System including functionally separated regions in electrophoretic system

Methods, apparatus, and applications for use of a stacked, reconfigurable system for electrophoretic transport are provided. In one embodiment, a system having a first chamber including at least a bottom support and an intermediate support, and a second chamber, said second chamber including a bottom support and a top member, the first and second chambers being coupled through a via. Electrophoretic, and optional electro-osmotic and thermal, transport is effected. In another aspect of this invention, three or more chambers are coupled by an electrophoretic buss. The electrophoretic buss includes driving electrodes and is adapted to receive fluid containing materials for transport. The chambers are coupled to the electrophoretic buss and serve as a tap from the buss for delivery of charged materials. In one embodiment, certain functions are performed in different chambers. For example, the first chamber may receive the sample and perform sample processing functions, the second chamber may perform amplification procedures, yet a third chamber may perform hybridization or other assays, and yet another chamber may perform immunoassays. By separating various functions to different chambers, speed and sensitivity may be improved. In yet another aspect of this invention, analysis from a earlier stage may be utilized in a subsequent stage to reconfigure the system for optimum use. In one application, analysis at a first level is utilized to determine an action at a second level, such as the synthesis of a compound. The synthesized compound in response to a biohazard may comprise vaccine or antidote.

FIELD OF THE INVENTION
 This invention relates generally to electronic devices for the movement of
 charged materials, especially charged biological materials. More
 particularly, it relates to microfluidic systems for the transport and/or
 analysis of electrically charged materials, especially biological
 materials including nucleic acids and biological pathogens or toxins.
 This application is related to the following applications filed on even
 date herewith, entitled "Stacked, Reconfigurable System for
 Electrophoretic Transport of Charged Materials", "Electrophoretic Buss for
 Transport of Charged Materials in a Multi-Chamber System", and "Apparatus
 and Method for Real-Time Configuration and Analysis in Detection System".
 BACKGROUND OF THE INVENTION
 Molecular biology comprises a wide variety of techniques for the analysis
 of nucleic acid and protein. Many of these techniques and procedures form
 the basis of clinical diagnostic assays and tests. These techniques
 include nucleic acid hybridization analysis, restriction enzyme analysis,
 genetic sequence analysis, and the separation and purification of nucleic
 acids and proteins (See, e.g., J. Sambrook, E. F. Fritsch, and T.
 Maniatis, Molecular Cloning: A Laboratory Manual, 2 Ed., Cold spring
 Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).
 Most of these techniques involve carrying out numerous operations (e.g.,
 pipetting, centrifugations, electrophoresis)on a large number of samples.
 They are often complex and time consuming, and generally require a high
 degree of accuracy. Many a technique is limited in its application by a
 lack of sensitivity, specificity, or reproducibility. For example, these
 problems have limited many diagnostic applications of nucleic acid
 hybridization analysis.
 The complete process for carrying out a DNA hybridization analysis for a
 genetic or infectious disease is very involved. Broadly speaking, the
 complete process may be divided into a number of steps and substeps. In
 the case of genetic disease diagnosis, the first step involves obtaining
 the sample (blood or tissue). Depending on the type of sample, various
 pre-treatments would be carried out. The second step involves disrupting
 or lysing the cells, which then release the crude DNA material along with
 other cellular constituents. Generally, several sub-steps are necessary to
 remove cell debris and to purify further the crude DNA. At this point
 several options exist for further processing and analysis. One option
 involves denaturing the purified sample DNA and carrying out a direct
 hybridization analysis in one of many formats (dot blot, microbead,
 microplate, etc.). A second option, called Southern blot hybridization,
 involves cleaving the DNA with restriction enzymes, separating the DNA
 fragments on an electrophoretic gel, blotting to a membrane filter, and
 then hybridizing the blot with specific DNA probe sequences. This
 procedure effectively reduces the complexity of the genomic DNA sample,
 and thereby helps to improve the hybridization specificity and
 sensitivity. Unfortunately, this procedure is long and arduous. A third
 option is to carry out the polymerase chain reaction (PCR) or other
 amplification procedure. The PCR procedure amplifies (increases) the
 number of target DNA sequences relative to non-target sequences.
 Amplification of target DNA helps to overcome problems related to
 complexity and sensitivity in genomic DNA analysis. All these procedures
 are time consuming, relatively complicated, and add significantly to the
 cost of a diagnostic test. After these sample preparation and DNA
 processing steps, the actual hybridization reaction is performed. Finally,
 detection and data analysis convert the hybridization event into an
 analytical result.
 The steps of sample preparation and processing have typically been
 performed separate and apart from the other main steps of hybridization
 and detection and analysis. Indeed, the various substeps comprising sample
 preparation and DNA processing have often been performed as a discrete
 operation separate and apart from the other substeps. Considering these
 substeps in more detail, samples have been obtained through any number of
 means, such as obtaining of full blood, tissue, or other biological fluid
 samples. In the case of blood, the sample is processed to remove red blood
 cells and retain the desired nucleated (white) cells. This process is
 usually carried out by density gradient centrifugation. Cell disruption or
 lysis is then carried out on the nucleated cells to release DNA,
 preferably by the technique of sonication, freeze/thawing, or by addition
 of lysing reagents. Crude DNA is then separated from the cellular debris
 by a centrifugation step. Prior to hybridization, double-stranded DNA is
 denatured into single-stranded form. Denaturation of the double-stranded
 DNA has generally been performed by the techniques involving heating
 (&gt;Tm), changing salt concentration, addition of base (NaOH), or
 denaturing reagents (urea, formamide, etc.). Workers have suggested
 denaturing DNA into its single-stranded form in an electrochemical cell.
 The theory is stated to be that there is electron transfer to the DNA at
 the interface of an electrode, which effectively weakens the
 double-stranded structure and results in separation of the strands. See,
 generally, Stanley, "DNA Denaturation by an Electric Potential", U.K.
 patent application 2,247,889 published Mar. 18, 1992.
 Nucleic acid hybridization analysis generally involves the detection of a
 very small number of specific target nucleic acids (DNA or RNA) with an
 excess of probe DNA, among a relatively large amount of complex non-target
 nucleic acids. The substeps of DNA complexity reduction in sample
 preparation have been utilized to help detect low copy numbers (i.e.
