CA2424941A1 - An integrated biochip system for sample preparation and analysis - Google Patents

Abstract

The invention includes a composition that is an integrated biochip system fo r processing and analyzing samples using sequential tasks that take place on o ne or more chips. The system preferably comprises one or more active chips, and can be automated. The invention also includes methods of using an integrated biochip for processing and analyzing samples. The methods include the application of a sample to the system and performing at least two sequential tasks on at least one chip surface. The method includes the use of physical forces, such as dielectrophoretic and electromagnetic forces to process and analyze samples, and includes the use of microparticles that can be coupled to sample components to be manipulated by dielectrophoretic and electromagnetic forces.

Description

AN )NTIECyRA'TED I$IC)C'I-IIP SY~')'LIi~I x'012 SAMPLE PRCPARA'hION AND
ANALYSIS
This application claims priority to Ullitcd States Provisional Aplalication Number 601239,299 (attorney docket number ART-00105.P.1) filed October 10, 2000, entitled '°An Integrated Biochip System for Sample Preparation and Analysis"
naming Chen g, et al. as inventors, and incorparated herein by reference in ils entirety.
'rECIINICAIJ hIELtD
The present invention relates generally to the field oC sample analysis, in particular to the processing and analysis o~ samples on chips. More particularly, the invention relates to the processing and analysis oC samples using an integrated system Ol' GhlpS, 111G1ud117g 017e Or Illore GhlpS 017 WhlGh Sa171p1e Gon7pOne17tS, e.g. blolOglcal cells and biomolecules, can be manipulated or processed using applied physical forces.
~?.C'IeCI2~I3NI3 The manipulation oC~ particles, especially biological material such as cells and molecules, Gan be used to advantage in a variety oC biomedical applications, The ability to manipulate individual cancer cells, l~or example, can allow a researcher to study the interaction o~ eith er a single cancer cell or a collection of cancer cells with selected drugs in a Garefillly controlled environment. Various kinds o~ forces Gall be used to manipulate particles. including optical, ultrasonic. mechanical, and hydrodynamic. For example. E7ow cytometry has been successfully used to sort and characterize cells. Another example is the centrifuge, which has been widely used in laboratories for processing biological samples.
A current trend in the biological and bion7edical sciences is the automation and miniaturization of hioanalytical cleviaes. ~L'h~ development o~so-GaIIGd hiochip-based microiluidic technologies has been ofi prn-ticular interest. A biochip includes a sokid substrate having a surCrlce on which bit~logical, bioGhe;mical, and chemical 1"eactions and processes can take place. Tlle substrate may be thin in one dimension and may have a Gross-secfion tleFned by the other dimensions in tile shal7e o1', for exa171p1C, a reCtaIlgIG, a G11'CIC:. a17 eIllpSC', 1O' COllel" ShapcS. A
hlOChlp 11711\° LIISO 111C1L1CIC

other structures, such as, For example, channels. wells, and electrode elements, which may be incorporated into or Cabricated on the substrate tar Facilitating biologicallbiochemical/chemical reactions or processes on the substrate. An important goal for researchers has been to develop fully automated and integrated devices that can perform a series of biological and biochemical reactions and procedures. Ideally, such an integrated device should be capable of processing crude, original biological sample (e.g., blood or urine) by separating and isolating certain particles or bio-particles tram the rest of the sample (e.g., cancer cells in blood, or fetal nucleated cells in maternal blood, or certain types of bacteria in urine). The isolated particles can then be further processed to obtain cellular components ~e.g., target cells are lysed to release biomolecules, such as DNA, mRNA and protein molecules). The cellular components of interest can then be isolated anti processed and analyzed (e.g., DNA molecules are separated and target sequences are amplified thraugh polymerase-chain-reactions, PCR). finally, a detection procedure may be 9 5 performed to detect, measure and/or quantify curtain reaction products (e.g., a hybridization may be performed on the PCR-amplified DNA segments with fluorescent detection then being used to detect the hybridization result).
Clearly, the ability of a biochip to manipulate and process various types of pauticles, including cells and cellular components from a particle mixture, would be of great signif cance.
Limited progress has been made to date in the manipulation of particles or bioparticles on a chip. Electronic hybridization technologies have been developed in which charged DNA molecules are manipulated and transported on an electranic chip ~e.g.. "Rapid Determination of Single Base Mismatch Mutations in DNA I-Iybrids by Direct Electric Field Control", ~osnowslci, R., et al., Prop. Nczll. Aca~'.
~ft~i., Volume 9~1, pages 1119-1 1?3, 1997; ''Clectric Field Directed Nucleic Acid Hybridization on Microchips", Edman, C., A°uc~L Acids Ri,s~., ?~: pages X907-X91 ~, I
99$, the disclosures of which are incorporated herein by reference in their entireties}. Also.
electrol:inetic pumping and separation technologies have been developed in which biomUlecules or other particles can be transported, manipulated. ancf separated through thr use ol~
elecfroosmosis and electrophoresis based l:inctlc effects (e.g., "Micromachining a miniaturized capillary electrophoresis-based chemical analysis system on a chip', (-larrison, D.J. et al, .fciem~t~. Volume 261, laa~~es: 895-$96, 1993; "I-Iigh-speed separation of antisense oligonucleotides on a micromachined capillary electrophoresis device', EITcnhauser, C.S. ct al.. W za/. (,'l~em. Volume (6, pages: 2949-2~)SS. 1994, the disclosures of which ai°c incorporated herein by reference in their entireties).
However, each ofthese devices suffers from limitations. Accordingly, there is a need for improved particle manipulation devices.
~~SC~t~T~o~ ~~ Tt~~ ~~~mtGs Figure ~A, is a schematic representation of a three-dimensional perspective view of a chamber that comprises a multiforce chip used in the system of the present invention.
1 a The chamber has inlet and outlet ports and a multiple force chip forming the bottom of the chamber. Not shown is a glass plate on the top (not shown). The chamber is connected to three neighboring chambers (not shown) for analyzing and detecting DNA, protein and mRNA, and small molecules. The multiple force chip comprises an acoustic layer, a magnetic layer, a particle switch layer, a DIJP electrode layer and a tap layer.
Figure 1>l~ is a schematic representation of a three-dimensional perspective view of the top layer of a multiple force chip. In this case the top layer can be, for example, a coating of BSA (Bavine Serum Albumin) or other coating that may minimize non-specific adhesion or binding o~ cells or other components of samples to the chip. The top Layer can also be a thin layer of SiO~ or other insulating materials.
Figure 1C is a schematic representation of a three-dimensional perspective view of the DEP electrodes an the DEP electrode layer of a multiple force chip. The rectangular-shaped DEP electrodes can be corrected to external signal sources (not shown).
Figure LD is a schematic representation of a three-dimensional perspective view ol~
particle switch electrodes o1~ the particle switch layer of a multiple force:
chip.

lFigure 1>l~ is a schematic representation of a three-dimensional perspective viwv ol~
the electromagnetic elements on the magnetic layer of a multiple Force chip.

~l Figure 1F is a schematic representation of a three-dimensional perspective view of the acoustic elements on thc~ acoustic layer o(~ a multiple Force chip.
Figure 2A is a schematic representation ota cross-sectional view ol~ a sample being inti°oducod into the chamber. The sample comprises target cells to be analyzed, non-target cells, and magnetic brads to which specit7c binding n.embers have been coupled. The speci~'ic binding members allow the target cells to bind to the magnetic beads.
Figure 2~ is a schematic r~:la~°esentation of a cross-sectional view of the sample that has been introduced into the chamber. The introduced sample comprises target cells, non-target cells, and magnetic beads.
Figure 3 is a schematic representation o~ a cross-sectional view of the sample in the chamber being mixed using acoustic forces to j~acilitate the bmdmg o~the magnetic beads to the target cells (energized acoustic layer depicted with thick bold lines).
Figure 4 is a schematic representation o~ a cross-sectional view oi~ the sample in the chamber when the magnetic beads are bound to the target cells following acoustic mixing and just prior to ma~~netic capture.
Figure SA. is a schematic represen ration ova three-dimensional perspective view o~
the target cells of the sample' in the chamber bound to magnetic beads with electromagnetic units bein g energized (enerf~iz~d magnetic layer depicted with thick bold lines). The energized electromagnetic units generate a magnetic ~icld distribution that causes the target cell-magneaic bead complexes to be collected towards these energized units.
Figure 5>S is a schematic. relaresen ration ot~ a three-dimension perspective view of'the chamber with the magnetic head-cell complexes or magnetic beads bein;~ crapped at the energized magnetic elements (energized magnetic layer depicted with thick bolts Bales), To illustrate that the magnetic bead comlalexes are collected as the en ergized 111aglletlC elelnentS, lndlvldLlal IllaglletlG e1e117e11t5 are 5Ghe111at1Cally s170w11. a1t11oL1g11 they would not be seen Fron7 the top oFthe chamber.
)Figure SC is a schematic rcpresen tation of a three-dimensional perspective view of the chamber with the nontarget cells being washed out of the chamber by fluid flow.
Target cells bound to magn Etie beads remain trapped at the energized magnetic elements.
Figure 6 is a schematic representation of a three-dimensional perspective view of the chamber with the target cells being de-coupled ~li°om the magnetic beads- The magnetic elements remain energized so that the magnetic beads remain trapped at the ends o~ the magnetic elements.
Figure 7A is a schematic representation of a cross-sectional view of the chamber with the DEf' electrode array energized by application of an AC electric signal (energized electrode layer depicted by thick bold lines).
»'igure 7~ is a schematic representation o~ a cross-sectional view of the chamber with the target cells being retained by dielectrophoretic forces produced by the non-uniform electric gelds generated by the D>JP electrode array. The magnetic beads are washed out of the chamber because the dielectrophoretic forces acting on these beads are small or negative.
Figure 8 is a schematic representation of a cross-sectional view of the chamber with 2S four different types of beads in a solution being introduced into the chamber. The 'Four types of the beads, type 1, type 2, type 3, and type 4 are used for capturing target mRNAs, target proteins, tar~~ot DNAs, and target small molecules, respectively.
)Figure 9A is a schematic r417resentation of a crass-sectional view of the chamb er with the target cells being lysed car disrupted to release their components.

Figure 9~ is a schematic representation oCa cross-sectional view of the chamber showing the released camponents of the lysecl target cells.
Figure 10 is a schematic representation of a cross-sectional view of the.
chamber with the acoustic elements being energized so that an acoustic mixing is provided to facilitate the binding of the molecules of interest to their respective beads (energized acoustic layer depicted by thick bold lines).
Figure 1~ is a schematic re~~resentation of a cross-sectional view of tile chamber with the molecules of interest being bound to their respective beads. Target protein molecules, DNA molecules. mRNA molecules and small molecules have been bound type 2, type 3, type 1 and type 4 beads, respectively.
Figure 1BA is a schematic representation o1' a cross-sectional view of the chamber with the molecule-bead complexes being collected to the bottom surface of the chamber under dielectrophoretic forces produced by energized DAP electrodes (energized DEP electrode layer shown by thick bold lines).
Figure ~2>a is a schematic represen ration of a crass-sectional view of the chamber with the molecule-bead complexes being collected to the central region of the bottom surface of the chamber under traveling-wave dielectrophoretic forces produced by energized DEP electrodes.
Figure ~3A is a schematic representation oFthe top view ol~ the chamber with the electrodes on the particle switch layer being energized.
Figure 13B is a schematic representation o1' the top view of the chamber looking through to the particle switch layer, illustrating the faur types of molecule-bead complexes being switched rind separated to the ends o~ three branches within a particle switch when the elec:irodes in the particle switch are energized with phase-shifted electric signals.

Figure 13C is a schematic representation ol=the top view ol=the chamber illustrating the tour types of molecule-head complexes switched and separated to three ends ol~
the chamber.
Figure 1~A is a schematic representation ol~ a three-dimensional perspective view ol~ a DNA-analysis chamber showing the DNA probe layer.
Figure ~~~ is a schematic representation ol~a three-dimensional perspective view o~
a DNA-allalySIS Chamber ShoW111g the traVellllg-Wave dleleGtrOphOreSlS (TW-DE~) Electrode layer. The detailed electrical connections a~such TW-DEP elECtrodes to a signal source that can produce at least 3 phase-shifted signals having the san 1e frequency are not shown.
Figure 1~C is a schematic rEpresentation of a three-dimensional perspective view of~ a DNA-analysis chamber showing the magnetic sensor layer. Th a letter "S"
represents "sensor".
Figure 1~1~ is a schematic representation of~ a three-dimensional perspective view of a DNA-analysis chamber showing that the traveling-wave dielectrophoresis layer being energized, and the energized traveling-wave dielectrophoresis electrodes moving the DNA-bead complexes into the chamber (Energized electrode layer depicted with thick bold lines). The DNA-analypsis chamber comprises a chip having a DNA probe layer (top layer), a traveling-wave DEP layer, and a magnetio sensor layer '?0 Figure .14E is a schematic representation ol~ a three-dimensional perspective view of~ a D ~ 'A-analysis chamber showing that the DNA-head complexes are dispersed into the chamber and target D ~ ~A molecules hybridized to the beads are also hybridized to the DNA probes on the chip.
Figure ~4F is a schematic representation of a three-dimensional perspective view o1' a ?5 DNA-analysis chamber showing that the single-stranded portions of the target DNA
molecules ell the DNA-bead complexes are hybridized to the DNA probes an the chip that are localized to magnetic sensors. The prGSCnce and the number ol'tIIG
magnetic beads are detected with the magnetic sensors (energized magnetic sensor layer depicted with thick bold lines). 1~o illustrate that magnetic sensors are responsive to the presence of the magnetic beads, individual magnetic sensors are schematically shown, although these sensor elements cannot be seen from the top of the chamber.
Figure SSA is a schematic representation oj~ a three-dimensional perspective view of the protein/mRNA-analysis chamber that comprises a chip showing the nucleic acid probel antibody probe layer (top layer) of the chip.
)figure ll~~ is a schematic representation ofi a three-dimensional perspective views of the protEI11lI11RNA-allalySIS ehambel' S110Wlllr' tllE tl'aVGllllg-WAVE
dlElectl'0(1110I'eSIS
electrode layer of the chip. 'I°he detailed electrical connections of such TW-DEP
electrodes to a signal source that can produce at least 3 phase-shifted signals having a same frequency are not shown.
Figure ASC is a schematic representation ol~ a three-dimensional perspective view ol=
the proteilllmRNA-analysis chamber showing that the protein-bead complexes and mRNA-bead complexes art dispersed into the chamber using traveling-wave dielectrophoresis (energized electrode layer depicted with thick bold lines).
Figure 15D is a schematic representation of a three-dimensional perspective view of the protein/mRNA-analysis chamber showing that the protein molecules and mRNA
molecules are decoupled or dissociated front the beads and have begun to bind specific binding partners on the chip surface.
Figure 15>C is a schematic representation of a three-dimensional perspective view of a proteinlmRNA-analysis chamber showing that the protein molecules and mRNA
molecules are bound to the antibody-probes and nucleic acid probes respectively, Detestably-labeled binding partners are bEint~ introduced to the proteilll111RN~A-allalySlS chamber' I'I'olll a pt51'l. ~rllC beads ll~lrC heell re111QVeC1 11'olll the I:11a111bC'.1' or' the ?5 detection regions or' the chamber by travslin~~-wave dielectrophoresis Forces by energizing 'f W-DGl' electrodes (not shown) or by fluid flow forces durin~~
the process of introduction of the detestably-labeled (fluol-s-scence-labeled) binding molecules (not shown).

