for the device. In an exemplary embodiment wherein the device includes a field effect fransistor (FET), the elecfrode is the gate or the channel of the transistor. For example, an ultratliin suspended silicon bridge, for example, a nano-wire, provides the FET channel and an active resonant sensing area. The suspended structure may be grown, for example, by tunnel epitaxial growth, where the growth path of the silicon is restricted and is allowed only in one direction in a cavity. During the formation of the cavity, an oxide layer forming a roof of the cavity can be made to collapse due to stiction and subsequent growth of silicon results in very thin single crystal silicon wires suspended between two islands or supporting structures, which may form the source and drain of the FET. Alternatively, by not doping the source and drain regions, the nano- wires can be simply used as resistors. Resistive nano-wires may provide detection of molecules binding on the wire by measuring resistance changes across the nano-wire. By oscillating the nano-wire channel using the FET gate, the current through the FET is modulated as a function of the distance between the suspended channel and the gate. Also, the current will be a function of the gate bias, as in typical FETs. The piezo-resistive effects on oscillating nano-wire channels also affect the modulation of current due to stress changes that the channel experiences due to mechanical oscillation. By monitoring the drain-source current through the FET, the amplitude and frequency of the oscillations can be determined, with high signal levels and high signal-to-noise ratios compared to typical capacitive sensing. Thus, an advantage to having the nano-wire or other suspended structure comprise the FET channel, rather than the FET gate, is the combined effect on the channel current of both the physical displacement of the channel relative to the gate and the piezo- resistive change from the bending of the channel. Whereas, if the nano-wire comprises the FET gate, the piezo-resisitive effect acts on the gate while the displacement effect acts on the channel. Because the oscillation frequency and amplitude can be sensed electically, and high signal-to-noise ratios are expected, very small changes in resonant frequncy, due to the added mass of particles on the nano-wires can be resolved by incorporating feedback. For a single crystal silicon (for example, E «162 GPa and density «2.33g/cm3) nano-wire beam, having a length of 5 μm , width of 3 μm, and thickness of O.Ol μm, the calculated resonant frequency is about 2.123340 MHz. Assuming a minimum detectable frequency of 1 Hz, the minimum detectable mass is estimated to be, 5.5 x 10"19g, which corresponds to the approximate mass of a single protein, therefore smaller than the mass of a single virus particle or cell. Alternatively, the exemplary device can be operated in other modes of electrical operation and/or mass detection, for example, as resistors, transistors, or capacitors with detection of a change in surface potential or a change in mass. Referring now to Fig. 1, one exemplary embodiment of the device may be an integrated micro-electromechanical analyte detection device 100 comprising integrated circuit 102 which has been fabricated to include all device components, for example, fluid channel 104, cantilever arrays 106, 108, and 110, DEP filters, concentrators, or sorters (which may be configured to filter, concentrate, or sort particles; however, herein sometimes collectively referred to as "filters") 112, 114, and 116, signal processing and control circuitry 118, and fluid intake filters 120 and exhaust filters 122. Integrated device 100 is capable of concentrating, capturing, and detecting the presence of selected chemical and/or biological analytes. Cantilevers 106, 108, and 110, or other suspended structure, for example, a bridge, has small enough dimensions to detect analytes having sub- 100 fg masses, for example, individual bacterial cells, viruses, or chemical molecules.
Integrated device 100 may include structures and circuitry 118 for measuring changes in surface stress, energy, deflection, or resonant frequency of cantilevers 106-110 induced by the added mass or other characteristic of an analyte bound or otherwise interacting with cantilevers 106-110. For example, referring to Figs. 2 and 3, the resonant mechanical oscillation (or vibration) frequency of cantilever 106, which is shown in Fig. 2 and is supported by support member 130, is decreased by a measurable quantity by the resting of analyte 132 on cantilever 106. For the example shown in Fig. 2, analyte 132 is a Vaccinia virus particle. Referring to Fig. 3, unloaded cantilever beam oscillation 134 has a peak resonant frequency of 1.27 megahertz (MHz) and loaded cantilever beam oscillation 136 has a peak resonant frequency of 1.21 MHz. As will be further discussed below, and as is generally known in the art, the shift in resonant frequency is directly related to the mass and therefore the identity of analyte 132 interacting with cantilever 106. Referring again to Fig. 1, individual or sets of cantilevers 106, 108, and 110 may be fiinctionalized in order to provide sensitivity to particular biological or chemical analytes. For example, cantilevers 106-110 may be coated with different antibodies, antigens, or other binding agents to promote binding of a particular analyte to the individual cantilever 106-110, or may be treated with an anti-fouling agent to prevent non-specific or undesirable specific particles from interacting with cantilevers 106-110. Additionally, DEP filters 112-116 may be utilized in conjunction with cantilevers 106-110 in order to selectively sort, concentrate or capture specific analytes or other particles and direct them toward or away from cantilevers 106- 110. Known nanomechanical sensors can detect single individual biochemical molecules (see for example, M. L. Roukes, Sci. Am. 285, 48 (September 2001, incorporated herein by reference). Operating as resonance detector based mass sensors, known microstructures can detect individual bacterial cells (see, for example, Ilic, et al, 2001). For cantilever beam 140, shown in Fig. 4, free end 142 is located opposite fixed end 144, which is mechanically coupled to support member 146. For cantilever beam 140, the change in mass as a function of change of resonant frequency. Assuming all the mass is added right at free end 142, is given as,
where k is the spring constant, f0 is the unloaded resonant frequency and f\ is the loaded resonant frequency. As can be understood, the way to improve the mass sensitivity (that is, larger frequency shift for a smaller mass loading) is to decrease k as well as increase the resonant frequency. This can be done by decreasing the size of the cantilever beam 140 as well as decreasing thickness 148 of the cantilever beam. Single-crystal materials are generally used to make sensor elements due to their high mechanical quality factor. Silicon, for example, can be used for fabricating sensor elements such as cantilever 140, due to advantages such as low stress and controlled material quality, using currently available VLSI circuit fabrication facilities, miniaturization of devices, high confrol of dimensions, and the economical advantage of batch fabrication. In addition, if piezo-resistive detection modes are preferred, for example, utilizing piezo-electric element 150 shown in Fig. 5, especially due to the need for arrays of cantilevers and detectors, then silicon provides the capability to realize such elements 150 to detect deflections. Cantilever beams were first introduced to the nanotechnology field with their use as force sensors in atomic force microscopy (AFM) (see Binnig, G., Quate, C. F. and Gerber,. Atomic force microscope, Physical Review Letters, 56:930- 933, 1986, incorporated herein by reference). They have also been used extensively as probes in various other imaging techniques, involving different interactions between the probe and the sample, (see, for example, Wickramasinghe, H.K. Progress in scanning probe microscopy , Acta Materialia, 48:347-358, 2000; Moy, V. T., Florin, E-.L. and Gaub, H. E. Intermolecular forces and energies between ligands and receptors , Science, 266:257-259, Oct. 1994; Lee, G.U., Chrisey, L.A. and
Colton, R. J. Direct measurement of the forces between complementary strands of DNA , Science, 266:771-773, 1994; Dammer, U., Popescu, O., Wagner, P., Anselmetti, D., Guntherodt, H-J. and Misevic, G. N. Binding strength between cell adhesion proteoglycans measured by atomic force microscopy , Science, 267:1173- 1175, 1995; Hinterdorfer, P., Baumgartner, W., Gruber, H. J., Schilcher, K. and Schindler, H. Detection and localization of individual antibody-antigen recognition events by atomic force microscopy , Proceedings of the National Academy of Sciences of the United States of America, 93:3477-3481, 1996; Rief, M., Gautel, M., Oesterhelt, F., Fernandez, J. M., an Gaub, H. E. Reversible unfolding of individual titin immunoglobulin domains by AFM, Science, 276:1109-1112, 1997; Radmacher, M. Fritz, H. G. Hansma, and P. K. Hansma, Direct observation of enzyme activity with the atomic force microscope , Science, 265:1577-1579, Sept. 1994; Gimzewski, J. K. and Gerber, C. A femtojoule calorimeter using micromechanical sensors , Review Sci. hist., 65:3793-3798, 1994; and Barnes, J. R., Stephenson, R. J., Woodbum, C. N., O'Shea, S. J., Welland, M. E., Rayment, T. , Gimzewski, J. K. and Gerber, C. A femtojoule calorimeter using micromechanical sensors , Review Sci. hist., 65:3793-3798, 1994; all of which are incorporated herein by reference). Additionally, surface-stress change induced by deflection of cantilevers has been noted (see, for example, Butt, H.-J. A sensitive method to measure changes in the surface stress of solids , J. Colloid Interface Science, 180:251-260, 1996; Fritz, J., Bailer, M. K., Lang, H. P., Rothuizen, H., Vettiger, P., Meyer, E., Guntherodt, H.-J., Gerber, C. and Gimzewski, J. K. Translating biomolecular recognition into nanomechanics , Science, 288:316-318, 2000; Berger, R., Delamarche, E., Lang, H. P., Gerber, C, Gimzewski, J. K., Meyer, E. and Guntherodt, H.-J. Surface stress in the self-assembly of alkanethiols on gold , Science, 276:2021-2024, 1997; and Wu, G., Datar, R.H., Hansen, K.M., Thundat, T., Cote, R.J. and Majumdar, A. Bioassay of prostate-specific antigen (PSA) using microcantilevers , Nature Biotech., 19:856-860, 2001; all of which are incorporated herein by reference), based on static bending 152 of the cantilever beam 140, as shown in Fig. 5. Alternatively, cantilever beam 140 deflections 154 can be measured in the dynamic mode allowing cantilever beam 140 to be used as a micro-mechanical oscillator sensor. Additionally, static 152 or dynamic 154 deflections of suspended structures may be measured by the change in capacitance or current flow associated with regions of the suspended structures and adjacent structures as will be further discussed below. Fabrication of exemplary embodiments of the present device may include selective epitaxial growth (SEG), epitaxial lateral overgrowth (ELO), and chemical mechanical polishing (CMP) for the micro-fabrication of thin single-crystal silicon cantilever beams or other suspended structures. The thin suspended structures can be integrated into silicon-based micro-systems such as flow sensors, pressure sensors, bio-chemical sensors and the like. The disclosed fabrication process can produce low stress sub-lOOnrn thickness cantilevers 140 for ultra-high sensitivity chemical and biological detection. The minimum detectable mass with a resonant cantilever beam will depend on the geometry and the material that the cantilever is made of, as well as the minimum detectable frequency change of the cantilever beam. Also, the location of the mass to be detected on cantilever 140 will effect the sensitivity. If the mass is placed at free end 142 of cantilever 150, the device can detect lower masses, since the effective mass will be higher closer to free end 142 of cantilever 140. The resonant frequency of cantilever 140 assuming small deflection on free end 142 of cantilever 140 (the mechanical system can be modeled as a mass and spring for small deflections) can be expressed as:
Figure imgf000021_0001
where meff is the effective mass of cantilever 140, and k is the spring constant. The spring constant will be dependent on the geometry of cantilever 140. For a rectangular cantilever with dimensions 1 as length 158, w as width 156, t as thickness 148, and a material modulus of E, the spring constant can be expressed as,
k = ^ (3) 4/3 J An effective mass is needed since the mass of cantilever 140 is not concenfrated at free end 142, but distributed. Effective mass can be expressed in terms of the cantilever mass by meJ^ = 0.24 • mcantilever . For a mass added to free end 142 of cantilever 140, the resonant frequency of the system will decrease. The new resonant frequency can be expressed as:
Figure imgf000021_0002
π y meff + dm where δm is the mass added at free end 142 of cantilever 140. h order to understand the dependence of the minimum detectable mass to the cantilever design, the expression should relate the minimum detectable mass to length 158, width 156, thickness 148, density and modulus of cantilever 140 and the minimum detectable mass. So, subtracting equation (4) from equation (2) we have,
Figure imgf000022_0001
to simplify further, and rearranging to solved for δm, we have
Figure imgf000022_0002
which shows that for lower detectable mass, minimizing length 158, width 156, density and the minimum detectable frequency would minimize the detectable mass. Thickness 148 of cantilever 140 largely affects the resonant frequency of the system. The resonant frequency, equation (2), can be expressed in terms of the cantilever properties as,
Figure imgf000022_0003
