WO2001057494A2 - Chemical sensor system utilizing microjet technology - Google Patents

Abstract

Microjet technology is used to print one or more indicator chemistries on an optically accessible surface. Each indicator chemistry contains one or more light energy absorbing dye(s) whose optical characteristics change in response to the target ligand or analyte. By spectrally monitoring these changes using fluorescence and/or absorption spectroscopy, sensitive detection and/or quantitation of the target ligand or analyte can be obtained.

Description

CHEMICAL SENSOR SYSTEM UTILIZING MICROTET TECHNOLOGY

The United States Government has rights in this invention pursuant to Contract No. W-7405-ENG-48 between the United States Department of Energy and the University of California for the operation of Lawrence Livermore National Laboratory.

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No.

60/177,105, filed 01/20/2000, entitled "Method for Creating Chemical Sensors Using Microjet Technology," which is incorporated herein by this reference.

BACKGROUND OF THE INVENTION Field of Endeavor

The present invention relates to chemical sensors, and more particularly, to chemical sensors utilizing microjet technology.

State of Technology

Over recent years, intensive research has occurred in the area of optical- based chemical sensor and detection methods for process control, environmental, occupational safety, quality control, biomedical applications, and other uses. The sensors typically consist of an indicator chemistry attached to the end of a fiber optic or other optically accessible surface, where the indicator chemistry is designed to change its optical properties (e.g. fluorescence, absorption) quantitatively in response to the presence of a target ligand or analyte.

Light of a suitable wavelength is used to illuminate the sensing zone. Some portion of this light is absorbed by the indicator chemistry (s). The absorption or re-emission of this radiation is then monitored via photosensitive detectors and the resulting signal used to make qualitative or quantitative determinations concerning a ligand or analyte of interest.

Most conventional optical-based chemical sensors, particularly those based on fiber-optic technology, require a separate sensor for each target ligand. Recently, however, researchers have developed a method for immobilizing multiple different indicator chemistries in discrete areas on the end of a fiber optic bundle. Each indicator chemistry can then be spatially discriminated from its neighbors via optical imaging techniques. This has several advantages over previous one indicator/one fiber sensors. The primary advantage of this technique is that different ligands in a very localized region can be simultaneously detected and/or measured in vivo.

This is particularly important for biomedical applications, where collection or interrogation of a small sample volume (i.e. blood, gingival crevicular fluid, saliva) is desirable or necessary. A secondary advantage of this multi- analyte sensor is the ability to use the same excitation and /or emission wavelength for all the different indicator chemistries, greatly simplifying the complexity and cost of the spectroscopic components and synthesis of the indicator chemistries. U. S. Patents Nos. 5,244,636; 5,250,264 and 5,320,814 to David R. Walt and

Steven M. Barnard, assigned to Trustees of Tufts College, patented June 14, 1994, incorporated herein by reference, describe a fiber optic sensor used in an apparatus for detecting at least one analyte of interest in a fluid sample. The detection of an individual analyte of interest is correlatable with an individual optical determination. The fiber optic sensor comprises a preformed, unitary fiber optic array comprising a plurality of individually clad, fiber optical strands disposed co-axially along their lengths and having two discrete optic array ends each of which is formed of multiple strand end faces. The preformed, unitary fiber optic array is of determinable configuration and dimensions. The two discrete ends of the preformed unitary fiber optic array presents two discrete optic array surfaces for introduction and conveyance of light energy. At least one sensing zone comprises not less than one light energy absorbing dye disposed as an uninterrupted deposit in aligned organization upon multiple strand end faces on one of the discrete optic array surfaces of the preformed, unitary fiber optic array. The different spatial positioning of each dye deposit is in aligned organization within said sensing zone on said discrete optic array surface and serves to identify and distinguish each light energy absorbing dye from all other light energy absorbing dyes disposed within said dye sensing zone. Each spatially positioned dye reacts with one analyte of interest. At least one sample viewing zone is adjacent to the dye sensing zone on the discrete optic array surface of the preformed, unitary fiber optic array. The sample viewing zone is formed of multiple strand end faces in aligned organization and in fixed spatial position on the discrete optic array surface.

The prior art chemical sensors were produced by immersing the optical fiber in a photopolymerizubic indicator chemistry and selectively "growing' the indicator chemistries on the end of the optical fiber strands. This process has numerous disadvantages. Among the disadvantages are the lack of reproducibility, the lack of uniformity, the need for individual calibration, the effect subsequent processing steps have on chemistries produced by earlier steps, inter and intra sensor variability, cost of chemicals, non-disposability, limited multi-plex ability by process. The main reason these prior art chemical sensors have never found wide-spread use is the difficulty inherent in reproducibly and inexpensively fixing indicator chemistries on an optical substrate. These limitations make disposable sensors impractical, greatly reducing the number of potential indicator chemistries and limiting the sensors' usefulness in clinical applications where infection control is necessary. Since reproducibly attaching precise amounts of a given indicator chemistry in a well- defined geometry on a fiber optic or other supportive surface is difficult, moreover, conventional methods of creating optical-based chemical sensors result in high inter- and intra-sensor variability. This variability significantly increases the complexity and cost of the manufacturing process and makes individual calibration for each sensor necessary.

