KR20010041147A - Advanced active devices and methods for molecular biological analysis and diagnostics - Google Patents

Abstract

Disclosed are an apparatus for manufacturing an active biological manipulation using a variety of structures capable of advantageously collecting and providing a charged biomaterial to a microposition array. In one embodiment, the device includes a focusing electrode that assists in directing and transporting the collection electrode to the array. Preferably, at least one intermediate transfer electrode is used, more preferably a transfer electrode of monotonically decreasing size between the collection electrode and the array is used to reduce current density mismatch. In another feature, a flow cell is used in the device that provides for the receipt of a solution containing the material to be analyzed. In another embodiment, a concentric ring design is provided. Various flip-flop embodiments are disclosed.

Description

ADVANCED ACTIVE DEVICES AND METHODS FOR MOLECULAR BIOLOGICAL ANALYSIS AND DIAGNOSTICS

Molecular biology includes a wide range of techniques for the analysis of nucleic acids and proteins. Many of these techniques and procedures form the basis of clinical diagnostic analysis and testing. Such techniques include nucleic acid hybridization assays, restriction enzyme assays, gene sequence analysis, and isolation and washing of nucleic acids and proteins (eg, EF Fritsch, and T. Maniatis, Molecular Cloning: A Laboratory Manual, 2 Ed., Cold spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989).

Most of these techniques perform various manipulations (eg, pipetting, centrifugation, electrophoresis, etc.) on large amounts of samples. These are often complex, time consuming, and require high precision. Many techniques limit their application to lack of sensitivity, specificity or reproducibility. For example, these problems limit various diagnostic applications of nucleic acid hybridization assays.

The complete process for conducting DNA hybridization assays for genetic or infectious diseases is very comprehensive. In general, a complete process can be divided into many steps and substeps. In the case of genetic disease diagnosis, the first step is to take a sample (blood or tissue). Various preprocessing is performed depending on the type of sample. The second step is to destroy or break down the cells and then to separate the raw DNA material along with other cellular components. In general, several substeps are required to remove cell debris and purify the raw DNA. At this point, there are several options for further processing and analysis. One option is to denature washed sample DNA and perform direct hybridization assays in one of many formats (dot blots, microbeads, microplates, etc.). A second option, called Southern blot hybridization, is to split the DNA with restriction enzymes, separate the DNA fragments on an electrophoretic gel, blot the membrane filters and then hybridize the blots to specific DNA probe rows. This procedure effectively reduces the complexity of genomic DNA samples, thus helping to improve hybridization specificity and sensitivity. Unfortunately, this procedure is long and difficult. A third option is to carry out a polymerase chain reaction (PCR) or other amplification procedure. PCR procedures increase the number of target DNA sequences compared to nontarget sequences. Amplification of the target DNA helps to overcome problems related to the complexity and sensitivity of genomic DNA analysis. All these procedures are time consuming, relatively complex, and greatly increase the cost of diagnostic tests. After this sample preparation and DNA processing step, the actual hybridization reaction takes place. Finally, detection and data analysis turn hybridization events into analysis results.

Sample preparation and processing steps are usually performed separately from other major steps of hybridization and detection and analysis. Indeed, various substeps, including sample preparation and DNA processing, are often performed as discrete operations separated from other substeps. Considering this substep in more detail, samples are obtained via any number of means, such as to obtain a complete blood, tissue, or other biological fluid sample. In the case of blood, the sample is removed of red blood cells and retains the desired aggregate (white) cells. This process is usually carried out by density gradient centrifugation. Cell disruption or digestion is then performed on the aggregated cells, preferably to separate the DNA by sonication, freezing / thawing techniques, or by addition of the digesting agent. The raw DNA is then separated from the cell debris in the centrifugation step. Prior to hybridization, double helix DNA is denatured into a single helix. Denaturation of double helix DNA is generally carried out by techniques involving heating (> Tm), salt concentration change, addition of base (NaOH) or reactive (urea, formamide, etc.) denaturation.

Nucleic acid hybridization assays generally involve the detection of a very small number of specific target nucleic acids (DNA or RNA) with an excess of probe DNA in a relatively large amount of complex non-target nucleic acid. Sub-steps of removing DNA complexity in sample preparation help to detect low copy numbers of nucleic acid targets (ie 10,000 to 100,000). DNA complexity is somewhat overcome 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). Although a huge number of target nucleic acid sequences are produced by amplification, which improves the successive direct probe hybridization step, amplification usually involves a long and cumbersome procedure that must be performed alone compared to other substeps. Performing the amplification step requires a substantially complex and relatively large piece of equipment.