which clearly indicates that, in order to keep the same resonant frequency when scaling down length 158 of cantilever 140 (which would scale down the minimum detectable mass), thickness 148 needs to be scaled down as the square of the scaling factor. So for example to scale down the length 100 times, thickness 148 would need to be scaled down 10,000 times in order to keep the same resonant frequency for cantilever 140. The value of the resonant frequency will play a role for the detection of the frequency. If the resonant frequency is too high, the deflection at fee end 142 will be very small, hence may not be detectable. Also circuitry 118 (Fig. 1) used for detection may not be feasible in the giga-hertz (GHz) range. However, keeping the resonant frequency too low is also not desirable, since this may cause interference with measurements from ambient noise. Thus the design of cantilever 140 should be made for the desired resonant frequency and minimum detectable mass, as shown graphically in Fig. 6 for exemplary cantilevers having width 156 of 1 μm and various thicknesses 148. By way of example, for illustrative purposes, the exemplary microfabrication and application of arrays of silicon cantilever beams as nanomechanical resonant sensors according to the present invention, are designed to detect the mass of individual virus particles. The dimensions of the fabricated cantilever beams 140 are in the range of 4-5 μm in length 158, 1-2 μm in width 156 and 20-30 nm in thickness 148. The virus particles used as an analyte for the exemplary embodiment were Vaccinia virus, which is a member of the Poxviridae family and forms the basis of the smallpox vaccine. The frequency spectra of cantilever beams 140, due to thermal and ambient noise, were measured using a laser Doppler vibrometer under ambient conditions. The change in resonant frequency as a function of the virus particle mass binding on the surface of cantilever beam 140 forms the basis of the detection scheme. A single Vaccinia virus particle has an average mass of 9.5 femto-grams (fg). The exemplary device can be very useful, for example, as a component of biosensors for the detection of air-borne virus particles or other analytes. Known macroscale quartz crystal micro-balance devices for the detection of virus particles require an external power supply and detection of the detachment of virus particles were measured (see, for example, M. A. Cooper, F. N. Dultsev, T. Minson, V. P. Ostanin, C. Abell, D. Klenerman, Nature Biotechnol. 19, 833, 2001, incorporated herein by reference). The below discussed exemplary embodiment according to the present disclosure includes nanomechanical devices formed on an integrated circuit, with a measurement set-up sensitive enough to measure thermal and ambient noise induced deflections and thus not requiring an external source to excite the cantilever beams (see, for example, B. Ilic, B. Uic, D. Czaplewski, M. Zalalutdinov, H. G. Craighead, P. Neuzil, C. Campagnolo, C. Batt, J. Vac. Sci. Technol. B 19, 2825, 2001, incorporated herein by reference), h order to fabricate exemplary device 200, shown in various stages of fabrication in Figs. 7A-E and 8A-E, P-type (100) 4" silicon-on-insulator (SOI) wafers 202 are used as the starting material, shown in Figs 7A and 8 A, for one exemplary fabrication method. The wafers have SOI layer 204 of 210 nm thickness and buried oxide (BOX) layer 206 thickness of around 390 ran. Wet oxidation followed by buffered hydrofluoric (BHF) etching is performed in order to thin SOI device layer 204 down to 30 nm. Photolithography followed by reactive ion etching (RLE) using Freon 115 to then etch SOI layer 204 and CHF3/O2 in order to then thin BOX layer 206, in order to pattern cantilever beams 208 as shown in Figs. 7F3 and 8B. After depositing a layer of plasma enhanced chemical vapor deposition
(PECVD) oxide as etch stop layer 210, etch window 212 was photolithographically patterned using BHF oxide etch, as shown in Figs. 7C and 8C. hi order to etch the underlying exposed silicon 204 and release cantilever beams 208, vapor phase etching using xenon difluoride (available from Xactix, Inc., of Pittsburgh, PA) can be used, resulting in device 200 as shown in Figs. 7D and 8D. After cantilever beams 208 are released, oxide 206 is etched in BHF, rinsed in DI water, immersed in ethanol and dried using critical point drying (hereinafter sometimes CPD), resulting in device 200 as shown in Figs. 7E and 8E. Although measurement of the unloaded and loaded cantilever resonant frequencies may be performed in many ways, one exemplary embodiment of system 220 associated with device 200 uses microscope scanning laser Doppler vibrometer 222 (Model #MSV-300 available from Polytec PI of Auburn, MA) with a laser beam spot size of around 1-2 μm. Vibrometer 222 may include, for example, monitor 224, CCD camera 226, microscope 228, scanner controller 230, vibration controller 232, oscilloscope 234, sensor head 235, laser 236, beam splitter 238, detector 240, reference signal 242, and measurement signal 244. The resonant frequencies of typical cantilever beams of length around 5 μm, width around 1.5 μm, and thickness around 30 nm are in the 1-2 MHz range with quality factor of around 5-7. After device 200 fabrication, cantilevers beams may be cleaned in a solution of H2O2:H2SO4=l : 1, rinsed in DI water, immersed in ethanol, and dried using CPD. The frequency spectra can be then measured in order to obtain the 'unloaded' resonant frequencies of cantilever beams 208. Next, analyte, for example, purified Vaccinia virus particles in DI water can be introduced over cantilever beams 208 and allowed to incubate for 30 min, following which the cantilever beams are rinsed in ethanol and dried using CPD so as to minimize stiction of the cantilever to the underlying sufaces. The resonant frequency of cantilever beams 208 are then measured again in order to obtain the 'loaded' resonant frequencies of cantilever beams 208 with the analyte. Using the mechanics of a spring-mass system, the added mass for the corresponding change in resonant frequency can be determined. The change in mass (placed right at free end 142 of cantilever beam 140 shown in Fig. 4) in relation to a change in resonant frequency can be given as,
Figure imgf000025_0001
where k is the spring constant of cantilever beam 208, f0 is the initial resonant frequency, and/} is the resonant frequency after the mass addition. Cantilever beams 208 can be calibrated by obtaining their spring constant, k, using the unloaded resonant frequency measurement f0, quality factor Q, and the plan dimensions (length and width) of the cantilever beam. The resonant frequency and the quality factor can be obtained by fitting the vibration spectra data to the amplitude response of a simple harmonic oscillator (SHO). The amplitude response of a simple harmonic oscillator (SHO) is given as,
Figure imgf000025_0002
where f is frequency in Hz, f0 is the resonant frequency, Q is the quality factor and Adc is the cantilever amplitude at zero frequency, as described by D. A. Walters, J. P. Cleveland, N. H. Thomson, P. K. Hansma, M. A. Wendman, G. Gurley, V. Elings, Rev. Sci. Instrum. 67, 3583, 1996, incorporated herein by reference). The measured spring constant of exemplary cantilever beams 208 is around 0.005-0.01 N/m. Virus particles (shown as 132 in Fig. 2) can be counted by observing cantilever beams 208 using a scanning electron microscope (hereinafter sometimes SEM). The change in frequency upon addition of mass can be detected by a laser Doppler vibrometer 246, as shown in Fig. 9. The effective mass contribution of the viruses can then be calculated based on their relative position from the fixed end of the cantilever beams (for example, fixed end 144 of cantilever 140, shown in Fig. 4). Using the measurements from various cantilever beams 208, the resonant frequency shift (decrease) versus the effective number of virus particles observed on the cantilever beam, can be plotted as shown in Fig. 10. The relationship can be found to be linear, as expected, to verify the validity of the measurements. Referring again to Fig. 3, the resonant frequency shift (Δf = 60 kHz) after the addition of a single virus particle is shown. Fig. 3 shows a 60 kHz decrease in the resonant frequency of a cantilever beam having a plan dimension of L = 3.6 μm and W = 1.7 μm. The unloaded resonant frequency f0 = 1.27 MHz, quality factor Q = 5, and spring constant k = 0.006 N/m. The resonant frequencies may be obtained by fitting the amplitude response of a simple harmonic oscillator to the measured data. hi one experiment using exemplary device 200 according to the above fabrication and method, the average dry mass for a single Vaccinia virus particle was measured to be 9.5 fg, which is in the range of the expected mass of 5-8 fg (see Bahr, G.F., Foster, W.D., Peters, D. and Zeitler, E.H. Variability of dry mass as a fundamental biological property demonstrated for the case of Vaccinia virions. Biophys. J. 29:305-314, 1980, incorporated herein by reference). The measured mass sensitivity of exemplary cantilever beams 208 for a 1 kHz frequency shift is 160 attograms (ag) added mass (6.3 Hz/ag). Another exemplary embodiment according to the present invention integrates device 200 with on-chip antibody-based recognition and concentrators, for ulfra-sensitive detection of air-bome virus particles, for example, device 100 shown in Fig. 1. Another exemplary micro-fabrication technique according to the present invention may be used for fabricating device 300 having ultra-thin cantilever beams 302 in single crystal silicon with no stress. The exemplary process utilizes a technique called MELO (Merged Epitaxial Lateral Overgrowth) and can be regarded as an extension of selective epitaxial growth (SEG) and epitaxial lateral overgrowth (ELO) of crystalline material. SEG is a form of vapor phase epitaxy (VPE), and is a variation on the conventional full wafer epitaxy process known in the art. In SEG, the epitaxial deposition conditions are adjusted to prevent silicon deposition on the insulator region 304, for example, Si02, while silicon epitaxial growth occurs only on the exposed silicon in the seed windows 305 (see, for example, Bashir, R., Venkatesan, S., Neudeck, G. W. and Denton, J. P. A polysilicon contacted subcollector BJT for a three-dimensional BiCMOS process. IEEE Electron Device Letters 13:392-395, 1992, incorporated herein by reference). Referring to Fig. 11, if the selective growth is allowed to continue beyond the point where the epitaxial silicon is at level with the top of the insulator region, the silicon will grow vertically as well as laterally across insulator mask 308 and under oxide layer 310, forming cantilever 302. Cantilevers with thickness ranging from 0.2-0.5 μm, maximum length of around 130 μm and widths of around 20 μm and 10 μm have been fabricated using the above described method and known surface micro-machining techniques. Mechanical characterization of device 300 can be perfonned by measuring the resonance frequency using thermal noise to excite unloaded cantilever 302 as shown in Figs. 13 and 14, and by adding known micro-sized particles 304 as shown in Figs. 15 and 16. See also, known particles located at free end 306 of cantilever 302 in Figs. 17A-D.Young's modulus, extracted from the added mass approach was found to be in the range of 80- 110 GPa and the mechanical quality factor was measured to be in the range of 20-50 in air. Such cantilevers 302 can be scaled to thickness of less than 100 nm and can be integrated into micro-fluidic channels (for example, channel 104 of Fig. 1 ) within the substrates for a wide variety of chemical and biological detection applications. To determine the mechanical characterization of cantilever beams 302, thermal mechanical noise is sufficient to oscillate cantilever beams 302 whose deflections can be detected by an AFM that employ the optical lever technique (see, for example, Meyer, G. and Amer, N. M. Novel optical approach to atomic force microscopy. App. Phys. Letters, 53:1045-1047, 1988, incorporated herein by reference). The advantage of this method over driving the cantilever using a piezoelectric or other exciter for mechanical characterization is that it does not excite other stiffer, higher mechanical resonance modes such as that of the supporting member to which cantilever 302 is coupled. In one exemplary system, the cantilever deflection signal was extracted from a Dimension 3100 SPM (see, for example, Meyer, G. and Amer, N. M. Novel optical approach to atomic force microscopy. App. Phys. Letters, 53:1045-1047, 1988, incorporated herein by reference), using the DI signal access module, and then digitized. The power specfral density (hereinafter sometimes PSD) of the signal was then evaluated using MATLAB software. The thermal spectra data was then fit to the amplitude response of a simple harmonic oscillator (hereinafter sometimes SHO), using equation (8) above, where f is frequency in Hz. The quantities f0 (resonant frequency), Q (the quality factor), and Adc (the cantilever amplitude at zero frequency) were extracted from the fit of equation 10 to the measured data. The technique was also used to then determine the mechanical properties such as stiffness (or spring) constant of the cantilever beams using the added mass (or Cleveland) method (see, for example, Cleveland, J. P., Manne, S., Bocek, D. and Hansma, P. K. A nondestructive method for determining the spring constant of cantilevers for scanning force microscopy , Review of Sci. Inst., 64:403- 405, 1993, incorporated herein by reference) was used. For example, polystyrene spherical beads 308-314 of known mass, shown in Figs. 17A-D, were placed at the free ends of cantilever beams 302 using, a micromanipulator. Spherical beads of diameter of around 5.48 μm and 3.18 μm were used in the exemplary system. Using the density of polystyrene of p = 1.05 • 10 kg/m , the masses of the beads can be calculated to be in the range of 90.5 picograms (pg) and 17.7 pg, respectively. Due to variation in the diameter of individual beads from the stated specifications of the manufacturer's values, the diameter can be measured using an optical microscope. The change in resonant frequency, fls due to addition of a single mass, M1? as shown in Fig. 19, can be measured and used to detect and extract the mass of the bead 308. The spring constant can be evaluated using, k = (2π)2 -, ^ (9) } (ι/Λ)2 - (ι/ )2 If masses are added right at the free end 306 of cantilever beam 302, giving M = k(2τf)~2 - ?n * , (10) where, M is the total added mass. It is also desirable to determine the minimum detectable mass that could be measured by cantilever beams 302. Assuming the minimum detectable frequency to be δf = 1 Hz and setting £χ = (f0 - δf) in equation (9), it is possible to determine the theoretical minimum detectable mass. Table I, below, presents the values of the measured spring constant, effective mass and mass resolution for previously microfabricated cantilever beams 320 and 330 shown in Figs. 21 and 22, respectively. These masses were detected by measuring the vibrations of the cantilever using optical means. Cantilever 320 shown in Fig. 21 was placed in a modified AFM system and the laser reflection off the cantilever surface was detected using a quad photo-detector, thus allowing us to measure the vibration of the cantilever 320, for example, as shown for cantilever 302 in Figs. 13 and 15, however, other techniques to detect the vibrations may be used. Table 1. Planar dimensions and measured values of spring constant and effective mass. Also listed are the extracted thickness from the effective mass, the extracted Young's modulus from the spring constant (assuming the given planar dimension), and the ideal calculated value of mass resolution from equation (9), assuming δf = 1 Hz for cantilever beams 320 and 330, shown in Figs. 21 and 22.