SUMMARY OF THE INVENTION The present invention relates to chemical sensors, production of chemical sensors, and operation of chemical sensors. More specifically, the present invention relates to a new chemical sensor, a new method of producing a chemical sensor, and a new method of operating a chemical sensor. The chemical sensor is used for detecting and /or analyzing at least one ligand or analyte of interest in a fluid or airborne medium. In the present invention, microjet technology is utilized to print one or more indicator chemistries on an optically accessible surface. Each indicator chemistry contains one or more light energy absorbing dye(s) whose optical characteristics change in response to the target ligand or analyte. By spectrally monitoring these changes using fluorescence and /or absorption spectroscopy, sensitive detection and/or quantitation of the target ligand or analyte is obtained. Multiple ligand-specific indicator chemistries are printed in a known pattern. Simultaneous detection and /or measurement of these ligands or analytes is accomplished using optical imaging techniques to spatially register each microdot. The present invention can be better understood by contrasting it with the prior art. In the prior art, chemical sensors were produced by"growing" the indicator chemistries on the end of the optical fiber strands. The "growin ' process consists of passing light through the optical fiber strands and exposing the ends of the strands to various chemical compositions. The indicator chemistries thus produced are not uniform, must be individually calibrated, and have difficulty remaining attached in the flowing stream (such as blood) when affected by mechanical vibration or stress. In the present invention microjet technology is used to print one or more of the indicator chemistries on the optically accessible surface. This allows exact duplicate copies of the indicator chemistries to be produced. A significant advantage is the uniformity of the indicator chemistries produced by the invention process. There is no need for individual calibration of the indicator chemistries. Because of the efficiency of mass production, chemical sensors produced by the present invention will be inexpensive compared to chemical sensors produced by the prior art. The prior art indicator chemistries are restricted by the effect of previous processing steps. The cost of chemicals and the disposability of the chemicals used in the prior art is avoided by the present invention. The limitations of the prior art makes disposable sensors impractical, greatly reducing the number of potential indicator chemistries and limiting the sensors' usefulness in clinical applications where infection control is necessary.

An advantage of the present invention is the ability to reproducibly attach precise amounts of a given indicator chemistry in a well-defined geometry on an optically accessible surface.

An aspect of the present invention is to provide sensors for a wide range of biomedical, environmental, occupational safety, process control, and bio war fare applications.

Another aspect of the present invention is the reproducibility and cost- effectiveness with which microjet fabricated sensors can be constructed.

Additional aspects, advantages, and features of the invention are set forth in part in the description that follows. Various aspects, advantages, and features of the invention will become apparent to those skilled in the art upon examination of the description and by practice of the invention. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic of a polymer printing station used in printing patterns of dye-doped microdots onto the tips of optical fiber. FIG. 2 shows a stroboscopically illuminated image of 50 μm droplets of fluid being emitted from a microjet print head with 50 μm orifice diameter operating at 2000 Hz.

FIGS. 3A and 3B are an illustration of seven 90 μm diameter microdots inkjet printed on a 500 μm diameter fiber optic bundle. FIGS. 4A and 4B are an illustration of a single 90 μm diameter microdot inkjet printed on a fiber optic bundle.

FIG. 5 is a ball and stick model illustrating indicator chemistry for measuring collagenase activity. FIG. 6 is an illustration showing that the intensity of the rhodamine signal decreases as the probe substrate is cleaved by the MMP enzyme.

FIG. 7 illustrates a configuration of an in vitro chemical sensor.

FIG. 8 illustrates the operation of a chemical sensor system. DETAILED DESCRIPTION OF THE INVENTION Referring now to the drawings and in particular to FIG. 1, an embodiment of a system constructed in accordance with the present invention is illustrated.

The present invention provides an optical-based chemical sensor system using microjet technology. The sensor system is useful for detecting and /or measuring at least one target ligand and/or analyte in a fluid or airborne sample. The system shown in FIG. 1 utilizes microjet technology to print one or more indicator chemistries on an optically accessible surface. Each indicator chemistry contains one or more light energy absorbing dye(s) whose optical characteristics change in response to the target ligand or analyte. By spectrally monitoring these changes using fluorescence and /or absorption spectroscopy, sensitive detection and /or quantitation of the target ligand or analyte can be obtained. If multiple ligand-specific indicator chemistries are printed in a known pattern, simultaneous detection and /or measurement of these ligands or analytes can be accomplished using optical imaging techniques to spatially register each microdot.

The system shown in FIG. 1 includes an automated microjet print head 2, a motion control systems 3 for printing accurately at pre-specified target sites and for shuttling the print head 2, and a UV-light pipe and target-viewing system 4. The printing axis is in the vertical plane, and a horizontal microscope 5 with stroboscopic illumination is utilized for viewing the microjetted droplets during microjetting process optimization for a particular material.