 10,000 to 100,000) of nucleic acid targets. DNA complexity is overcome to
 some degree by amplification of target nucleic acid sequences using
 polymerase chain reaction (PCR). (See, M. A. Innis et al, PCR Protocols: A
 Guide to Methods and Applications, Academic Press, 1990). While
 amplification results in an enormous number of target nucleic acid
 sequences that improves the subsequent direct probe hybridization step,
 amplification involves lengthy and cumbersome procedures that typically
 must be performed on a stand alone basis relative to the other substeps.
 Substantially complicated and relatively large equipment is required to
 perform the amplification step.
 The actual hybridization reaction represents the most important and central
 step in the whole process. The hybridization step involves placing the
 prepared DNA sample in contact with a specific reporter probe, at a set of
 optimal conditions for hybridization to occur to the target DNA sequence.
 Hybridization may be performed in any one of a number of formats. For
 example, multiple sample nucleic acid hybridization analysis has been
 conducted on a variety of filter and solid support formats (See G. A.
 Beltz et al., in Methods in Enzymology, Vol. 100, Part B, R. Wu, L.
 Grossman, K. Moldave, Eds., Academic Press, New York, Chapter 19, pp.
 266-308, 1985). One format, the so-called "dot blot" hybridization,
 involves the non-covalent attachment of target DNAs to filter, which are
 subsequently hybridized with a radioisotope labeled probe(s). "Dot blot"
 hybridization gained wide-spread use, and many versions were developed
 (see M. L. M. Anderson and B. D. Young, in Nucleic Acid Hybridization--A
 Practical Approach, B. D. Hames and S. J. Higgins, Eds., IRL Press,
 Washington, D.C. Chapter 4, pp. 73-111, 1985). It has been developed for
 multiple analysis of genomic mutations (D. Nanibhushan and D. Rabin, in
 EPA 0228075, Jul. 8, 1987) and for the detection of overlapping clones and
 the construction of genomic maps (G. A. Evans, in U.S. Pat. No. 5,219,726,
 Jun. 15, 1993).
 New techniques are being developed for carrying out multiple sample nucleic
 acid hybridization analysis on micro-formatted multiplex or matrix devices
 (e.g., DNA chips) (see M. Barinaga, 253 Science, pp. 1489, 1991; W. Bains,
 10 Bio/Technology, pp. 757-758, 1992). These methods usually attach
 specific DNA sequences to very small specific areas of a solid support,
 such as micro-wells of a DNA chip. These hybridization formats are
 micro-scale versions of the conventional "dot blot" and "sandwich"
 hybridization systems.
 The micro-formatted hybridization can be used to carry out "sequencing by
 hybridization" (SBH) (see M. Barinaga, 253 Science, pp. 1489, 1991; W.
 Bains, 10 Bio/Technology, pp. 757-758, 1992). SBH makes use of all
 possible n-nucleotide oligomers (n-mers) to identify n-mers in an unknown
 DNA sample, which are subsequently aligned by algorithm analysis to
 produce the DNA sequence (R. Drmanac and R. Crkvenjakov, Yugoslav Patent
 Application #570/87, 1987; R. Drmanac et al., 4 Genomics, 114, 1989;
 Strezoska et al., 88 Proc. Natl. Acad. Sci. USA 10089, 1992; and R.
 Drmanac and R. B. Crkvenjakov, U.S. Pat. No. 5,202,231, Apr. 13, 1993).
 There are two formats for carrying out SBH. The first format involves
 creating an array of all possible n-mers on a support, which is then
 hybridized with the target sequence. The second format involves attaching
 the target sequence to a support, which is sequentially probed with all
 possible n-mers. Both formats have the fundamental problems of direct
 probe hybridizations and additional difficulties related to multiplex
 hybridizations.
 Southern, United Kingdom Patent Application GB 8810400, 1988; E. M.
 Southern et al., 13 Genomics 1008, 1992, proposed using the first format
 to analyze or sequence DNA. Southern identified a known single point
 mutation using PCR amplified genomic DNA. Southern also described a method
 for synthesizing an array of oligonucleotides on a solid support for SBH.
 However, Southern did not address how to achieve optimal stringency
 condition for each oligonucleotide on an array.
 Concurrently, Drmanac et al., 260 Science 1649-1652, 1993, used the second
 format to sequence several short (116 bp) DNA sequences. Target DNAs were
 attached to membrane supports ("dot blot" format). Each filter was
 sequentially hybridized with 272 labeled 10-mer and 11-mer
 oligonucleotides. A wide range of stringency condition was used to achieve
 specific hybridization for each n-mer probe; washing times varied from 5
 minutes to overnight, and temperatures from 0.degree. C. to 16.degree. C.
 Most probes required 3 hours of washing at 16.degree. C. The filters had
 to be exposed for 2 to 18 hours in order to detect hybridization signals.
 The overall false positive hybridization rate was 5% in spite of the
 simple target sequences, the reduced set of oligomer probes, and the use
 of the most stringent conditions available.
 A variety of methods exist for detection and analysis of the hybridization
 events. Depending on the reporter group (fluorophore, enzyme,
 radioisotope, etc.) used to label the DNA probe, detection and analysis
 are carried out fluorimetrically, calorimetrically, or by autoradiography.
 By observing and measuring emitted radiation, such as fluorescent
 radiation or particle emission, information may be obtained about the
 hybridization events. Even when detection methods have very high intrinsic
 sensitivity, detection of hybridization events is difficult because of the
 background presence of non-specifically bound materials. A number of other
 factors also reduce the sensitivity and selectivity of DNA hybridization
 assays.
 In conventional fluorimetric detection systems, an excitation energy of one
 wavelength is delivered to the region of interest and energy of a
 different wavelength is remitted and detected. Large scale systems,
 generally those having a region of interest of two millimeters or greater,
 have been manufactured in which the quality of the overall system is not
 inherently limited by the size requirements of the optical elements or the
 ability to place them in optical proximity to the region of interest.
 However, with small geometries, such as those below 2 millimeters, and
 especially those on the order of 500 microns or less in size of the region
 of interest, the conventional approaches to fluorimeter design have proved
 inadequate. Generally, the excitation and emission optical elements must
 be placed close to the region of interest. Preferably, a focused spot size
 is relatively small, often requiring sophisticated optical designs.
 Further, because it is usually desirable to maximize the detectable area,
 the size of the optical components required to achieve these goals in
 relation to their distance from the region of interest becomes important,
 and in many cases, compromises the performance obtained. Accordingly, a
 need exists for an improved fluorescent detection system.