Figure 15F is a schematic representation oi~ a three-dimensional perspective view o(~ a proteinlmRNti-analysis chambei° showing that the fluorescence-labeled binding molecules are bound to the protein molecules and to the mIZNA molecules that have bound to the probes on the chip.
Figures ~6A and I$ are schematic representations of a three-dimensional perspective view of a small-molecule analysis chamber comprising a chip at the bottom. The chip has a fluidic channel layer (~), and a traveling-wave DEP layer (B). The detailed electrical connections of the traveling-wave UEP electrodes to a signal source that can generate at least 3 please-shi ited signals having tile same frequency are not shown.
Figure 1.6C is a schematic representation of a three-dimensional perspective view of the small-molecule analysis chamber showing that the small-molecule-bead campiexes are moved to the central regions of the channel using traveling-wave dielectrophoresis (active electrode layer depicted with thielc bald lines).
Figure Jt6d) is a schematic representation of a three-dimensional perspective view oi' the small-molecule analysis chamber showing that the small molecules are de-coupled or dissociated from the beads. The beads have been moved out of the chamber by traveling-wave dielectrophoresis (not spawn). The molecules are then labeled with florescence molecules (not shown).
Figure ~6;IJ is a schematic representation of a three-dimensional perspective view of small-molecule analysis chamber showing that the small molecules are directed through the channel under electrophoresis ar Llectro-asmosis elects.
Figure 16F is a schematic representation of a three-dimensional perspective view of small-molecule analysis opamber showing that the small molecules are directed through the channel and art' detected by an ol~(=chip iluoresoence detector_ 2~ Figure 17 depicts a single chip integrated biochip system, in which the Chip is part o(~
a chamber, and the cover ol~ the chamber has inlet ports for the application ol~ a sample and the addition of reagents. ante outlet ports for the outflow oi~ waste.
'1'hrc~e separate axeas of the chip are used for sample processin~~ (areas A and 13) and analysis (C), and each area ofthe chip has dil~lcrent functional or~r~s.
Figure 18 depiots a single chip integrated biochip system, in which the chip is part of a chamber, and the cover ol~ the chamber has inlet ports for the application o1' a sample acid the addition of reagents. and outlet ports I-or the outflow of waste.
'T'11e chip comprises a particle switch that can direct sample components to different areas of the chip for further pracessing and analysis tasks.
Figure >19A is a top view of a multiple force chip capable of producing dielectrophoretic foi°ces from an upper layer having interdigitated Electrodes and electromagnetic forces from a lower layer having electromagnetic elements.
Figure )19~ is a tap view through the chamber comprising the multiple lorcc chip showing a diluted blood sample introduced into the chamber.
Figure J19~ is a top view through the chamber comprising the multiple force chip showing white blood cell collected at the edgea of the interdigitated microelectrode array by positive dielectrophoretio forces.
)Figure ~.9D is a top view through the chamber comprising the multiple irrcc~
chip just after the addition of a lysis buffer that contains magnetic beads with oli~~c>-Cd°C)~~
modified surfaces.
Figure I9>E is a top view through the chamber comprising the multiple foi°ce chip showing the capture of the magnetic beads at the poles of activated magnetic elements.
lNigu~~e ><9F is an image of tm agarose gel showing an RT-PCTZproduct ~aoneratiy li~om mRNA recovered from the captured magnetic beads.
JO

SUMMARY
The present invention recognizes that analytical techniques that can he uselul in medical diagnosis, forensics, genetic testing, In°ognosties, and pharmacoge«omies, and research often require extensive preparation of complex biological samples.
Preparation of biological samples such as blood samples can require multiple steps such as centrifugation, filtering, and pipeting, and steps that involve lysis procedures, incubations, enzymatic treatments, gel purification of nucleic acids or proteins, etc.
Such steps are time-consuming, labor intensive, and diffcult to standardize.
The present invention recognizes that an automated integrated system that can perform both sample preparation and sample analysis can standardize and streamline testing procedures from sample to result, representing, i~z effect, a "lab on a chip"
that requires minimal manual intervention. In addition, such systems can be designed to analyze multiple sample components at once, reducing the need for multiple samples to be taken from a single source, greatly accelerating the process of diagnasis, assessment, or investigation.
The present invention also recognizes that the ability to manipulate particles, such as culls and microparticles bound to sample components using applied physical forces, can be utilized to automate, streamline; sample processing and analysis. These methods of manipulating sample components for sample processing (or sample ?0 preparation) and analysis can be utilized for a variety of purposes, such as the detection of particular molecules, compounds. or nucleic acid sequences in samples, for use in the diagnosis or prognosis of disease states, conditions, or infection with etiological agents, in the identification of subjects, in the genetic screening of subjects, and other applications.
'?5 A first aspect of the invention is an integrated biochip system that comprises a single chip, wherein the chip can perform at least two sequential taslcs, and at least one of the tasks functions in the pracessing ol~ a sample. frcferably, at least one task is performed by the application of physical forces that are in part generated by micro-scale structures that are built info or onto a chila. freferabIy, at least one to s1: is 30 performed by the manipulation of binding partners that are coupled to a sample moiety. An integrated biochip system is preferably automated.

l A second aspect of the invention is an integrafed biochip system that comprises two ar more chips and can perform at (east two sequential tas(a using two or more chips of the integrated system, wherein at least one of the chips aC'the system can pei°form at least one task in the preparation of a sample.
Preferably, un integrated S biochip system comprising two or more chips is automated, and at least two of the chips of the system can be in fluid communication with one another.
Translocation of sample components from at least one chip oi= the integrated biochip system to at least one other chip of the integrated biochip system is preferably by a mechanism other than fluid flow, mast preferably through tho application of physical forces.
Preferably, at least one task is performed by the application of physical farces that are in part generated by micro-scale structures that are built into ac onto a chip, al least one task can be performed by the manipulation of binding partners that are coupled to a sample moiety.
A third aspect ofthc invention is a method ofusin g a system of integrated 1.S chips for processing and analyzing samples. ~hhe method includes the application of a samplo to the system and performing at least two sequential tasks in the processing and, optionally, analysis, of a sample. At least one processing task can be performed by the integrated system using applied physical forces that are in part generated by microscale structures an the surface of a chip aF the system. Preferably but optionally the processing stop can include the manipulation of sample moieties coupled to micraparticles.
DETAILED DESCRIPTION Oi° 'I"IiE INVENTION
DEFINITIONS
2S Unless donned otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein and the manufacture or laboratory procedures described below are well known and commonly employod in the art. C:anventional mothods art used far these procedures. such as those provided in the art and various general re ferences. 'I'crms of orientation such as "up" and "down" or "upper's or °'lower" and the like refer to orientation of~ parts during use of a device. Where a term is provided in the singular, the inventors also li contemplate the plural of tlntt term. The nomenclature used herein is well known and commonly employed in the nrt. Where there are discrepancies in terms and definitions used in references that are incorporated by reference, the tern ~s used in this application shall have the dclunitions given herein. As employed throughout the disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:
An "integrated chip system'', "integrated biochip system", a "system of integrated chips", a "system of integrated biochips" ar "system" is at least one chip that can perform at least two sequential tasks in the processing and analysis of a sample, in which at least one task performed by the integrated biochip system is a processing task.
A "task'" is a function in the processing or analysis of a sample. A task can comprise more than one stela. For example, a separation task can comprise mixing and binding steps that facilitate the separation.
A "fun coon" performed by a chip o(= a system of the present invention can be a task, such as a processing ar analysis task, or can be another function that occurs between tasks or as part of a task and facilitates the performance of the task. One example of a non-task function is a mixing function, such as a mixing function that is performed by acoustic forces on a chip that facilitates dispersion andlor binding of sample components. Another example of a non-task function is a translocation of moieties from one chip to another chip, or from one area of a chip to another area of a chip, such as by electrophoresis. dielectrophoresis, traveling wave dielectrophoresis, or traveling wave magnetophoresis.
A "processing fiask'~ is a procedure in the processing of a sample. ( Processing '?5 of a sample is also referred to as sample preparation,) Generally a processing task serves to separate components of a sample, translocate compon ents of a sample, focus, capture, isolate, con ccntrate, or enrich components of a sample. at least partially purify components of a sample, or disrupt or structurally alter ctunl~anents ol~
a sample (for example, by Ivrsis, denaturation. chemical modification, or hintling of components to reagents). A processing step can net on one type of sample component to release, expose, modify, c,r generate another type of sample component that can be used in a further processing or analysis task. li"c,r example, a cell can be Ivpscd in a 1 ~l processing step to release nucleic acids that can be separated in a further processing task and detected in a subsccluent analysis task. Binding or coupling can he a step in a pi°ocessing task, where binding or coupling, particularly the coupling ofa sample component to a binding partner such as a microparticle, facilitates the se:paralion, translocation, capture, isolation, focusing, concentration, enrichment, structural alteration, or at least partial purification of at least one component of a sample, Mixing can also be a step in a processing task, where mixing facilitates the binding, separatian, translacation, concentration, structural alteration, or at least partial purification of at least one component of a sample.
An "analysis task" is a task that determines a result of a sample larocessin g and analysis procedure, and can be an assay, such as a binding assay, a biochemical assay, a cellular assay, a genetic assay, a detection assay, etc. Generally an analytical task determines the presence, amount, or activity of a sample component. Binding ar coupling can be a step of an analysis task, where binding or coupling facilitates the detection or assay of at least one component of a sample. Mixing can also be a step of an analysis task, where mixing facilitates the binding, detection, or assay ol=at least one component of a sample.
"Sequential" means following a particular order, where followings a particular order of tasks, for example. is necessary to ~~chieve the desired final result. In an 2Q integrated biochip system ol~ fihe present invention, taslcs are performed sequentially to abtain a final result. When two tasks are performed sequentially, a second task uses one ar more products of the first task, where ''product" can mean a sample component that was separated, at least partially purified, or concentrated in the first step, or a sample component that was the result of a denaturing or lysing step, was subjected to a biochemical reaction ar away. became bound to a reagent, etc., in a previous task.
As used herein. "first" and "second" do not rcFer to their absolute order in the integrated system, but rather to their relative order, where a process performed on the second chip occurs after a process performed on the first chip.
A 'xcllip'~ is a surFacr on which at least one manipulation or process. such as a iranslocation, separation, capture, isolation, lr~cusing, enrichment, concentration.
physical disruptian. mixing. binding, or assav° cm be performed. A
chila can he a solid or semi-solid substrate, porous or non-porous on which certain processca, such as 1i physical, chemical, biological, biophysical or biochemical processes, eic..
can be carried out. A chip that performs more than one lunation can have combinations of one or more different Functional elements such specific binding members, substrates, reagents, or different types of micro-scale structures that provide sources of different physical forces used in the processes carried out on the chip. Chips can be multiple force chips, in which differs nt functional elements can be pravided on the same surface, or in different structurally linked substrates or layers (where a layer is a surface that supports substrates, micra-scale structures, or moieties to be manipulated) that are vertically oriented with respect to cue another. For descriptions of multiple I 0 force chips, see United States Application IW unber 091679,024 having attorney docket number 4718~12000~100, Entitled "Apparatuses Containing Multiple Active I~orce Generating Elements and Uses Thereof' filed October ~l, 2000, herein incorporated by reference in its entirety.
Micro-scale structures such as but not limited to channels and wells, electrode elements, electromagnetic elements, and piezoelectric transducers are incorporated into, fabricated on, or otherwise attached to the substrate for facilitating physical, biophysical, biological, biochemical, or chemical reactions or processes on the chip.
Tlve chip may be thin in one dimension and may have various shapes in other dimensions, for example, a rectangle, a circle, an ellipse, or other irregular shapes.
The size of the major surface of chips of the present invention can vary considerably, e.~:, from about 1 mm'' to ahout 0.25 m~. Pre:ferably, the size of the chips is from about ~. mm2 to about 25 cm~ with a characteristic dimension from about I mm to about 5 cm. The chip surfaces may be flat, or not flat. The chips with non=Ilat surfaces may include channels or wells fabricated on the surfaces.
~5 '°Micro-scale structures" are structures integral to or attached on a chip or chamber that have characteristic dimensions of scale for use in microfluidic applications ranging ti~om about 0.1 micron h~ about 20 nun. hxample o 1 micro-scale structures are wells, channels, scaffolds, electrodes, electromagnetic units, piezoelectric transducers, metal wires or films. feltier elements, micro lubricated pumps or valves, microfabricated capillaries car tips, or optical elements. A
variety ol~
micro-scale structures are disclosed in United States Patent Application Number 09I67t),024, having attorney docket number ~718~12000~10(l. en titled c'Ahparatuses Containing Multiple Active: force Generating ~;lements and uses Thereof-"
lined October 4, 2000, herein incorporated by reference in its entirety. Micro-scale structures that can, when energy, such as an electrical signal, is applied.
generate physical forces useful in the present invention, can be referred to as "physical Force-s generating elements" "physical force elements'", ''active Force elements'-, or "active elements".
"Substrate'" refers to fine surface of a chip where a moiety to be manipulated can be held and manipulated. A substrate can be hydrophobic or hydrophilic, or a combination thereof and can comprise materials such as silicon, rubber, glass, one or snore ceramics, plastics, polymers, or copolymers. The substrate can be soled or semi-solid, can comprises one or more channels or wells, and can support micro-scale structures and functional elements such as specific binding members, substrates, reagents, or catalysts.
An "electrode" is a structure of highly electrically conductive material. A
highly conductive material i5 a material with a conductivity greater than that of surrounding structures or materials. Suitable highly electrically conductive materials include metals, such as gold. chromium, platinum, aluminum, and the like. and can also include nonmetals, such as carbon and conductive polymers. An electrode can be any shape, such as rectangular, circular, eastellated, etc. Electrodes can also comprise doped semi-conductors, where a semi-conducting material is mixed with small amounts of other conductive materials.
A "chamber" is a structure that that is capable of containing a t"luid sample and preferably comprises at least a portion of a chip.
A "port" is an opening in a chamber through which a Fluid sample can enter or exit the chamber. A port can be of any dimensions, but preferably is of a shape and size that allows a sample to he translocated through the porn by physical Forces, or dispensed through the port 1y> means of a pipette. syringe, or conduit. or other means of applying a sample.
A "conduit"' is a means For fluid to be transported From a container to a chamber of the present invention. Preferably a conduit enga~~es a porfi in a chamber. A
conduit can comprise any material that permits the passage of a l7uid tlzrc~ut~h it.
Preferably a conduit is tubin~~, such as, for t~~tunhle, rubber, "I"el7on {polytetral7uoroethylene), or tygon tubing. A conduit can be of any dimensions. buff preferably ranges From 10 microns to 5 millimeters in internal diameter.
A "well" is a structure in a chip, with a lower surface surrounded on at least two sides by one or more walls fihafi extend I-rom fihe lower surface of the well or channel. The walls can extend upward from the lower surFace of a well ar channel at any angle or in any way. The walls can be o(~an irregular conformation, fihafi is, they may extend upward in a sigmoidal or otherwise curved or multi-angled C~ashiou.
The lower surface of the well or channel can be at the same level as the upper surface of a chip or higher than the upper surface of a chip, or lower than fine upper surface of a chip, such that fihe well is a depression in the surf ace of a chip. 'The sides or walls of a well or channel can comprise materials ofiher fihan thaw that make up the lower surface of a chip. In this way the lower surface of a chip can comprise a thin material fihrough which electrical (including electromagnetic) forces can be transmitted, and fine walls of one or more wells andlor ono or more channels can optionally comprise I S other insulating materials that can prevent the: transmission of electrical Forces. The walls of a well or a channel of a chip can comprise any suitable material, including silicon, glass, rubber, and/or one or more polymers, plasfiics, ceramics, or metals.
A "channel" is a structure in a chip wifih a lower surface and afi least two walls that extend upward from the lower surface oFthe chamlel, and in which the length of 2Q two opposite walls is greater fillan fine distance between the two apposite walls. A
channel therefore allows fan Ilow of a fluid along its internal length. A
i:hannel can be covered ~a "tunnel") or open.
An "active chip" is a chip that comprises micro-scab structures that are built into or onto a chip that when energized by an external power source can generate at Z5 least one physical Force that can perform a prooessing step or task or an analysis step or task, such as, but not limited to, mixing, iranslocation, Focusing, separation, concentration, capture, isolation, or enrichment, An active chip uses applied physical forces to promote, enhance, or facilitate desired biochemical reactions or processing steps or flasks or analysis steps or tasks. On an active chip, "applied physical Forces ' 30 are physical farces thafi, when energy is provided by a power source that is external fio an active chip, are generated by micra-scale structures built into or onto a chip.