Figure imgf000029_0001
As shown in Table 1, cantilevers 320 and 330 are capable of detecting masses down to about lOfg using a detection resolution of 1Hz change in frequency. However, the detection of such a small frequency change likely requires driving the cantilevers using forced excitations rather than thermal noise sources. For example, a piezoelectric film connected to an external signal generator to sweep the signal frequency and measure the resonant frequency of cantilevers 320 and 330 could be utilized. The design of cantilever beams is critical to the overall system design and the ability to detect single virus particles or other minute analytes. Using equations (6) and (7), the minimum detectable mass can be calculated as a function of the cantilever geometry, for example as shown in Fig. 6. As the length, and correspondingly the mass, is decreased, the minimum detectable mass is decreased. To be able to detect single virus particles, for example, the size of a cantilever would need to be reduced to 3um wide x about lOum long. For these dimensions, the resonant frequency would be larger than 1-5 MHz if a thickness of 0.5um is used. However, if the thickness is reduced down to 20nm, them the resonant frequency is also reduced to below 100kHz, which likely would be easier signal to detect and process. There are at least three alternatives for the fabrication of the silicon cantilevers, for example, silicon-based cantilevers which can included integrated piezo-resistive elements to provide an electrical output. One option, perhaps the most practical one, is to use commercially available SOI (silicon-on-insulator) wafers, which can have thicknesses down to O.lum (lOOrrm). These wafers can then be used to fabricate surface micro-machined cantilevers. The main drawback of this approach is that the silicon material might have residual stress and the cantilevers might be curled and stressed upon final release. Another option is the use of the above discussed technique using merged epitaxial lateral overgrowth (MELO) and chemical mechanical polishing (hereinafter sometimes CMP) of silicon. Yet another fabrication option is 'tunnel epitaxy' shown in Figs. 11 and 12, which allows the fabrication of cantilevers down to a thickness of less than lOnm. In this case, a tunnel is defined using deposited films and selective growth of silicon is performed to fill the tunnel with single crystal material, forming an ultra-thin cantilever or other suspended structure. The films around the silicon can then be removed, thus releasing these nano-mechanical structures. Another exemplary device 400, shown in Fig. 23, according to the present invention may, include piezo-resistive element(s) or fransistor region element(s) 404 integrated into suspended member 402. Element 404 may be used for the detection of the deflection, vibration, or surface stress or energy of suspended member 402. Element 404 can be grown selectively at the anchored or fixed end 406 where the stress is maximized, specifically, where suspended member 402 joins support member 408 of device 400, especially at the surfaces of the junction of members 402 and 408. The growth of selective elements 404 enables a device such as device 100 (Fig. 1) or the devices shown in Figs. 25A-D having an array of cantilevers or other suspended members for detecting selective analytes. In another exemplary embodiment of the present disclosure, using standard integrated circuit and MEMS processing technologies, cantilevers or other suspended structures can be fabricated with lateral dimensions limited only by the resolution of the photolithography used, and thickness defined by thin silicon-on- insulator (SOI) layers, formed by wafer bonding, separation by implanted oxygen (SIMOX), or confined lateral selective epitaxial growth (CLSEG). These cantilevers, which typically have a mass of less than one nanogram (ng), have fundamental resonant mode frequencies in the range of 100 kHz, and are easily stimulated through room-temperature thermal energy. By monitoring the resonant frequency of these cantilevers, the attachment of particles with extremely small masses can be detected, and the total attached mass quantified. By using on-chip signal processing and control, integrated piezo- resistive or fransistor detection methods using, for example, device 400 shown in Fig. 23, eliminate the need for external components by electrically measuring the surface strain or other characteristic induced by deflection or vibration of cantilever 402. Referring to Fig. 24, in exemplary device 400 having a piezo-electric element 404, the resistance of element 404 changes in response to strain, which is induced in cantilever 402 by deflection. When cantilever 402 is deflected, a strain gradient is induced throughout the body of the cantilever, with zero strain at the center. One surface is in tension (positive strain), while the opposite surface is in compression (negative strain). Since the response of piezo-electric element 404 to strain is approximately symmetric about zero strain, an element that penetrates through the entire cantilever would not respond to deflection. The response of the top half of the element would be equal and opposite to that of the bottom half, resulting in no net response. Therefore, to be most effective piezo-electric element 404 must be restricted to the near surface region of cantilever 402. Nvhile this is simple to accomplish by implantation and/or diffusion into relatively thick cantilevers, it can be very difficult to achieve in sub-100 nm thick cantilevers. Alternative, exemplary device 500 (shown in Figs. 26 A-B) having transistor element 510 at the junction of cantilever 502 and support member 508 offers a possible solution to this limitation. For example, device 500 may utilize ultra-thin channel 510 of field effect fransistor (FET) 512, for example a MOSFET also having gate 504, source 514, drain 516, and source/drain region 511, as a piezoelectric sensing element. Conductive channel 510 of FET 512 may be confined to within a few nanometers of the surface, thus in the region of maximum strain upon deflection of cantilever 502. Furthermore, it has recently been demonsfrated that the mobility of the carriers in this channel 510 can be significantly enhanced by tensile strain, especially close to the surface of cantilever 502, as shown in Fig. 27. Thus the combination of these two characteristics, piezo-electric resistance and carrier mobility, makes a device such as a MOSFET an advantageous deflection sensor for ultra-thin cantilevers. Fig. 27 was generated for device 500 by simulation using SCRED 2.0, a tool which self-consistently solves the Schrόdinger and Poisson equations. The structure simulated is a dual gate n-channel MOSFET with a background concentration of 1016 cm"3, a body thickness of 10 nm, an oxide thickness of 1.5 nm, and a metal gate with a work function of 4.6 eN. The back gate was held at a constant zero bias, while the top gate was swept from zero to two volts. As Fig. 27 indicates, the channel carriers are contained within the top half of the structure, with the peak concentration occurring at approximately 1 mn from the oxide interface. This level of confinement should enable MOSFET-based piezoresistive detection in cantilevers as thin as 10 nm. A MOSFET based strain sensor can be accurately located at this point of maximum strain by using sidewall processing techniques. An exemplary fabrication method for device 500 shown in Fig. 26A-B may be: 1. Begin with an SOI wafer. 2. Thin the SOI in the area where cantilever 502 will be formed using an anisotropic etch. This forms an abrupt step between support member 508 and the top surface of cantilever 502. 3. Oxidize the silicon surface to form gate oxide 518. 4. Conformally deposit polysilicon gate material 504. 5. Anisofropically etch polysilicon gate material 504 through the entire deposited thickness. This leaves a sidewall of polysilicon on any exposed vertical surface, with a lateral width approximately equal to the deposited thickness. 6. Implant the source 514 and drain 516 regions appropriately, using polysilicon gate 504 as an implantation mask. 7. Release cantilever 502 by wet or dry etching. Another potential advantage of using a MOSFET-based deflection sensor such as device 500 is that by externally modulating the gate bias it may be possible to directly obtain the vibrational frequency spectrum of cantilever 504, through a known technique, for example heterodyne mixing, or other known signal processing techniques. Heterodyne mixing uses the fact that the product of two sinusoidal signals is the sum of two sinusoids with frequencies equal to the sum and difference of the original sinusoidal frequencies. By filtering out the sum portion of the signal, the result is a lower frequency signal with the same information content as the original signal. Heterodyne mixing is well know from radio receivers that "mix down" the incoming RF signal to an "intermediate frequency" that is more easily processed by downstream circuitry. Another example of the use of mixing is the lock-in amplifier, in which a reference signal is used to stimulate a device under test (DUT). The output of the DUT is then multiplied, or "mixed," with the reference signal, and subsequently filtered to remove the sum component. Assuming the response of the DUT is linear, the result will be a DC signal that is proportional to the magnitude of the transfer function of the device at the reference frequency. The small signal drain 516 current of MOSFET 512 operating in the saturation region is i*Q) = gmvt(t) (11) where gm is the transconductance of MOSFET 512, given at low frequencies by Sm = kμc {VG -Vτ) (12) where k is a proportionality constant, μc is the channel mobility, VG is the DC gate voltage bias, and Vris the threshold voltage. Assuming that channel 510 mobility is linearly dependent on the strain of cantilever 502, the transconductance will also be linearly dependent on this strain, through the channel mobility term. Therefore the transconductance can be written as gm(t) = kμc(t)(VG -Vτ) = kμ0 l + &-S(t) (rG -vT) l+^S(t) SmO > μ0 (13) where gmo is the unperturbed fransconductance, μs is the linear coefficient of channel mobility strain dependence, and S(t) is the strain in the channel 510 region. Note that the strain and therefore the fransconductance are explicitly written as a function of time, ubstituting this expression for fransconductance in drain 516 current expression yields:
, <?) •■
Figure imgf000034_0001
The first term in equation (14) is the unperturbed response of MOSFET 512 under the application of a small signal. The second term is the mixing term that is of interest in this discussion. The Fourier transform of (4) is (P = gmoVg(®) +—gm0 [S(ω)®vg(co)] μ° (15) If we assume that gate 504 voltage is pure sinusoidal with a frequency of ωo, then vg(ω) = vg fπ/2 (δ(ω-ω0) + δ(ω+ ωo )) (16) and
S(ω) ®vg(ω) = S(Ω) vg (ω-Ω)dΩ
= vg jπ/2 (S(ω-ω0) + S(co + ω0)) (17) Substituting (7) into (5) yields
Figure imgf000034_0002
Applying a low-pass filter with a cut-off frequency less than ωo eliminates the unperturbed response and the sum response (noting that
S(-ω) = S(ω)), leaving
Figure imgf000034_0003
Therefore the filtered response has a DC component that is proportional to the amplitude of the vibrational strain at a frequency of ωo. By slowly sweeping the frequency of the voltage applied to gate 504 the entire vibrational frequency spectrum can be obtained directly. Note also that the result is proportional to the transconductance of MOSFET 512, which provides gain to amplify the small vibrations of cantilever 502 that are of interest. In one alternative embodiment including FET 512, fransistor element 510 is located on another suspended structure, such as a bridge, rather than cantilever 502. In another alternative embodiment including FET 512, fransistor element 510 or 511 located on cantilever 502 (or another form of suspended structure) may be a gate which cooperates with a channel located on another portion of device 500 such that the distance between the gate and channel as cantilever 502 deflects induces changes in the channel current. Dielectrophoresis (DEP) may also be utilized in an exemplary embodiment of the present invention. DEP is the franslational motion of neutral particles in a non-unifonn field region (see, for example, Pohl, H. A. The motion and precipitation of suspensoids in divergent electric fields, J. Appl. Phys. 22 (1951) 869- 871, incorporated herein by reference). DEP has been demonstrated to be able to capture and separate biological materials in fluid using micro-fabricated elecfrodes with ac electric fields (see, for example, Li, H. and Bashir, R. Dielectrophoretic separation of live and heat-inactivated Listeria on microchips. Sensors and Actuators, hi press, 2002a, incorporated herein by reference). Neutral particles (including, for example, biological cells) become polarized due to the presence of electric fields. DEP forces can occur on cells when a non-uniform electrical field interacts with the field-induced electrical polarized particles, as shown in Fig. 28. The time-averaged DEP force F for a dielectric sphere immersed in a medium in constant field phases in space is given as:
Figure imgf000035_0001