 Attempts have been made to combine certain processing steps or substeps
 together. For example, various microrobotic systems have been proposed for
 preparing arrays of DNA probe on a support material. For example, Beattie
 et al., in The 1992 San Diego Conference: Genetic Recognition, November,
 1992, used a microrobotic system to deposit micro-droplets containing
 specific DNA sequences into individual microfabricated sample wells on a
 glass substrate.
 Various workers have addressed fluid handling in microfluidic and mesoscale
 devices. A subclass of those efforts involve electronic and/or magnetic
 forces to aid in the movement of charged materials. For example, Pace U.S.
 Pat. No. 4,908,112 discloses a generally channel shaped structures
 containing a plurality of electrodes. Substrates such as silicon are
 suggested, and an optional covering is suggested for containment. Soane et
 al. U.S. Pat. No. 5,126,022 discloses a tube like system having a
 plurality of electrodes by which electrical or magnetic (via current
 application) fields are generated. Chow et al. (Caliper Technologies) U.S.
 Pat. No. 5,800,690 discloses a system having a number of fluidic pathways.
 Finally, Wilding et al. U.S. Pat. Nos. 5,304,487 and 5,587,128 describe
 various channel based systems for mesoscale devices including flow
 channels, reservoirs and mixing areas.
 These and other systems having suffered from various limitations or
 deficiencies. Generally, the prior devices have been limited in their
 ability to provide easy fabrication in the z-direction (i.e.,
 perpendicular to the plane of the device). Most microfluidic systems are
 difficult to scale in the z-direction due to the requirements for fluidic
 structures such as channels and vias which do not lend themselves to
 integration in the vertical direction. Generally, the photolithographic
 and etching techniques used is microengineering are best suited to create
 essentially planar structures. Yet a further limitation on such systems is
 the fact that fixed fluidic structures impose limitations on flexibility
 and functionality.
 Generally, the prior art processes have been extremely labor and time
 intensive. For example, the PCR amplification process is time consuming
 and adds cost to the diagnostic assay. Multiple steps requiring human
 intervention either during the process or between processes is suboptimal
 in that there is a possibility of contamination and operator error.
 Further, the use of multiple machines or complicated robotic systems for
 performing the individual processes is often prohibitive except for the
 largest laboratories, both in terms of the expense and physical space
 requirements.
 As is apparent from the preceding discussion, numerous attempts have been
 made to provide effective techniques to conduct multi-step, multiplex
 molecular biological reactions. However, for the reasons stated above,
 these techniques are "piece-meal" and limited. These various approaches
 are not easily combined to form a system which can carry out a complete
 DNA diagnostic assay. Despite the long-recognized need for such a system,
 no satisfactory solution has been proposed previously.
 SUMMARY OF THE INVENTION
 Apparatus, methods and modes of operation for a stacked, reconfigurable
 electronic system for the electrophoretic transport of materials is
 provided. In one embodiment, a multiple chamber, reconfigurable system is
 provided. In one implementation, the system includes a first chamber
 having at least a bottom support and an intermediate support, and a second
 chamber, said second chamber including a bottom support and a top member,
 the first and second chambers being coupled through a via. Transport
 between the first chamber and second chamber may be unidirectional or
 bidirectional. Various modes of transport may be utilized in conjunction
 with the electrophoretic transport, such as electrosmotic transport and/or
 thermal transport. A plurality of individually controllable electrodes are
 provided within the chambers to permit reconfiguration of the system. A
 control system is provided for control of said electrodes.
 The vias may be controlled by an associated electrode. Preferably, the
 electrode is formed adjacent, for example, circumferentially surrounding
 the via. Optionally, an electrode may be disposed within the chamber on a
 wall opposite from the via, so as to receive a signal generating a
 repulsive force to the charged materials of interest thereby providing an
 electrophoretic motion towards the via. The combination of electronic
 attraction to the via, coupled with electronic repulsion away from the
 wall opposite the via results in enhanced electrophoretic flow.
 The stacked, reconfigurable system is preferably formed from planar,
 sheet-like materials. For example, the first chamber may be formed from a
 relatively thin bottom layer and intermediate layer, such as 1 mil
 Kapton.TM., while being separated by a spacer having a relatively thicker
 dimension, e.g., 5 mils. Preferably, the spacer is die-cut so as to form a
 chamber then formed by the coaction of the bottom layer, intermediate
 layer and edges of the spacer. Preferably, the spacer is at least five
 times thicker than the intermediate or bottom layer.
 The chambers may include various materials within them. For example, one or
 more collection electrodes may be disposed within the chambers, optionally
 near a tap location. Affinity or other filter materials may be included
 within the chambers. Optionally, a permeation layer may be disposed
 adjacent any electrode, or within a via, to reduce the damage to
 biological materials from contact with the electrode.
 In yet another aspect of this invention, three or more chambers may be
 coupled via an electrophoretic buss. The electrophoretic buss comprises a
 chamber region which spans more than two chambers. Driving electrodes are
 disposed at substantially opposite ends of the electrophoretic buss.
 Optionally, an input may be coupled to the electrophoretic buss, which if
 present, permits use of an electrode through region within the electrode
 adjacent the input. The electrophoretic buss utilizes the free space
 nature of the electrophoretic transport, to enhance transport and permit
 the tapping or selecting removal of materials from the electrophoretic
 buss. Preferably, collection electrodes are disposed adjacent the
 periphery of the electrophoretic buss, aiding in the tapping or otherwise
 removing of material flowing through the electrophoretic buss.
 In yet another aspect of this invention, various functions are performed in
 different chambers, such as at different levels. By segregation of various
 functions, typically biological processing or analysis functions,
 processes may be optimized for those functions, resulting in a more
 focused, sensitive and specific system. In the preferred embodiment, a
 first chamber is adapted for sample preparation of biological materials. A
 second chamber is adapted for sorting of the biological materials, which
 are obtained at least in part from the first chamber. A third chamber is
 adapted for analysis of the biological materials, which are obtained at
 least in part from the second chamber. The first, second and third
 chambers are in fluidic coupling with each other through vias, or by a
 electrophoretic buss. Optionally, the system includes a chamber adapted
 for amplification of the biological materials. Optionally, the sample
 preparation chamber may be disposed central to the device, whereby charged
 materials of a first state are moved in one direction, and those charged
 materials of an opposite state are moved in an opposite direction.