A ''passive chip" is a chip that daes not utilize externally applied physical forces to manipulate or control molecules and particles for chemical, biochemical, or biological reactions. Instead, the reaction process on a passive chip involves thermal diffusion of molecules and particles and involves naturally occurring farces such as the earth's gravity.
An "electromagentic chip" is a chip that includes at least one electromagnetic unit, such as a micro-electromagnetic unit. The electromagnetic unit can be on the surface of a chip, or can be provided integrally ar at least partially integrally, within said chip. her example, an electromagnetic unit can be provided on the surface of a I 0 chip or can be imbedded within a chip. Optionally, an electramagnetic unit can be pal-tially imbedded within a chip. Preferred electromagnetic chips are those disclosed in United States Patent Application Serial Number 09/399,299 (attol°ney docket number ART-OOl O~.P.1), tiled September 17, 1999, entitled, "Individually Addressable Micro-Electromagnetic Unit Array Chips" and United States Patent 15 Application Serial Number t)9/685,410 (attorney docket number ART-001 p4.P.1.1 ), filed October 10, 2000, entitled, "Individually Addressable Micro-Electromagnetic Unit Array Chips in Horizontal Configurations', both herein incorporated by reference in their entireties.
"Particle switch chip'" refers to the chip disclosed in United States Application 20 Number 091678,263 (attorney docket number AI2TLNC0.002A), entitled "Apparatus for Switching and Manipulating Particles and Methods of Use Thereof' laled on October 3, 2000, incorporated by reference in its entirety, comprising al least three sets of electrodes that are independent of one another, that can translocatc particles using traveling wave dielectrophoresis or traveling wave electrophoresis, and that can ?5 be used to move particles alr~ng different pathways connected at a common branch point when the sets afelectrodes are connected to out-of=phase signals.
A "multiple force chip" or "multiforcc chip" is a chip that generrltes physical force fields and that has at least two different types of built-in structures each of which is, in combination with an external power source, capable oj~ generating one 30 type of physical 1-7eld. A ful I description of th c multiple Force chip is provided in United States Application Number 091679,0'' having attorney docket number ~~18i~?000~00, elltltled "<~l~yal'atLISC~ COllttllillll~~ Mllltlple ActIVe f"()1'eC.' (it'll('I'~1t111 1~
Elements and Uses Thereol-' Filed Uctober ~. ~t)00, herein incorporated by rclcrence in its entirety.
"Mixing" as used herein means the use of physical forces to cause particle movement in a sample, solution, or mixture (such as a mixture of sample and sample solution, or a mixtm°e or moieties and binding partners), or to cause mavement of sample, solution or mixture that is contained in a chamber such that components of the sample, solution, or mixture become interspersed. Preferred methods of mixing for use in the present invention include use of acoustic forces and thermal convection.
''Disruption" as used herein means changing the struotural state of a sample component. Examples of disruption are cell lysis, denaturation of proteins, and dissociation of subunits of complexes, such as, for example, ribosomes.
Disruptions can be effected through the use of physical forces, such as for example, high voltage electric fields or acoustic forces, or by use of reagents such as denaturing agents, chelating agents, surfactants, or enzymes.
"Piezoelectie transducers" are structures capable ol=generatiug an acoustic field in response to an electrical signal. Preferred piezoelectric transducers arc piezoelectric ceramic disks or piezoelectric thin films covered on both surfaces with a metal film.
"Electromagnetic units" are structures that, when connected to a source of electric current, can produce a magnetic hold and exert a magnetic force on magnetic or paramagnetic particles. L;lectromagnetic units preferably include a core that is preferably magnetic or maf~netizable, and a means, such as a conducting coil, for conducting an electric current about said magnetic core.
"Fluid flow" refers tc~ the mass flow ol~ (laid by means such as by electrophoresis or mechanic,.il force, such as laressure or thermal convection forces.
"Autamated" means not requiring manual procedures, such as pipeting or other manual transfer of samples or reagents. inversion or vortexing of tubes, placing samples in a centrifuge, incubator, etc. by a practitioner, anel the like. IAn automated system may, however, require manual application oi~th~ sample to the system (i.e., by 3(~ laipeting or injecting), or manual recovery ol~sample components that have been fully processed by tl~e system (i.c.. by pipeting tiwm a chamber, or collecting iv a tube that a conduit leads into). An automated system many c7r may not require a practitioner to Ca17t1'Ol pOWel'-dl'IVell Sy5tel77s f01' j~Llld flaw. to Galltl'O1 powel'-dl'1Ve11 SyStel115 la l' gellel'atlllg phySlGa1 farCeS 1a1' tile pel'tar111a17CG aI' pl'oCeSSlllg alld allalySIS tEiSl<S, to control power-driven systems For generating physical forces for the translocation oI~
sample canlpon ents, and the like, during the operation of the integrated chip system.
5 An automated system, such as an automated integrated biochip system of the present invention, is preferably but optionally programmable.
As used herein, "physical field," e.g., used itself ar used as "physical field in a region of space" ar "physical field is generated in a region o~ space" means that the region of space leas fallowin~~ characteristics. When a Illoiety of appropriate 10 propel'ties is introduced into the region of space (i.e. into the physical field), Forces are produced on the moiety as a result of the interaction between the moiety and the field.
A moiety Gan be manipulated within a field via the physical forces exerted on the moiety by tile field. Dxemplary fields include electric, magnetic, acoustic, optical and velocity fields. In the present invention, physical field always exists in r1 medium in a 15 region of space, and the moiety to be manipulated is suspended in, ar is dissolved in, ar mare generally, is placed in the medium. 1"ypically, the medium is a fluid such as aqueous or noel-aqueous liquids, or a gas. Depending an the field con fiiguratian, an electric field may produce electrophoretiG forces on charged moieties, or may produce conventional dielectcophoretic forces and/or traveling wave dielectrophoretic forces 20 on charged and/or neutral moieties. Magnetic fuelds may produce magn etic forces on magnetic moieties (inclLldint~ paramagnetic moieties, or tra~~eling-wave magnetophoretic farces on magnetic moieties. Acoustic field may produce acou stic radiation forces on moieties. Optical field may produce optical radiation farces on moieties. Velocity field in the medium in a region of space refers to a velocity distribution of the medium that moves in the reunion a~ the space. Various mechanisms play be responsible far causing the medium to move and the medium at different positions may exhibit different velocities, thus generating a vGloGiiy field. A
velocity field may exert mechanical Farces on moieties in the n7ediun7.
As used herein, "phi°aical force" ref~ra to any Force that moves l~hrv moieties or their binding partners withcmlt chemically or biologically reacting with t17~
moieties and the binding partners, or with minimal chemical or biolo~~ical reactions with the binding partners and the moieties sa that the bialogicallchenlical Functions/praperties of the binding partners and the moieties are not substantially altered as a result of such reactions. Throughout the typlication, the team of "Forces'" or ''physical li~rcc~s"
always means the "Forces" ~~r "physical forces" e:xeuted on a moiety ar moieties. 'I"he "forces" or ''physical forces" are always generated through "fields" or ''physical Gelds". The forces exerted on moieties by the 1-fields depend on the properties of the moieties. Thus, for a given lueld or physical field to exert physical forces on a moiety, it is necessary for the moietv° to lave certain properties. While certain types of fields may be able to exert farces on different types of moieties laving different properties, other typos of fields may be able to exert forces on only limited type of moieties. Far example, magnetic field can exert forces or magnetic forces only on magnetic particles or moieties having certain magnetic laroperties, but not on other particles, e.g,, polystyrene beads. On the other hand, a non-uniform electric field can exert physical forces on many types of moieties such as polystyrene beads, cells, and also magnetic particles.
As used here in, "electric forces" (or ''electrical Forces") are the Forces exerted on moieties by an electric (or electrical) field.
"Electric field pattern" refers to the field distribution, which is function oFthe frequency of the field, the magnitude of the field, the geometry of the electrode structures, and the frequency and/or magnitude modulation of fine field.
"Dielectric properties" of a moiety are properties fihat determine, at least in part, the response of a moiety to a dielectric iaeld. The dielectric properties of a moiety include the effective electric conductivity of a moiety and the efFective electric permittivity of a moiety. T'or a particle of homogeneous composition, for example, a polystyrene bead, the effective conductivity and effective pcrmifitivity arc independent ?5 of the frequency of the electric field. For moieties of nonhomogeneous composition, For a example, a cell, the ei-1-active conductivity and eFfective permittivity are values that take into account the el~lective conductivities and effective permittivities of both the surface (membrane) and internal portion c~f~ the cell, and can vary wish fine frequency of the electric i~iclcl. In addition, the dielectric Force experience by a moiety 3b in an electric field is dependent on its size; therefore, the coverall sire ol'moietyr is herein considered to be a dielectric property oFa moiety. Properties of a moiety that contribute to its dielectric properties include the net charge on a moiety:
ihc composition of a moiety (including the distribution of chemical groups or moieties on.
Wlthln, Or t171'OLIghOLlt a 11701Cty~; SILe Of a II7C)Iety; 5u1'faCe CClIIt1gL11'at1017 ()1'L117101ety;
surface charge of a moiety; and the conformation of a moiety.
A "dielectrophoretic force" is the force that acts on a polarizable particle in a nonuniform AC electrical IUc:ld. As used herein "dielectrophoresis" is the n7ovement of moieties in response to dielectric forces.
"Dielectrophoresis''_ sometimes called ''conventional dielectropl7oresis, is the movement of polarized particles in nonuniform electrical fields. There are generally two types ofdielectrophoresis, positive dielectorphoresis and negative dielectrophoresis. In pasitive dielectrophoresis, particles are moved by dielectrophoresis toward the strong field regians. In negative dielectrophoresis, particles are moved by dielectrophoresis toward weak field regions. Whether moieties exhibit positive or negative clielectrophoresis depends on whether particles are more or less polarizable than the surrounding medium.
"Traveling-wave dielectrophoretic (DGP) ford" refers to the force that is generated on particles or molecules due to a traveling-wave electric field. An ideal traveling-wave field is characterized by the distribution of the phase values ofAC
electric field components, being a linear function of the position of the particle. A
traveling wave electric field can be established by applying appropriate AC
signals to the microelectrodes appropriately arranged oll a ch 1p. For generating a iraveling-wave-electric field, it is necessary to apply at least three types of electrical signals each having a different phase value. An example to produce a traveling wave electric field is to use four phase-quardrature signals (0. 90, 180 and 270 degrees) to energize Four linear, parallel electrodes patterned on the chip surfaces. Such four electrodes may be used to form a basic. repeating unit. Depending on the applications, there may be more than two such units that are located next to each other. This will produce a travelin l; electric field in the spaces above or near the elecirodca. As long as electrode elements are arr~lngod Following certain spatially sequential orders, applying phase-seduenced signals will result ill r°atablisl7ing traveling electrical (welds :30 in the region close to the electrodes.
As used herein, '°traveling wave dielecirophoresis"' is the movement of moieties in response to a traveling wave electric field.

As used herein, "magnetic Forces" are the F01'GeS exerted on moieties by a magnetic field.
"Traveling wave electromagnetic Force" refers to the Force that is generated on particles or molecules due to a traveling magnLtiG field or a traveling nla~~n~tio wave.
"Traveling wave magnetophoresis" rulers to the movement of a magnetic particle or magnetizable particle under the influence of a traveling magnetic held or a traveling magnetic wave generated by an array of electromagnetic units. The individual electromagnetic units are arranged according to specific spatial relationships among the units. >~or example. individual electromagnetic units may be of rectangular geometry and of equivalent lengths, alld microfabricated on chips so that the units are aligned and parallel to Each other, as depicted, for example, in Figure 2~B oFUnited States Patent Application Serial I~lumber 09IG85,~10 and having attorney docl~et number AR'f-00104.P.1.1, bled October 10, 2000, entitled, "Individually Addressable Micro-Electromagnetic Unit Array Chips in I-Iorizon cal Configurations", which is incorporated by reference ill its entirety.
Traveling wave magnetophoresis can be synchronized or continuous. In synchranized maglletOphOre5lS, a I'C CLII'1'CSlt IS LlSed t0 I11ag11et1Ze lndlVIClLIal eleGtl'Olllag11Gt1C L1111tS
within an array such that the electramagnetic units can be addressed sequentially. The sequentially addressed electromagnetic units arc energized in an order, sue( as a ~0 predetermined order, such that a magnetic particle or magnetizable particle trans~el's ti°om one location to another, In continuous magnetophoresis, an AC
Gurrcnt is used such that the electromagnetic units are addressed using currents that are out o-F phase, such as, but not limited to, about 90 degrees out of phase. Alternative phase shifts can also be utilized. The phase :shifts cause a travelin g magnetic wave or traveling magnetic 'field to form.
As a sed herein, "acoustic forces (or acoustic radiation Forces)" are the forces eYel°ted on moieties by an acoustic Feld.
As used herein, "optical (or optical raLliation) farces" are the Forces exerted on moieties by an optical field.
~0 A "sample'° is any fluid (roll which components are to be separated or analyzed. A sample can be From any source. such as an organism, '~rouh ol~cr~~unisnls 1i'om the same or differont spGGics, from th-r c:nvirollmed, such as l~ronl a htoiy oI~

water or from the soil, or from a Food source or an industrial source. A
sample can be an unprocessed or a processed sample. A sawplc can be a gas, a liquid, or a semi-solid, and can be a solution or a suspension, A sample can be an extract, for example a liquid extract of a sail or food sample, an extract of a throat or genital swab, or an extract of a fecal sample.
A "blood sample" as used herein can refer to a processed or unprocessed blood sample, i.e., it can be a centrifuged, filtered, extracted, or otherwise treated blood sample, including a blood sample to which one or more reagents such as, but not limited to, anticoagulanCs or stabilizers have been added. A blood sample can be of any volume, and can be from any subject such as an animal or human. A
preferred subject is a human.
"Subject" refers to any organism, such as an animal or a human. An animal can include any animal, such as a feral animal, a companion animal such as a dog or cat, an agricultural animal such as a pig or a cow, or a pleasure animal such as a horse.
A "white blood cell" is a leukocyte, or a cell of the hematopoietic lineage that is not a reticulocyte or platelet and that can be found in the blood of an animal.
Leukocytes can include lymphocytes, such as I3 lymphocytes or T lymphocytes.
Leukocytes can also include phagocytic cells, such as monocytes, macrophages, and granulocytes, including basophils, eosinophils and neutrophils. Leukocytes can also comprise mast cells.
A "red blood cell" is an erythrocyte.
"Neoplastic cells" refers to abnormal cells that grow by cellular proliferation more rapidly than normal and can continue to grow after the stimuli that induced the new growth has been withdrawn. hleoplastic cells tend to show partial or complete lack of structural organization and functional coordination with the normal tissue, and may be benign or malignant_ A "malignant cell" is a cell having the laroperty of locally invasive and destructive growth and metastasis.
A ''stem cell" is an undifferentiated cell that can give rise, through one or more cell division cycles, to at least one dil~lcr~ntiated cell type.

A "progenitor cell" is a committed but undifferentiated cell that can give rise, through one or more cell division cycles, to at least one differentiated cell type.
Typically, a stem cell gives rise to a progenitor cell through one or more cell divisions in response to a particular stimulus or set oI~ stimuli, and a progenitor gives rise to one 5 or more differentiated cell types in response to a particular stimulus or set o(~ stimuli.
An 'ketiological agent" refers to any etiological agent, such as a bacteria, virus, parasite or prior that can infect a subject. An etiological agent can cause symptoms or a disease state in the subject it infects. A human etiological agent is an etiological agent that can infect a human subject. Such human etiological agents may be specific 10 for humans, such as a specific human etiological agent, or may infect a variety of species, such as a promiscuous human etiological agent.
A "component" of a sample or "sample component" is any constituent of a sample, and can be an ion, molecule, compound, molecular complex, organelle, virus, cell, aggregate, or particle ol=any type, including colloids, aggregates, particulates, 1 ~ crystals, minerals, etc. A component of a sample can be a constituent entity of a sample that has been exposed or: altered by processes performed before application of the sample to a system of the present invention, or by the methods of the present invention, such as methods performed by a system of the present invention. A
component of a sample can l)e soluble or insoluble in the sample media or a provided 20 sample buffer or sample solution. A component ofa sample oar be in gaseous.
liduid, or solid form. A component of a sample may he a moiety or may not be a moiety.
A ''moiety" or "moir''ty of interest" is rely entity whose manipulation in a system of the present invention is desirable. A moiety can be a solid, including a suspended solid, or can be in soluble form. A moiety can be a molecule.
lVlolecules 25 that can be manipulated include, but are not limited to, inorganic molecules, including ions and inorganic compounds, or can be orf~anic molecules. including amino acids, peptides, proteins, glycoproic:in s, lipoproteins, glycolipoproteins, lipids, lots, sterols, sugars, carbohydrates, nucleic acid molecules. small organic molecules, or complex organic molecules. .~ moiety can also be a nlolccular complex, can be all organelle, can be one or more cells, including prokaryotic and eulcaryotic cells, or can be one or more etiological agents. including viruses, parasites, or priors, or portions tll~reof~. A
11101ety Call alSO bC a CL'yStal. 111111e1'al, COl1o1C1_ ll'ag111el1t, lIlyCC111C, dl'oplCi, 17u1)ble. t)I' the like, and can comprise one or more inor~~nllic materials such as polynleric materials, metals, minerals, Mass, ceramics, and the like. Moieties can alsc7 be aggregates of molecules, complexes, cells, organelles, viruses, etiological agents, crystals, colloids, or fragments. Cells can be any cells, including prokaryotic and eul<aryotic cells. Eukaryotic cells can be of any type. Of particular interest are cells such as, but not limited to, white blood cells, malignanf cells, stem cells, progenitor Dells, fetal cells, and cells inFected with an etiological agent, and bacterial cells.
Moieties Gan also be artificial particles such polystyrene microbeads, microbeads of other polymel° compositions. magnetic micorbeads, carbon nlicrobeads~
As used herein, "intracellular moiety" refers to any moiety that resides or is otherwise located within a cell, i.~,, located in the cytoplasm or matriX
o~l=cellular organelle, attached to any intracellular membrane, resides or is otherwise located within periplasm, if there is one, or resides or is otherwise located on cell surface, i.e"
attached on the outer surface: of cytoplasm membl°ane or Gell wall, if there is one.
As used herein, "nlalnipulation" refers to moving or processing at- the moieties, which results ill one-, two- or three-dimensional movement of the moiety, in a Ghip format, whether within a single Ghip ar between or among multiple chips.
Moieties that are manipulated by the methods of the present invention can optionally be coupled to binding partners, such as microparticles. Non-limiting eXamples of the manipulations include transportation, capture, Focusing, enrichment, concentration, aggregation, trapping, repulsion, levitation, separation, isolation or linear or other directed motion oCthe moieties. par effective manipulation of moieties coupled to binding partners, fine bindings partner and the physical force used in the method must be colnpatlble. ~'pl" eXanlpl(:. bllldlllg pal'tllel'S wlth InagIletIG
propertleS IllLlSt be LISed with magnetic force. Similarly, binding partners with certain dielectric properties, c:.g,, plastic particles, polystyrene microbeads. Must be used with dielectrophorelic force.
As used herein, "th moiety to be manipulated is substantially coupled onto surface of the binding partner" means that a majority of the moiety to be mvnipLllat~d 3p is coupled onto surface of the binding partner anti can be manipulated by a suitable physical force via manipulation of the bindill~.~ partner. Ordinarily, at least 1 t% of the moiety to be manipulated is coupled onto surf-ace of the binding partner, I'rGlerably, at least 5°l0, 10%, 20°r'°, 30°; r~.
~10°~'°, 50%. 60'~~, 70%, 80°l° or 90°f° of the moiety to be manipulated is coupled onto surFacc oFthe binding partner.
As used herein, "the moiety to be manipulated is completely coupled onto surFace of the binding partner" means that at least 90°l0 of the moiety to be S manipulated is coupled onto surface of the binding partner. Preferably, at least 91 °Jo, 92%, 93°,~0, 9~t°J°, 95%, 96°f,.
97°r'°, 98°,~°, 99°/~ or 100°~° oFthe moiety to he manipulated is coupled onto surface of the binding partner. A "solution that selectively modifies red blood cells" is a solution that alters non-nucleat~:d red blood cells such that they do not interfere with the diclectrophorctic separation o~l~ other cells or components of a bland sample, without substantially altering the integrity of white blood cells, or interfering with the ability oFwhite blood cells to be dielectrically separated from other components of a blood sample.
"Binding partner" reFers to any substances that both bind to the moieties with desired affinity or specificiiv and ace manipulatable with the desired physical force(s).
l5 ~ ~on-limiting examples of the binding partners include cells. cellular organelles.
viruses, microparticles or an aggregate or complex thereof , or an aggregate or complex of molecules.
A "microparticle" or "particle" is a structure of any shape and of any composition, that is manipulatable by desired physical force(s). The mieroparticles used in the methods could hove a dimension from about 0.01 micron to about ten centimeters. Preferably, the microparticles used in the methods have a dimension from about 0.1 micron to about several thousand microns, Such particles or microparticles can be comprised of any suitable material, such as glass or ceramics, and/or one or mare polymers. such as, For example, nylon, polytetrafi7uoroethylene 2~ (TEFLON ~~~~, polystyrene, holyacrylamide, scpaharose, agarose, cellulose, cellulose derivatives, or dextran, and~"or can comprise metals. Examples of microparticles include, but are not limited to, plastic particles. ceramic particles, carbon particles, polystyrene microbeads. glass beads, magnetic beads, hollow glass spheres, metal particles, pauticles of complex compositions. n~icroFabricated or micromachined particles, etc.

