Abstract:
A device includes an inlet for receipt of a sample. A first chamber is coupled to the inlet and includes at least one affinity region. A second chamber is disposed adjacent to the first chamber. The first chamber and the second chamber share a common intermediate member, the intermediate member having at least one via formed in the common intermediate member. The second chamber includes an assay chip comprising an array of addressable electrodes. An outlet is coupled to the second chamber. The device may be used to selectively amplify and elute nucleic acids for subsequent detection and analysis.

Description:
RELATED APPLICATION INFORMATION  
       [0001]    This application is a continuation of application Ser. No. 09/204,324 filed Dec. 2, 1998, now U.S. Pat. No. 6,638,482, which is a continuation-in-part application of application Ser. No. 08/753,962, filed Dec. 4, 1996, entitled “Laminated Assembly for Active Bioelectronic Devices”, now U.S. Pat. No. 6,287,517, which is a continuation-in-part application of Ser. No. 08/534,454, filed Sep. 27, 1995, entitled “Apparatus and Methods for Active Programmable Matrix Devices”, now issued as U.S. Pat. No. 5,849,486, which is a continuation-in-part of application Ser. No. 08/304,657, filed Sep. 9, 1994, entitled, as amended, “Molecular Biological Diagnostic Systems Including Electrodes”, now issued as U.S. Pat. No. 5,632,957, which is a continuation-in-part of application Ser. No. 08/271,882, filed Jul. 7, 1994, entitled, as amended, “Methods for Electronic Stringency Control for Molecular Biological Analysis and Diagnostics”, now issued as U.S. Pat. No. 6,017,696, which is a continuation-in-part of application Ser. No. 08/146,504, filed Nov. 1, 1993, entitled, as amended, “Active Programmable Electronic Devices for Molecular Biological Analysis and Diagnostics”, now issued as U.S. Pat. No. 5,605,662, and application Ser. No. 08/709,358, filed Sep. 6, 1996, entitled “Apparatus and Methods for Active Biological Sample Preparation”, now issued as U.S. Pat. No. 6,129,828, all incorporated herein by reference as if fully set forth herein.  
         [0002]    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 “System Including Functionally Separated Regions in Electrophoretic System”. 
     
    
     
       FIELD OF THE INVENTION  
         [0003]    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.  
         BACKGROUND OF THE INVENTION  
         [0004]    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).  
           [0005]    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.  
           [0006]    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.  
           [0007]    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.  
           [0008]    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.  
           [0009]    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).  
           [0010]    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. 757758, 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.  
           [0011]    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).  
           [0012]    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.  
           [0013]    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.  
           [0014]    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□° C. to 16□° C. Most probes required 3 hours of washing at 16□° 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.  
           [0015]    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, colorimetrically, 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.  
           [0016]    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.  
           [0017]    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.  
           [0018]    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.  
           [0019]    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.  
           [0020]    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.  
           [0021]    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  
         [0022]    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.  
           [0023]    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.  
           [0024]    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™, 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.  
           [0025]    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.  
           [0026]    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.  
           [0027]    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.  
           [0028]    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.  
           [0029]    It is therefore an object of this invention to provide for an improved integrated, reconfigurable, multifunctional system.  
           [0030]    It is yet a further object of this invention to provide a sensitive, adaptable low-cost diagnostic system.  
           [0031]    It is yet a further object of this invention to provide a system having an improved mode of fluidic communication within a multichamber device.  
           [0032]    It is yet a further object of this invention to provide a system having improved sensitivity and specificity.  
           [0033]    It is yet a further object of this invention to provide systems having dynamic reconfigurable components for the analysis of materials. 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0034]    [0034]FIGS. 1A and 1B show an active, programmable electronic matrix device (APEX) in cross-section (FIG. 1A) and in perspective view (FIG. 1B).  
         [0035]    [0035]FIG. 2 is a cross-sectional view of a multilayer structure including two chambers and interconnecting vias.  
         [0036]    [0036]FIG. 3 is a cross-sectional view of a multilayer structure including at least three chambers, multiple vias and an electrophoretic buss.  
         [0037]    [0037]FIGS. 4A and 4B show a multilevel, stacked, reconfigurable system for transport and analysis of charged materials in perspective view (FIG. 4A) and in cross-section (FIG. 4B).  
         [0038]    [0038]FIG. 5 is a flowchart showing steps from the receipt of a sample through to the result of the intervening action steps.  
         [0039]    [0039]FIG. 6 is a plan view of an assay level in a multilevel, reconfigurable system including an electrophoretic buss and multiple reagent dispensers.  
         [0040]    [0040]FIG. 7 is a plan view of an electrode configuration including one embodiment having taps from a principle transport pathway.  
     
    
     DETAILED DESCRIPTION  
       [0041]    [0041]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  12 A,  12 B,  12 C and  12 D. 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  16 A,  16 B,  16 C and  16 D to correspond with the identification of the electrodes  12 A-D, respectively.  
         [0042]    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 functionalized specific binding entity  20  towards the electrode  12 . For example, if the electrode  12 A were made positive and the electrode  12 D negative, electrophoretic lines of force  22  would run between the electrodes  12 A and  12 D. The lines of electrophoretic force  22  cause transport of charged binding entities  20  that have a net negative charge toward the positive electrode  12 A. Charged materials  20  having a net positive charge move under the electrophoretic force toward the negatively charged electrode  12 D. When the net negatively charged binding entity  20  that has been functionalized contacts the attachment layer  16 A as a result of its movement under the electrophoretic force, the functionalized specific binding entity  20  becomes covalently attached to the attachment layer  16 A.  
         [0043]    [0043]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.  
         [0044]    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 .  
         [0045]    [0045]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 .  
         [0046]    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.  
         [0047]    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 .  
         [0048]    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 .  
         [0049]    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.  
         [0050]    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.  
         [0051]    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 Kapton□. 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.  
         [0052]    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.  
         [0053]    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.  
         [0054]    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.  
         [0055]    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.  
         [0056]    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.  
         [0057]    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.  
         [0058]    [0058]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 .  
         [0059]    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 .  
         [0060]    [0060]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.  
         [0061]    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 .  
         [0062]    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.  
         [0063]    With respect to the structures of FIGS. 2, 3,  4 A and  4 B, 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 .  
         [0064]    [0064]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.  
         [0065]    [0065]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 8×8 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.  
         [0066]    [0066]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 .  
         [0067]    The systems described herein have numerous applications. Without limiting the generality of the foregoing description, various particularly advantageous applications will be described herein.  
         [0068]    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.  
         [0069]    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.  
         [0070]    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 micro locations.  
         [0071]    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 .  
         [0072]    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.  
         [0073]    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.  
         [0074]    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.  
         [0075]    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.  
         [0076]    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.  
         [0077]    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.