 Additional chambers for the processing of those respective materials are
 then disposed adjacent the sample preparation chamber on the respective
 sides.
 In yet another aspect of this invention, a system is provided for
 performing analysis on a pathogen wherein the pathogen is analyzed in a
 first chamber to determine at least certain information regarding the
 pathogen, and then transferred to a second chamber, wherein the second
 chamber is electrically reconfigurable to permit action with respect to a
 plurality of pathogens, the reconfigurable system being configured at
 least in part upon the analysis conducted at the first level. By way of
 example, when analyzing for a biological pathogen, the first level may
 perform an initial determination broadly as to the type of pathogen, and
 that information then is used in the configuration of the second chamber
 for more specific analysis or counteraction with respect to the pathogen.
 In one embodiment, the response to the pathogen includes a chamber wherein
 a compound may be synthesized, such as a vaccine or an antidote to the
 pathogen. In one implementation, that synthesized material is then
 provided in an injectable structure. Optionally, an air handling system is
 utilized in conjunction with the pathogen analysis system.
 It is therefore an object of this invention to provide for an improved
 integrated, reconfigurable, multifunctional system.
 It is yet a further object of this invention to provide a sensitive,
 adaptable low-cost diagnostic system.
 It is yet a further object of this invention to provide a system having an
 improved mode of fluidic communication within a multichamber device.
 It is yet a further object of this invention to provide a system having
 improved sensitivity and specificity.
 It is yet a further object of this invention to provide systems having
 dynamic reconfigurable components for the analysis of materials.

DETAILED DESCRIPTION
 FIGS. 1A and 1B illustrate a simplified version of the active programmable
 electronic matrix hybridization system for use with this invention.
 Generally, a substrate 10 supports a matrix or array of electronically
 addressable microlocations 12. For ease of explanation, the various
 microlocations in FIG. 1A have been labeled 12A, 12B, 12C and 12D. A
 permeation layer 14 is disposed above the individual electrodes 12. The
 permeation layer permits transport of relatively small charged entities
 through it, but limits the mobility of large charged entities, such as
 DNA, to keep the large charged entities from easily contacting the
 electrodes 12 directly during the duration of the test. The permeation
 layer 14 reduces the electrochemical degradation which would occur in the
 DNA by direct contact with the electrodes 12, possibility due, in part, to
 extreme pH resulting from the electrolytic reaction. It further serves to
 minimize the strong, non-specific adsorption of DNA to electrodes.
 Attachment regions 16 are disposed upon the permeation layer 14 and
 provide for specific binding sites for target materials. The attachment
 regions 16 have been labeled 16A, 16B, 16C and 16D to correspond with the
 identification of the electrodes 12A-D, respectively.
 In operation, reservoir 18 comprises that space above the attachment
 regions 16 that contains the desired, as well as undesired, materials for
 detection, analysis or use. Charged entities 20, such as charged DNA are
 located within the reservoir 18. In one aspect of this invention, the
 active, programmable, matrix system comprises a method for transporting
 the charged material 20 to any of the specific microlocations 12. When
 activated, a microlocation 12 generates the free field electrophoretic
 transport of any charged finctionalized specific binding entity 20 towards
 the electrode 12. For example, if the electrode 12A were made positive and
 the electrode 12D negative, electrophoretic lines of force 22 would run
 between the electrodes 12A and 12D. The lines of electrophoretic force 22
 cause transport of charged binding entities 20 that have a net negative
 charge toward the positive electrode 12A. Charged materials 20 having a
 net positive charge move under the electrophoretic force toward the
 negatively charged electrode 12D. When the net negatively charged binding
 entity 20 that has been functionalized contacts the attachment layer 16A
 as a result of its movement under the electrophoretic force, the
 functionalized specific binding entity 20 becomes covalently attached to
 the attachment layer 16A.
 FIG. 2 is a cross-sectional diagram of a laminated, stacked, reconfigurable
 structure 30 according to one embodiment of this invention. Broadly, the
 stacked, reconfigurable structure 30 includes a plurality of chambers, in
 FIG. 2 showing two chambers, a first chamber 40 and a second chamber 50. A
 chamber comprises a bounded volume for providing controlled flow via
 electrophoretic, electroosmotic, thermal or other modes of transport,
 typically having one or more points of connection (e.g., such as by a via
 or buss) with one or more other chambers. The chamber may be closed except
 for the presence of vias, and/or a buss, or may have one or more open
 sides while still defining a volume useable consistent with the goals and
 objects of this invention.
 The first chamber 40 is defined by a bottom support 42, an intermediate
 member 44 and a spacer 46. The spacer 46 includes edges 48 which provide
 boundary walls for the first chamber 40 on the left and right ends. The
 second chamber 50 is defined on the bottom by the intermediate member 44,
 preferably the same intermediate member 44 which serves to define the top
 of the first chamber 40. The upper portion of the second chamber 50 is
 formed by the top member 52, which may optionally be transparent or
 translucent. Spacer 56 includes edges 58 which serve to define the left
 and right boundaries of the second chamber 50.
 FIG. 2 shows various mechanisms by which the first chamber 40 and second
 chamber 50 of the stacked, reconfigurable structure 30 interface with the
 external world and between chambers. An inlet port 60 permits fluidic
 coupling from external to the device 30 into the first chamber 40 via
 aperture 64 formed through the bottom support 42. Optionally, the inlet
 port 60 may include a mating lock 62, such as a Luer lock. The outlet port
 70 couples to aperture 74 formed in the top member 52 and provides for
 (fluidic and possibly gas) output from the stacked, reconfigurable
 structure 30. While the ports 60, 70 have been labeled inlet and outlet,
 respectively, they may be reversed without loss of generality. The first
 chamber 40 and the second chamber 50 are further fluidically (and for gas
 flow) coupled through the first via 80 and second via 82. The vias 80, 82
 are formed through the intermediate member 44, and as shown, through the
 electrodes 54.
 A plurality of electrodes 54 are provided within the reconfigurable
 structure 30 in shapes and positions to achieve the functionality
 described herein. Electrodes 54 preferably have a generally sheet-like or
 planar structure, at least at certain portions of the electrode 54. The
 electrode 54 includes an upper surface 54' and lower surface 54". In
 certain of the electrodes, an electrode through region 84 may be located
 in the electrode 54. In the preferred embodiment, the electrode through
 region 84 is a hole, that is, the electrode 32 completely circumscribes
 the electrode through region 84. However, the electrode through region 84
 need not be formed as a hole, and may only be bounded by or partially
 surrounded by the electrode 54, or may be set back from the hole as in an
 annulus.