The equipment, print head device configuration, and printing process utilized in the system shown in FIG. 1 are similar to those used for the printing of micro-optical elements. U. S. Patent No. 5,707,684 To Donald J. Hayes, et al, patented January 13, 1998, assigned to MicroFab Technologies, Inc., incorporated herein by reference, shows a method for direct printing of micro- optical components onto optical substrates or active devices to create optical circuit elements as well as micro-optical components and systems, such as plano-convex circular, cylindrical or square lenslets, anamorphic lenslets, waveguides, couplers, mixers and switches and monolithic lenses deposited directly onto optical components such as diode lasers and optical fibers. Individual components of the system include oscilloscope 7, waveform generator 8, LED drive 9, PWR Amp 10, PC Control 3, TV 4, data input 11, Z- axis stage 12, UV pipe 13, print head 2, microjet orifice 14, shuttle stage 15, substrate 16, substrate vacuum chuck 18, substrate XY stages 19, jet viewing microscope 20, TV 21, TV 22, and fume exhaust 23.

Referring now to FIG. 2 the time and spatial precision and reproducibility of the microjetting process of the present invention is illustrated. The system of the present invention includes a drop on demand printing system wherein a droplet is emitted from the device orifice every time an appropriate driving pulse produces a displacement of the piezoelectric element in the device. The image 24 shown in FIG. 2 is the superposition of about 1000 droplets, indicating by its clarity the time and spatial precision and reproducibility of the microjetting process. The image 24 shown in FIG 2 is a stroboscopically illuminated image of superposition of 50 μm droplets of fluid being emitted from the microjet print head 2. The microjet print head 2 has a 50 μm orifice 14 diameter operating at 2000 Hz. The micrograph of FIG. 2 demonstrates the time and spatial precision and reproducibility of the microjetting process.

A clean-air /exhaust system 6 is used to prevent airborne particulate contamination of the substrate and to evacuate any fumes arising from heating of typical polymeric formulations to the temperatures required (up to 200°C) to reduce their viscosities to the 20-30 cps level needed for microjet printing. The fluids contained in the print head reservoir are typically kept under a nitrogen atmosphere prior to being emitted by the print head device orifice, in order to prevent any degradation from oxygen-driven reactions during heating.

Upon striking a target substrate, a deposit consisting of one or more droplets spreads to an equilibrium diameter. This diameter depends heavily on the characteristics of the material being printed, the degree of wettability of the substrate surface by the material and the speed with which its flow may be arrested by initiation of solidification, e.g., by in-situ UV curing or cooling. Control of the dimensions and aspect ratio of a printed element to a given specification is obtained by adjusting the following variables: (a) number and diameter (via device orifice size) of droplets deposited at a target site;

(b) characteristic contact angle of the low-wet coating applied to the substrate;

(c) temperature of the substrate during printing. Results of an initial study of the system 1 are useful in explaining the system. Half a dozen fiber bundles with a six-around-one pattern of fluorescein microdots on the end were produced. This is shown in FIGS. 3A and 3B. FIG. 3A is a top view and FIG. 3B is a side view of seven 90 μm diameter microdots 25 inkjet printed on a 500 μm diameter fiber optic bundle 26. Each microdot 24 contains a fluorophore contained in a UN curable polymer matrix.

Referring now to FIG. 4A, which is an expanded top view, and FIG. 4B, which is a side view, an illustration of a single 90 μm diameter microdot inkjet 27 printed on a fiber optic bundle 28. The polymer matrix acts as a lens from the top view, providing a clear image of the underlying fiber bundle. The diameter of the circle through the centers of the circumferentially printed microdots 24 is 260 μm, indicating this pattern easily fit on the end of the 480 μm fiber bundle. The deviation between the central microdot and fiber bundle axis is approximately 2 μm.

The reproducibility of the fiber printing process was excellent for both microdot diameter and roundness. All fiber-average and standard deviations for these data are:

Microdot diameter = 93.3 +/- 2.2 μm and

Microdot roundness [(max. diameter - min. diameter )/ avg. diameter] = .00072 +/- .00023.

Spectroscopic measurements were made using an imaging spectrometer. Microdot intensity, both on a single fiber bundle and between different fiber bundles, varied by less than 2%. The foregoing clearly demonstrated the capabilities of microjet technology for reproducibly printing a pattern of identical but spatially discrete sensing regions on a fiber optic bundle.

A preferred embodiment of a system for detecting microjet-based chemical sensors is illustrated in Figure 8. The system is designated generally by the reference numeral 40. Indicator chemistries contained in a polymer matrix are microjetted on the tip of a unitary fiber optic array 42. The Indicator chemistries are designated by the reference numerals 50 and are located on the end 41 of the fiber optic array 42.