where e0 is the vacuum dielectric constant, r is the particle radius, Eπns is the root mean square value of the electric field, and ep and em * are the relative complex permittivities of the particle and medium, respectively (see, for example, Pohl, H.A. The motion and precipitation of suspensoids in divergent electric fields, J. Appl. Phys. 22 (1951) 869-871, incorporated herein by reference). Depending on the relative size of the dielectric constant of the particle with respect to the medium, the particle can exhibit positive or negative DEP. Advantageously, according to the present invention, DEP may be incorporated into exemplary device 600, as shown in Fig. 29. Suspended, silicon members, for example, opposed cantilever pair 602 and 604 are used for a dual purpose: (1) as electrodes for DEP in order to capture air-borne or aerosolized virus particles and (2) as a mass sensor in order to detect the virus particles. Cantilevers 602 and 604 are scaled down in size by the exemplary processes described above to provide the mass sensitivity necessary for detection of individual virus particles. When an ac electric field or another predetermined waveform is applied to cantilevers 602 and 604, which may be electrically coupled to elecfrodes 608 and 606, respectively, biological cells 616 and 618, or other analytes, will be captured at regions 614 with the largest gradient of the field. Advantageously, the largest gradient of the field is located at free ends 610 and 612 of cantilever beams 602 and 604, respectively, as shown in Figs. 29 and 30, which, as discussed above, is also the preferred mass location for maximum change in resonance frequency for cantilevers 602 and 604. The technique of DEP and manipulation of analytes by electrical forces provides a unique means to confrol the separation dynamics of biological agents and other particles. The method has numerous biological and medical applications, e.g., identification and characterization of individual cells, purification of cell subpopulations from mixture suspension, etc., especially, for example, with integrated use of DEP separation and trapping of analytes such as air-borne infectious agents, combined with the detection of the analytes using micro-cantilevers. Since the dielectric properties of different species of particles are different and the dielectric constants of both particle and medium are functions of frequency, different species of particles at a given frequency may have opposite DEP responses, e.g., positive and negative DEP, respectively. By choosing a predetennined waveform of proper frequency and a suspending medium so that two different particles with different dielectric properties may experience positive and negative DEP respectively, a very useful method to selectively separate particles of different dielectric properties arises. Biological entities such as cells, proteins and DNA consist of adjacent structures of materials that have very different electrical properties and exhibit large induced boundary polarizations that are highly dependent on the applied field frequency as well as their physiological states. For example, the cell membrane consists of a very thin lipid bi-layer containing many proteins and is highly insulating with a conductivity of around 10"7 S/m, while the cell interior contains many dissolved charged molecules, leading to a conductivity as high as lS/m. Upon death, the cell membrane becomes permeable and its conductivity can increase by a factor of 104 due to the cell contents exchanging freely material freely with the external medium through the small pores on the membrane. This large change in the dielectric properties on cell death indicates a large change in the dielectric polarizability. Hence a large difference in DEP responses (positive and negative respectively) and a selective separation can be achieved between live and dead cells. DEP is particularly useful in the manipulation and separation of microorganisms and has been employed successfully in isolation and detection of sparse cancer cells, concentration of cells from dilute suspensions, separation of cells according to specific dielectric properties, and trapping and positioning of individual cells for characterization (see, for example, Wang, X., Huang, Y., Gascoyne, P. R. C. and Becker, F. F. IEEE Transactions On Industry Applications 33:660-669, 1997; Huang Y, Hδlzel R, Pethig R, Wang X-B, Differences in the ac electrodynamics of viable and nonviable yeast-cells determined through combined dielectrophoresis and electrorotation studies. Phys. Med. Biol., 37:1499-1517. 1992; Markx GH, Huang Y, Zhou X-F, Pethig R (1994) Dielectrophoretic characterization and separation of microorganisms. Microbiol. 140:585-591; Becker et al, 1994; and Stephens et al, 1996, incorporated herein by reference). Continuous separation can also be achieved by combining with a technique similar to field-flow-fractionation (see, for example, Markx GH, Huang Y, Zhou X-F, Pethig R (1994) Dielectrophoretic characterization and separation of microorganisms. Microbiol. 140:585-591; all of which are incorporated herein by reference). Yet, the reports that deal with the applications of DEP in separation of viruses and small bacteria are rare. The first report of molecular-scale particle manipulation was that of Washizu et al,, 1990, where they reported DNA fragments of 48.5 kilobase pairs (or about 30 mega-daltons (mDa) in molecular weight, which would be ~30 nm radius if the molecule were closely packed into a sphere were trapped and stretched (approximately 16μm long) by positive DEP. A more recent work demonsfrated that the same technique can be used to precipitate DNA and proteins as small as 25 kilodalton (kDa) (see Washizu, M., Suzuki, S., Kurosawa, O., Nishizaka, T., Shinohara, T., Molecular dielectrophoresis of biopolymers. IEEE Trans hid Appl 30:835-843, 1994, incorporated herein by reference). The step downward in size has been accelerated by advances in fabrication technology such as the use of electron beam lithography, which allows the manufacture of electrodes with feature sizes of the order lOOnm. Another study demonstrated that molecules of the 68kDa protein avidin can be concentrated from solution by both positive and negative DEP (see Bakewell, D.J.G., Hughes, M.P., Milner, J.J. andMorgan, H. Dielectrophoretic mampulation of Avidin and DNA, Proc. 20th Ann. Int. Conf. Of the IEEE Engineering in Medicine and Biology Society (Piscataway, NJ: IEEE), 1998, incorporated herein by reference). Attachment of the analytes, for example, proteins, on micr -fabricated surfaces is important to the success of applications such as protein chips, and the attachment and capture of cells on micro-fabricated surfaces, such as the cantilevers of the Exemplary embodiments according to the invention. The attachment of proteins on surfaces is complex when compared to the attachment of DNA to surfaces. Proteins have to be attached in such a way that their structure and functionality should be retained. The attachment of antibodies and proteins has been demonstrated on micro-fabricated surfaces using functional groups such as silane (see, for example, Britland, S., Arnaud, E. P., Clark, P., McGinn, B., Connolly, P., and Moores, G., Micropatterning proteins and synthetic peptides on solid supports: A novel application for microelectronic fabrication technology, Biotechnol Prog. 8, 155, 1992; and Mooney, J. F., Hunt, A. J., Mclntosh, J. R., Liberko, C. A., Walha, D. M., Rogers, and C. T., Patterning of functional antibodies and other proteins by photolithography of silane monolayers, Proc. Natl. Acad. Sci., 93(22), 12287, 1996, all of which are incorporated herein by reference), amine (see, for example, Nicolau, D.V., Taguchi, T., Taniguchi, H., and Yoshikawa, S., Micron-sized protein patterning on diazonaphthoquinone/novolak thin polymeric films, Langmuir, 14(7), 1927, 1998, incorporated herein by reference), carboxyl (see, for example, Williams, R. A. and Blanch, H. W., Covalent immobilization of protein monolayers for biosensors applications, Biosensors and Bioeledronics, 9, 159, 1994, incorporated herein by reference), and thiols (see, for example, Lahiri, J., Isaacs, L., Tien, J., and Whitesides, G. M., "A strategy for the generation of surfaces presenting ligands for studies of binding based on an active ester as a common reactive intermediate: a surface plasmon resonance study," 71, 777, 1999a, incorporated herein by reference). Attachment of avidin on micro-fabricated surfaces using a bovine serum albumin (BSA) layers has also been demonstrated in such a way that the avidin retains its binding ability to biotin and hence any biotinylated protein (see, for example, Bashir, R., Gomez, R., Sarikaya, A., Ladisch, M., Sturgis, J., and Robinson, J. P. Adsoφtion of Avidin on Micro-Fabricated Surfaces for Protein Biochip Applications, Biotechnology and Bioengineering, 73, 4, 324-328, May 2001, incoφorated herein by reference). Patterning of ligands has also been demonstrated using alkenethiolate SAMs, which were produced on Au layers and then ligands such as biotin was printed on the SAMs using micro-contact printing (see, for example, Lahiri, J., Ostuni, E., and Whitesides, G. M., Patterning ligands on reactive SAMs by microcontact printing, Langmuir, 15, 2055, 1999b, incoφorated herein by reference). Proteins micro-arrays have been demonstrated where proteins were immobilized by covalently attaching them on glass surfaces that were treated with aldehyde-containing silane reagents (see, for example, MacBeath, G. and Schreiber, S. L., Printing proteins as microarrays for high-throughput function determination, Science, 289, 1760, 2000, incoφorated herein by reference). These aldehydes react with the primary amines on the proteins such that the proteins still stay active and interact with other proteins and small molecules. 1600 spots were produced on a square cm using robotic nano-liter dispensing where each site was about 150-20Oum in diameter. All of the above approaches take a protein and devise a technique to attach it to a micro-fabricated surface by functionalizing one end of the protein with chemical groups that have affinity to that particular surface. Regarding binding on antibody to silicon suspended members, the silicon cantilever surface will always form a native oxide layer, on which the antibodies will need to be attached. We have demonstrated the adsoφtion of the protein Avidin on patterns of silicon dioxide and used of fluorescent microscopy to detect binding of biotin (see Bashir, R., Gomez, R., Sarikaya, A., Ladisch, M., Sturgis, J., and Robinson, J. P. Adsoφtion of Avidin on Micro-Fabricated Surfaces for Protein Biochip Applications, Biotechnology and Bioengineering, 73, 4, 324-328, May 2001, incoφorated herein by reference). The silicon dioxide microchip was formed using plasma enhanced chemical vapor deposition while platinum was deposited using radio-frequency sputtering. After cleaning using a plasma arc, the chips were placed into solutions containing Avidin or bovine serum albumin. The Avidin was adsorbed onto the microchips from phosphate buffered saline or from PBS to which ammonium sulfate had been added. Avidin was also adsorbed onto bovine serum albumin (BSA) coated surfaces of oxide and platinum. Fluorescence microscopy was used to confirm adsoφtion of labeled protein, or the binding of fluorescently labeled biotin onto previously adsorbed, unlabeled Avidin. When labeled biotin in PBS is presented to Avidin adsorbed onto a BSA coated microchip, the fluorescent signal was significantly higher than for Avidin adsorbed onto the biochip alone. The results show that a simple and low cost adsoφtion process will deposit active protein onto a chip in an approach that has potential applications in the development of protein biochips for the detection of biological species. In one exemplary embodiment, biotinylated BSA, which adsorbs strongly onto a C18 modified silica surface at pH 7.2 through hydrophobic interactions, has been used for analyte specific bonding. Its activity is maintained, as indicated by the strong adsoφtion of streptavidin, whiph was validated through fluorescence microscopy (see, for example, Huang, T., Gaba, A., Gomez, R., Bashir, R., Sturgis, J., Robinson, J. P., Ladisch, M. R, Submitted to Biotechnology and Bioengineering, 2002, incoφorated herein by reference). Subsequent capture of fluorescently labeled biotin by streptavidin indicated that biotinylated antibody could be successfully attached to the surface. Recent binding studies indicate the biotinylated BSA has low non-specific binding to IgG antibody and Listeria cells while streptavidin alone binds both L. monocytogenes and L. innocua. However, when the streptavidin is blocked with BSA, the non-specific adsoφtion is significantly reduced. Then when biotinylated antibody is fixed to the surface specific binding of L. monocytogenes occurs. Attachment of proteins to silica (SiO2) surface of chips has carried out (see, for example, Mooney, J. F., Hunt, A. J., Mclntosh, J. R., Liberko, C. A., Walba, D. M., Rogers, and C. T., Patterning of functional antibodies and other proteins by photolithography of silane monolayers, Proc. Natl. Acad. Sci., 93(22), 12287, 1996, incoφorated herein by reference) where the functionality of biotinylated goat antibodies was demonstrated using fluorescently labeled mouse immunoglobulin IgG as reporter molecules. The SiO2 surfaces are first derivatized with octadecyltrichlorosilane (ODTS) to form a C18 surface. Biotinylated bovine serum albumin (BSA) is then adsorbed onto a C18 modified hydrophobic silica surface. After washing the