 This electrode structure is particularly advantageous to aid in the
 movement, processing and analysis of materials in the system 30. The
 various vias 80, 82 and apertures 64, 74, preferably have adjacent
 electrodes 54 formed in the manner described in the preceding paragraph.
 Such electrodes serve as a conductive structure adapted to receive a
 signal from a control system or source serving to provide an
 electromagnetic environment adjacent the through paths through the
 apertures 64, 74 and vias 80, 82 to control the flow in the manner
 desired. Optionally, electrodes 54 may be disposed on the chamber 40, 50,
 in a position opposite to the via 80, 82 or aperture 64, 74.
 By way of example, the first via 80 may be disposed opposite an electrode
 54 formed on the bottom support 42 directly across from the via 80. Such
 an electrode may be energized to provide a repulsive force so as to drive
 materials from the volume of the first chamber 40 towards the first via
 80, through and into the second chamber 50. In combination with this
 repulsive force, the electrode 54 adjacent the left-most electrode through
 region 84 may be biased attractive to the desired materials to aid in
 drawing those materials from the first chamber 40 to the second chamber
 50.
 In this way, the signals provides to the electrodes 54 may generate a
 reconfigurable flow pattern as required for the operation of the system.
 FIG. 2 shows arrows depicting possible flow directions. While the first
 via 80 shows flow from the first chamber 40 to the second chamber 50, the
 direction of flow may be opposite given appropriate biasing of the
 electrodes 54. Similarly, the second via 82 is shown having bi-directional
 arrows. In actual operation, the flow of charged materials may be of but a
 single direction (e.g., from the second chamber 50 to the first chamber
 40) or may indeed be of both directions simultaneously, such as where both
 positively charged and negatively charged materials are present within the
 solution contained in the device 30. Such a bi-directional flow may occur
 in the presence of DNA (negatively charged) and proteins (positively
 charged). Thus, the arrows shown in the drawing of FIG. 2 are merely for
 expository convenience, and not intended to provide a limiting depiction
 of directionality.
 The various structures including the bottom support 42, intermediate member
 44, top member 52 and spacers 56 are preferably formed of a sheet-like
 material. These materials are generally planar, having an upper and a
 lower surface. Generally, this sheet-like material has lateral extension
 which is significantly (at least 10:1 times) greater than the thickness of
 the material. The spacer 46, 56, is preferably formed from a relatively
 thicker (e.g., 5 mil) sheet-like material. While these thicknesses are
 currently preferred, the actual thickness may be chosen based upon
 availability and functional requirements. Preferably the chambers 40, 50
 are formed via die cutting of the overall sheet.
 The preferred sheet-like material for structures, e.g., the bottom support
 42 and spacer 46, is polyimide. One source for sheet polyimide is DuPont
 who currently sells materials generally ranging from 1 mil to 1.5 mm thick
 under the trademark Kaptonr.TM.. Generally, it is desired that these
 materials have relatively low swelling (preferably less than 10%, more
 preferably less than 5% and most preferably less than 2%) in the presence
 of fluids, preferably have relatively low inherent fluorescence, are
 substantially inert in an acidic environment (most preferably to a pH of 2
 and more preferably to a pH of 1), are electrically insulative or
 nonconducting. Utilizing currently available materials, relatively thin,
 e.g., 1 mil thickness sheets, may be patterned with 1 mil wide lines and 1
 mil wide spaces.
 While polyimide is the preferred material, other materials meeting one or
 more of the criteria include: polymethylmethacrylate (PMMA),
 polytetrafluorethylene (PTFE-Teflon), polyester (Mylar), polystyrene,
 polycarbonate and like materials. Further, various layers in the laminated
 structure 30 may be selected from different materials to optimize the
 performance of that layer or the laminate structure 30. For example, the
 exposed surfaces in the chambers 40, 50 may optionally be selected for low
 adhesion to biological materials. The support may be chosen for its
 inherent low specific binding with biological materials or the surface may
 be altered to that purpose. One or more layers may be chosen for high
 reflectivity, low reflectivity (such as through the use of black or
 absorbing materials), having a desired texture (e.g., low texture for
 bonding purposes and surface chemistry optimization), or have hydrophobic
 or hydrophilic properties. Preferably, the layers are nonporous. The
 laminated structure 30 is generally preferred to be impermeable to fluids,
 such as water.
 The electrodes 54 are preferably formed on or integral to a sheet, such as
 a polyimide sheet. The electrode materials are preferably noble metals,
 most preferably gold. Generally, it is preferred that no base metals which
 would adversely affect biological materials to be supplied to the
 laminated structure 30, such as DNA, are exposed in the electrode 54. Most
 preferably, it is desirable to avoid copper and iron, and to a lesser
 extent lead and tin in the materials, or at least, avoiding the exposure
 of those materials or their ions if present to the biological materials.
 The electrode 30 should be formed from a material, and result in a
 structure, which is generally noncorrosive, is bondable, adheres to other
 materials, serves to minimize or avoid leakage currents, generates
 relatively low amounts of electrochemistry and has a relatively high
 electrochemical voltage at which the surface of the electrode emits
 constituents materials. Other desirable electrodes may be formed from
 nichrome, platinum, nickel, stainless steel or indium tin oxide (ITO), ITO
 being advantageously used when optical detection, especially from the back
 side, is utilized.
 In the preferred embodiment, when polyimide sheets are utilized, the
 preferred adhesive is DuPont acrylic adhesive, or polyester adhesive.
 Generally, it is desirable that the adhesive have low squeeze out
 properties such that during the lamination process, excessive amounts of
 adhesive do not exit such as at the interior edge 48, 58, lest excessive,
 and unpredictable, amounts of adhesive reside on the electrode 54.
 Generally, the adhesive is on the order of 1 mil thick.