"coupled" means bound. I~or example, a moiety can be coupled tc~ a micraparticle by speciFic or nonspecific binding. As disclosed herein, the binding call be covalent or noncovalent. reversible or irrcvcrsible.
A "specific binding member" is one of two different molecules having an area on the surface or in a cavity which specifically binds to and is thereby defined as complementary with a particular spatial and polar arganization of the other molecule.
A specific binding member can be a member of an immunological pair such as antigen-antibody, can be biotin-avidin or biotin streptavidin, ligand-receptor. nucleic acid duplexes, IgG-protein A, DNA-DNA, DNA-IZNA, RNA-RNA, and the like.
A "nucleic acid molecule°' is a polynucleotide. A nucleic acid molecule can be DNA, RIA, or a cambinatitm of both. A nucleic acid molecule can also include sugars other than ribose and deoxyribose incorporated into fine backbone, and thus can be other than DNA or RNA. A nucleic acid can oomprise nucleobases that are Naturally occurring or that do not occur in nature, such as xanthine, derivatives of Nucleobases, such as 2-aminoadenine, and the life. A nucleic acid molecule ofth a pl'eSent lllvelltlOn Gall have I I111CageS OtheI' than phOSphOdleSter IInICageS. A llLlclele acid molecule of the present invention can be a peptide nucleic acid molecule, in which nucleobases are linked to a peptide backbone. A nucleic arid molecule call be of any length, and can be single-stranded, double-stranded, or triple-stranded, or any combination thereof.
"I-Iomogeneous manipulation" refel's to the manipulation of pal'ticles in a mixture using physical farces, wherein all particles of the mixture have the same response to the applied forcL~.
"Selective manipulation" refers to the manipulation of parficles using physical ?5 l:orces, ill which differen t particles in a mixture have diflerl:llt responses to the applied Force.
"Separation'" is a process in which oNe or more components of a sample is spatially sEparated from one or more other components of a sample. A se l~aration can be performEd such that one or more moieties oFinterest is iranslocatcd to one or more areas of a separation apparatus and at least se~nlc of the remaining comlaonents are translocated away from the clrea or areas where the one or more moieties of interest al'e traIlSlOCated t0 alld/~OI' rCtallled 111, Or I11 W111Ch One OI' 11101'e 111OIetleS 1;; I'Etalll(:d 111 c~
one or more areas and at least some or the remaining components are removed ti'om the area or areas. Alternativ~c;ly, one or more components c>l~a sample can be translocated to andlor retained in one or more areas and one or more moieties can be removed from the area or areas, and optionally collected. It is also possible to cause one or more moieties to be translocated to one or more areas and one ar more moieties of interest or one or more components of a sample to be translocated to one or more other areas. Separations can be achieved through the use of physical, chemical, electrical, or magnetic forces. Examples of (or ces that can be used in separations are gravity, mass flow, dielectrophoretic forces, and electromagnetic forces.
"Capture" is a type of separation in wi~ich one or mare moieties is retained ill one or more areas of a chip. A capture can be performed using a specif c binding member that binds a moiety of interest with high affinity.
An "assay" is a test performed on a sample or a component of a sample. An assay can test for the presence of a conlponeni'. the amount or concentration of a component, the composition of a component. the activity of a component. etc.
Assays that can be performed in conjunction with the compositions and methods o(= the present invention include biochemical assays, binding assays, cellular-assays, and genetic assays.
A "reaction" is a chemical or biochemical process that changes the chemical ar biochemical composition of one or more molecules or compounds or that changers the interaction of one or snore molecules with one or more other molecules or compounds. Reactions of thG present invention can be catalyzed by enz~~nlcs, and can include degradation reactions, synthetic reactions, modifying reactions. or binding reactions.
A "binding assay" is an assay that tests l~or the presence or concentration of an entity by detecting binding ol~ the entity to a sheciFic binding member, or that tests the ability of all entity to bind another entity, or tests the binding affinity ol~ one entity for another entity. An entity can be an organic or inorganic molecule, a nlolccular t;omplex that comprises, or'~cmic, inorganic. t>r a combination of~ organic ante inorganic GO111pOLlIIdS, all OI'gallelle, a VII'L15, 01' a Cell. 13111c11ng aSSayS Call LISe detectable IabelS
or signal generating systems that give rise to detectable signals in the presence ol~ the bound entity. Standard binding assays incluclc those that rely on nucleic acid ~0 hybi°idization to detect speci tic nucleic acid sequences, those that rely on antibody binding to entities, and thaw that rely an ligands binding tt~ receptors.
A "biochemical assay" is an assay that tests for the presence, concentration, or activity of one or more components of a sample.
A "cellular assay' is fm assay that tests For a cellular process, such as. but not limited ta, a metabolic activity, a catabolic activity, an ion channel activity, an intracellular signaling activity, a i°eceptor-linlceci signaling activity, a transcriptional activity, a translational activity, or a secretary activity.
A "genetic assay" is an assay that tests For the presence or sequence of a genetic element, where a genetic element can be any segment of a DNA or R~ ~A
molecule, including, but not limited to, a gene, a repetitive clement, a transposable element, a regulatory element, a telomere, a centromere, or DNA or RNA of unknown function. As nanlimiting examples, genetic assays can use nucleic acid hybridization techniques, can comprise nucleic acid sequencing reactions, or can use one or more polymerases, as, For example a genetic assay based on PCR. A genetic assay can use one or more detectable labels, such as, but not limited to, I7uorochromes.
radioisotopes, or signal generating systems.
A °'detection assay ~ is an assay that can detect a substance, sLicl~
as an ion, molecule, or compound by producin g a detectable signal in the presence oFthe substance. Detection assays can use specific binding members, such as antibodies or nucleic acid molecules, and detectable labels that can directly or indirectly bind the specif c binding member or the substance or a reaction product of the substance.
Detection assays can also use signal producing systems, including enzymes or catalysts that directly or indirectly praduce a detectable signal in the presence oFthe ?5 substance or a product oFthc substance.
A "detectable label's is a compound ar molecule that eau be detected, or that can generate a readout, such as fluorescence, radioactivity, color, chemiluminescence or other readouts known in the art or later developed. The readouts can be based on i7uorescence, such as by Iluorescent labels, such as but not limited to. C~v-s, Cy-5, p phycoerythrin, phycocyanin. allophycocyanin. 1' ITC, rhodamine. ar lanthanides: and by tloureseent proteins such as, but nat limited to. green Iluorescent protein (Gl~ P).
'Fhe readout can be based on cnzvmatic activity such as, but not limited to, the Jl activity of beta-galactosidase, beta-lactamaso. horseradish peroxidase, alkaline phosphatase, or luciFerase. The readout can be based on radioisotapes Csuch as ~~P, ~I-1 , ~'~G, ~'S, ~Z~I, ~''P or ~~~1 ). A label optionally can be a base with modified mass, such as, Ior example, pyrimidines modified at the ~5 position or purines modified at the N7 position. Mass modiFyin g groups can be, (or examples, halogen, ether or polyether, alkyl, ester or polyester, or of the general type XR, wherein X is a linking group and R is a mass-moth lying group. One of skill in the art will recognize that there axe numerous possibilities for mass-modifications useful in modifying nucleic acid molecules and oligonucleotides, including those described in Oligonu cleotides and Analogues: A Practical Approach, Eclcstein, ed. (1991 ) and in PGTiUS9~1100193.
A 'signal producing system" may have one or more components. at least one component usually being a Labeled binding member. The signal producing system includes all of the reagents required to produce or enhance a measurable signal including signal producing means capable oI~ interacting with a label to produce a signal. The signal producing system provides a signal detectable by external means, often by measurement of a change in the wavelength of light absorption or emission.
A signal producing system pan include a cllromopharic substrate and enzyme, where chromophoric substrates arc enzymatically converted to dyes which absorb light in the ultraviolet or visible region, phosphors or lluorescers. However, a signal producing system can also provide a detectable signal that can be based on radioactivity or other detectable signals.
The signal producinf~ system can include at least one catalyst, usually at least one enzyme, and can include: at least one substrate, and may include two or mare catalysts and a plurality of u.ibstrates, and may include a combination of onrvmos, ~5 where the substrate olvone enzyme is the praduct of the other enzyme.
'I'IZe operation of the signal producing system is to produce a product which provides a detectable signal at the predetermined site, related to the presence of label at the predetermined site.
In order to have a dc:toctahle signal, it way be desirable to provide moans For amplifying the signal produced by the presence oFthe label at the predetermined site.
Therefore. it will usually ho preFerable For the label to be a catalyst ar luminescent compound or radioisotope. most prcFerablv a catalyst. PreFerably. catalysts arc 3~
enzymes and coenzymes which can produce a multiplicity of~ signal generating molecules from a single label. An enzyme or coenzyme can be emhloycd which provides the desired amplification by producing a product, which absarbs light, for example, a dye, or emits light upon irradiation, for Example, a fluoresces.
S Alternatively, the catalytic reaction can lead to direct light emission, l:or example.
chemiluminescence. A large number of enzymes and coenzymes for providing such products are indicated in U'.S. Pat. No. ~,275.1~9 and U.S. Pat. No.
x,318,9$0, which disclosures are incorporated herein by reference. A wide variety of non-enzymatic catalysts which may be employed are found in LT.S. Pat. No. 4,160,d~15, issued July 10, 1979, the appropriate portions of which are incorporated herein by reference.
The product of the enzyme reaction will usually be a dye or t7uorescer. A
large number of illustrative Eluorriscers are indicated in U.S. Pat. No, ~t,275,1~1C). which disclosure is incorporated h crein by reference.
Other technical terms used herein have their ordinary meaning in the art that they are used, as exemplified by a variety of technical dictionaries.
A SYSTt;ld9 OF I~'TE(:Itr~TGD CHIPS FOR TI-IE i'ROCESSING AND ANAL~I'SIS OF A
SAIViPI~E
The present invention includes an integrated biochip system for the processing and analysis of a sample. Bv '°integrated biochip system" is meant a system that: 1 J
comprises at least one chip. ?) is capable of laerForming at least two seduential tasks on a sample, wherein at least one task is a processing task. Preferably, at least one task performed by a system of integrated chips of the present invention rccluires the application at physical force by a source that 1s in part external to a chip and in part intrinsic to a chip, and prel~rably but optionallv, at least one sample com~~onent is manipulated through the use ofspecitie bindinr~ partners. such as microhrtrticles. in a task performed on at least one chip of a system ol' the present invention.
The present invention includes at least one chip, where a chip has a surface on which at least one separation, trap slocation. capturing procedure, assay, or acoustic mixing ox physical disrupticw process can bc~ le;rt'ormed. A chip can comprlsc silicon, glass, rubber, photoresist. or one or more mcials. ceramics. polymers.
cop>Ivmers. or WO 02/30562 . . . PCT/USO1/42601 ~3 plastics. A chip can comprise one or more flexible materials. A chip can he from about 1 mnh to about 0,25 :o~. Preferably, the sne of the chips useable in the present methods is ti-om about 4 mm~ to about 25 cm~. 'l'he steal?e ol~ the chips useable in the present methods can be regular shapes such as square, rectangular, circular, or oval, or can be irregularly shaped. The active surface ol~ a chip need not be flat, but can be curved, angled, etc. Chips useable in the methods of the present invention can have one or mare wells or one or more channels that can be etched or bored into a chip or built into or onto the surface: of a chip.
A chip can be part or' a chamber, can cn4~age a chamber, or can be at least partially enclosed by a chamber, but this is not a requirement of the present invention.
A chamber of the present invention is a structure that call contain a fluid sample. A
chamber can be oh any size or dimensions, and preferably can contain a Iluid sample of between 0.001 mioroliter and 50 milliliters, more preferably between about 0.1 microliters and about 25 milliliters, and most preferably between about I
microliter and about two milliliters. Preferably, a chamber comprises at least a portion of at least one chip. A chamber can comprise more than anc chip, or several chambers may comprise, contact, or engage the same chip. A chamber can comprise any suitable material, for example, silicon, glass, metal, ceramics, polymers, plastics, etc. and can be of a rigid or flexible material. Preferred materials for a chamber include materials that do not interfere with the manipulation aF moieties in a sample, for example, insulating materials that do not bind charged or polarized molecules, such as certain plastics and polymers, for example, acrylic, or glass.
A chamber that coral?rises at least a portion of a chip useable in the methods of the present invention can comprise one or more ports, or openings in tile:
walls of a 2~ chamber. A port can be of any appropriate shape or size for the transport or dispensing of a sample, sample components, bur°fers, solutions, or reagents through the port. A port can be permanently open, or can comprise a flap or valve:
that allows the part to be reversibly closed. A part can al?banally be an opening in a wall that is a common wall between two chambers. Alternatively, a port call provide an opening in a wall of a chamber- for the dispensing of sample into the chamber by, far extlmple, dlSpells111g 01' 111jeCtloll.

3 ~1 A part can engage a conduit. A conduit can be any tube that allows for the entry of a fluid sample, solution, or reagent into the chamber, or allows for the translocation of sample component or microparticles from one chamber to another chamber. Preferred conduits for use in the present invention include tubing, for example, rubber or polymeric tubing, e.g., tygon or Teflon~~~M
(polytetrafluoroethylene) tubing. Conduits that engage one or snore ports of a chamber can be used to introduce a sample, solution, reagent, or preparation by any means, including a pump for example, a peristaltic pump or infusion pump), pressure source syringe, or gravity feed.
Preferred chips in a system of the present invention include active chips.
Preferably, at least one chip in an integrated bioehip system of the present invention is an active chip. Active chips are chips that comprise micro-scale structures that can generate a physical force when energy is supplied to them From, for example, a pawer supply. Thus, the applied physical forces used in the methods of the present invention require an energy source (sometimes called a "signal source") and a structure capable of converting the energy to a type of force useFul in the present invention.
Active chips are therefore described as chips that supply at least in part, a source of a physical farce used in the methods of the present invention. Micro-scale structures that can convert the applied energy to a type of force useful in the present invention can be, as nonlimiting examples, electrodes for generating electrophoretic and dielectrophoretic Forces, electromagnetic units for generating electroma~~netic or magnetophoretic or magnetic forces, and piezoelectric transducers far generating acoustic forces. Depending on the type of micro-scale structure they comprise, they can be referred to as, for example, Electrophoresis or dielectrophoresis chips (comprising electrodes), electromagnetic chips (comprising electromagnetic units) or acoustic chips (comprising piezoelectric transducers). Chips can also comprise optical elements, micro-capillaries or tips, heating e;lcments (e.g., metal wires), 1'eltier elements, micro-valves, or micro-pumps.
~n active chip can ht: constructed by huildin g physical Force elements (e.g., electromagnetic units, pie~oe:lectric transducers, or electrades) onto or into the chip surface, or by applying functional layers such as, Car example, oligonuclc:otidc arrays or protein arrays onto the surface of the chila to make, for example, a pasalve chip.

Other materials that can be provided on passive or active chips of the present invention include speciFc binding members, including, but not limi ed to avidin, streptavidin, or biotin, antibodies, and nucleic acid molecules; enzymes.
catalysts, or substrates (including, but not limited to enzymes, catalysts, and substrates used for 5 detection); reagents, including insulating Layers. or coatings or layers of substances provided to prevent nanspecitic binding or interaction of one or- more sample components to a chip surface; complexes; and even viruses and cells. These materials can optionally be provided in wells or channels of a chip of a system of the present invention. Materials that can be used as coatings or layers to prevent nonspecific or 10 undesirable interactions of one or more sample components with a chip surface (including micro-scale structures on the chip) can form a i~top layer" of the chip, and can be thin (less than 100 Angstrom) layers of polymers, compaunds such as silicon dioxide, surfactants, ax biomolecules, such as BSA.
Examples of active clips include, but are not limited to, the dielectrophoresis 15 electrade array on a glass substrate (c.g., Dielectrophoretic Manipulation of Particles by Wang et ul., in IEEE Transactian on Industry Applications, Vol. 33_ No. 3, May/June, 199'7, laages 660-669''), the individually addressable electrode array on a microfabricated bioelectronic chip (e.g., Preparation and LLybridization Analysis of DNAIRNAfrom E. coli on Microfabricated Bioelectronic Chips by Cheng cl crl., L0 Nature Biotechnology, Vol. 16, 1998, pages '?ill-546), the capillary electropharesis chip (e.g., Combination of sample-Preconceniration and Capillary >electrophoresis On-Chip by Lichtcnberg, e~ cal., in Micro Total Analysis Systems 2000 edited by A.
van den Berg et crl., pages 3t>7-310), the acoustic force chips disclosed in U~.S. Patent No. 6,029,518, the electromagnetic chips disclosed in l~.S. Patent Application Serial ?5 No. 09/399,299 (attorney docket number AR'f-00104.P. I ), Bled September 17, 1999, herein incarporatetl by reference, and United States Application Number ()9/6$5,410 (having attorney docket number AIZT-00104.!.1.1 ), Fled October 10, ?O()(), entitled c'Individually Addressable Micro-I~IECtroma~~nctic Unit Array Chips in Horizontal Configurations", also incorporated by reference.
30 For dielECtrophoresl;~ chips, including, chips that are used for conventional and traveling wave dielectrophoresis. electrodes ~~n a chip can be: of any sh~y~_ such as rectangular, castellatetl, triani~ular, circular, tmcl the lilce. hlcctrodes can be arranged in vat°ious patterns, for example, spiral, parallel, interdigitated, polynomiU. etc.
Clectrode arrays can be l~abr~cated on a chip by methods known in the art. for example, electroplating, spattering, photolithography or etching. Examples oFa chip comprising electrodes include, but are not limited to, the dielecti°ophoresis cleotrode array on a glass substrate (c~.~., Dielectrophoretic Manipulation of Particles by Wang et al., in LEES Transaction on Industry Applications, Vol. 33, No. 3, May/.lune, 1997, pages 660-669), individually addressable electrode array on a microfabricated bioelectronic chip (e.g., Preparation and Hybridization Analysis of DN'AIRNA
from E. Goli on Microfabricated lBioelectronic Chips by Chen g of crl., Nature Biotechnology, Vol. 16, 19O$, pages 541-546). and the capillary electrophoresis chip (e.g., Combination of Sample-Preconcentration and Capillary Electrophoresis On-Chip by Lichtenberg, el crl., in Micro Total Analysis Systems 2000 edited by A. van den Berg et al-, pages 307-~ 10).
Other preferred chips that find usefulness in the present invention are described in United States ~\pplioation Number 09167$,263 (attorney doclcot number ARTLNC0.002A), entitled "Apparatus for twitching and Manipulating Particles and Methods of Use Thereof' f'lled on October 3, '2000 and United States Application Number 09/679,024 (havinf~; attorney docket number 471$42000400), entitled "Apparatuses Containing Multiple Active horee Generating Elements and Uses Thereof" filed October 4, 200D, also herein incorparated by reference.
Single Chip ~5~~.slems~
Ln one aspect of the present invention. an integrated biochip system comprises a single chip. In this aspect. a single-chip into~~rated biochila system comprises an 2~ active chip that can perform at least two sequential tasks. Preferably, an active chip of a single-chip system comprises different functional elements to perform at least two sequential tasks.
A chip that performs more than one Cvnction can have combinaii~ws o(one or more different functional elements such specific binding members, substrates.
reagents, or diFferent types t(~ micro-scale structures, including micro-scale structures that provide, at least in part. one or more sources oFphysical forces used in processes or tasks carried OLIt on the chip.