A light source 43 is spectrally filtered (filter 44), coupled into the fiber array 42, and used to illuminate the sensing region. After being exposed to a sample containing the analyte of interest, optical changes in the indicator chemistry 50 are returned through fiber array 42, spectrally filtered (filter 45), and detected using a two-dimensional detector (detectors 48 and 49), such as CCD cameras. Appropriate focusing elements including mirrors 46 and 47 are used for collimating, focusing, and coupling the light between the source, sensor, and detector elements. The focusing elements are well know in the art. Spatial orientation of the detected light is used to uniquely identify each microjetted region, while the intensity and wavelength of the fluorescent indicator emission is used to detect and measure the targeted analytes of interest. The components and operation of the system and associated apparatus of the present invention will now be described. The components of the sensor system and associated apparatus include the following: 1. Indicator Chemistries One or more indicator chemistries microjet printed on the surface of an optically accessible surface where each indicator chemistry contains at least one light energy absorbing dye whose optical characteristics change in response to the target ligand. Light absorbing dyes are typically divided into two different classes: fluorophores - those compositions that emit light energy after absorption; and chromophores - those compounds that absorb light energy and internally convert this energy to kinetic or heat energy. These dyes can, in addition, be linked to other materials such as enzymes substrates and antibody conjugates that interact with the target ligand. Specific examples are provided below. a. Chromophores

Some absorptive dyes are the family of triphenylmethanedyes, such as malachite green and phenolpthalein, and the family of monoazo dyes that include the mordant browns, oranges, yellows and reds. b. Fluorophores The are many fluorescent dyes used in chemical assays. The most common are the xnathine dyes (fluroescein and rhodamine), oxazine dyes (nile blue and cresyl violet), the coumarins, and the more recently developed bimanes. Direct measurement of PH, for example, can be made using fluorescent dyes. c. Fluorescent antibody conjugates

Antibodies are proteins synthesized by an animal in response to a foreign substance, called an antigen. Antibodies have specific affinity for the antigens elicited by their synthesis, with the capability to discriminate differences of a single residue on the surface. Fluorescent antibody conjugates can therefore be used in a solid phase immunoassay to quantitate the amount of a protein or other antigen. These tests, currently referred to as enzyme-linked immunosorbent assays (ELISA), are fairly rapid and convenient. During an ELISA assay, an antibody is attached to a polymeric support and exposed to the target protein. After washing the support to remove any unbound molecules, a second antibody specific for a different site on the antigen is added. The amount of second antibody added to the support is proportional to the quantity of targeted antigen in the sample. This second antibody is also linked to an enzyme, such as alkaline phosphatase, that can rapidly convert a colorless substrate into a colored product, or a nonfluorescent substrate into a fluorescent product. The primary limitations of this technology are the multiple washing and steps necessary to reach a fluorescent product and the nonspecific binding that occurs with some antibody substrates. These limitations make creation of an in vivo device challenging. The benefit, however, of using ELISA assays is the relatively huge number of antibody based tests already available for many target diseases (such as pregnancy, HIV, etc.). d. Fluorescent enzyme substrates

Enzyme substrates are highly specific both in the reaction catalyzed and in their choice of reactants, called substrates. An enzyme usually catalyzes a single chemical reaction (such as cleaving a peptide chain) or a set of closely related reactions.

A specific example of an indicator chemistry containing an enzyme substrate with two dyes is illustrated in FIG. 5. Here the targeted ligand is collagenase, a destructive enzyme that participates in the breakdown of the major protein components of the extracellular matrix. The activity of these proteinases can be determined by the rate at which the enzyme cleaves a specific amide linkage that binds two amino acids of a particular sequence in the protein substrate. However, rather than determine the rate at which an intact protein is cleaved, sensitive assays have been developed which use a short amino acid sequence that represents the substrate portion of protein recognized by the collagenase. These sequences are usually only six to ten amino acids long. The polypeptide is prepared with two different fluorescent dyes (rhodamine and fluorescein), one at each end of the substrate molecule. See Nagase, H. and Fields, G. B., Human Matrix Metalloproteinase Specific studies Using Collagen Sequence-Based Synthetic Peptides, Biopolymers

(Peptide Science) 1996, 40: 399-416, incorporated herein by reference. These dyes are specially chosen because they form an energy transfer (ET) pair, such that when the dye molecules are within a minimal distance from one another, energy absorbed by fluorescein (the donor) is transferred directly to the nearby rhodamine (the acceptor).

The efficiency of the transfer process is dependent on several factors, but two important requirements are: (1) that there be overlap between the emission spectrum of fluorescein and the excitation spectrum of rhodamine, and (2) that the dye molecules be located within a limited distance of one another, generally less than 4 nm. In the absence of enzyme activity, fluorescein absorbs blue light.

However, rather than lose this energy as fluorescence, the energy is efficiently transferred to the nearby rhodamine attached just a few amino acids away on the short polypeptide. When the substrate molecule is subjected to collagenase activity, the molecule will be cleaved at a specific amino acid sequence between the two dyes of the ET pair as shown in FIG. 5.