 The laminated structures are preferably formed by methods which permit the
 high yield, low cost manufacturing of high quality devices. The various
 holes, such as vent holes, sample through holes and electrode through
 regions may be formed through any known technique consistent with the
 objects and goals of this invention. For example, microminiaturized drills
 may form holes as small as 3-8 mils, while laser drilled holes may be as
 small as 4 mils, or photolithographically patterned holes may be formed to
 substantially 1 mil. Generally, utilizing current technology, the thinnest
 sheets permit the formation of the smallest diameter holes. Optionally,
 chemical etching may be utilized to remove debris from the holes. This
 technique is particularly advantageous after laser drilling of holes, so
 as to reduce or remove previously ablated materials. After the electrodes
 are patterned on the support, and various layers are fabricated, the
 laminated or composite structure 30 is adhered together. Generally, it is
 desirable to have minimal or no squeeze out of adhesive to avoid
 nonuniformity in terms of exposed electrode area. In one embodiment,
 relatively larger holes are first formed, and then relatively smaller
 holes are drilled through the larger holes. Alternately, the supports
 including vents and holes may be formed first, and then aligned, such as
 through optical alignment, prior to the setting of the adhesive.
 The electrodes in the various embodiments may optionally be in contact with
 or adjacent to a permeation layer. Generally, the permeation layer serves
 as a medium to prevent or reduce the amount of sample which may directly
 contact the electrode surface. Various permeation layers include polymer
 coatings, or other materials compatible with these goals and objects. In
 yet another structure configuration, a polymer layer or permeation layer
 may be disposed within a via or electrophoretic buss. Such a structure may
 form essentially a miniature separation column to provide separation, for
 example, of such species as DNA and proteins.
 Yet other regions of the device may be decorated with affinity materials.
 For example, the transport of charged polymers or ions through the vias
 could be used to form purification by separation on the basis of
 charged-to-mass ratio or attraction to an affinity matrix which could be
 coated onto or near an electrode, in the via or electrophoretic buss. In
 yet another aspect, small charged species may be separated from
 macromolecules by using molecular weight cut-off membranes. Such membranes
 may be located in the vias or in the electrophoretic buss. Yet further
 structures for assays or functional analysis may be performed by including
 functional groups corresponding to said assays or analysis in the coating
 on the electrodes. For example, DNA probes or antibodies may be attached
 to the permeation layers which are in turn attached to or adjacent the
 electrodes.
 FIG. 3 is a cross-sectional view of a multichamber system. Here, a first
 chamber 100, second chamber 102 and third chamber 104 are stacked one on
 top of the other. Structurally similar features between FIG. 3 and FIG. 2
 are present, and the comments regarding one figure apply with equal force
 with respect to other figures. Thus, an inlet port 110 and an outlet port
 112 each include an aperture which provides for fluidic (and possibly gas)
 communication from external of the device to the various interior
 portions. The device itself is preferably fabricated with stacked
 laminates, such that from the bottom to the top the system would include a
 bottom support 120, a first intermediate support 122, a second
 intermediate support 124 and a top member 126. These structures are
 separated by the presence of a first spacer 130, second spacer 132 and
 third spacer 134. The spacers 130, 132 and 134 are preferably relatively
 thick (e.g., five times thicker, and more preferably substantially ten
 times thicker) than the thickness of the other support members 120, 122,
 124 and 126. It will be appreciated that all support members need not be
 of a uniform thickness (and therefore chambers 100, 102, 104 of uniform
 volume), but may be varied as desired to serve the required
 functionalities. Vias 136 are located between the first chamber 100 and
 second chamber 102, and between the second chamber 102 and third chamber
 104. Apertures 138 coupled to the ports 110, 112 so to provide coupling
 between external to the device and internal to the device. As shown,
 outlet port 112 is optionally disposed at a portion of the third chamber
 104 which is away from the electrophoretic buss 140, to thereby induce
 flow through said third chamber 104.
 An electrophoretic buss 140 typically consists of a chamber region 142
 which spans more than two chambers 100, 102, 104. Driving electrodes 144,
 146 are disposed at substantially opposite ends of the electrophoretic
 buss 140. Driving electrode 144 disposed on the bottom support 120
 preferably includes an electrode through region 148 adjacent the aperture
 138 whereby flow through the surrounding electrode 144 may be effected.
 The driving electrode 146 disposed on the top member 126 may be uniform or
 may include an electrode through region if necessary to promote fluidic or
 gas transfer through the region containing the driving electrode 146. The
 electrophoretic buss 140 serves to provide a volume in which the free
 space nature of the electrophoretic transport in the device may permit the
 easy transport of materials to the desired chamber 100, 102, 104.
 Preferably, collection electrodes 150 are disposed adjacent the periphery
 of the electrophoretic buss, aiding in the tapping or otherwise removing
 of the material flowing through the electrophoretic buss 140 into the
 chamber (e.g., chamber 104). Optionally, upper electrodes 152 may be
 disposed within the chambers 100, 102, 104 to aid in the tapping or
 movement of materials. By activation of the collection electrodes 150, and
 optionally the upper electrodes 152, materials may be removed from the
 electrophoretic buss 140 at the time when desired materials are in
 proximity thereto. In the structure of FIG. 3, the "tap" consists of
 selecting material from the electrophoretic buss 140 having a first
 direction flow into a flow direction which is substantially perpendicular
 thereto, namely, into and through a chamber 100, 102, 104.
 FIGS. 4A and 4B show perspective and cross-sectional (along the plane
 A--A') of a full stacked assay system. The structures having similarity to
 those described in the preceding figures apply with equal force here. The
 relatively thin base layer 160, first intermediate layer 162, second
 intermediate layer 164, third intermediate layer 166 and tap number 168
 are separated by the series of first spacer 170, second spacer 172, third
 spacer 174 and fourth spacer 176. An input port 180 is connected to an
 aperture 184 in the base layer 160 by an optional pathway 186. The output
 port 182 is coupled through the top member 168. One or more vias 188 may
 be included.
 In one aspect of this invention, the various chambers may have different
 principle functionalities. For example, first chamber 190 may be
 principally for sample preparation, such as through filtering, affinity
 membranes and dilution. Further, the first chamber 190 may include a
 sorting level, such as through the use of dielectrophoresis for cell
 sorting and initial screening. At least some of the materials from the
 first chamber 190, such as DNA obtained from the cell sorting and initial
 screening is provided via the electrophoretic buss 200 or vias 188 to
 other chambers or levels. For example, at least a portion of the output of
 the first chamber 190 may be transported through via 188 into the second
 chamber 192 wherein DNA amplification (e.g., PCR, SDA, enzymatic
 amplification, or other linear or exponential amplification technique) may
 be utilized. The third chamber 194 may provide functions such as DNA
 assay. Optionally, the assay may be performed on an assay chip 202, the
 output of which passes through via 188 to the fourth chamber 196. The
 fourth chamber 196 may optionally perform other, different, processes or
 analysis, such as an immunoassay at assay site 204.