In embodiments where a system ofthc present invention comprises a chip that has different lvnctional elements, the regions oI~ the chip having different functional elements can be in close proximity, such that sample components are fi~ce:ly and readily diffusible among the different functional elements (see, for example, >f~'igure 17), and preferably but optionally, the different functional elements are at least partially interspersed with one another. Alternatively, in a multiple force chip, different functional elements, in particular different physical force-generating elements, can be provided in different structurally linked substrates that are vertically oriented with respect to one another. For examples of multiple force chilas see United States Application Number ()9/679,021 (having attorney docket number d718~2000400), entitled "Ahparatuses Containing Multiple Active Foi°ce Generating Elements and Uses Thereol~~ filed October ~, 2000, herein incorporated by reference.
Lt is also possible to have different functional elements on a chip of a system of the present invention that are not in immediate proximity. Preferably, such chips f5 are multiple force chips that comprise functional elements that eau generate physical forces that can be used to translocate sample components From one area of a chip to another area of a chip. Preferred physical force-generating elements of a chip for translocating sample components are electrodes and electromagnetic units. In preferred embodiments of the present invention, functional elements sunk as electrodes and electromagnetic units that are used in translocating a sample component from one area ol~ a chip to another area oFa chip are arranged such that they can generate traveling wave dielectrophoretic forces or traveling wave electromagnetic forces.
The order of sequential tasks performed on the same chip can be regulated by 2~ the selective activation of functional elements; by controlled translocation of sample components and binding partners, optionally but preferably including microparticles coupled to sample components; by the regulatc:cl addition of reagents, including, but not limited to, detergents, el~rymes, and spocil!c binding members; or combinations thereof.
(referred chips and preferred active layers of chips oFthe present invention for translocatin g sample compr~nents From one functional area of a chip to another include those described in 1 ~nitod States Application t~I~umhe:r 09/678,'?OS
(having 3g attorney docket number AR'ILNC0.002A), entitled 'Apparatus for Switching and Manipulating (articles and Methods of Use "IhereoF' filed on October s_ ?()00, herein incorporated by reference. such particle switch chips and particle switch active layers of chips can be used for translocating sample campanents from one area of a chip to another area of a chip, where different areas aF a chip can have different Functional elements for performing different tasks. Particle switch chips and particle switch active layers of chips can also be used for translocating sample components from one chip of a system to another chip of a multiple chip system, where different chips of the sysfem can have different functional elements for performing different tasks.
It is also possible to Dave one or more sources of a force used to translocate sample components or microparticles on or intrinsic to a chamber, such as a chamber that comprises a chip. For example, electrades used as a source of an electric field used to translocate particles can be incorporated into a chamber wall, or extend from a chamber wall (including the top wall) in any direction. It is also passible to have one or more source elements that are external to a chip, or chamber of the present invention, but this is not pre (erred.
Multiple Chip S,~~stc~nzs In one aspect of the present invention. an integrated biochip system comprises multiple chips. In this aspect. a multiple chip integrated biochip system comprises al least one active chip and can perform at least two sequential tasks.
Where an integrated biochip system of the present invention comprises more than one chip, preferably at least one task in the processing of a sample can be performed on at least one chip of the present invention and at least one other task can be performed on at least ono other chip of the 1?resent inven tian.
In these aspects, pre(crably at least two chips are, For at least a portion of the time that the system is operating, in fluid connnunication with one anotl?cr.
ITluicl eammunication in this sense means that rluid con move Dram the surface c~l'onc chip a0 to the surface aFanather chil?. and in particular that sample components cmci microparticles. in soluble ~~r suspended form in a h~luid (that is, a liquid car a has), can be tl°anslocated from the sur(hce aFone chip is the surface of another chip, by means other than collecting and dispensing a Fluid From one chip to another chip such as by pipeting or withdrawing and injecting.
Chips that are in fluid communication with one another are preFerallly positionally and Functionally order°ed such th~lt a "second" chip can receive Ci'om a "first" chip a sample, samplr= component, or sample product that is the product of a separation, translocation, capture, assay, mixing or disruption process perl:ormed on the "first" chip, and the "second°' chip can perl:orm a function that is a further step in the processing or analysis o(~ the sample. (As used herein, "first" and "second" do not refer to their absolute order in the integrated system, but rather to their relative order, where a process performed on the second chip occurs immediately after a process perfol°med on the first chip. ) ThLls, the first and second chips in the example are preferably positionally ordered such that a sample, sample component, or sample product (including, for example, a sample camponent coupled to microparticles) can be translocated from the first chip to the second chip. Preferably, in this example, the 6xst and second chips are adjacent or in close: proximity.
Preferably, the translaort of sample components from one chip to another chip, or from one chamber to another chamber, does not require manual transfer. but is accomplished through fluid l'low (using force generated by a pump, for example) or by using applied physical forces.
In a multiple chip system, forces used to translocate sample components ar microparticles from one chip of the system to another chip ofthe system can have one or more sources that are but It onto or into a chip. Thus, active chips of the multiple chip system can be used for transporting sample components by, far example, traveling-wave dielectropln.~resis or traveling-wave magnetophoresis for one chip to another chip. ~IAhe particle switch chip described in United States Application Number 09/678,263 (having attorney docket number AIZTLNC0.002A), entitled "apparatus for Switching and Manipulating Particles and Methods al~ Use Thereoi'~ filed on October 3, 2000, herein incorporated by reference, can be used in this rGt~ard. Particle switch chips Gan also be uscol Far translocatin~.~ sample eolnponents from one Area of a i0 Ghlp t0 anOthel' al'ea of a Chlp 111 a Inllltlple Clllla UI' Single Chlp System, \~r11(:1'C C111Te1'ellt ill'eaS Ofa chip Call have dll'1C1'ellt tL111ct1o11a1 C1C111entS for pel'tol'llling Clll~lC1'Cllt tCISICS.

~10 The multiple force chips described for the single-chip system and described in United States Application Number Q91679,02~ (having attorney docket number 471812000400), entitled "Apparatuses Containing Multiple Active Forcn C;enerating Clements and Uses 'hhereol~~ tiled October ~. 2000, herein incorporated by reference, can also end use in multiple chip systems ofthe present invention. For example, a multiple force chip can be used to separate components oI a sample using dielectrophoretic and magnetic forces, and then the separated components can be directed to one or more other chips o1>' the system f=or one or more analysis tasks.
A multiple chip system of the present inventian can also optionally comprise one or more passive chips whose function does not require an applied physical force.
Passive chips that are a part ot~ a system of the present invention can be used for a variety o~assays and detection s, such as but not limited to binding assays.
biochemical assays, cellular assays, genetic assays, sandwich hybridizatians, etc.
~Segaaenlial Ta.~'k5' in lhc Pnot~ce~a~ing a~zd ~ncrh,sis of cr ~fanaplc An integrated biochip system of the present invention is capable of performing at least two sequential tasks in the processing and analysis of a sample.
~equontial tasks are tasks that are performed in a particular order to achieve the desired t7nal result. When two tasks are performed sequentially, a second task uses one or more direct or indirect products ot~the first task, where "praduct" can mean a sample component that was separated, at least pal'tially purified, or concentrated in a first step, or a sample component that was the result of a denaturing or lysing step. was subjected to a biochemical reaction or assay, became bound to a reagent, etc., in a previous task. 13y "Fist" and ''second" is meant the relative order and not the absolute order, o~ tasks performed in the integrated system.
At least ane function that can be performed by a chip of the system of the present invention is a processing task, in which a processing task is any procedure that prepares a sample For analysis and can include as nonlimiting examples, a separation, translocation, focusing, capture, isolation, enrichment. concentration, enrichment.
pal'tlal Ol' SLlbstatltlal pul'IflCtit1011, StI'LlctLlral altel'atlOll 0I' phySlCal dlsl'Llptloll: a17C1 Call include as part of the task chrrmical reactions. including ~.?nzynlatic reactions alld binding reactions, such as binding ofsamplc ct~nlponents to microparticlcs"

~pt1011aI1y, at least tile Othel' tL111Ct1017 hel'1Or111ed by a Ghlp Ot rl SVStC171 Ol the preSellt lnVellt1011 Call be all allalySIS ta5lC. fall allalySlS taSlC lS ally 1L111Ct1017 that leads t0 a result of a processing and analysis procedure. Nonlimiting examples ol~
analysis procedures are assays, such as biochemical, cellular, genetic, and detection assays.
Detection assays can also include binding reactions and enzymatic reactions.
In cel-tain preferred embodiments in which a system comprises a single chip, at least one processing task and at least one analysis task can be performed on the single chip. In other preferred embodiments where an integrated biochip system of the present invention comprises more than one chip, preFerably at least one processing task can be performed on at least one chip of the present invention and at least one analysis task can be performed on at least one other chip of the present invention, but this is not a requirement of the present invention.
Where an integrated biochip system oI~ the present invention comprises more than one chip, preferably at least two Ghips are, f:ar at least a portion of the tinge that 1 S the system is operating, in fluid communication with one another. Fluid communication in this sense: means that fluid can move from the surface of one chip to the surface of another chip, and in particular that sample components and microparticles, in soluble or suspended form in a fluid (that is, a liquid or a gas), can be translocated from the sul'f'ate of one chip to the surface of anofher chip, by means other that collecting and dispensing a fluid from one chip to another chip such as by pipetting or withdrawing and injecting.
ships that are in fluid communication with one another are preferably positionally and functionalli' ordered such that a "second" chip can receive from a "first" chip a sample, sample component, or sample product that is the product of a separation, translocation, capture, assay, mi;~ing or disruption process performed on the "first" chip, and the "second"' chip can pcrl-orm a function that is a l~urlller step in the processing or analysis ol~the sample. (As used herein, ''lurst" and "second" do not refer to their absolute order ill the integrated system, bu t lather to their relative older, where a process perfarlned «n the second chip occurs immediately alter a process performed on the first chip. ) 'fllus, the first and second chips in the exan7ple arc preferably positionally ordered such that a san1111e, sample component, or sample product (including, For exan7ple, a sample component coupled to microptlriiclesl can be translocated ti-om the first chip to the second chip. Preferably, in this example, the first and second chips are adjacent or in close proximity.
The inventors contemplate that in preferred embodiments of the present invention, an integrated system of the present invention can perform at least two sequential tasks in the processing and analysis oFa sample while the sample remains continuously within the integrated system. 'That is, a sample applied to the integrated biochip system can remain continuously within said integrated system from the beginning of the first of the sequential tasks until the end of the last of the sequential tasks performed by the integrated system.
1Q Preferably, the sample and sample components are moved within the system withouf manual transfer from one location to another within the system. sample and sample components, as well as, optionally, solutions, buffers and reagents, can be moved within the integrated system using, for example, fluid flow generated by power-driven pumps (such as syringe pumps or peristaltic pumps). In preferred embodiments of the present example (some of which are illustrated in Fi;ures Jl - JL3), sample components axe translocated from one area of a chip to another area of a chip, or from one chip or chamber to another chip or chamber, using applied physical forces.
In especially preferred embodiments, an integrated biochip system of the 2p present invention is automated, such that the tasks are performed by tile integrated system sequentially without manual intervention, such as, Cor example, transfer of sample or sample components from one chamber to another chamber. 1~n automated system may, however, require manual application of the sample to the system (i.e., by pipeting or injecting), or manual recovery oh ~ sample components that have been fully processed by the system (i.c., by pipeting ti-om a chamber, or collecting laroccssed components in a cube that a conduit leads into). ~n automated system oCthe present invention may or may not require a practitioner to control power-driven systems for Iluid flow, to control power-driven systems for generating physical forcc:~s for the performance ofprocessing and analysis tasla_ to control power-driven systems for 3() generating physical forces t~c~r the translocation ol~ sample components, and the like, during the operation of the integrated chip syrst~m. An automated integrRcd biachip system ofthe present invention, is preferably hut optionally programmublc.