The fragments that result from this activity separate in solution substantially beyond the minimal distance allowed for energy transfer to occur. Consequently, the energy absorbed by fluorescein is not transferred to rhodamine but rather is emitted as fluorescence from fluorescein's emission manifold with a maximum at 512 nm. The change in the ratio of light emitted from fluorescein (512 nm) and from rhodamine (564 nm) is a measure of enzyme activity as shown in FIG. 6. FIG. 6 illustrates that the intensity of the rhodamine signal decreases as the probe substrate is cleaved by the MMP enzyme. With the rhodamine no longer attached to the probe, it diffuses away leaving fluorescein to loose its absorbed energy through fluorescence. The intensity of the rhodamine emission diminishes and the florescence emission from fluorescein increases as the energy-transfer-pair-linked substrate is cleaved by enzyme.

This approach could be used to measure the activity of metalloproteinases other than collagenase. Because each metalloproteinase enzyme recognizes a different substrate amino acid sequence, indicator chemistries could be developed that separately assay the activity of each of the targeted metalloproteinases. This could be particularly valuable for a wide range of diseases that activate an undesirable immune response. In particular, this method would be valuable for detection of periodontal disease activity, where measurement of a single biomarker is often inadequate to make an accurate diagnosis. Table 1 lists fluorogenic substrate probes that have been evaluated and used to measure the activity of several matrix metalloproteinases. See Nagase, H. and Fields, G. B., Human Matrix Metalloproteinase Specific studies Using Collagen Sequence-Based Synthetic Peptides, Biopolymers (Peptide Science) 1996, 40: 399-416, incorporated herein by reference.

Table 1. Fluorogenic substrates for various MMPs. MMP Family Specific Enzyme Mwt(kDa) Probe Sequence

MMP-1 collagenase interstitial collagenase 42 Dnp-Pro-Leu-Ala-Leu-Trp-Ala-Arg- NH₂ MMP-2 gelatinase gelatinase A 72 Mca-Arg-Pro-Lys-Pro-Tyr-Ala-Nva-Trp-

Met-l_ys(Dnp)-NH₂

MMP-3 stromelysin stromelysin-1 45 Dnp-Pro-Tyr-Ala-Tyr-Trp-Met-Arg-OH

MMP-7 gelatinase matrilysin 19 Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH₂

MMP-8 collagenase PMN collagenase 65 Dnp-Pro-Leu-Ala-Tyr-Trp-Ala-Arg~NH₂

MMP-9 gelatinase gelatinase B 92 Dnp-Pro-Leu-Gly-Met-Trp-Ser-Arg- NH₂

e. Optically Responsive Particles

Particles, in some embodiments, possess both the ability to bind an analyte of interest and to create a change in the optically detected signals. In general, these particles can be conveniently organized into three classes: polymer-based, inorganic crystals, and quantum dots. The first type of particle consists of a polymeric material, such as polystyrene, acrylamide, dextrose, etc. These polymer beads can be optically encoded (e.g. with organic dyes) to provide unique signatures. In one embodiment several different bead sets could each be doped with different amounts of a single organic dye, allowing unique optical identification based solely on the strength of the detected fluoresecent signal. Further complexity can be added by doping these polymeric beads with combinations of optical dyes, where each dye has a given spectral emission. This method is commonly used in flow cytometry instruments to provide mobile sensing platforms. The polymer particle itself, the organic dye used to dope the particle, or the attached indicator chemistry (e.g. antibodies, oligopeptides, DNA, etc.) can all serve as the indicator chemistry that responds to the target analyte. One preferred embodiment, for example, uses optically encoded microbeads with attached recognition antibodies that respond optically to an analyte of interest.

The second type of particle, optically active inorganic crystals, can also be used as sensors or sensor-containing platforms. One class of these crystals that has desirable characteristics for this type of application are upconverting phosphors. These compounds convert light of longer wavelengths into higher energy, lower wavelength phosphorescence. This is desirable since longer wavelength light sources, particularly diode based laser systems, are much more available and inexpensive than lower wavelength sources. It is possible to create multiple upconverting phosphors with distinct spectral characteristics, allowing unique identification of each crystal set. These can then behave in a similar fashion to organically dyed polymeric microbeads as sensors, or sensor- containing supports. They have the advantage, when compared to organic dyes, of being much more optically stable and less suspectible to light or temperature-based degradation (i.e. photob leaching).

The third class of chemically sensitive particles, quantum dots, are relatively new and have great promise as optical labels. These particles are typically 1-100 nm in size, composed of materials such as silicon, germanium arsenide, and other semiconductor-type materials. Quantum dots interact with light in a very different method than fluorescent-based dyes, with several advantages. While fluorescent emission typically has a relatively broad spectral bandwidth (20-60 nm), quantum dots in theory can have sub-nanometer type spectral bandwidths. This aspect makes them very attractive for spectral multiplexing schemes, where each quantum particle is easily identified by the wavelength of light it emits. In addition, quantum particles of the material but different size emit light at different wavelengths, but can all be excited at a single wavelength. This has very practical advantages when designing sensor instrumentation, since a single light source can be used to produce multiplexed signals. The spectral emission of quantum particles is very susceptible to surface effects. These effects can be used as a sensor medium, where interaction with different analytes of interest produce shifts in the spectral emissions of the particles, or the surface can be inactivated and the particles used as optically- active labels for other recognition moieties, such as antibodies, oligonucleotides, etc. 2. Polymer matrix