 Optionally, detection of the conditions at the assay chips 202 and/or the
 assay site 204 may be performed optically, in which case it is desirable
 to have optical access through the top member 168, and as necessary,
 through other intermediate support layers, such as the third intermediate
 layer 166. Various detection systems may be utilized, including systems
 disclosed in "Scanning Optical Detection System", filed May 1, 1997,
 published as PCT US98/08370 U.S. Ser. No. 08/846,876, incorporated herein
 by reference. Optionally, the various assay chips 202 or assay sites 204
 may be formed on chips, such as silicon chip based technology (See, e.g.,
 FIG. 1), and may optionally be mounted on the intermediate support layers
 162, 164, 166 through various attachment technologies, such as flip-chip
 attachment techniques. Heaters/electrodes 206 are disposed at the right
 most portion of the chambers 190, 192, 194 and 196, and may comprise
 reagent delivery regions.
 With respect to the structures of FIGS. 2, 3, 4A and 4B, described above,
 it will be appreciated that alternative terminology may be utilized to
 describe structural or functional attributes. For example, the lowest
 intermediate support (intermediate member 44 in FIG. 2, first intermediate
 support 122 in FIG. 3, and first intermediate layer 162 in FIGS. 4A and
 4B) could also be referred to as a top member for the first chamber as it
 is disposed above the chamber space and bottom. Likewise, that same
 structure could also be termed the base layer or bottom support or like
 terminology when used in context of the next higher chamber. Stated
 otherwise, the first intermediate support 122 of FIG. 3 may be both termed
 a top member for the first chamber 100 as well as the bottom support or
 base layer for the second chamber 102.
 FIG. 5 shows a flow chart of a structure and implementation such as in
 FIGS. 4A and 4B. The description will compare the functional steps of the
 flow chart of FIG. 5 with the structure shown in FIGS. 4A and 4B. Sample
 210 is provided to sample preparation region 212 from input port 180 to
 first chamber 190 wherein the screening/sorting 214 occurs. Optionally,
 amplification 216 may occur if transfer through via 188 into the second
 chamber 192 is effected. Otherwise, the screening/sorting step 214 leads
 to DNA hybridization 218 via the electrophoretic buss 200, as is the case
 with the output of the amplification step 216 from the second chamber 192.
 Some or all of the output of the screening/sorting step 214 may be
 supplied to the immunoassay step 220 such as from the output of the first
 chamber 190 via the electrophoretic buss 200 to the fourth chamber 196.
 DNA hybridization 218 may occur in the third chamber 194, which may be
 reached via the electrophoretic buss 200. Monitoring of the output of the
 system, such as through optical monitoring of the assay site 204 and assay
 chips 202 results in read out and data reduction 222. From this, the
 result 224 is obtained.
 FIG. 6 is a plan view from the top of the assay level (e.g., the third
 chamber 194 in FIG. 4B). The electrophoretic buss 230 is disposed to the
 left of the structure. A collection electrode 232 is preferably disposed
 on the substrate 234 to aid in the removal or tapping of materials to the
 electrophoretic buss 230. Reconfigurable array 236 is shown as an
 8.times.8 array of sites, though the number may be larger or smaller as
 required. Two columns of vias 238 may be selectively utilized for
 transportation between various levels. Assay chips 240 are then disposed
 to the right of the reconfigurable array 236. Optionally, focusing
 electrodes 242 may be disposed adjacent the assay chips 240. (See, e.g.,
 Ser. No. 09/026,618, entitled "Advanced Active Electronic Devices for
 Molecular Biological Analysis and Diagnostics and Methods for Manufacture
 of Same", filed Feb. 20, 1998, incorporated herein by reference as if
 fully set forth herein, specifically with respect to focusing electrode
 designs.) Additional vias 244 provide for fluidic or gas transport between
 various levels. Reagent containers 246 are fluidically coupled to the
 remainder of the chamber to the left.
 FIG. 7 is a plan view of an electrode configuration for electrophoretic
 free field transport including electronic taps. Driving electrodes 250
 provide for a net overall electrophoretic force in the direction of the
 arrow. Focusing electrodes 252 serve to provide a constraining force for
 charged materials in the direction of the flow represented by the arrow. A
 tap electrode 254 is disposed above the gap 256 formed by separation
 between adjacent focusing electrodes 252. In operation, materials being
 electrophoretically transported between the driving electrodes 250 may be
 cause to move with a force component in a direction transverse to the line
 between the driving electrodes 250, towards the cap electrodes 254.
 The systems described herein have numerous applications. Without limiting
 the generality of the foregoing description, various particularly
 advantageous applications will be described herein.
 Turning now to the operation of the systems described, above, in typical
 operation, the fluidic system would first be filled with an appropriate
 buffer. Next, the sample of interest would be injected into the input
 port. By selective activation of the electrodes, the desired materials
 would be attracted to the input electrodes. Damages to the sample may be
 avoided if a permeation layer is utilized, so as to prevent the unimpeded,
 direct contact of the materials with the electrode. The charged species
 then moves between electrodes in the planes of the structure to perform
 various functions. For biological samples, these functions may include
 some or all of the following: cell sorting, such as by dielectrophoresis,
 electronic lysis, and extraction of DNA, RNA or proteins from the lysed
 cells, electric-field driven amplification, sequence enrichment by
 hybridization, hybridization assays, protein binding assays, chemical
 sample processing, including mixing and synthesis steps. After processing
 is completed in the initial level, the appropriate species may then be
 transported through vias or the electrophoretic buss to the next or higher
 level in the stack. This is also optionally achieved electrophoretically
 by biasing an electrode below the vias so as to repel the species of
 interest and by biasing the ring electrode above the via so as to attract
 the species. In this way, chemical species may be mapped from one level to
 the next level. Sample preparation may advantageously be performed in an
 intermediate or middle level. This is so since proteins (typically having
 a positive charge) will move in an opposite direction to DNA (typically
 having a negative charge) to promote efficient separation.