II. METIiODS OP USING A ~IrSTEM OE INTE(:RA'1'ED CHIPS I~OR TI-IE PRO('I:SSINC
t\~VD
ANALYSIS OF A SAnil'LI:
A system of the present invention can be used to process and optionally S analyze a sample. Pi°acessing a sample can involve: separating components of the sample, translocating components of a sample, capturing components of a sample, isolating components oFa sample, focusing components of a sample, at least partially purifying con ~ponents of a sample, concentrating components of a sample, enriching components of a sample. disrupting components of the sample, disrupting components l 0 of the sample, with or without added solutions, reagents, or preparations, analyzing a sample can involve: detecting components of a sample, quantitating components of a sample, or measuring the activity o~ components of a sample (where activities can be, for example, regulatory, catalytic or binding activities, or activities whose mechanisms are known or unlazovi°n, such as cytotoxic activities, mitogcnic activities, 15 transcription-stimulating activities, etc.).
The method includes: application of a sample to a system of inte~~rated chips of the present inventions and performing at least two sequential tasks in the integrated system, in which at least one of the sequential tasks is a pracessing task. IA
processing task can include: separating components of the sample, translocating components of a 20 sample, capturing components of a sample, isolating components of a sample, focusing components of a sample, at least partially purifying components of a sample, concentrating components ol~a sample, enriching components of a sample"
disruptin~~
camponents oFthe sample, disrupting components of the sample, with or without added solutions, reagents, or preparations. ~lacciluc nonlimiting examples oI~
25 processing taslts are: separating white blood cells from a blood sample or a bufFy coat preparation of a blood samlale, separating fetal ells from a maternal blood sample or a maternal amniotic tluid sample, separatint.; malignant cells from a blood sample, separating a stem cell from a bone marrow sample, lysing white blood calls (that have been separated From a blood ,ample), conce~~irating bacterial ells from a urine 30 sample, and separating mRi~A molecules from a lysate of target cells.
The method can also include the translocation ofsample components from one area oFa chip to another area of a chip, wherein at least two different tasla arc ~l~
performed in the dif~Ferent areas of the chip, or translacation of sample components Ii-om chip to another chip, milercin at least tv.~~o cii lferent tasks are performed on the different chips.
S ~polication of~~'anzple A sample can be any fluid sample, such as an environmental sample, including air samples, water samples, Food samples, and biological samples, including extracts of biological samples. A sample can optionally be at least partially processed. her example, a sample can be a centrifuged sample, or a sample to which a detergent has been added. A sample may lave been heated or chilled before being used in the methods of the present invention. A sample can also have reagents added to it, such as, but not limited to stabilizers, including chclators, reducing agents, surfactants, anti-coagulants, glycerol, DMSO, and the like. A sample can be a sample that has been stored, including samples that have been stored at low temperature, including 1 ~ samples that have been frazcn. Biological samples can be blood, serum, saliva, urine, semen, occular fluid, pleural fluid, cerebrospinal fluid, amniotic fluid, ascites fluid, extracts of nasal swabs, throat swabs, or genital swabs or extracts of fecal material.
Qiological samples can also be samples of organs, tissues, or cell cultures, including both primary cultures and cell lines. A preferred sample is a blood sample.
A blood sample can be any blood sample, recently taken from a subject, taken from storage, or removed from a source external to a subject, such as clothing, upholstery, tools, etc. A blood sample can therefore be an extract obtained.
for example, by soaking an article containing blood in a buffer or solution. A
blood sample can be unprocessed. processed, or partially processed, for example, a blood sample that has been centrifuged to remove serum, dialyzed, subjected to C7ow cytometry, had reagents added to it, etc. A blood sample can be of any volume.
For example, a blood sample can be less than 0.(>5 microliters, or more than ~
milliliters, depending on the application.
A sample can be applied to an integrated chip system by any appropriate .30 means, fm- example, by dispensing the sample on to a chip or into a chamber of a system by pipeting or injection. The application of sample can opfionallv be through a conduit that engages a poi°t ofa chamber that comprises a chip of a system of the 4>
preSellt 111VEIltlol7 alld Call opt1011a11y LISe a plllllp, SLICK aS a(1 II1)ECtlon pL1177p OI' peristaltic pump, c»' gravity I~~:d.
One or more reagents. compounds, buFFers, or solutions can be added to a sample before adding the sample to an integrated chip system of the present invention.
Mixing of compounds or solution s with a sample can optionally occur in one or more conduits leading to an integrated chip system, or in one ar more reservoirs connected to conduits. Sample solutions that play be useFul in particular' aspects of the present invention include solutions that can modify tl7e dielectric properties of at least one component of a sample, and solutions that prefr:rentially lyse red blood cells. Such solutions are disclosed in L.Pnited States Patent Application Serial I~la.
09686,737 (attorney docket number ART-00102.P.1), Oiled October 10, 2000, entitled "Compositions and Methods For Separation of Moieties on Chips", herein incorporated by reference. (one or more solutions, buffers, reagents, compounds, or preparations, including preparations of microparticles, can also be added to a chamber or chip of a system of the present invention at any point during the processing and analysis of a sample on a chip. Such solutions. buFfers, reagents, compounds, and preparations cars be added to a chamber or chip by any means, such as but not limited to dispensing, fluid flow, or translocation using physical forces, including.
for example, dielectrophoretic and electromagnetic Forces for the movement of particles, ?0 Solutions that call find use in the present invention and their methods of use include those disclosed in t~.S. Patent Application Serial ~lo. 09/686,737 (attorney docket number ART-0010?.I'.I), entitled "Con7positions and Methods ~lvr separation of Maieties on Chips", incorporated by reference in its entirety.
Tll~o on tljlore Sc~c~ztcjilial Tcr,v°lt,~~
Preferably, at least one processing task, including, but rat limited to a separation, translocation, capture, isolation. purification, enrichment, Focusing, structural alteratian, or disruption procedure that takes place on a chip oFthe system of the present inven tion is (l7rough the application of physical forces.
Alalalfcation ol~
physical Forces to eFfect a processing task is l7reFErably b}r means that arc in part intrinsic to chips of the systL m of the present iwYention and fn part external to chips of the present invention. "hhe exact mechanism ol~ the application of forcES
dcllends on ~O
the forces employed. For example, acoustic, optical, electromagnetic, dielectrophoretic, and electrophoretic forces can be generated by applying electric signals using a power supply connected to piezoelectric transducers, optical units, Pettier elements, metal wires, microcapillarics, micro-tips, micro-valves, micro-s pumps, electromagnetic units or electrodes that are built onto or into a chip. The physical forces that can be used in the invention are described in the following applications: United States Patent Application Serial Number 091636,104 lined August 10, 2000, entitled "Methods for Manipulating Moieties in Microl7uidic Systems"; United States Application Number 091678>263 attorney docket number ARTLyNC0.002A), entitled ''Apparatus for Switching and Manipulating Particles and Methods of Use Thereof' filed on October 3, 2000; United States Application Number 091679,021 (attorney docket number X71842000400), entitled "Apparatuses Containing Multiple Active Force Generating hlements and Uses Thereol" Filed October ~l, 2000, United States Patent Application Serial Number 09/399,299 (attorney docket number AIZT-00104.P.1 ), 171ed September 17, I 999, entitled, "Individually Addressable Ivticro-Electromagnetic Unit Array Chips"; and United States Application Number ()9/685.410 (attorney docket number ART-001 O~.P.1.1 ), filed October 10, 2000, entitled "Individually Addressable Micro-Electromagnetic Unit Array Chips in Horizontal Configurations°', all of which are in corporated by reference in their entireties, A chip capable of producing acoustic Forces and conventional dielectrophoretic forces may be used to exert these two types offoi°ces simultaneously on moieties such as cells, or microparticles on the same chip surface.
Alternatively, two different types of physical force can perform sequential tasks, and the tasks can take place on the same or differen t chips. 'The physical forces can be exorted on a plurality of moieties sequentially or' simultaneously. For example, a chip oT
a system oTthe present invention capable ot~ producin~~ acoustic forces and conventional dielecti°ophoretic forces mew be used to exert those two types of forces simultaneously on two typos oT moieties such as calls and microbeads. "Thus, both types of moieties experience acoustic forces and conventional dielectrophoretic Tortes. In another example. a system capable of producing magnetic forces and traveling wave dielectrophorotic forces mav' he used to exert these two types of forces simultaneously, and on two types of moieties such as magnetic beads and certain types of biological cells, respectively. These functions can occur on the same chip of the system or in parallel on separate chips of the system. 'I"hus, magnetic forces are exerted only on magnetic microbeads and traveling wave clielectrophoi°c~tic forces may be exerted only on biological cells. In still another example, a system can produce magnetic forces and traveling wave dielectrophoretic forces sequentially an different chips. First, the magnetic force generating elements are turned on so that magnetic microbeads bound to a particular sample moiety experience magnetic forces for a specified length of time and are captured on one chip. The non-captured sample I O components are transferred to a second chip, where traveling wave dielectrophoretic force generating elements arc turned an so that biological cells that are sample components experience traveling-wave dielectrophoretic forces.
Of particular relevance to the methods of the present invention is the ability to control the application of physical forces using one or more external energy or signal sources that preferably are connected to micro-structures on a chip of a system of the present invention that generate the physical force on the chip. For example, one or more electrical signal sources can produce one or more electric signals in a particular sequence to apply current to a set of electromagnetic units, to apply an electric field generated by an electrode array, etc. These different functional units can be on the '?0 same or different chips. Alternatively, more than one type of functional element can be turned on at the same time, such as, for example, piezoelectric transducers for producing acoustic forces and electrodes for producing conventional dielectrophoretic forces, where the two types of functional elements are interspersed or overlapped on the same chip and can provide, for example, simultaneous mixing and scparatian. It is also possible to sequentially apply a power signal to subsets offuncfional elements on the same chip as for example, in traveling wave magnetophoresis, or to apply electrical signals of different pleases to different subsets of electrodes, as Ior example, in traveling wave dielectrolahoresis. Preferably, the application of physical Fields through one or more power or signal sources is controlled by a power supply control 3(~ system or signal generator control system that leas an automatable and pro~~rammable switch mechanism, Preferably, a power supply control system or signal ~~~:nerating control system also allows the operator to re~~ulate and modulate parameters of~ the output power or the generafied signals . Where electric fields are used, fihcse parameters can include the signal frequency. signal phase, signal amplitude, and signal modulation mode.
At least one of the procedures in fihe present system can be a processing task or an analysis task that is perFormed on a sample by manipulating sample components in a chip format. Moieties to be manipulated can be cells, cellular organelles, viruses, molecules or an aggregate or complex thereo(~. Moieties to be manipulated can be pure substances or can exist in a mixture of substances wherein the target moiety is only one of the substances in the mixture. For example, cancer cells in fine blood from leukemia patients and metasfiatia cells in the blood patients with solid tumors can be the moieties to be manipulated. Similarly, various blood cells such as red and white blood cells in the blood can be the moieties to be manipulated.
Non-limiting examples of manipulatable cells include animal, plant, Fungi, bacteria, recombinant or cultured cells. l~or animal cells, cells derived I'uom a pauicular tissue or organ can be manipulated. Preferably, cells derived lu°om an internal animal organ such as brain, lung, livLr, spleen, bone marrow, thymus, hearfi, lymph, blood, bone, cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis, ovary, uterus, rectum. nervous system, gland, internal blood vessels, etc. can be manipulated. Further, cells derived from any plants, fungi such as yeasts, bacteria such as eubacteria ar archaebacteria can be manipulated. Recombinant cells derived From any eucaryofiic or prokaryotic sources such as animal, plant, fungus or bacterium cells can also be manipulated. Body fluid such as blood, urine, saliva, bone marrow, sperm or other ascitic Fluids. and subfractions thereof i.~~;, serum or plasma, can also be manipulated.
2~ Manipulatable cellular organelles include nucleus, mitochondria, chloroplasts, ribosomes, ERs, ~olgi apparatuses, lysosomes, proteasomes, seoretorv vesicles.
vacuoles or microsomes. Manipulatable viruses, whether intact viruses or any viral structures, ca.~>., viral particles, in fine virus lily cycle can be derived from viruses such as Mass I viruses. Mass I1 viruses, Class III viruses, Class 1V viruses, Class V viruses 3~ or Class VI viruses.
Manipulatable intracellular moieties include any moiety that resiclc~; or is ofiherwise located within a cell, i.c~" located in the cytoplasm: or matrix of~ccllvlar organelle; attached to any intracellular membrane; resides or is otherwise located within periplasma, iCthere is one; or resides or is otherwise located on cell surface, i.e., attached on the outer surface of cytoplasm membran a or cell wall, if there is one.
Any desired intracellular moiety can be isolated from the target cell(s).
I~~'or example, cellular organelles, molecules or an aggregate or complex thereof can be isalated.
Non-limiting examples of such cellular organelles include nucleus, mitochondria, chloroplasts, ribosomes, ERs, Golgi apparatuses, lysasomes, proteasomcs, seeretory vesicles, vacuoles or microsomes, membrane receptors, antigens, enzymes and proteins in cytoplasm.
Manipulatable molecules can be inorganic molecules such as ions, organic molecules or a complex thereof. Non-limiting examples of manipulatable ions include sodium, potassium. magnesium, calcium, chlorine. iran, copper, zinc, manganese, cobalt, iodine, molybdenum, vanadium, nickel, chramium, Iluorine, silicon, tin, boron or arsenic ions. Non-limiting examples of manipulatable organic molecules include amino acids, peptides, proteins, nucleosides, nucleotides, oligonucleotides, nucleic acids, vitamins, monosaccharides, oligosaccharides, carbohydrates, lipids or a complex thereof.
Far any moieties that cannot be directly manipulated with the desired physical farces, binding partners that themselves can be directly manipulated with the desired physical forces can be coupled to the moieties and the manipulation of such moieties can be effected through the manipulation of coupled binding partner-moiety complexes. Any binding partners that both bind to the moieties with desired a-ffinity or specificity and are manipulatable with the compatible physical forces) can be used in the present methods. The binding partners can be cells such as animal, plant, fungus or bacterium cells, cellular organelles such as nucleus, mitochondria, ehloroplasts, ribosomes, CRs. Golgi apparatuses, lysosomes, protcasomcs, secretory vesicles, vacuoles or microsomes; viruses, microparticles, or an aggregate or complex thereof. Cells, cellular organelles and viruses can also be used as bindim~
partners.
Preferred binding partners are microlaarticles. The microparticles used in the methods have a dimension from about 0.01 micron to about ten centimeters.
Preferably, the microparticles used in the present method have a dimension li-om about 0.01 micron to about several thousand microns. Also larefcrably _ Ihc microparticles used are plastic particles, pol~rstyrene microbeads, glass beads, magnetic beads or hollow glass slaheres, laarticlc;s of comlalcx compositions, micxofabricated free-standing microstructures.
In preferred embodiments ofthe present invention, at least one sample 5 component to be manipulated in a processing or analysis task can be coupled to the surface of the binding partner, such as a microparticle, with any methods known in the art. Fox example, fine moiety can be caupled to the surface of the binding parfner directly or via a linker, preferably, a cleavable linker. The moiety can also be coupled to the surface of the binding partner via a covalent ox a non-covalent linkage.
10 Additionally, the moiety can be coupled to the surface of the binding partner via a specific or a non-specific binding. Preferably, the linkage between the moiety and the surface of the binding partner is a cleavable linkage, e.g., a linkage that is cleavable by a chemical, physical or an enzymatic treatment. Also lareferably, the methods for coupling and/or decoupling the moieties to their binding partners disclosed in the co-15 pending U.S. Application entitled "Methods For Manipulating Moieties in Microfluidic Systems" (US application No. 091636,104; attorney docket number X7184-2000100), filed on August 10, 2000 and incorporated by reference in its entirety, can be used. Preferably, the moiety to be manipulated is substantially coupled onto surface of the binding partner.
20 Preferably, the methods far manipulating the moieties through the use of binding partners disclosed in the co-pending U.S. Application No. 09/636,104 entitled "Methods for Manipulating Moieties in MicroL7uidic Systems" (attorney docket number ~I718~I-2000100), filed on August 1 p, 2p00 can be used for manipulating moieties that cannot be directly manipulated with the desired physical forces.
2S The moiety can be manipulated in a liduid, or gaseous state/medium, or a combination thereof. Preferably, the moiety is manipulated in a liquid medium.
The liquid medium can be a suspension, a solution or a combination thereof.
The present method can be used to manilaulate a single moiety at a time, and can also be used to manipulate a plurality ol~moieties simultaneously. In some cases, 30 the moiety to be manipulatccl can be con taro ed in a mixture and the moiety is selectively manipulated. Selective manipulation refers to the manipulation larocess that the moiety that is beinf~ manipulated is selectively larocessed, and/or is selaarated from the mixture, and/or is caused to experience different manipulation forces or manipulation procedures from other moieties or other particles or other molecules in the mixture. In other cases. the moiety to be manipulated constitutes a mixture and the entire mixture is manipulated. The moieties to be manipulated include the ones that can be manipulated directly by various physical forces and the ones that cannot be manipulated directly by various physical Forces and have to be manipulated through the manipulation oh the binding partner-moiety complex. In specihic embodiments, moieties to foe manipulated are cells, cellular organelles, viruses, molecules or an aggregate or complex thereof, The present methods can use any type of manipulations. Non-limiting examples of the manipulations include transportatian, focusing, capture, enrichment, concentration, aggregation, trapping, repulsion, levitation, separation, fractionation, isolation or linear or other directed motion ofthe moieties.
Preferably, in the method of the present invention the first task performed on a chip is a separation, translocation, capture, mixing, or disruption procedure that functions in the processing of a sample, but that is not a requirement of the present invention. Thus, in nonlimiting examples of the processing procedures that can be used on a sample comprisin4~ cells, cells of interest can be separated from other cells, for example, by conventional dielectrophoresis, or can be translocated from cellular debris of lysed cells of other types, for example, by traveling wave dielectrophoresis, ar can be captured, for example, by binding to electromagnetic units (where a preparation of magnetic microparticles has been added to the sample), or can be mixed, for example, with specific binding members, using, for example, acoustic elements, or can be disrupted, for example, by electronic lysis. In ce~~tain preferred embodiments of the present invention, at least two sequential analysis tasks can be performed on different types of sample components, for example, a first separation task can be performed on cc Ifs, and a second separation task can be performed on proteins, or a first separation task can be performed on proteins, and a second separation task can be performed on RNA molecules.
~ljzcrl>>.si,~~ ~'u.s~k Preferably but optionally, in a system of the present invention, at Icast one analysis task of a simple ohihe present invention occurs alter at least one processing 5?
task. Analysis tasks performed on chips of a system of the present invention can use mixing or binding steps, and preferably include detection assays, biochemical assays, cellular assays, binding assays or genetic assays. One or more analysis tasks can be performed sequentially of in parallel using the methods of the present invention. T'or example, a detection assay For protein and a detection assay for RNA molecules can be performed simultaneously, and in some aspects on the same chip (see, for example, Fig. ~ 5>E).
An analysis task can optionally include an assay, including, without limitation biochemical, cellular, genetic, and detection assays, and can include a mixing procedure or a reaction, such as a binding, chemical, or enzymatic reaction.
In some embodimenfs of the present invention, a method of using a system oir integrated chips includes the use of detection assay on at least one chip of the system.
Preferred detection methods include binding of a sample component to a specific binding member, such as for example, an antibody or nucleic acid molecule that is attached to the surface of a chip. In some preferred aspects of these detection methods the sample component to be detected has been manipulated by physical Forces when coupled to a micraparticle, and prior to the detection step, fine sample component to be detected is decoupled from the binding partner. Reversible linlcers for coupling moieties to microparticles are disclosed in United States Patent Application Serial Number 09/636,104 (attorney docket number 471$4-2000100) filed August 10, 2000, entitled "Methods for Manipulating Moieties in Microfluidic Systems", incorporated by reference. The sample component bound to specific binding partners attached to the surface of a chip can be detected in several ways. The component can be labeled prior to binding the specific binding member with a detectable label.
Aliernafively, a sandwich hybridization can be performed, in which a third molecule (typically an antibody or oligonucleotide> that is detectably labeled is bound to the bound sample component. Other methods oFdetectian can be envisioned, such as enzymatic reactians that add detectable labels to bound sample components (e.g., 'Till-in polymerase reactions on bound nucleic acid moleculesj. See, for example tl~nited States Patent Application Number 09/64$,0t; l lattorney docket number AID°f-0010i.P.1) entitled "Methods and ~'omposit~ions for ldentilying Nucleic Acid Molecules Using Nucleolyiic Activities and 1-yPhridization~~. filed on Auf~ust 25, ?000, herein incorporated by referonce. Preferably, detectable labels used in thoso detection methods are t7uorescent, or spectrophotometrically detectable. In such cases a chamber that encloses a detection chip has a transparent cover, such as a glass cover, to permit detection.
Other mechanisms of detection are also contemplated. Far example, moieties bound to magnetic beads can bind specific binding members attached to the surface of a chip that are in proximity to magnetic heads on fihe chip that are connected to detectors that produce signals generated by the presence of magnetic particles. In another example, the moieties bound to microparticles can bind specific binding I O members that are linked to weight sensing systems, such as cantilevers.
The weight of a particle can be sensed by the cantilever and a signal can be transmitted to a display or recording device.
It is also possible to detect fluorescence emitted by labeled moieties translocated through an aperture, such as the port of a chip. Moieties eau be directed through a port by, for example, fluid flow.
Trarzslocation of Sarnple C'orrrponents, fi~ona crl lc~crst one Chip of the ~fys~I~rn to. trl lecrsl orze Others Chip of ~lh~ Sy~lcna Sample components. including sample components coupled to speci~7c 2(1 binding partners such as microparticles, oars be translacated from one chip of the system to another chip of the system by any means, including fluid flow ( including mass flaw through the application of mechanical force, such as by a syringe pump or peristaltic pump, or convection forces), but preferably translacatian of sample components (including sample components bound to micraparticles) from at least one of the chips of a system of the present invention to at least one other chip of the system is by application of physical farces such as, but not limited to, eloctrophoretic forces, dielectrophoretic forct;s (including convon banal and traveling wave dielectrophoretic Forces) or olectromagnetic Ibrces. Cspecially preferred meahods far translocatian ol~ a sample component from ono area of a chip to another urea of a chip, or from one chip to another chip ofa system are traveling wave dieleetralaharesis and traveling wave magnetopharosis. In preferred embodiments, sample components coupled to microparticles o(~the present invention are translocated from ono are of a 5~1 chip to another area ofa chip, or From one chip to another chip of the present invention using traveling wove dielectrophorcsis or traveling wave ma~nntophoresis.
Of particular relevance to the methods of the present invention is the ability to control the application of physical forces using one or more external energy or signal sources that preferably are connected to micro-structures on a chip or chamber of the system of the present invention That generate the physical forces responsible for translacating sample components from one area of a chip to another area of a chip or From chip to chips Thus the direction of sample components from one area of a chip to another area of a chip or fi°om one chip to another to allow for the step-wise sequence I 0 of functions performed by the system, can be controlled by controlling the power source that directs the sample components ti-om chip to chip, or from one area of a chip to another area of a chip. It is also necessary in some applications, to sequentially apply a power signal to subsets of functional elements on the same chip as in traveling wave magnetophoresis, or to apply electrical signals of different phases to different subsets of electrodes, as for example, in traveling wave dielectrophoresis.
Preferably, the application of physical Fields thi°ough one or mare power or signal sources is controlled by a power generator control system or a signal generator control system that has an automatable and programmable switch mechanism. Preferably, a power generating control system or signal generator control system also allows the operator to regulate and modulate parameters of the power outputs and generated signals , such as, for example in the case ol~ electrical forces, the signal frequency, signal amplitude, signal phase, and signal modulation mode.
Translocation of sample components and micropartieles from one chip to another chip of a system of the present invention can occur through a part in a chamber that comprises one oFthe chips, optionally through a conduit, but this is not a requirement of the present invention. Translocatian of sample components and micropartieles from one arch of a chip to another area of a chip or Pram one:
chip to another chip of a system of the present invention can occur thi°augh Iluici Claw, including mass flaw and electrophoresis, but preC'erably, the translocation r~C~ sample components and micraparticles that occurs thraLigh physical farces occurs by conventional or traveling wave dielectrophoresis or electromagnetic forces.
including traveling wave magnetophorosis_ In the puelcrrcd modes aCtranslocation oC~
sample components and microparticles from one area oFa chip to another area o(~ a chip or From one chip to another chip oFthe system, hreFerably at (cast one oFthc sources of the force used to eFfect the iranslocation is integral to at least one chip o1= the system or at least one chamber of the system. Sample components, including sample components coupled to microparticles, are translocated sequentially from one chip to another chip of a system of the present invention, so that processes in tile processing and analysis of a sample arc performed in an order that allows for a desired final result. For example, components of a sample that are cells of a specific type can be separated on a first chip, and then translocated to a second chip where they are lysed 10 to expose other sample components that are intracellular moieties, and where the sample components are mixed with a preparation of specific binding parfners such as microparticles. Sample components coupled to microparticles can then be translocated, for example using traveling wave dielectrophoresis, to a third chip where, for example, a detection assay can be IaerFormed.
15 Sample components. including sample components coupled to microparticles, can also be translocated from one chip to more than one other chip of a system of the present invention, so that subsequent processes in the processing and analysis of a sample can be performed in parallel. The sample components can be translocated simultaneously or sequentially to more than one chip. Preferably, differ°ent sample ?0 components are translocated to different chips, but this is not necessarily the case. For example, a protein sample component can be transferred to one chip, a nucleic acid sample camponent can be transferred to a second chip, and a steroid hormone can be translocated to a third chip. In the alternative. I~~I~A and protein sample components can be directed to the same detection chip, For example. In preferred embodiments, 25 the transfer of different components to different chips or to different areas of a chip can be achieved through the coupling of different companents to microparticles with different properties, For example difFeren t dielectric properties. In this way, microparticles will respond differently to physical forces alaplied to the chip and will be directed in diFferent directions, Ior example. directing dihFeren t samlalc component 30 through different ports to enter diFFerent chambers, or by directing the microparticles to different areas of the same chip.