streptavidin can be adsorbed onto the biotinylated BSA. The role of streptavidin is to capture biotinylated monoclonal antibody and orient it in a way that enables the antibody to contact and capture Listeria monocytogenes. While there are many covalent attachment schemes for biotin or streptavidin, an exemplary embodiment according to the present disclosure addressed a sandwich scheme for the C18 modified surface using non-specific binding of Listeria monocytogenes and Listeria monocytogenes binding to biotinylated antibody cl le9 using PBS buffer at pH 7.2 and incubated for 2 hours, the comparisons for which are shown in Fig. 31. An additional exemplary embodiment according to the present disclosure includes covalent attachment techniques of Immobilizing IgG C11E9 antibodies on a silicon dioxide surface for use in a biosensor to detect capture of pathogen Listeria monocytogenes (see, for example, Gaba, A., Sturgis, J., Robinson, J. P., Gomez, R., Bashir, R, Ladisch, M. R. Immobilization of IgG Cl 1E9 on a silica surface for use in a biosensor to detect capture of pathogen Listeria monocytogenes, in Press 2002, incoφorated herein by reference). Thus, the development of a platform for placing more selective antibodies for L. monocytogenes and other target cells has been demonsfrated and can be extended to the detection of virus particles. While the physiology of living cells and viral particles are obviously different, the basic fabrication steps are similar. In both cases non-specific adsoφtion must be blocked, so that binding to the antigen is not masked by binding of similar, non-target species to the cantilever's surface itself. Various surface derivatization approaches can be utilized to anchor coronavirus specific antibodies to silicon cantilevers. For example, utilizing several heterobifunctional cross-linkers and micro-patterning of cantilever surfaces with the coronavirus specific antibodies. Although exemplary cantilevers according to the present disclosure include well depths of about 14-16 μm, the problem of stiction could occur and hence surface modifications can be introduced to minimize such stiction of cantilevers to subsfrate after the BHF etching of the oxide encompassing the cantilevers. The exemplary embodiment utilizes a coating including hydrophobic self-assembling mono layers (SAM) films. For example, 1.0 mM of octadecyltrichlorosilane (OTS) in 2,2,4-trimethylpentane (isooctane) may be used as the solvent to form the SAM coatings. Other anti-stiction methods and surface modifications may alternatively be utilized. Another exemplary embodiment utilizes covalent attachment techniques of Immobilizing IgG C11E9 antibodies on a silicon dioxide surface for use in a biosensor to detect capture of pathogen Listeria monocytogenes (see Gaba, A., Sturgis, J., Robinson, J. P., Gomez, R, Bashir, R., Ladisch, M. R. Immobilization of IgG Cl 1E9 on a silica surface for use in a biosensor to detect capture of pathogen Listeria monocytogenes, in Press 2002, incoφorated herein by reference). Thus, a platform for placing more selective antibodies for L. monocytogenes and other target cells may be extended to the detection of virus particle. Eliminate the non-specific adsoφtion of the virus particles in crucial. Therefore, anti-fouling or blocking layers such as BSA and other bio-chemical layer may be utilized. Additionally, DEP forces for the movement and manipulation of the virus particle may be utilized, if they are non-specifically adsorbed on the antibody coated cantilevers. An ac signal or other waveform may be pulsed at these elecfrodes in such a way that could effectively sweep the virus particles away from or toward the cantilevers, as desired. At resonance, the vibrations of the cantilever could also provide a novel method to detach the biological entities captured on the cantilever beam surfaces. The non-specifically bound species could be detached first, for example, at approximately 0.1-1.0 pN when an anti-fouling agent is utilized, while the specifically bound entities will not be removed, as shown in Fig. 32. As the magnitude of the ac signal or other wave form is increased, the amplitude of vibrations will also increase, resulting in the release of specifically bound entities, for example, at approximately 200 pN. An overall system device, for example device 100 shown in Fig. 1, may utilize many of the above described exemplary embodiments, including those further described below. Additionally, as shown in Fig. 33, antigen-binding sites, antibodies, linking molecules, and blocking agents may be used in conjunction with the suspended member in order to functionalize it for a specific entity. Referring again to Fig. 1, device 100 may be micro-fabricated with input port tube 124 with mechanical filter 120 that only lets particles less than 0.5 urn, for example, pass through. Once particles are in the air sfream in fluid channel 104 of integrated circuit 102, and they will contact DEP potential wells from the particle sorters 112 and 114, where they would get diverted into the appropriate fluid channel chambers having an array of cantilevers 106-110. Analytes with different dielectric properties or size will pass through the first sorter 112 and can be selected by the second sorter 114. Only one sorter 112 and 114 and one cantilever chamber may also be used. Cantilevers 106-110 may be coated with antibodies for specific virus or other analytes and will therefore capture these specific analytes. Another DEP filter 116 after the cantilevers can be used to confine or concentrate the analytes in the region close to cantilevers 106-110 to maximize capture by cantilevers 106-110. Optical or electrical measurement of the resonant frequency change can detect the binding of single virus particles, for example, electrical measurement utilizing signal processor and confrol circuitry 118 implementing any of the above discussed embodiments for electromechanical sensing. Mesh filters 122 at the output port of device 102 is useful to ensure that there is a mechanism to contain analytes inside device 102 if infectious agents are found in the air sample. HEPA type filters and the rotary rod method for capturing viral particles that are in the range of 0.5um or less in size my be used to concentrate analytes. For example, an exemplary apparatus for validating the inventive concept may consist of a closed plastic box where known quantities of a virus can be introduced into the air, and the air then circulated over the filter or capture device. The selection of the type of blower which will push the air across the filter will be important as will the size of the plexiglass box, which may be initially fabricate to contain half a cubic foot of air. Viruses are generally 0.05-0.1 microns and hence, the exemplary apparatus may use a surrogate viruses (that are not pathogenic) and focus on particle sizes in the 0.05-0.1 micron range. Calibration of the system may be done using inert particles (that are typically used for calibrating particle size instruments) in order to begin to identify optimal linear velocity through the filter/membrane, and determine the effectiveness of particle capture. Operation using surrogate virus may use standard culture methods. In addition, a labeling compound and fluorescence microscopy can be used to identify virus that are trapped in the filter or membrane. Air-spray with aerosolized virus particles may also be directly injected into device 100 at controlled rates for the puφose of performing proof-of-concept validation. Airborne microorganisms (bacterial, fungal spores, viral particles and pollen) are known to cause various health effects, including infection, hypersensitivity, toxic reactions, irritations, and inflammatory response (see, for example, Agranovski, I. E., N. Agranovski, T. Reponen, K. Willeke, and S. A. Grinshpun, Development and Evaluation of a New Personal Sampler for Culturable Airborne Microorganisms, Atmospheric Environment, 1-10, 2001, incoφorated herein by reference). Virus particles can range from 0.05-0.1 μm in size and are highly resistant to extreme environmental conditions. To detect a few harmful spores from the air in a regular-sized office room represents a very challenging technological endeavor. The airborne particles need to be first pre-processed in order to isolate the bio-aerosol. The air samples in a room need to be pre-processed in order to isolate the airborne microorganism. A coarse filter can be used to filter out the larger airborne particles (see, for example, Battarbee, J. L., N. L. Rose, and X. Long, "A Continuous, High Resolution Record of Urban Airborne Particulates Suitable for Retrospective Microscopical Analysis," Atmospheric Environment, 31(2), 171-181, 1997, incoφorated herein by reference). However, many other non-biological particles also have μm-size characteristics. There are several methods including cylindrical traps, impingers, and centrifugal type collectors for filtering or sorting particles. Cylindrical traps work by coating the inner surface of a cylinder fist with a sticky film such as cellulose. Then, the pre-processed air is feed onto the surface. The viral capture efficiently is heavily influenced by the airflow velocity (see, for example, Griffiths, W. D., I. W. Stewart, S. J. Futter, S. L. Upton, and D. Mark, "The Development of Sampling Methods for the Assessment of Indoor Bioaerosols," J. Aerosol Sci., 28(3), 437-457 (1997); Maus, R., and H. Umhauer, Collection Efficiencies of Coarse and Fine Dust Filter Media for Airborne Biological Particles, J. Aerosol Sci., 28(3), 401- 415 (1997); and Mullins, J., and J. Emberlin, Sampling Pollens, J. Aerosol Sci., 28(3), 365-370, 1997; all of which are incoφorated herein by reference). the impactor technique, a microscope slide is used as the standard sampling surface in the volumetric viral trap developed by Hirst. In Hirst's sampler, the pre-processed air is drawn through a slit with the same dimensions as the second stage of the cascade impactor. The particles then contact the sticky surface of a glass slide, moving past the orifice at 2 mm h"1. Thus, over the course of 24 hr, a trace that is 48 mm long is obtained with the deposit at any point representing the mean concentration over a 1 hr sampling period. This sampler has been adopted as the standard for most pollen sampling networks in Europe (see, for example, Mullins, J., and J. Emberlin, Sampling Pollens, J. Aerosol Sci., 28(3), 365-370, 1997, incoφorated herein by reference). The rotary sample method was devised by Perkins (1957) for short-term sampling and is available through Aerobiology Research Laboratories of Nepean, Ontario, Canada. In this sampler, airborne spores are impacted onto the leading edges of a 'U' -shaped rod made of 1.6 mm square cross- section brass rod, with arms 6 cm long and 8 cm that are rotated through air at 2500- 3000 rpm. Advantageously, DEP filters or valves, which are selective to any object having a dielectric constant different from the medium, for the capture of microorganisms may be utilized instead of the above mentioned approaches. For example, polystyrene beads can be separated from buffer, and cells or spores can be separated from water (see, for example, Li, H. and Bashir, R. Dielectrophoretic separation of live and heat-inactivated Listeria on microchips. Sensors and Actuators, In press, 2002a; Li, H. and Bashir, R. Dielectrophoretic separation of live and heat- inactivated cells of Listeria on microfabricated devices with interdigitated electrodes, Proceedings of the Spring MRS 2002b. San Fransisco, CA; and Gomez, R., Bashir, R., Bhunia, A.K. and Ladisch, M.K. "Microfabricated device for impedance-based detection of bacterial metabolism", Proceedings of the Spring MRS 2002. San Fransisco, CA; all of which are incoφorated herein by reference). This filter trap can be switched on/off by the ac fields and does not clog, unlike mechanical mesh-like filters. These filters can be placed at the end of the fluidic detection chambers as shown in Figs. 34 and 35. DEP separation of live and heat-treated Listeria innocua cells has been achieved as shown in Figs. 36A-C. This was the first report of separation of Listeria in water by DEP with about 90% efficiency by application of a IV and 50KHz signal. It was observed that the DEP behaviors of live 162 and dead 164 cells differed in the frequency range from ~30KHz to -lOOKHz in the selected medium (water). Both live 162 and dead 164 cells can collect either on the top centers of the electrodes 160 in negative DEP, shown in Fig. 36 A, or at elecfrode 160 edges in positive DEP, shown in Fig. 36B, for the special electric field configuration of the interdigitated microelecfrodes used. As shown in Fig. 36C, at 50 kHz, live cells 162 located to elecfrode 160 edges and dead cells 164 located to elecfrode 160 centers. The viability of the cells was verified by a rapid epifluorescence staining method using the LINE/DEAD Bacterial Viability Kit (BacLight, available from Molecular Probes of Eugene, OR) and the live 162 and dead 164 cells can be monitored simultaneously in the experiments. It is envisioned that the separation and manipulation of microorganisms and viruses on biochips using DEP might become very useful method in sample preparation and preprocessing and in diagnostic applications. Referring to Figs. 37A and 37B, an exemplary device may utilize DEP filter 700 to selectively capture a particle of interest inside the integrated circuit.