The actual hybridization reaction represents one of the most important and central steps in the overall process. The hybridization step includes contacting the prepared sample with a particular reporter probe in a series of optimal conditions for hybridization occurring in the target DNA sequence. Hybridization can be performed in any of a number of formats. For example, many sample nucleic acid hybridization assays are performed on various filters and solid support formats (GA 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 called so called dot blot hybridization involves non-covalent attachment to a filter of target DNA, where the attached DNA is hybridized by a radioisotope label probe. Dot blot hybridization has been widely used and many versions have been developed (MLM Anderson and BD Young, in Nucleic Acid Hybridization-A Practical Approach, BD Hames and SJ Higgins, Eds. IRL Press, Washington, DC Chapter 4, pp. 73 -111, 1985). It was developed for multiplex analysis of genomic mutations (D. Nanibhushan and D. Rabin, in EPA 0228075, July 8, 1987) and for overlapping clones and for mapping genomics (GA Evans, in US Patent No. 5,219,726, June 15). , 1993).

New techniques have been developed for conducting multiple sample nucleic acid hybridization assays in microformat multiplex or matrix devices (eg DNA chips) (M. Barinaga, 253 Science, pp. 1489, 1991; W. Bains, 10 Bio / Technology, pp. 757-758, 1992). These methods usually attach specific DNA rows to very small specific regions of solid supports, such as microwells of DNA chips. This hybridization format is a micro scale version of conventional dot blot and sandwich hybridization systems.

Microformat hybridization can be used to perform sequencing by hybridization (SBH) (M. Barinaga, 253 Science, pp. 1489, 1991; W. Bains, 10 Bio / Technology, pp. 757-758, 1992). ). SBH uses all possible n-nucleotide oligomers (n-mers) to identify n-mers in unknown DNA samples, which are then aligned by algorithmic analysis to generate DNA sequences (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 RB Crkvenjakov, US Patent # 5,202,231, April 13, 1993).

There are two formats for doing SBH. The first format is to create an array of all possible n-mers on the support and then hybridize to the target rows. The second format is to attach the target row to the support and then probe all possible n-mers. Both formats present a fundamental problem of direct probe hybridization and additional difficulties associated with multiplex hybridization.

Southern, United Kingdom Patent Application GB8810400, 1988; E.M. Southern et al., 13 Genomics 1008, 1992 propose a method for analyzing or sequencing DNA using a first format. Southern used PCR amplified genomic DNA to identify known single point mutations. Southern also describes a method for synthesizing oligonucleotide arrays on solid supports for SBH. However, Southern did not address how to achieve optimal persuasive conditions for each oligonucleotide on the array.

At the same time, Drmanac et al., 260 Science 1649-1652, 1993 sequenced several short (116 bp) DNA sequences using a second format. Target DNA was attached to the membrane support (dot blot format). Each filter was hybridized sequentially with 272 labeled 10-mer and 11-mer oligonucleotides. A wide range of persuasion conditions were used to achieve specific hybridizations for each n-mer probe, with wash times ranging from 5 minutes to overnight, with temperatures ranging from 0 degrees to 16 degrees. Most probes required a 3 hour wash at 16 degrees. The filter had to be exposed for 2-18 hours to detect the hybridization signal. The false total negative hybridization rate was 5% despite the simple target sequence, reduced oligomeric probe set and the most convincing conditions possible.

There are various methods for the detection and analysis of hybridization events. Depending on the reporter group (fluoropores, enzymes, radioisotopes, etc.) used to label the DNA probes, detection and analysis is performed in a fluorometer manner, in a colorimeter manner, or in an automated radiograph. Observing and measuring emission radiation, such as fluorescent or particulate radiation, can provide information about hybridization events. Even in a detection method with very high inherent sensitivity, detection of hybridization events is difficult due to the background presence of non-specific boundary materials. In addition, many other factors reduce the sensitivity and selectivity of DNA hybridization assays.

Attempts have been made to combine certain processing steps or substeps together. For example, many microrobotic systems have been proposed for preparing arrays of DNA probes on support materials. For example, Beattie et al., In The 1992 San Diego Conference: Genetic Recognition, November, 1992, used microrobotic systems to insert microdrops containing specific DNA rows into individual microfabricated sample wells on glass substrates.

In general, the conventional process was extremely labor and time intensive. For example, PCR amplification processes are time consuming and diagnostic analysis adds cost. Many steps that require human intervention during or between processes are not optimal because of the potential for contamination and operator error. In addition, the use of multiple machines or complex robotic systems to perform individual processes is often forbidden in terms of cost and physical space requirements except in the largest laboratory.

Attempts have been made to enhance the overall sample introduction into the sample preparation analysis process. Often given the relatively small amount of sample material possible, an improved process is required for efficient sample preparation, sample transport and effective sample analysis. Various proposals have been made, but certain systems enjoy relative advantages in certain environments.

Another area of interest is electrical addressing for relatively large arrays. If the array grows relatively large, efficient operation of the system becomes more important. Efficient interfacing of array based systems with electrical connection off-chip causes pin or contact limiting problems. In addition, limitations on effective chip or array size create problems with the choice of components to include on the chip or substrate, and their size. Often, various choices must be made to provide optimization of effective benefits in the overall design.