When forming and depositing each indicator chemistry in a microdot, it is desirable to combine the absorbing dye with monomer formulations to create a polymerizable mixture. A variety of different polymerization processes are known, including thermal techniques, photoinitiated methods, ionization methods, plasma methods, and electroinitiation methods. The most commonly used methods in microjet processes use thermal and/or photoinitiated methods. There are several key characteristics a polymer formulation should have if it is to be used in microjet printing indicator chemistries. The polymer formulation selected should have the appropriate chemical and physical properties (such as polarity and viscosity) for forming small, evenly distributed microdots on a given optical substrate. In addition, the chosen polymer matrix allows intimate interaction with the target ligand maximizing sensor sensitivity and minimizing sensor response time. By selecting polymers that are wettable, only slightly cross-linked, and biologically compatible, it is possible to minimize the effects of substrate immobilization and maintain a solution-phase-like environment. There are several types of polymers to choose from which are compatible with enzymes, including polyacrylamides, polyhydroxyethylmethacrylate, and various phosphazene polymers. 3. Optical substrate

The optical substrate on which the indicator micodots are placed should have several basic properties. The surface of the substrate must be accessible to incident and emitted light energy. In the case of a transmission based measurement, the substrate would necessarily consist of a transparent media such as glass and some plastics. A transparent substrate is not necessary, however, for a reflection based measurement, since the indicator microdots could be accessed from either side of the optical substrate. The surface of the substrate should be designed to permit minimal spreading of the microdots during the printing process while still maintaining good adhesion between the microdot and the optical substrate. Some type of surface preparation, such as glass silanization, could be necessary to make this feasible. Finally, the optical substrate chosen should be capable of being mass-produced inexpensively. Examples include injection molded plastics, fiber optics, and preformed glass components. The choice of an optical substrate is also largely dependent on the desired sensing application. Two types of measurements are generally made: in vivo, where the measurement is made directly in the sample volume; and in vitro, where a sample volume is collected and then exposed to the sensing apparatus. a. In vivo applications

For in vivo applications it is desirable to have the sensor portion contained in a probe capable of accessing the desired sample. The sensor, for example, could be incorporated in a mechanical periodontal probe for sampling the gingival crevicular fluid and saliva; a needle for accessing tissue; a catheter, endoscope, or guidewire for monitoring blood constituents; a cone penetrometer for making soil gas measurements; or a down well sampler for ground water monitoring, among others. A fiber optic is a natural choice for these applications, since fibers can guide light long distances with minimal loss of intensity and are very compact. If individual fiber optics are used as the optical substrate (identifying fiber-independent indicator chemistries must rely on spectroscopic differentiation) since light returned from the sensing regions on the fiber tip would not be spatially coherent. These fibers could conceivably be bundled together to create a multi-component sensor. Alignment of these fibers during the microjetting process, however, could be challenging. A simpler approach is to use a fiber optic bundle, such as those used for endoscopic imaging applications. A standard fiber imaging bundle may contain over a 1000 individual fibers optics in a small diameter bundle (<500 μm). Since each microdot overlays at least one imaging fiber the orientation (i.e. rotation) of the bundle tip relative to the microjet element becomes less important, making sensor manufacture much easier and allowing many more indicator microdots to be placed in a given area. The microdots could either be printed directly on the distal end of the fiber bundle or printed on the tip of a disposable sleeve (e.g. plastic) that could be slipped over the end of the imaging fiber bundle. b. In vitro applications

Although the physical constraints on in vitro sensor design are less than for in vivo probes, it is still usually desirable to analyze only a small sample volume at one time. There are several reasons for this. First of all, a small volume implies precise sampling from a specific location. This is important for measuring changes that may only occur in a very localized region. For example, periodontal disease activity can vary significantly in a single patient depending on what part of the oral cavity is probed. Secondly, it is often difficult to obtain a large sample volume. This is particularly true of biomedical applications (such as blood glucose monitoring). Finally, a small sample volume allows multiple measurements to be made at the same site without significant risk of sample dilution. Sensors constructed using the method described in this invention are ideally suited for measuring multiple constituents in a small sample volume, since each indicator microdot occupies such a small area. Inexpensive inkjet printers, for example, are capable of 600 dpi resolution. This translates to approximately 30 μm diameter microdots (onto absorbing paper only; on glass or plastic a 2x diameter spread would be expected). At this resolution (60 μm diameter) approximately 166 different indicator chemistries, each separated by a full microdot diameter of space, could be placed in a channel 1 mm deep x 1 mm wide x 10 mm long, with less than 10 μl of sample volume required to make the measurement. A simple configuration for an in vitro sensor is shown in FIG. 7 to illustrate this concept. A sample 29 is flowed through microchannels