Polystyrene beads 704 (coated with antibodies selective to target species) are flowed through the integrated circuit. An array of elecfrodes 702 may be used to generate an AC electric field at a frequency on the order of 1 MHz, and a peak intensity of at least 106 V/μm. When beads 704 approach electrodes 702, they will experience a DEP force which repels them away from regions where the gradient of the electric field is maximum (at the edges of electrodes 702). If this force is equal to or larger than the drag force exerted on beads 704 by the liquid flow, beads 704 remain trapped in the chamber (along with the bacterial cells they carry) while everything else in the sample flows out of the chip. Fig. 37A shows 2.38 μm (diam.) beads 704 flowing freely through the chip while the electric field is off. Fig. 37B shows beads 704 accumulating in the middle of electrodes 702 when the field is turned on (beads 704 cannot cross the edges of electrodes 702, where the DEP force if maximum). The techniques described above can be used to concentrate bacteria, cells, viruses, DΝA, or proteins, as long as their dielectric constant is different than the dielectric constant of the medium that they are suspended in. Hence, this technique can be used to separate virus particles from other particles in air, or used to concentrate particles of a particular type in a micro-chamber of interest. Fig. 38 shows an optical image of a device (0.75cm x 0.75cm), which was used to perform the above-mentioned studies of cells and beads in water and buffers. Exemplary interdigitated microelecfrodes according to the present invention can be manufactured on silicon subsfrate using standard photolithography. The electrode material can be gold, 1000 A thick, magnetron sputtered onto a 100 A thick seed layer of chromium. The width of the elecfrodes and the spacing between two adjacent electrodes can be simulated so as to produce enough DEP force to stop the particle in the air-flow. For proof-of-concept validation, the electrodes can be connected to an arbitrary wavefonn generator as the AC signal source by attaching two conducting wires to the contact pads. Fluorescently labeled aerosolized samples can be allowed to enter into the DEP chambers and a sinusoidal signal with varying voltage and frequency values can be applied to the electrode array. The electrokinetic behaviors can then be viewed on a TV monitor through a CCD digital camera on a fluorescence microscope. The filters can be designed using ANSYS or other finite element software, which will allow the modeling of the ac fields as a function of the electrode geometry and spacing. The DEP forces will be calculated. Another exemplary device 700 according to the present invention includes an ultra-thin suspended silicon member 702, for example, a nano-wire, providing an active resonant sensing area, for example a fransistor channel (or gate) in the case of device including a field effect transistor. The suspended member 702 may be grown, for example, by tunnel epitaxial growth, where the growth path of the silicon is restricted and is allowed only in one direction in a cavity. During the formation of the cavity, an oxide layer forming a roof of the cavity can be made to collapse due to stiction and subsequent growth of silicon results in very thin single crystal silicon wires suspended between two islands, which may form the source and drain of the FET. Alternatively, by not doping the source and drain regions, the nano- wires forming suspended members 702 can be simply used as resistors. Resistive nano-wires provides detection of molecules binding on the wire by measuring resistance across the nano-wire. By oscillating the nano-wire member 702 using the FET gate, the current through the FET, specifically the FET channel formed by member(s) 702 is modulated as a function of the distance between the suspended member 702 and the gate. Also, the current will be function of the gate bias, as for typical FETs. The piezo-resistive effects on oscillating nano-wire members 702 also affect the modulation of current due to stress changes that the nano-wire member experiences from mechanical oscillating. By monitoring the drain-source current through the FET, the amplitude of the oscillations and the frequency can be determined, with high signal levels and sinal-to-noise ratios compared to pure capacitative sensing. Because the oscillating frequency and amplitude is sensed electically, and high signal-to-noise ratios are expected, very small changes in resonant frequncy, due to the added mass of particles on nano-wire members 702 can be resolved by incoφorating feedback. For a single crystal silicon (for example, E = 162 GPa and density=2.33g/cm3) nano-wire member, having a length of 5 um, width of 3 urn, and thickness of 0.01 um, the resonant frequency is 2.123340 MHz. Assuming a minimum detectable frequency of 1 Hz, the minimum detectable mass is estimated to be, 5.5xl0"19g, which corresponds to the approximate mass of a single protein, therefore smaller than the mass of a single virus or cell. Alternatively, the novel device can be operated in other modes of electrical operation and/or mass detection, for example, as resistors or transistors, with detection of a change in surface potential or a change in mass. If a resistor is fabricated, then binding of entities on the surface of the nano-wire members 702 will result in change in electrical resistance of the resistor due to change in surface properties. Members 702 can be thermally excited and the mechanical resonance can be measured electrically using capacitive technique and the bottom gate. Then once a mass is added (by a binding event, for example as discussed for exemplary embodiments above), the resonance frequency will change. The resonance could also be measured using the changes in resistance value of the nano-wire member. Alternatively, nano-wire 702 can be capacitively excited using the bottom gate and the mechanical resonance can be measured electrically using the changes in resistance value of the nano-wire upon mass addition to the nano-wire member. If device 700 includes an FET, then binding of analytes on the surface of the suspended nano-wire members 702 will result in change the source/drain current due to changes in surface potential.. Once the suspended nano-wire member vibrates (thermal or capacitive excitation), the piezo-junction effect will change the source drain current and that could be electrically detected. In addition, the thermal vibrations can also be detected capacitively using the bottom FET gate. Alternatively, the suspended nano-wire member 702 can be driven into resonance using the bottom gate and the changes in source/drain current can be measured as a means to detect the vibration and thus change in resonant frequency, as discussed above for cantilever devices. Although carbon nano-tubes and silicon nano-wires have been demonstrated as single molecule biosensors, the fabrication methods that have been used for creating those devices are typically not compatible with modem semiconductor manufacturing techniques and their large scale integration is problematic. The exemplary method of fabrication of silicon nano-wires at precise locations described below overcome those limitations. The exemplary method of fabrication allows for the realization of truly integrated sensors capable of production of dense arrays. Sensitivity of these devices to changes in the ambient gas composition has also been demonstrated. Miniaturization of biological and chemical analysis tools to the level of the "lab on a chip" decreases the analysis time and the sample size needed for specific detection in genomic and proteomic applications as well as in detection of warfare agents and environmental pollutants. In general, as the sensor dimensions shrink down to the size of the analyte, the sensitivity of the device increases. Specifically, suspended nano-wire type sensors are very attractive because their large surface area to volume ratio results in high sensitivity. Carbon nano-tubes and silicon nano-wires have been demonsfrated as single molecule biosensors (see, for example, K. Besteman, J.L. Lee, F.G.M. Wiertz, H.A. Heering, C. Dekker, Nano Lett, 3, 727(2003); Y. Cui, Q. Wei, H. Park, CM. Lieber, Science, 293, 1289, 2001, incoφorated herein by reference), but the fabrication methods that have been used for creating these devices are typically not compatible with modem semiconductor manufacturing techniques and their large scale integration has been quite problematic (see, for example, J.F. Klemic, E. Stem, M. Reed, Nature Biotechnol, 19, 924, 2001, incoφorated herein by reference). However, the exemplary fabrication method according to the present invention and initial fabrication test results on a silicon nano- wire sensors using top-down microelectronics processing techniques overcomes the former problems. A process known as confined lateral selective epitaxial growth (CLSEG) (see, for example, P.J. Schubert, G.W. Neudeck, IEEE Elec. Dev. Lett., 11, 181, 1990, incoφorated herein by reference) was utilized to obtain single crystal silicon nano-plates that are as thin as 7 nm and nano-wires as small as 40 nm in diameter at precise locations. The exemplary fabrication method allows for the realization of truly integrated dense array of sensors. Initial testing of the devices showed sensitivity towards oxygen ambient, suggesting the possibility of using these sensors for chemical and biological detection. Fabrication of exemplary device 800 may be perfonned on p-type low doped silicon wafers 802. A 2000 A thick oxide 804 can be grown by wet oxidation for substrate isolation. A sacrificial layer 806 of amoφhous silicon with 100 A of thickness can next be deposited and defined lithographically on the silicon dioxide layer 804, as shown in Fig. 40A. Another 4000 A thick oxide layer 810 can then be deposited using plasma enhanced chemical vapor deposition (PECVD). Via holes 808 that will subsequently be filled with silicon and act as the FET source and drain regions, can be etched using a reactive ion etch, as shown in Figs. 40 A and 41 A. The sacrificial layer of polycrystalline silicon can then be removed selectively by wet etching using tefra-methyl-ammonium-hydroxide. The removal of the sacrificial layer defines a gap between the thermally grown oxide and deposited oxide. Due to the surface tension of the liquid after the rinse step, bridge formed by the top oxide collapsed, leaving via holes 812 near the anchors of the oxide bridge 814, as shown in Fig. 41 C. Via holes 812 are then used as a mold for the epitaxial silicon to grow through which will later form the suspended nano-wire members. A seed hole 816 was wet etched in thermal oxide 804 on the source side down to the silicon surface 802, as shown in Fig. 40D, in order to grow epitaxial silicon by a CLSEG with no intentional doping. The exemplary fabrication process yields good quality single crystal silicon with low n-type doping (~ 1016 cm"3). Epitaxial silicon grows through via holes 812 at the edges of the collapsed oxide bridge 814 as well as at the interface of the collapsed region. The silicon grown in the interface region between via holes 812 forms a 6-7 nm thick plate. The excess silicon remaining on the surface can be removed by chemical-mechanical polishing as shown in Figs. 40E and 41E. A high dose n-type blanket implant can be performed in order to form conductive source and drain regions 820 and 822, respectively. After depositing a 2000 A thick PENCD oxide insulation layer, a high temperature anneal can be performed to activate and drive in the implanted dopant, and to densify the PECND oxide. Contact holes can next be wet etched in the oxide to access the silicon source and drain regions 820 and 822. A 200 A of chromium, followed by 2500 A of gold was evaporated and patterned to define elecfrical contacts, as shown in Figs. 40E and 4 IE. The final step in the exemplary fabrication process is to uncover (thus suspending) the silicon nano-wire members 824 by removing the encapsulating oxide by un-patterned wet etching in buffered hydrofluoric acid, shown in Fig. 40F. The fabrication method yields a film of silicon about 7 nm thick in the collapsed regions, and 50 nm diameter suspended nano-wire members s at the edges of the film between the anchors. Etching in BHF for 6 minutes removes the oxide covering on the device, leaving the plate and wires in place, with a supporting oxide layer below, as shown in Fig. 45. A longer BHF etch (14 min) removes all remaining oxide as well as the thin silicon film 823 (Fig. 45) between the wires, resulting in the formation of suspended nano-wire members 824 (shown in Fig. 46, and shown in Fig. 39B as 702). After rinsing, the samples can be soaked in methanol without drying to ensure the complete displacement of the water, and then air dried. Experiments have been performed on both the plate and wire structures in order to verify the feasibility of using the structures as field effect sensors. While fluidic detection of chemical and biological analytes is conceived, gas phase measurements were initially performed due to simpler experimental setup. All measurements were performed in a closed chamber where the ambient gas composition and pressure was controlled. A 1 kHz 10 mV peak-to-peak sinusoidal probe signal superimposed on a variable DC bias was produced by a function generator (Model DS345, available from Stanford Research Systems, of Sunnyvale, CA) and fed into the source elecfrode of device 700. The resulting drain current was amplified through a low-noise current preamplifier (Model SR570, also available from Stanford Research Systems) and detected using a lock-in amplifier (Model SR850, also available from Stanford Research Systems). Dry nitrogen was used to purge the test chamber, and 20% oxygen in argon was used as the analyte gas. Using the described setup, we were able to directly collect the small signal conductance (dl/dN) of the device as a function of time and DC bias. The effect of ambient gas on the conductance of the devices was investigated. As confrol measurements, we tested devices that were not released and found no reaction to oxygen ambient. Released devices exhibited up to a 9% decrease in conductance when exposed to 20% oxygen in argon. The decrease in conductance of the wire is believed to be attributed to the physiosoφtion of oxygen species on the nano-wire members resulting in a decrease of the work- function of the silicon surface. It is known that the work- function of the silicon surface reduces upon exposure to oxygen, while it does not change upon exposure to inert gases. A decrease in the work-function will result in an increased energy barrier between the heavy doped n-type contact regions and the low doped wire, and result in a reduction of the current through device 700. The oxygen molecule, which is a diradical and