SG
A preferred chip for the differential translocation of sample components to differen t chips is the particle switch chip, disclosed in U.S. Patent Application United States Application Number C)9/G78,263 (attorney docket number ARTL1~1C'0.002A), entitled "Appal°atLis for Switching and Manipulating Particles and Methods ol~ Use Thei°eof ' filed on October l . ?000, herein incorporated by reference.
'>Ah~ particle switch chip translocates microparticles using traveling wave electrophoresis or conventional or traveling wave dielectrophoresis. Microparticles that respond to different field frequencies can be directed to different locations, and can be made to migrate clang different paths, using different electrical signals applied to the particle switches.
Operation ofczzz Izzlegrated l3ioclZip System In the methods of the present invention, at least two tasks axe performed sequentially. This means that at least one tasl. is performed on a sample component that is a product or result of an earlier task performed on a sample.
Preferably, tasks performed by the system occur in an order that allows progressive purification or enrichment, or in some cases alteration, of a sample component that can then be analyzed. In this respect, use of an integrated biochip system to process and analyze a sample leads from "sample to answer".
Although it is preferred that at least two of the tasks performed on a system of the present invention be performed sequentially, it is not a requirement of the present invention that all tasks be performed in a sequential order. for example, it can be preferred in some embodiments, for example to have certain analysis steps performed in parallel, where one analysis step is for detecting one type of sample component (for example, RNA), and another analysis task is far detecting anothei° type of~ sample component (for example, protein).
The operation of a svfstem can be exemlalitied by reference to the figures, which are provided far illustration, and not by way of limitation:
lFego ~ shows a chamber that compris~:s a multiforce chip used in the system of the present invention. DifFere;nt geometries ohthe DEP electrodes may be used, for example, spiral electrode arrays, as described in "Dielectrophoretic manipulation of cells using spiral electrodes by Wang et al., l3iophys. ,L, Vol. 72, pages:

( 1997)" may he used instead of the rectangular array shown in pig. 1 B. All of the functional elements (acoustic, DEP electrode. electromagnetic elements.
particle switch elements) spawn in I~'ig. 1 B-1 E require electrical connection to edcrnal signal sources. Far clarity, nave t>(~ the electric connections were shown. The details of these connections can be found in U.S. Patent Application Serial No.
09/399,299 (attorney docket number AI~T-0010~.P.1 ), filed September 17, 1999; United States Application Serial No. Number 09/685,110 (having attarney docket number ART-OOIO~.P.I .1), filed October l p, 2000, entitled Celndividually Addressable Micro-Electromagnetic Unit Array Chips in Horizontal Configurations"; United States Application Serial No. 09/678,263 (attorney docket number ARTLNC0.002A), entitled °'Apparatus for Switching and Manipulating Particles and Methods of Use Thereof' filed on October 3, 2000; and United States Application Serial No.
09/679,024 (having attorney docket number ~718~2000~00), entitled 'Apparatuses Containing Multiple Active horce Generating Elements and Uses Thereat'' filed October ~1, 2000, all herein incorporated by reUeren ce.
A sample, such as a blood sample, to which a preparation of microparticles coupled to specific binding members has been added, is introduced into the chip by pumping the sample through a port of a chamber (Fig. 2A and >g).
The chip comprises acoustic elements, and mixing of the sample is performed using acoustic forces ('fig 3). The acoustic forces are produced by energizing the acoustic elements within the acoustic layer using AC electric signals. Under the applied AC electrical signals, the acoustic elements exhibit mechanical vibration due to the piezoelectric effects. Such mechanical vibration at the same frequency as that of the applied electric signals is coupled into the chamber and produces an ~coListic wave or acoustic field within the chamber. The resulted acoustic field or wave exerts Forces on the culls and beads in the chamber and also exerts forces on the suspending medium in the chamber to result in an acoustic-field-induced mixing. Where paramagnetic micropartielcs comprising spe:cilic binding members are tnc:d is the system of the present invention, acoustic forces can increase the efficiencv° ol~
microparticle binding to specific components ol'the sample tFig. ~).

hollowing binding to specific components of a sample, the paramagnetic microparticles can be used in separation methodologies. I-Iere, the microparticles can be poi°amagnetic particles comprising antibodies specific for a specific cell type, and a multi-force chip used in the system of the present invention can comprise 5 electromagnetic units. The energized electromagnetic elements are used to collect and trap the magnetic bead-cell complexes, while other call types and sample components are washed out of the chamber (Fig. 5A and SB, and SC), I~or example, by mass flow of fluid pumped tlu-ough the chamber. The microparticles can then be dissociated from the moieties of interest (Fig. 6), for example by chemical cleavage of linkers, and in a further process, the moieties at interest can be dielectrophoretically separated from the microparticles (fig. 7A and 7B). The magnetic microparticles, having different dielectric properties from those of the target cells, can he:
I7ushed From the chamber, for example, by fluid flow, Dielectrophoretic retention can be achieved by application of an electric signal to an electrode array to produce a nonuniform electric field. The electric field pattern, the composition of the suspending medium, and the composition of the magnetic microparticles is such that moieties of interest are retained at electrode surfaces, and magnetic microparticles are not retained at electrode surfaces.
~ther solutions, suspensions, preparations, or reagents can be added to the chamber that contains dielectrophoretically retained moieties of interest. Car example.
a suspension of different types of microparticle is introduced to the chamber in F'ig. 8.
Each type of miccoparticle has a different specific binding member attached thereon, in which the different speciluc binding members can bind different components of the moiety of interest. Toe example, one type oi~ particle can be coupled to antibodies to a particular type of protein, another type of particle can be coupled to antibodies to a small molecule such as a steroid molecule, another type aF microparticlc can be coupled to an oligo dT nucleic acid that can bind the poly A tail of mRNAs, and another type of microparticlc can be coupled to a single-stranded DN~1 molecule that is complementary to a sequence that is known or suspected of being present in a moiety of interest, such as a cell of interest. 'IAhe moiety ol~ interest can be disrupfed to expose or contact components of the moiety oC interest to reagents or preparations, such as one or more preparations of microparticlcs. hoe example, a cell can be lysed to allaw internal moieties ol~a cell to be released into the medium and contact preparations ofmicropai°ticles coupled to specific binding members (Fib;. 9A and l~).
Lsysis of cells can occur, for example, by adding a hypotonic solution or a solution comprising a detergent or other lysing agents to the chamber. Mechanical forces (such as agitation), or electric or acoustic forces can aptionally be applied using functional elements on a chip to cause disruption of the cells. The application of acoustic forces can promote efficient mixing of the sample comprising components of the disrupted moieties (e.g., components of lysed cells) and the preparation of different types of microparticles (Fig. 10). This increases the el~iciency of binding of the components to 1 C) the microparticles ()Fig. ~A), I-Iere, mRNA derived from lysed target cells binds to Type 1 beads, a target protein derived from lysed target cells binds Type '~
beads, DNA derived from lysed target calls binds to Type 3 beads, and a target small molecule derived from lysed target cells binds °fype 4 beads.
In this example, the different types of microparticles (beads) exhibit positive dielectrophoresis in response to an applied electric field pattern (shown in L~ igs. 12A
and B), but this need not be the case. The microparticles of different types bound to different moieties of interest can be dielectrophoretically focused to the central regions on a multi-force chip by applying an electric field across a plurality of electrodes that are on one layer of the multiple force chip (fig. 12 B). In this case, phase-shifted signals can be applied to DEP electrodes in the chamber so that generated traveling-wave electric fields travel either towards the center or towards the periphery of the electrode array. To generate a traveling wave electric held, the electrodes are grouped such that each group receives the same phase of an AC
signal, and electrodes of each group are interspersed with electrodes of each of the other groups (receiving different phase signals). At bast three groups of electrodes are required with at last three different phase si~~nals applied to generate a traveling wave electric held. In one example, every Ii(th of the rectangular electrodes (counted from the innermost one) arc connected together to farm ~ groups of electrodes:
i.e., group 1: electrodes 1, 5, and 9; group ?: electrodes 2, 6, and 10; group 3: 3, 7, and 1 1;
and group ~: electrodes d, ~. and 12. The four groups of electrodes can be:
applied with AC signals of same Frequency bLlt phased at 0, 90, 180 and 270 de~~ra-rs, or 0, -90, -180 and-270 degrees. Multi-layer fabrication is required For making such electrode configurations. Alternatively, the spiral electrodes, described.
described in "Dielectrophoretic manipulation oi~ cells using spiral electrodes by Wang et al., Biophy.s. .L, Vol. 72, pages: 1887-1899 (1997)" may be used.
Microparticles that are retained in one car more areas oi' a chip can be separated 5 on a particle switch chip, described in United States Application Number 09/678,263 (attorney docket number AIZTLNC0.002A), entitled "Apparatus for Switching and Manipulating Particles and Methods of Use Thereof' fled on October 3, ?000, herein incorporated by reFerence. Microparticles, including microparticles coupled to moieties o~ interesfi, can be translocated on a particle switch chip using traveling wave 10 dielectrophoresis (F'ig. ~3 A, >I$, and C~. At the branch point, application oFa non-zmiform and traveling-wave field directs one type of microparticle in one direction, and another type o~F microparticle in another direction. 'plze movement oi~
different types o~Inicroparticles to diFl'erent directions in the particle switch may occur simultaneously under a given electrical signal application condition.
Alternatively, 15 certain signal combinations are applied first to move are type ("the first type") of~
microparticles in one direction in the particle switch while other types of microparticles remain stationary or essentially stationary. After "the f rst Type" o~
microparticles reaches the required position in the particle switch, different signal combinations are applied to move the other types of microparticles in ofher directions 20 in the particle switch. The microparticles can be directed through di~Ferent ports of a chamber comprising a particle switch chip to different chips tar further separation, analysis, or detection, or can be directed to diFf'erent areas of a chip for Further separation, analysis, or detection.
One method of detection uses electromagnetic signals generated by the 25 binding ova magnetic particle to a region of a chip that comprises an oligonucleotide array. In this aspect, depicted in F'ig. lib A, l~, and ~, a preparation oi~
magnetic micropartieles coupled to nucleic acid molecules is used. A given micraparticle is coupled to a species o~ nucleic acid molecule known to be or suspected of being present in a sample being tested. A set o~ such microparticles is allowed to hybridize 30 to nucleic acid molecules in a sample. Hybridization occurs such that the nucleic acid molecule i'rom the sample ihat is hybridized to the nucleic mid coupled to the microparticle has a single-stranded overhang that is capable of binding to an oligonuclcotide on the chip. I1'nbound nucleic acid molecules of the sample can be removed, for example, by v,rashing the chamber following electromagnetic capture of the magnetic microparticles. The magnetic microparticles that are bound to nucleic acid molecules of the sample can bind oligonucleotides on the array, thereby binding a magnetic microparticle to o particular location on the array. The presence of magnetic microparticles at that position can be detected on the chip by certain magnetic field sensors or by cantilever-type pressure detecfors, for example.
For example, the sensor technology described in "A biosensor based on magnetoresistance technology", in Biosens. Bioelectron. Vol: 13, pages 731-739, 1998, by Baselet et al, can be used to detect the presence of the magnetic particles.
Detection can also be by the binding aF tluarescen t molecules fo nucleic acids or proteins (Fig SSA-D). In this case, miaraparticles bound to moieties of interest can be translocated by conventional or traveling wave dielectrophoresis onto or across a chip that: comprises specific binding members such as, for example, single:-stranded nucleic acid molecules and antibodies. The moieties of interest baund to micraparticles (for example. proteins or interest or RNAs of interest) can be decoupled from the microparticles before or during dielectrophoretic translocation of the microparticles. The dissociated moieties of interest are then available to bind specific binding members attached to the chip. The chamber can optionally be flushed with a solution to remove any unbound moieties. A "sandwich" hybridization is then performed, with fluorescent molecules attached to molecules that are specific binding members specific for the moieties of interest. The fluorescent molecules will thus become attached to areas oC~the chip that correspond to particular- moieties of interesf.
and can be detected by any slandai°d fluorescence detectioj~ methods.
Detection can also be by means of generation of a fluorescence sf~~ual that occurs when moieties of interest flow through a chazmel or port. For example, small molecules such as, for example, steroids that Dave been sep~.irated from rather moieties and sample components dielcctrophoretically using microparticles can be translocated and focused in a channel ofn chip (16 A, 13). 'fhc microparticles can be:
clccoupled from the moiety of interest and the moiety of interest can be labeled, for example with 6'' a fluOreSGent label, alld dll"('.Cted throLlgh the Cllallllel, Ion' exalllple, by l~Llld l7ow ( 16 C, 1), and ~) and detected «si~lg optical light sources.
In fhe examples depicted in Figures 1~A-1~F, and 15A-F, traveling-wave dielectrophoresis (TW-DEP) electrodes are energized to move and disperse microparticles with bound molecules of interest into the chamber. In this case, traveling-wave dielectrophoretic forces are used. Phase-shifted signals cal be applied to the TW-DEP electrodes so that traveling-wave electric fields are praduced to exert traveling-wave dielectrophoretic forces to move and dispense the microparticles. To generate a traveling wave electric held, the electrodes are grouped such that eaoh I 0 group receives the same phase of an AG signal, and electrodes of each group are interspersed with electrodes of each of the other groups (receiving different phase signals). At least three groups of electrodes are required with at least three different phase signals applied to generate a traveling wave electric f end. In one e~anlple, every fourth of the semicircular electrodes (counted from the innermost one in Figure 14B and TSB) are connectecC together to form 3 groups of electrodes: i.e., ~~roup 1:
electrodes l, ~, and 7; group ?: electrodes 2, 5, and 8; group 3: 3, 6, and 9.
The three parallel line electrodes may also be connected into the above mentioned three groups of electrodes. The three groups of electrodes can be applied with AC signals of same frequency but phased at 0, 1 '?0 and 2~0 degrees, or 0, -I'?0, -2~Ip degrees.
Mufti-layer fabrication is 1°equired for nlalcing such electrode configul°afiions.
Figure L7 depicts a single chip integrated biochip system, in which the chip is part of a chamber, and the cover of the chamber has inlet ports for the aplalication of a sample and the addition of reagents, and outlet ports for the outflow of waste. Three separate areas of the chip are: used for sample processing (areas A and 13 ) and analysis (~), and each area of the chip has differEnt functional areas or layers.
Figure 18 depicts a single chip integrated biochip system, in which the multiple force chip is part ol-mulfiiple chanlbcrs_ Bind the cover of the challlbers has inlet ports for the application of a sample and the addition of reagents, and outlet ports for the autllow of waste. The chip comprises a particle switch that can tlireci sample conlpollents to different areas ofthe chip for further processing and anal°sis tasks.