30 containing indicator chemistry microdots 31. Sample measurement is performed by imaging 32 and spectrally filtering 33 the emitted light from the flow cell microdots. 4. Microjet printing The process for microjet printing indicator chemistries on an optical substrate is similar to that used to produce micro-optical components. The method provides a means of precisely printing many different materials in a given pattern and a wide variety of microdot geometries. Utilizing data-driven, drop-on-demand inkjet technology, droplets of polymeric material may be deposited onto substrates such as the tips of optical fibers to form arbitrary patterns of arbitrarily sized optical elements. By incorporating multiple print heads and a fiber mounting substrate into a polymer jetting platform such as that illustrated in FIG. 1, different indicator chemistries may be printed into a pattern on the same optical fiber. Fiber printing throughput rates in excess of 5 fibers/ minute, for example, would be readily achievable on a production microjet printing platform depositing four indicator chemistries per fiber. 5. Illumination and detection of the sensing site

Some type of light energy must be transmitted to the sensing site for optical changes in the indicator chemistry to be observed. The simplest types of light sources include light emitting diodes (LEDs), lasers, laser diodes, and filament lamps. These sources can be used in conjunction with optical filters, diffraction gratings, prisms, and other optical components to provide a specified spectral component of light. Alternative forms of radiation such as bioluminescence, phosphorescence, and others could also potentially be employed. Although typical fluorophores require excitation wavelengths in the visible portion of the spectrum (300-700 nm wavelength), other wavelengths in the infrared and ultraviolet portion of the spectrum could also prove useful for illuminating the indicator chemistry (s). The transmitted, reflected, or re- emitted light from the sensing region must then be propagated to an optical apparatus for detection and/or some type of spectral and spatial filtering. a. Spectral filtering

The same techniques as those described above (i.e. optical filters, diffraction gratings, etc.) can be used to spectrally process changes in the light returning from the sensing region. There are several ways this spectral information available from each illuminated indicator microdot can be used. First, it could be used to register the spatial position of the specific indicator chemistry. A very simple approach, for example, would be to design one indicator microdot to emit blue light in the presence of a particular biomarker and to design a second indicator chemistry that emits green light in the presence of a different biomarker. The intensity of the emission from each microdot could then be correlated to the concentration of their respective targeted biomarkers. It may also be desirable to use fluorescently labeled microbeads, each with a unique spectral signature, with specific indicator chemistries attached. The limitation of using spectral filtering for registration purposes is the potential overlap that will occur between multiple emission wavelength bands. In addition, if multiple biomarkers are targeted, each will require its own specific dye with a corresponding spectral processing scheme and possibly different excitation wavelength. A simpler approach for registration of each indicator microdot is to use their spatial location on the optical substrate, as described below. The second and more practical use of spectral filtering is to separate the desired component of the emitted light from the incident radiation. In the case of fluorescence, this amounts to separating the incident excitation band from the transmitted or reflected emission band. This method is also intended to incorporate more complex spectral processing schemes of single and multiple dye conjugates, including multivariate analysis, ratioing, and other standard spectroscopic techniques. b. Detection and spatial processing

The spectrally filtered light from the sensing region can be detected using photosensitive detectors such as photodiodes or photomultiplier tubes. Spatial filtering of the light is also possible with two dimensional detectors such as charge coupling device cameras (CCDs) and video cameras. The use of a two dimensional detection system allows direct registration of multiple indicator microdots, eliminating the need to use spectrally diverse absorbing dyes and their associated spectral filtering components. This greatly simplifies the optical apparatus necessary to measure changes in the indicator chemistry (s). If the geometry of the microdot pattern is axis symmetrical (such as the six-around- one pattern), it is necessary to include (or exclude) a "reference" microdot to determine the positions of the other indicator chemistries (other than the central microdot). These detection schemes may or may not be coupled to fiber optic /fiber optic bundles depending on the need to remotely access the sensing sites. The data from the selected detector system can then be acquired, processed, and displayed to the user using available data acquisition/processing systems. Depending on the application, these systems could range from a very simple detection scheme where a positive identification lights an LED to much more complicated systems using a computer interface to process image information for simultaneous real-time monitoring of multiple constituents.

The system of the present invention has a wide range of uses. Examples of some of the uses are listed below to more fully illustrate the invention. There are additional uses of the present invention that are not described.

(1) Biomedical Applications — Biosensor systems constructed in accordance with the present invention could be used as measure biomarkers for infectious diseases, blood gas levels (0₂, C0₂, etc.), electrolyte concentrations (K⁺, Ca⁺, Li⁺, etc.), periodontal disease (metalloproteinases), polymerase chain reaction (PCR) products, and other clinically important parameters (pH, glucose, etc.).

(2) Environmental Applications — Chemical sensor systems constructed in accordance with the present invention could be used for monitoring hazardous materials such as heavy metal, hydrocarbons, and chlorinated hydrocarbons in both the groundwater and soil of contaminated sites.

(3) Occupational Safety — Chemical sensor systems constructed in accordance with the present invention could be used for making accurate dosimetry measurements of hazardous materials, such as carcinogens or mutagens present in hostile or potentially hostile environments. These could include compounds that are traditionally detected using flame ionization detectors (FID) or portable gas chromatographs.