very reactive, effectively induces a net negative charge on the surface of the wires. This results in a net depletion of the silicon nano-wire/native-oxide interface, causing an effective decrease of its electrical diameter. These hypotheses are confirmed by measurement results. Confrol experiments were also performed on released devices with pure argon gas to ensure the conduction change was indeed due to oxygen. Devices which responded to the argon-oxygen mixture did not respond to pure argon. It was also seen that the decrease in conductance was reversible and repeatable. Fig. 42 shows a decrease in the conductance of the plates upon exposure to oxygen, and recovery of the conductance after purging in nitrogen. However it can also be seen that the baseline conductance of device 700 shifts for the same amount of recovery time, indicating some irreversibility of the adsoφtion. In order to fully recover the conductance of devices 700, they were heated in vacuum at elevated temperatures (80-90°C). A similar set of experiments was also performed on the nano-wire members 702. The nano-wire members 702 showed similar response to exposed oxygen, however simply purging the test chamber was not sufficient to recover the conductance of the wire device 700. Device 700 conductance continued to decrease at a slower rate after the nitrogen purge was started until the chamber was evacuated. After evacuation of the chamber the conductance stabilized at a constant value, hi order to desorb the oxygen, the nano-wire device 700 was heated up to 80°C in vacuum and cooled back to room temperature. Upon this procedure device 700 conductance increased due to desoφtion of oxygen gas from the wire surface. A device cycling experiment is shown in Fig. 43. The results obtained from gas phase measurements are very encouraging, and suggest that the use of a top-down nano-fabrication technique as disclosed above is capable of producing nanoscale sensors for the detection of very low concentrations of analytes. By analyte specific receptors or ligand functionalizing and placement of these sensors in a micro-fluidic channel, highly specific and high throughput analysis systems can be realized in a well-integrated fashion. Referring to Fig. 47, exemplary device 900 includes a suspended structure, for example cantilever 902, coupled to support structure 906 and having free end 904 and elecfrode 908. Electrode 908 is capable of functioning as a terminal for DEP. Cantilever 902 may comprise a semiconductor, a metallic conductor, or a non-conductor, or any combination thereof. Electrode 908 may be coupled with or defined by cantilever 902. For example, elecfrode 908 may comprise the entire extent of or a portion of cantilever 902. For example, elecfrode 908 may comprise a thin metal or degeratively doped semiconductor layer or otherwise defined portion of cantilever 902. For example, electrode 908 my include a conductive material extending to free end 904 of cantilever 902, forming a terminal for concentrating the selected analyte at free end 904.

Claims

CLATMS:
1. Apparatus comprising: a filter structure adapted to be coupled to an alternating current voltage source, the filter structure including at least one electrode having a feature which promotes establishment of at least one of a region of increased electric flux and a region of decreased electric flux relative to an adjacent region to select a first species susceptible of dielectrophoretic selection, a cantilever structure upon which the first species is collected, and at least one device for causing the cantilever structure to oscillate, and for receiving as an output from the cantilever structure the cantilever structure's frequency of oscillation, the at least one device adapted to be coupled to means for determining from the output the mass of the first species collected on the cantilever structure. 2. The apparatus of claim 1 , further including the means for determining from the output the mass of the first species collected on the cantilever structure, the means for determining from the output the mass of the first species collected comprising means for determimng from the output the concentration of the first species collected on the cantilever structure. 3. The apparatus of claim 1, further including the means for determining from the output the mass of the first species collected on the cantilever structure, the means for determining from the output the mass of the first species collected comprising means for determining from the output the identity of the first species collected on the cantilever structure. 4. The apparatus of claim 1, wherein the filter structure includes multiple elecfrodes arranged side-by-side to select the first species susceptible of dielectrophoretic selection. 5. The apparatus of claim 1, wherein the feature which promotes establishment of at least one of a region of increased electric flux and a region of decreased electric flux relative to an adjacent region comprises a feature which promotes establishment of a region of increased electric flux relative to an adjacent region to select the first species susceptible of dielectrophoretic selection by being attracted to said region of increased electric flux. 6. The apparatus of claim 5, wherein the feature comprises an edge of the elecfrode. 7. The apparatus of claim 1, wherein the feature which promotes establishment of at least one of a region of increased electric flux and a region of decreased electric flux relative to an adjacent region comprises a feature which promotes establishment of a region of increased electric flux relative to an adjacent region to select the first species susceptible of dielectrophoretic selection by being repelled by a region of decreased electric flux. 8. The apparatus of claim 7, wherein the feature comprises an edge of the electrode. 9. The apparatus of claim 1, wherein the feature which promotes establishment of at least one of a region of increased electric flux and a region of decreased electric flux relative to an adjacent region comprises a feature which promotes establishment of a region of decreased electric flux relative to an adjacent region to select the first species susceptible of dielectrophoretic selection by being repelled by a region of increased electric flux. 10. The apparatus of claim 1, wherein the feature which promotes establishment of at least one of a region of increased electric flux and a region of decreased electric flux relative to an adjacent region comprises a feature which promotes establishment of a region of decreased electric flux relative to an adjacent region to select the first species susceptible of dielectrophoretic selection by being attracted to said region of decreased electric flux. 11. The apparatus of any preceding claim wherein the filter structure and cantilever structure are provided on a common subsfrate. 12. The apparatus of any preceding claim wherein the filter structure, cantilever structure and the at least one device for causing the cantilever structure to oscillate are provided on a common substrate. 13. The apparatus of any preceding claim wherein the filter structure, cantilever structure and the at least one device for causing the cantilever structure to oscillate, and for receiving as an output from the cantilever structure the cantilever structure's frequency of oscillation are provided on a common subsfrate.
14. The apparatus of any preceding claim wherein the first species exhibits an affinity for a second species, the cantilever including the second species for selectively capturing the first species. 15. The apparatus of claim 14, wherein the first species includes a poison and the second species includes a substance which binds the poison. 16. The apparatus of claim 14, wherein the first species includes a toxin and the second species includes a substance which binds the toxin. 17. The apparatus of claim 14, wherein the first species includes an antigen and the second species includes an antibody to the antigen. 18. A integrated micro-electromechanical analyte detection device, compnsmg: a substrate; a support member coupled with said subsfrate; a cantilever having a fixed end coupled to said support member and a free end; and a first electrode coupled to said subsfrate and positioned adjacent said free end of said cantilever, such that said free end and said electrode are excitable terminals for dielectrophoresis. 19. The detection device of claim 18, further comprising a piezo- resistive element defined by said cantilever. 20. The detection device of claim 18, further comprising a piezo- resistive element coupled to said cantilever. 21. The detection device of claim 18, further comprising piezo- resistive element defined at a junction of said fixed end and said supporting member. 22. The detection device of claim 18, further comprising a coating on at least a portion of said cantilever, said coating capable of facilitating binding of a specific analyte. 23 . The detection device of claim 18, further comprising an anti- fouling coating on at least a portion of said cantilever, said coating capable of reducing binding of non-analytes. 24. The detection device of claim 18, further comprising a fluid channel coupled to or defined by said subsfrate, said cantilever positioned within said fluid channel, said fluid channel structured to accommodate flow of at least one of a liquid, gas, solid, water, and air. 25. The detection device of claim 18, further comprising an array of said cantilevers. 26. The detection device of claim 25, wherein each said cantilever includes one of a plurality of coatings each binding a different specific analytes. 27. The detection device of claim 18, further comprising a dielecfrophoresis filter coupled with said substrate; and a fluid channel coupled to or defined by said substrate and in communication with said filter and said cantilever. 28. The detection device of claim 18, further comprising an array of oppositely oriented pairs of cantilever such that said free ends of each said pair are adjacently located. 29. The detection device of claim 18, further comprising a transistor having at least one region defined by a junction of said fixed end of said cantilever and said support member. 30. The detection device of claim 18, further comprising a transistor having a region defined by said cantilever. 31. The detection device of claim 18, wherein said first elecfrode includes a second cantilever having a fixed end coupled to said subsfrate and a free end adjacent said free end of said first cantilever. 32. The device of claim 18, wherein said cantilever comprises two beams each having first ends and second end, said first ends coupled to said support member and said second ends coupled together. 33. The device of claim 18, further comprising a second electrode at least one of coupled to and defined by at least a portion of said cantilever. 34. The device of claim 33, wherein the analyte is at least one of a chemical molecule, protein, biological species, virus particle, cell, spore, mold, yeast, and microorganism. 35. An integrated micro-electromechanical analyte detection device, comprising: a substrate; a support member coupled to said substrate; a cantilever having a fixed end coupled to said support member and a a transistor having at least one region formed on at least one of said free end of said cantilever and a portion of said support member 36. The detection device of claim 35, wherein said at least one region is located adjacent or across a junction of said free end and said support member. 37. The detection device of claim 35, further comprising a piezo- resistive element defined by said cantilever. 38. The detection device of claim 35 , further comprising a piezo- resistive element coupled to said cantilever. 39. The detection device of claim 35, further comprising piezo- resistive element defined at a junction of said fixed end and said supporting member. 40. The detection device of claim 35, further comprising a coating on at least a portion of said cantilever, said coating capable of facilitating binding of specific analytes. 41 . The detection device of claim 35, further comprising an anti- fouling coating on at least a portion of said cantilever, said coating capable of facilitating non-binding of non-specific particles. 42. The detection device of claim 35, further comprising a fluid channel coupled to or defined by said subsfrate, said cantilever positioned within said fluid channel. 43. The detection device of claim 35, further comprising an anay of said cantilevers. 44. The detection device of claim 43, wherein each said cantilever member includes one of a plurality of coatings each binding a different specific analytes. 45. The detection device of claim 35, further comprising a dielectrophoresis filter coupled with said subsfrate; and a fluid channel coupled to or defined by said substrate and in communication with said filter and said cantilever. 46. The detection device of claim 35, further comprising an array of oppositely oriented pairs of cantilever such that said free ends of each said pair are adjacently located.
47. The detection device of claim 35, wherein said region includes a gate of said transistor. 48. The detection device of claim 35, wherein said region includes a channel of said transistor. 49. The device of claim 35, further comprising: a source of said transistor defined in said support member; a drain of said transistor defined in said support member; and a channel defined in the cantilever and electrically coupling said source and drain. 50. The device of claim 35, further comprising a gate of said transistor defined at a junction of said support member and said cantilever 51. The detection device of claim 35, further comprising an elecfrode coupled to said subsfrate such that said cantilever and said elecfrode include excitable terminals for dielecfrophoresis. 52. The detection device of claim 51 , wherein said elecfrode comprises a second cantilever. 53. The device of claim 35, where in said cantilever comprises two beams each having first ends and second end, said first ends coupled to said support member and said second ends coupled together. 54. The device of claim 35, wherein the analyte is at least one of a chemical molecule, protein, biological species, virus particle, cell, spore, mold, yeast, and microorganism. 55. An integrated micro-electromechanical analyte detection device comprising: a substrate; a support member coupled to or defined by said substrate; a suspended member having first and second ends, at least said first end coupled to said support member such that at least a portion of said suspended member is movable relative to said subsfrate; and a field effect fransistor having a channel defined by said suspended member. 56. The detection device of claim 55, further comprising a piezo- resistive element defined by said suspended member.