111 aI7 eXe177plary LISC Ol'the 5111g1e C171p SySteI77 117 )Fl~Ture 118, a flllid 5a177p1e comprising target and non-target cells is introduced to chamber A. T17E target cells are separated from the non-target cells in chamber A, and alter removal ~~C~thE
nontarget cells by fluid flown, tl7e target cells are lysed to release their intracellular components. Two types of micropal-ticles are then introduced into chamber A:
one type ofmicroparticles that binds to InRNA molecules and another type c>f microparticles that bind to target protein molecules. The cell separation and cell disruption of target cells to obtain intracellular moieties performed in chamber A is similar to the methods illustrated in )Figures 1 -13.
Using the particle switch on tl7e chip, microparticles with bound mRNA
molecules are directed to chamber B 1 and n7icroparticles with bound target protein molecules are directed to chamber B2 (Figure 18). Thus, mRNA molecules and protein molecules are separated from od7ex intracellular components into two separate chambers. mR ~ ~A molecules and protein molecules on the microparticles are then labeled with fluorescent molecules introduced into chambers B 1 and B2 through the inlet and outlet ports connected to cl7alnber B l and B2. The fluorescent molecules are coupled to specific binding members that can bind to the n7RNA molecules and protein molecules on the mfcroparticles. The labeled mRNA molecuies and protein molecules are then de-coupled or dissociated from microparticle surfaces, and are then transported via fluid fi7aw to chambers C 1 and C2, respectively.
The top surface of chamber G1 has in7mobilized nucleic acid probes that can bind to target mRNA molecules, and hybridization can occur between the bound probes and target mRNA n7olecules under con trolled stringency conditions.
Similarly.
the top surface of chamber (.'2 has innnobilized antibody probes, and binding aftarget ?5 proteins to the bound antibodies can occur vender controlled stringency conditions.
The stringency control is provided by the components of the hybridization or binding buffers and wash buffers introduced into chambers Cl and C2 via the inlet and outlet parts connected to chambers Cl and C2, reslaectively. Tl)E intensity of the fluorescent signal emanating li-om the chip after washing off unbound label provides duan iitative information on the mRNA molecules and protein molecules from the tar~~el cells in tl7e original sample.

CXAMf P L>G
Use of an integp-~ted system for separastion of white blood cells from a Mood s~~nple and lE$10IA isolation.
MZCltiple F'or"ce C.'hi/~
A multiple force chip of dimensions 1 cm by 1 cm was constructed on a silicon substrate. The chip had two active layers, as shown in Figure 19A: an upper layer of interdigitated microelectrodes, and a lower layer of having a microfabricated electromagnetic coil. The microelectrodes are made of chromium (100 Angsti°om thick) as a seed layer and 0.? micron thick gold film as the top layer and have a 50 micron width and ~0 ixiicran gap. The electromagnetic units contained a magnetic core having dimensions 50 micron (width) by 200 micron (length) by 5 - 10 micron (thickness). (Detailed descriptions of fabrication procedures for malting these electromagnetic units on a chip is disclosed in United States Patent Application Serial Number 091685,410 filed October 10, 2000, entitled, "Individually Addressable Micra-Electromagnetic Unit Array Chips in Horizontal Configurations"".
incorporated by reference in its entirety. ) Dielectric insulation between the microelectrodes and the electromagnetic elements was achieved using deposited, thin, dielectric Films (e.g.
SiO~, S to 20 micron thick).
A chamber was constructed around the multiple force chip. In this case, a molded plastic rectangular enclosure (having four sides but no top or bottom) was glued onto the chip to make the chamber walls, The chamber walls had a thickness of about 600 microns. A piece of thin glass was then glued to the top edges oI~
the plastic enclosure to make a top for the chamber. 1-Ioles were molded on two opposite ~5 plastic walls ofthe chamber. and Teflon tubing of diameter Ill6 inch was glued to the plastic chamber walls at the holes, and used as the "inlet tubing'" and the ''outlet Cubing". Samples were introduced into the chamber via ono piece of tubing (the "inlet tubing") connected to one end of the chamber and removed from the chamber via the other piece of tubing (the ''t>utlet tubing"') cannected to the other end oi~
the chamber.
Dieleclroplmrelic a'c/acrralicm ~f~l'I'hile Bloucl ('ells' from cc l3loocl Scn~~/~lc~
Peripheral blood samples ol~about 10 microliters volume were diluted in a hypotonic sucrose solution f 4 2% sucrose in weight-to-weif~ht ratio) with a ratio of 1:19 of blood to hypotonic sucrose solution. A diluted blood sample ol=?00 microliters was then introduced to the chamber via a syringe pump with the syringe connecting to the inlet tubing. The chamber was pre-tilled with an isotonic sucrose buffer (8.5°~'° sucrose plus 0.3 °~'o dextrose) prior to the introduction of the blood S samples. During the sample: introduction, AC' electrical signals of up to ~
V peals-to-peak at frequencies between 1 - 6 MHz were applied to the electrodes using a power supply. Under these electric field conditions, white blood cells in the flow-introduced samples experienced positive dielectrophoretic Forces and were collected by the microelectrodes at the electrode edges despite continuous fluid flow through the i 0 chamber Figure ,~9~).
The flow rate through the chamber was adjusted to optimize white blood cell separation. High fluid flow rates through the chamber resulted in losses oFwhite blood ells, and different flow rates resulted in diffErent percentages of white blood cells being collected at the electrode edges. ~hhe flow rates used were between 0.5 15 mLlhour and 2 mLlhour. The introductian ol~ blood sample into the chamber and the collection of white blood cells at the electrode edges continued for several minutes (e.g. 5 minutes), while excess buffer and sample components that did not collect at the electrodes were removed by fluid flow through the outlet tubing, so that a sufficient number of white blood cells was collected on the chip by dielectrophoresis (shown in 20 )Figure 19C). figure 19C demonstrates the use of dieleatrophoresis an a multiforce chip for a processing task, i.e., separating /collecting white blood cells from a diluted blood sample.
After collecting white blood calls at the electrode surfaces, a lysis~binding solution was introduced into the chamber via the inlet tubing with the electrical 25 signals (e.g., I-h MI-Iz at ~ ~ V poak-to-peal:) applied on the microelectrodes ()Figure 19~). The lysislbinding solution (I00 mM Tris-I-LCI, pLI 7.5; 500 mM hiCl.
IOmM
EDTA; 1°J° LIDS and SmN1 dithiothreitol (D"I"T)), contained magnetic microbeads of 2.8 microns in diameter copied with Oligo (dT)~; (supplied by Dynal). A('ter a volume ofthe solution similar to the volume o(~ the chamber (about 30 microliters) 30 was intraduced_ the fluid flow was stopped. 'l9hc sample, now a cell lysnte_ v=as allowed to incubate; with the lysislbinding solutian that contained magnetic beads for - 10 minutes to allow released mRNAs from lysed white blood cells to hybridize to Oligo (dT)~~ on the surfaces of the magnetic beads.
Eleclr'onaagnelic ~'uplzare,for I,~'olcrlioh of~rrrIZN~I
DC electrical current was applied to electromagnetic units on the lower layer of the multiple force chip so that each unit was energized with a current value of 100 - 200 mA. The applied DC current to the electromagnetic units praduces a non-uniform magnetic field distribution around these electromagnetic units, and as a result, the magnetic beads collect at the strongest field region corresponding to tile two poles at the ends of the major axis of the electromagnetic coil (>Figure >i 9>B).
After the magnetic beads were collected with applied DC current for 1 - s minutes, a flow of washing buffer A ( I 0 mM Tris-HC 1, pI-I 7.5; 0.17 M LiCI, 1 mM LDTA, 0.1 °~'o LiDS) was applied into the chamber to wash off unbound molecules such as DNA, proteins, and other biomolecules that exited via the outlet tubing.
After pumping washing buffer A through the chamber to remove molecules such as DNA, proteins and other malecules that were not bound to the magnetic beads, a flow of washing bul~ter B (10 mM 'hris-I-ICI, p1~ 7.5; 0.17 M LiCI. 1 mM
EDTA) was used to wash the bound beads. The volume of washing buffer A and B
pumped through the chamber was 30 to 100 microliters at flow rates below 3 mLlhour. At these flow rates, magnetic beads remained on the two ends of the electromagnetic elements/coils.
After the flow was stopped, the electric ctn°rents that wEre applied to electromagnetic elements were turned off so that the magnetic buds were no longer subjected to a strong attractive magnetic i~icld to immobilize them on the poles of the 2S electromagnetic units. A buffer was pumped into the chamber through the inlet tubing and magnetic beads were removed from the chamber via the outlet tubing and collected into a microfuge tube.
PCR ~1,~'s'~r~.~ of 'h~'ulcrfccl rnR~:1 Collected magnetic heads were then ~ulojected to au ohf-chip rE~~Lrse-transeription reaction to generate cDNA molecules, The cDNAs were liirihcr simplified in a I'C12 reaction using a pair ol~l-~rimers hybridizing to houscl:eeping gen a G3PDH. The PCR mixture contained 0.2 ).~M primer, 1.5 mM MgCI~, t).? mM dNTP, mM Tris-I-IC1 (pH=8.3). ~0 mM KCl and t),t)01°lo gelatin, and the PCIZ
was performed at temperature cycles of 9~1 "C (30 rocs) followed by 60 "C (60 secs) followed by 72 "C (60 secs). A total of 30 cycles were used. The reactions were loaded on an agarose gel, and amplified G3PDI-I products were detected after electrophoresis and ethidium bromide staining ai~ the gel (Figure ~9)~).
The strongly stained band corresponding to the size of amplified G3PDH gene segment in the right lane o~ the gel demonstrated that the magnetic beads captured anRNA molecules corresponding to the G3PDH Bones. The negative control loaded 10 in the middle lane of the gel shows the PCR results when magnetic beads introduced into the chamber did not have coated oligo-(dT)25 molecules Figure 1)F), or magnetic beads introduced into the chamber that was not pre-used far separating white blood cells from blood samples.
r All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.
All publications, including patent documents and scientific articles, referred to in this application and the bibliography and attachments are incorporated by reference in their entirety foi° all purposes to the same extent as if each individual publication were individually incorporated by reference.

9$I33I~I1~GI~APH~' United States Provisional Application Number 601239,299 (attorney docizet number ART-001 OS.P.I) tiled October 10, 2000, entitled "An Integrated Biochip System for Sample Preparation and Analysis" naming Cheng, et al. as inventors.
United States Patent Application Serial No. ()9/399,299 (attorney docket number ART-OOIO~.P.I), tiled September l7, 1999, entitled "Individually Addressable Micro-Electromagnetic Unit Array Chips".
United States Application Serial No. 091685.110 (having attorney docket number ART-0010~.P.1.1 ), fled October 10, 2000, entitled "Individually Addressable Micro-Clectromagnetic Unit Array Chips in Horizontal Configm°ations".
United States Application serial No. 09/678,263 (attorney docket number ARTUNC.O.002A), filed on October 3, 2000, entitled "Apparatus for Switching and Manipulating Particles and Methods of Use 'I~l~ereof'.
United States Application Serial No. 091679,02 (having attorney docket number I 5 ~171842000~100), " Filed October ~1, 2000, entitled "Apparatuses Containing Multiple Active Force Generating Elc~:ments and Uses "IAhereol".
United States Patent Application Serial No. 09186,737 (attorney docket number ART-00102.P.1), Filed October 10, 2000, entitled "Compositions and Mcahods rar Separation of Moieties on Chips".
United States Patent Application Serial No. ()9/636, I 0~1 (attorney docket number 4718-2000100), tiled on August 10, 2000, ~:ntitled "Methods for Manipulating Moieties in Microtluidic Systems".
United States Patent Serial Applicatian No. t)~)I6~18,081 (attorney docket number ART'-00101.P_ 1 ), filed oia ~\ugust 25, 2000, entitled ''Methods and Compositions for Identifying Nucleic Acid Molecules Using l~lucleolytic Activities and I-Ivbridization'".
U.S. Patent No. X1.1 GO,G~S

U.S. Patent No. 4,275,149 U.S. Patent No. 4,318,980 U'.S. Patent No. 6,029,518 Baselet et al. (1998) "A Biosensor Based on Magnetoresistance Technology" , Biosens. Bioelectron. Vol. 13, pages 731-739.
Cheng et al. (1998) "Preparation and Hybridization Analysis of DNAIR~~A From E.
coli on Microfabricated Bioelectronic Ghips'~ Nature Biotechnology, Vol. 16, pages 541-546.
Edman, C. (1998) "Electric llield Directed Nucleic Acid Hybridization on Microchips", Nz~cl. ~lcids~ Rc~.s.. 25: pages 491)7-4914.
Effenl~ausor, C.S. et al. {19~~~1) "I-Iigh-speed separation of antisense oligonucleotides on a micromachined capillary electrophoresis device", ~Ij~crl. Ch~f~i. Volume 66, pages 2949-2953.
I S Hawison, D..1. et al. (1993) "Micromachining a miniaturized capillary electrophoresis based chemical analysis system on a chip", ~S'4~lr~l~Ce, Volume 261, pages:
895-896.
Sosnowski, R., et al. (1997) "Rapid Determination of Single Base Mismatch Mutations in DNA Hybrids by Direct Electric Meld Control", Pros. Null. tlcarl.
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Volume 94, pages i I 19-I 1?3.
Wang et al. (1997) "Dielectrophoretic Manipulation of Particles by in IL;LC
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Claims (42)

We claim:

1. An integrated biochip system for sample preparation and analysis, comprising at least one chip, wherein said integrated biochip system can perform two or more sequential tasks, wherein at least one of said two or more sequential tasks is a processing task.

2. The integrated biochip system of claim 1, comprising at least one chamber.

3. The integrated biochip system of claim 1, wherein said at least one chip is an active Chip.

4. The integrated biochip system of claim 3, wherein one or more sample components can be moved from at least one area of a chip to at least one other area of a chip is by a mechanism other than fluid flow electrophoresis, or electro-osmosis.

5. The integrated biochip system of claim 4, wherein sample components can be moved from at least one area of a chip to at least one other area of a chip by traveling wave dielectrophoresis or traveling wave magnetophoresis.

6. The integrated biochip system of claim 3, wherein a sample applied to said integrated biochip system can remain continuously within said integrated system from the beginning of the first of said two or more sequential tasks until the end of the last of said two or more sequential tasks performed by said integrated system.

7. The integrated biochip system of claim 6, wherein said integrated biochip system is automated.

8. The integrated biochip system of claim s, wherein said at least one chip is a multiple force chip.

9. The integrated biochip system of claim 6, comprising two or more chips, wherein said integrated biochip system can perform two or mare sequential tasks using at least W a of said two or more chips, further wherein at least one of said two or more sequential tasks is a processing task.

10. The integrated biochfp system of claim 9, comprising at least one chamber.

11. The integrated biochip system of claim 9, wherein at least two of said two or more chips are active chips.

12. The integrated biochip system of claim 11, wherein at least one of said active chips is a particle switch chip.

13. The integrated biochip system of claim 9, wherein one or more sample components can be moved from at least one area of a chip to at least one other area of a chip is by a mechanism other than fluid flow, electrophoresis, or electro-osmosis.

14. The integrated biochip system of claim 13, wherein sample components can be moved from at least one area of a chip to at least one other area of a chip by traveling wave dielectrophoresis or traveling wave magnetophoresis.

15. The integrated biochip system of claim 9, wherein at least one of said active chips is a multiple force chip.

16. The integrated biochip system of claim 9, wherein said at least two of said two or more chips can be for at least a part of the time during the operation of the integrated biochip system, in fluid communication with one another.

17. The integrated biochip system of claim 16, wherein one or more sample components can be moved from at least one chip to at least one other chip is by a mechanism other than fluid flow, electrophoresis, or electro-osmosis.

18. The integrated biochip system of claim 17, wherein sample components can be moved from at least one chip to at least one other chip by traveling wave dielectrophoresis or traveling wave magnetophoresis.

19. A method of using an integrated biochip system of claim 5, comprising:

a) applying a sample to an integrated biochip system; and b) performing two or more sequential tasks in said integrated biochip system, wherein at least one of said two or more sequential tasks is a processing task.

20. The method of claim 19, wherein said sample is a water sample, a blood sample, ascites fluid, pleural fluid, cerebrospinal fluid, or amniotic fluid.

21. The method of claim 19, wherein said at least one processing task is a separation, translocation, concentration, purification, isolation, enrichment, focusing, structural alteration, or disruption.

22. The method of claim 19, wherein at least one processing task is performed using the application of one or more physical forces that are in part generated by micro-scale strictures integral to a chip.

23. The method of claim 22, wherein said applied physical forces are acoustic forces, dielectrophoretic forces, magnetic forces, traveling wave dielectrophoretic forces, or traveling wave magnetophoretic forces.

24. The method of claim 22, wherein said at least one processing task comprises the manipulation of moieties by applied physical forces.

25. The method of claim 24, wherein said applied physical forces are dielectrophoretic forces, magnetic forces, traveling wave dielectrophoretic forces, or traveling wave magnetophoretic forces.

26. The method of claim 25, wherein said manipulation of moieties by applied physical forces is by manipulation of binding partners.

27. The method of claim 26, wherein said binding partners are magnetic beads.

28. The method of claim 22, wherein at least one processing task is per formed by the application of more than one type of physical force.

29. The method of claim 19, further comprising performing an analysis task.

30. A method of using an integrated bichip system of claim 9, comprising:

a) applying a sample into au integrated biochip system: and b) performing two or more seduential tasks in said integrated biochip system, wherein at least one of said two or more tasks is a pracessing task.

31. The method of claim 3p, wherein said sample is a water sample, a blood sample, ascites fluid, pleural fluid, cerebrospinal fluid, or amniotic fluid.

32. The method of claim 30, wherein said processing task is a separation, translocation, concentration, purification, isolation, enrichment, focusing, structural alteration. or disruption.

33. The method of claim 32, wherein at least two pracessing tasks arc performed using the application of physical forces that are in part generated by micro-scale structures integral to a chip.

34. The method of claim 32, wherein said applied physical forces are acoustic farces, dielectrophorotic forces, magnolic forces, traveling wave dielectropharetic forces, or traveling wave magnetophoretic forces.

35. The method of claim 34, wherein said at least ane processing task is accomplished througt the manipulation of moieties by applied physical forces.

36. The method of claim 35, wherein said applied physical forces are diolectrophoretic forces, magnetic forces, traveling wave dielocirophoretic Forces, or traveling wave magnetophorctic forces.

37. The method of claim 36, wherein said manipulation of moieties by applied physical forces is by manipulation of binding partners.

38. The method of claim 37, wherein said binding partners are magnetic beads.

39. The method of claim 33, wherein at least one processing task is performed by the application of more than one type of physical force.

40. The method of claim 30, wherein sample components can be moved from at least one chip to at least one other chip by a mechanism other than fluid flow, electrophoresis, or electro-osmosis.

41. The method of claim 40, wherein sample components can be moved from at least one chip to at least one other chip is by traveling wave dielectrophoresis or traveling wave magnetophoresis.

42. The method of claim 30, further comprising performing an analysis task.