(4) Process Control — Sensors systems constructed in accordance with the present invention could be implemented in assembly line type configurations for quality and process control type applications. Examples include measurements of gases emitted from fruits and vegetables and detection of contaminants in soft drink or bottled water solutions.

(5) Chem/ Bio warfare Applications — Sensors systems constructed in accordance with the present invention could be developed for detection /early warning of airborne or water-based chemical and biowarfare agents such as anthrax.

Changes and modifications in the specifically described embodiments can be carried out without departing from the scope of the invention, which is intended to be limited by the scope of the appended claims.

Claims

THE INVENTION CLAIMED IS

1. A method of producing a chemical sensor, comprising: using microjet technology to print one or more indicator chemistries on an optically accessible surface.

2. The method of Claim 1 wherein said one or more indicator chemistries is a light absorbing dye.

3. The method of Claim 2 wherein said light absorbing dye is a light absorbing fluorophore.

4. The method of Claim 2 wherein said light absorbing dye is a light absorbing chromophore.

5. The method of Claim 2 where including the step of linking said light absorbing dye to enzyme substrates.

6. The method of Claim 2 including the step of linking said light absorbing dye to enzyme antibody conjugates.

7. The method of Claim 1 including the step of linking said one or more indicator chemistries to an optically active particle.

8. The method of Claim 1 including the step of linking said one or more indicator chemistries to optically encoded microbeads

9. The method of Claim 1 including the step of linking said one or more indicator chemistries to quantum dots.

10. The method of Claim 1 including the step of linking said one or more indicator chemistries to phosphorescent crystals.

11. The method of Claim 1 where the indicator chemistry is intrinsic to an optically active particle.

12. The method of Claim 1 wherein said optically accessible surface is the tip of a single fiber optic strand.

13. The method of Claim 1 wherein said optically accessible surface are the tips of multiple fiber optic strands.

14. The method of Claim 1 wherein said optically accessible surface is the tip of a preformed unitary fiber optic array.

15. The method of Claim 1 wherein said optically accessible surface is an optically transparent disposable material that fits over the end of a single fiber optic strand.

16. The method of Claim 1 wherein said optically accessible surface is an optically transparent disposable material that fits over the end of a preformed unitary fiber optic array.

17. The method of Claim 1 including the step of using the spectral characteristics of each indicator chemistry to determine its identity.

18. The method of Claim 1 including the step of printing the indicator chemistries at specific locations on said optically accessible surface.

19. The method of Claim 1 including the step of using the location of each indicator chemistry on the optically accessible surface is used to determine its identity.

20. A method of producing a chemical sensor, comprising: printing one or more indicator chemistries on an optically accessible surface through the use of microjet technology.

21. The method of Claim 20 wherein said one or more indicator chemistries is a light absorbing dye.

22. The method of Claim 20 including the step of linking said one or more indicator chemistries to an optically active particle.

23. The method of Claim 20 wherein said optically accessible surface is the tip of a single fiber optic strand.

24. The method of Claim 20 wherein said optically accessible surface are the tips of multiple fiber optic strands.

25. The method of Claim 20 wherein said optically accessible surface is an optically transparent disposable material that fits over the end of a single fiber optic strand.

26. The method of Claim 20 wherein said optically accessible surface is an optically transparent disposable material that fits over the end of a preformed unitary fiber optic array.

27. A chemical sensor, comprising: a fiber optic strand with an indicator chemistry printed on an optically accessible surface of said fiber optic strand using microjet technology.

28. A chemical sensor, comprising: multiple fiber optic strands with indicator chemistries having substantially the same shape on optically accessible surfaces on said fiber optic strands.

29. A sampling method for detecting and /or analyzing a sample in a fluid, comprising: utilizing microjet technology to print one or more indicator chemistries whose optical characteristics change in response to the sample on an optically accessible surface, contacting said one or more indicator chemistries whose optical characteristics change in response to the sample and said fluid, and optically monitoring changes in the response of said one or more indicator chemistries whose optical characteristics change in response to the sample to detect and/or analyze the sample.

30. The method of Claim 29 wherein said monitoring is conducted in vivo.

31. A sampling method for detecting and/or analyzing a sample in an airborne medium, comprising: utilizing microjet technology to print one or more indicator chemistries whose optical characteristics change in response to the sample on an optically accessible surface, contacting said one or more indicator chemistries whose optical characteristics change in response to the sample and said airborne medium, and optically monitoring changes in the response of said one or more indicator chemistries whose optical characteristics change in response to the sample to detect and /or analyze the sample.

32. The method of Claim 31 wherein said monitoring is conducted in vivo.

33. A sampling method for detecting a sample in a fluid or an airborne medium, comprising: utilizing a fiber optic strand with an indicator chemistry printed on an optically accessible surface through the use of microjet technology, contacting said one or more indicator chemistries whose optical characteristics change in response to the sample and said fluid or airborne medium, and optically monitoring changes in the response of said one or more indicator chemistries whose optical characteristics change in response to the sample to detect and/or analyze the sample.

34. The method of Claim 31 wherein said monitoring is conducted in vivo.