57. The detection device of claim 55, further comprising a piezo- resistive element coupled to said suspended member. 58. The detection device of claim 55, further comprising piezo- resistive element defined at a junction of said fixed end and said supportive member. 59. The detection device of claim 55, further comprising a coating on at least a portion of said suspended member, said coating capable of facilitating binding of specific analytes. 60 . The detection device of claim 55, further comprising an anti- fouling coating on at least a portion of said suspended member, said coating capable of facilitating non-binding of non-specific particles. 61. The detection device of claim 55, further comprising a fluid channel coupled to or defined by said substrate, said suspended member positioned within said fluid channel. 62. The detection device of claim 55, further comprising an array of said suspended members. 63. The detection device of claim 62, wherein each said suspended member includes one of a plurality of coatings each binding a different specific analytes. 64. The detection device of claim 55, further comprising a dielectrophoresis filter coupled with said subsfrate; and a fluid channel coupled to or defined by said subsfrate and in communication with said filter and said suspended member. 65 The detection device of claim 55, wherein said second end is coupled to said substrate. 66 The detection device of claim 55, further comprising an electrode coupled to said substrate such that said suspended member and said electrode are excitable oppositely charged nodes for dielectrophoresis. 67 The device of claim 55, where in said suspended member comprises two beams each having first ends and second end, said first ends coupled to said support member and said second ends coupled together. 68. The device of claim 55, wherein the analyte is at least one of a chemical molecule, protein, biological species, virus particle, cell, spore, mold, yeast, and microorganism.
69. The device of claim 55, wherein said suspended member comprises a nano-wire. 70. An integrated micro-electromechanical detection device, comprising: a substrate; a first suspended member having first and second ends, at least said first end fixed with said subsfrate; a dielectrophoresis filter coupled to said subsfrate; and a fluid channel defined by said subsfrate and communicating fluid between said first suspended member and said dielectrophoresis filter. 71. The detection device of claim 70, further comprising a transistor having a region defined by said first suspended member. 72. The detection device of claim 70, further comprising a transistor having a region defined by one of said first suspended member, and a junction of said first suspended member and said subsfrate. 73. The detection device of claim 70, further comprising a second suspended member having a third and fourth end, said third end fixed with said subsfrate and said fourth end located adjacent said second end of said first suspended member. 74. The detection device of claim 73, wherein said second end and said fourth end comprise terminals for dielecfrophoresis. 75. The detection device of claim 70, wherein said suspended member includes a coating for binding a specific analyte. 76. The detection device of claim 70, further comprising a piezo- resistive element defined by said first suspended member. 77. The detection device of claim 70, further comprising a piezo- resistive element coupled to said first suspended member. 78. The detection device of claim 70, further comprising a piezo- resistive element defined by a junction of said first end of said first suspended member and said substrate. 79. The device of claim 70, wherein the analyte is at least one of a chemical molecule, protein, biological species, virus particle, cell, spore, mold, yeast, and microorganism.
80. The detection device of claim 70, wherein said fluid channel is structured to accommodate at least one of a liquid, gas, solid, air, and water. 81. A method of detemiining a characteristic of an analyte interacting with an integrated detection device, the device including a subsfrate and a suspended member having first and second ends, at least the first end coupled with the subsfrate, the method comprising: applying an electric signal to the suspended member to perform dielecfrophoresis on the analyte; and determining a change in a parameter of the suspended member. 82. The method of claim 81 , further comprising determining the characteristic of the analyte based on the change in the parameter of the suspended member. 83. The method of claim 81 , wherein the characteristic includes the mass of the analyte. 84. The method of claim 83, wherein the characteristic includes the biological or chemical identity of the analyte. 85. The method of claim 81, wherein the parameter includes the resonant mechanical oscillation frequency of the suspended member. 86. The method of claim 81 , wherein the parameter includes a capacitance between the suspended member and another portion of the device. 87. The method of claim 81 , wherein the parameter includes piezoelectric characteristic of the suspended matter. 88. The method of claim 81 , wherein the parameter includes a current signal characteristic conducted by the suspended matter. 89. The method of claim 81 , wherein the characteristic includes the mass of the analyte and the method further comprises determining the mass of the analyte based on the change in resonant frequency. 90. The method of claim 81 , wherem the parameter includes a current signal characteristic conducted by a region located approximately at the junction of the first end of the suspended member and the substrate. 91. The method of claim 81 , wherein the suspended member comprises a region of a transistor and the parameter includes a current signal conducted by the region.
92. The method of claim 91 , wherein the region includes a fransistor channel. 93. The method of claim 91 , wherein the region includes a transistor gate. 94. The method of claim 81 , further comprising exciting the suspended member into resonant oscillating motion. 95. The method of claim 81, further comprising associating a analyte specific binding agent with the suspended member. 96. The method of claim 95, wherein analyte specific binding agent includes at least one of an antibody, antigen, protein, and small molecule. 97. The method of claim 81, further comprising associating an anti- fouling agent with the suspended member to reducing binding of non-specific particles. 98. The method of claim 81, wherein applying an electric signal includes electrostatic excitation. 99. The method of claim 81 , wherein applying an electric signal includes piezo-electric excitation. 100. The method of claim 81 , wherein applying an electric signal includes excitation by at least one of thermal noise and ambient noise. 101. The method of claim 81 , further comprising counting and locating the relative positions of analytes bound to the suspended member, and wherein the step of calculating the first characteristic is further based on the number and position of the analytes. 102. The method of claim 81 , further comprising vibrating the suspended member through a range of frequencies in order to determine the resonant frequency of the suspended member with a bound analyte. 103. The method of claim 81 , further comprising vibrating the suspended member at a predetermined frequency or frequency range in order to remove non-specific particles from the suspended member while minimizing removal of specific analytes . 104. The method of claim 81 , further comprising exposing the suspended member to a flow of fluid including the analyte.
105. The method of claim 104, further comprising flowing the fluid including the analyte through a dielectropheresis filter. 106. A method of determining a characteristic of an analyte interacting with an integrated detection device including a substrate, an elecfrode and a suspended member having first and second ends, at least the first end coupled with the substrate, the method comprising applying an electric signal to the electrode and the suspended member to perform dielectrophoresis on the analyte. 107. A method of determining a characteristic of an analyte interacting with an integrated micro-electromechanical detection device, the device including a substrate, a support member coupled with the substrate and a suspended member having first and second ends, at least the first end coupled to the support member, the method comprising detennining a change in a current signal conducted by a transistor region defined at least in part by one of the suspended member and the junction of the first end of the suspended member and the support member, upon the analyte interacting with the suspended member. 108. The method of claim 107, further comprising measuring the current signal upon the suspended member oscillating at resonant frequency. 109. The method of claim 107, wherein the change in current signal is related to a change in resonant frequency of the suspended member induced by the analyte interacting with the suspended member. 110. The method of claim 107, wherein the characteristic includes the mass of the analyte. Ill The method of claim 107, wherein the characteristic includes the biological or chemical identify of the analyte. 112. The method of claim 107, wherein the transistor region includes a gate. 113. The method of claim 107, wherein the fransistor region includes a channel. 114. The method of claim 107, further comprising associating a analyte specific binding agent with the suspended member. 115. The method of claim 107, wherein analyte specific binding agent includes at least one of an antibody, antigen, protein, and small molecule.
116. The method of claim 107, further comprising associating an anti-fouling agent with the suspended member to reducing binding of non-specific particles. 117. The method of claim 107, further comprising exciting the suspended member into resonant motion. 118. The method of claim 117, wherein exciting the suspended member includes electrostatic excitation. 119. The method of claim 117, wherein exciting the suspended member includes piezo-electric excitation. 120. The method of claim 117, wherein exciting the suspended member includes excitation by at least one of thermal noise and ambient noise. 121. The method of claim 107, further comprising counting and locating the relative positions of analytes bound to the suspended member, and calculating the characteristic based on the number and position of the analytes and the change in the current signal. 122. The method of claim 107, further comprising the suspended vibrating the suspended member through a range of frequencies in order to determine the resonant frequency of the suspended member with a bound analyte. 123. The method of claim 107, further comprising vibrating the suspended member at a predetermined frequency or frequency range in order to remove non-specific particles from the suspended member while minimizing removal of specific analytes. 124. The method of claim 107, further comprising exposing the suspending member to a flow of fluid including the analyte. 125. The method of claim 107, further comprising flowing the fluid including the analyte through a dielectropheresis filter. 126. A method of fabricating an integrated circuit on a substrate, the integrated circuit including a cantilever, the method comprising: providing a silicon-on-insulator (SOI) wafer having a buried oxide layer (BOX); thinning the SOI layer to less than approximately 30 nm; photolithographically patterning and etching the SOI layer to define the cantilever; thinning the exposed portions of the BOX layer; depositing an etch stop layer to the exposed portions of the SOI and BOX layers; photolithographically patterning an etch window extending laterally approximately from the free ends of the cantilever and vertically to at least the depth of the base of the SOI layer; and etching the BOX layer below the cantilever, thereby releasing an end of the cantilever. 127. A method of fabricating an integrated circuit on a subsfrate, the integrated circuit including a cantilever, the method comprising: patterning a seed window using an insulator mask on substrate; providing an oxide layer displaced vertically above the seed window and laterally overlaying the seed window and at least a portion of the insulator mask; growing silicon selectively from the base of said seed window, vertically to the top of the insulator mask, and laterally between the insulator mask and the oxide layer; and etching at least a portion of the oxide layer and the insulator mask, thereby releasing a suspended end of the selectively grown silicon from the remaining structure. 128. A method of fabricating an integrated circuit on a substrate, the integrated circuit including a cantilever and a field effect transistor, the method comprising: providing a silicon-on-insulator (SOI) wafer; thinning the SOI in the area where the cantilever will be formed using an anisotropic etch, thereby fonning an abrupt step adjacent the top surface of cantilever and the junction of the remaining structure; oxidizing the silicon surface of the cantilever and the step to form transistor gate oxide; depositing polysilicon gate material conformally along the top surface of the cantilever adj acent the step; anisotropically etching the polysilicon gate material through the entire deposited thickness such that a sidewall of polysilicon remains on any exposed vertical surface, the sidewall having a lateral width approximately equal to the deposited thickness; implanting a source and drain region using the polysilicon gate as an implantation mask; and etching to release a suspending portion of the cantilever on the end opposite the fransistor gate. 129. A method of fabricating an integrated circuit on a substrate, the integrated circuit including a field effect fransistor (FET), the method comprising: providing a silicon wafer; thermally growing a first thick oxide layer for subsfrate isolation; lithographically patterning and depositing a sacrificial silicon layer on the oxide layer; depositing a second thick oxide layer using plasma enhanced chemical vapor deposition (PECND); etching via holes to define the FET source and drain regions; etching to selectively remove the sacrificial silicon layer, the removal defining a bridge formed by a portion of the second oxide layer suspended over the thennally grown oxide layer; collapsing the oxide bridge to leave nano-scale via holes near the anchors of the oxide bridge; wet etching a seed hole in the first oxide layer in the source region via hole, the seed hole extending down to the silicon layer surface growing silicon epitaxially through the via holes; and removing silicon remaining on the surface by chemical mechanical polishing (CMP); implanting the source and drain regions; etching to remove the oxide encapsulating the epitaxially grown silicon, the silicon forming suspended nano-wires forming the FET channel and a thin connecting silicon plate between the wires; and etching to remove the remaining oxide covering on the device and the thin silicon plate between the wires.
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