 In operation, it may be highly advantageous to separate or allocate various
 biological or chemical functions to distinctly different levels or
 chambers. By creating a layered system, it is possible to segregate
 various biochemical functions to different layers so as to optimize the
 electrical and chemical environments and to perform the series of
 operations necessary to produce a meaningful identification of viral,
 bacterial, and toxic agents. By way of example, if an initial layer
 includes the sample preparation functions, that layer may be used to
 filter out extraneous material from the target sample. Filters and
 affinity membranes may be utilized, among other structures, to clean the
 sample at an initial, e.g., crude, level. A next level may utilize sorting
 an screening for pathogenic cells. At such a level, optionally,
 dielectrophoresis may be utilized to perform cell sorting and electronic
 cell lysis to extract DNA and target proteins from the crude sample. After
 cell lysis, the released DNA, charged chemical and biological toxins, and
 other molecules of interest may be transported electrophoretically to a
 series of diagnostic levels. Some DNA may be directed to an amplification
 level through appropriate vias, while proteins of interest could be moved
 to the electrophoretic buss where the larger potentially available
 currents may results in more rapid movement of the proteins. Since
 proteins usually move more slowly than DNA, the proteins final destination
 will depend in part on the time of flight actuation of collection
 electrodes on the appropriate levels.
 In yet another aspect of this device, the system may be reconfigured as a
 result of an initial analysis on a sample. Thus, the stacked system may
 perform directed assays, that is, where the system may sort cells, screen
 for pathogens and then perform specialized analysis on reconfigurable
 arrays based on the screening information. Significant improvements in
 both the speed and accuracy result for multiplexed tests. Furthermore,
 different kinds of biochemical information relating to DNA sequence and
 toxin repertoire may be collected from specific microorganisms, helping to
 identify the threat and select appropriate countermeasures. By
 electronically configuring the assay arrays based upon initial analysis,
 sensitivity may be optimized for the appropriate DNA sequence and antigens
 present at those locations. The assay process would be streamlined, also
 resulting in a significant enhancement of sensitivity and specificity by
 choosing appropriate probe sets and redundancy from a large array of
 available microlocations.
 In yet another aspect of this system, the electronic tap may be used to
 selectively remove material from one region of transport, to yet another
 region or chamber. Optionally, a second power supply may be utilized so as
 to effect a lateral force vector on the ions of interest, as supplied from
 an electrode coupled to the second power supply. Optionally, an electronic
 gate may be utilized to regulate the flow of ionic species between
 chambers or levels. For example, a mesh electrode may be placed between
 the driving electrodes 144, 146 (FIG. 3) or at or in vias 188.
 In yet another application, drug discovery may be performed through the
 synthesis of various products which are then mapped to potential binding
 sites. Synthesis products, e.g., peptides, may be mapped to potential
 binding sites for drug discovery. The use of an array of electrodes and
 vias to map the products of a number of synthesis reactions performed on a
 first level on to an array of analysis sites on another (second) level may
 be utilized.
 Considering the synthesis reaction in more detail, the system is able to
 concentrate reagents to enhance the reaction kinetics, create pH gradients
 at the electrodes under bias which can be utilized to deprotect various
 reaction groups, and move in reactive groups with good control of their
 type and quantity to precisely control microchemical reactions. This sort
 of reaction control could, for example, be used to synthesize
 oligonucleotides and oligopeptides. For oligopeptide synthesis, a strategy
 could be employed that utilizes amino acid building blocks with fMoc
 protecting groups which are also acid labile. In addition, the permeation
 layer would contain amino groups blocked with acid labile tBOC groups.
 Selective deprotection of sites and attachment would be accomplished using
 acid cleavage to expose hydroxyl groups. To allow attachment at a specific
 site, the electrode benefit it would be positively biased at a sufficient
 potential or current to create acidic conditions. At the appropriate
 current level our data shows that the low pH is limited to a region near
 the activated electrode, so cross-talk between microlocations is minimized
 and specific control of the synthesis at individual reaction sites can be
 achieved. A variety of chemical ligation procedures are available for
 peptide assembly. These reactions may be made both highly concentration
 dependent and highly pH dependent, two parameters which may be programmed
 and carefully controlled using the disclosed (and incorporated) electrode
 technology. Rapid combinatorial assembly of preformed peptide epitope
 building blocks can be achieved. Linkage will be designed to take
 advantage of electric field mediated concentration and acidification which
 occurs over positively biased electrodes on the chip.
 In yet another application, medical diagnostic assays may be performed. By
 segregation of various functionalities to different levels, the speed and
 precision of operation of the system may be enhanced.
 In yet another application, the system may be utilized in the detection of
 pathogens, such as may occur in biological warfare applications. The
 stacked, reconfigurable system may perform directed assays, such as to
 sort cells, screen for pathogens, and then perform specialized assays on
 reconfigurable arrays based on the screening information. This selection
 and specialization of the arrays results in significant improvements in
 both the speed and the accuracy of the multiplexed tests. The different
 kinds of biochemical information relating to DNA sequence and toxin
 repertoire can be collected from specific microorganisms, helping to
 identify the threat and select appropriate counter measures. In yet a
 further optional aspect, the system may be adapted to generate the counter
 measures. For example, based upon the initial assay or other analysis, it
 is possible to perform directed peptide synthesis which results in the
 on-chip synthesis of vaccines to respond to the biological threats.
 Optionally, a detachable support may be anchored over one or more
 electrodes which may be used as the starting material for linkage of
 peptides. The resulting synthetic peptide may be used as a vaccine, or for
 drug synthesis. Optionally, the peptides may be anchored to the detachable
 support, which may be removed from the chip for injection. For use as a
 drug, for example, to block binding of a neurotoxin, the peptides may be
 attached to a cleavable linker such as a disulfide.
 Optionally, such a detection system may be modified to detect airborne
 pathogens. Advanced sample collection techniques including air handling
 and sampling techniques may be utilized. To capture the airborne pathogens
 when admixed with significant amounts of spurious background material, an
 optional pre-filtering step may be utilized to minimize the volume of
 background material relative to the pathogens. In one implementation,
 electrostatic methods may be utilized for particulate attraction, which
 may then be utilized in conjunction with the electrophoretic techniques
 described herein to separate species according to their charge.
 Although the foregoing invention has been described in some detail by way
 of illustration and example for purposes of clarity and understanding, it
 will be readily apparent to those of ordinary skill in the art in light of
 the teachings of this invention that certain changes and modifications may
 be made thereto without departing from the spirit or scope of the appended
 claims.