One proposed solution for the control of an electrode array using up to one individual dedicated connection per electrode or test location is reflected in the Kovacs United States Patent Application Serial No. 08 / 677,305, entitled "Multiplexed Active Biological Array", filed July 9, 1996. The array includes a plurality of electrode positions, representative electrode positions including the electrodes, a drive element coupled to the electrode for applying electrical stimulation to the electrode, and a signal indicative of the magnitude of the electrical stimulus to be applied to the electrode. It consists of combined local memory. Various embodiments are disclosed for selectively coupling value signals through interaction of storage row lines and column lines in local memory. In this way, the values at the various electrodes in the array can be different.

Fiaccabrino, GC et al., " Array of Individual Addressable Microelectrodes", Sensors and Actuators B, 18-19, (1994) 675-677 is, n 2 n the pin array of the ^two electrodes for signal output and the bulk bias And two additional pins. The matrix signal drives the transistors connected in series to supply a signal value to the working electrode. Such a system cannot switch two or more electrodes simultaneously to different potentials.

Kakerow, R. et al., "A Monolithic Sensor Array of Individually Addressable Microelectrodes", Sensors and Actuators A, 43 (1994) 296-301, provide monolithic single-chip sensor arrays for measuring chemical and biochemical parameters. Is disclosed. The sensor cells are addressed in series by the sensor control unit. One horizontal and one vertical shift register controls the selection of the sensor cell. Only one sensor cell is selected at a time. As a result, multiple positions cannot be driven simultaneously.

Another concern is the ability to inspect an electronic device before providing a conductive solution on the electronic device. As devices or chips become more complex, the likelihood of manufacturing or process errors generally increases. Although visual inspection of the circuit can be carried out, it is possible to ensure that the operating device is provided to the end user by another inspection.

As is evident from the foregoing description, various attempts have been made to provide effective techniques for conducting multistage multiplex molecular bioreactions. However, for the reasons mentioned above, these techniques are slow, limited and have no effective optimized solution. These various approaches do not form a system that can be easily combined to perform a complete DNA diagnostic analysis. Despite the long recognized need for such a system, no satisfactory solution has conventionally been provided.

<Summary of invention>

Disclosed are a device suitable for use in an active electronic device used for biological diagnosis and a method of manufacturing the same. In particular, various layouts or embodiments involving the selection of components are used in the beneficial combinations to provide a useful device. Various structures, shapes and combinations of the electrodes and the co-operation with various applied signals (voltage, current) result in the useful preparation, transport, diagnosis and analysis of biological or other charged materials. Various beneficial protocols are described.

In a first preferred embodiment, an active biological manipulation electronic device comprises a support substrate; A microposition array disposed in a first region on the substrate; A first collection electrode disposed on the substrate; And first and second focusing electrodes disposed on the substrate, wherein the first and second electrodes are at least partially disposed adjacent to the microposition array, and the spacing between the first and second electrodes adjacent to the array is Less than a distance between said first and second electrodes in another region away from an array; And an opposite electrode disposed on the substrate. In one embodiment, a V or Y structure is used to collect the charged biomaterial in the desired area. Preferably, the focusing electrode has a proximal end disposed adjacent to the microposition array and a distal end disposed away from the array. The distance between the near ends of the first and second electrodes is shorter than the distance between the distal ends of the first and second electrodes.

In operation of this embodiment, a solution containing DNA or other biomaterial to receive the response command signal is provided on the substrate of the device. As a typical initial step, the focusing electrode and the return electrode are driven to collect charged biomaterial by moving over or near the collection area. In a preferred embodiment, the focusing electrode and the returning electrode or electrodes send response commands to a relatively large amount of sample. Typically, the collecting electrode and the counter electrode are disposed on the substrate such that the line of electric movement of force extends over almost all of the flow cell volume. For example, the focusing and return electrodes can be placed near the periphery of the trace of the flow cell. In other embodiments they may be disposed at opposite ends of the flow cell. In another embodiment, the return electrode surrounds the trace of the flow cell with the centered collection electrode. Effective response command origination for a sample in a flow cell is one desired result. Once the sample is modified, the focusing electrode can be manipulated to further focus the material towards the micropositional array. As the material moves from the focusing electrode towards the array, the space reduction between the first and second focusing electrodes focuses the analyte and other charged material into a smaller space. In this way, more efficient material transport from a relatively large focusing electrode region to a relatively small microposition array region can be achieved.

In another feature of this embodiment of the present invention, one or more transfer electrodes are provided, the transfer electrodes being disposed on a substrate and disposed between the first collection electrode and the array. In a preferred embodiment there are at least two transfer electrodes, and also the transfer electrodes differ in size, preferably the ratio between the large and the small is at least 2: 1. In this way, a relatively large area facing the collection electrode can be gradually moved to a smaller location near the analysis area of the device. This structure aids in the transition from a relatively large area of the collection electrode, but the step characteristic of this embodiment reduces current density mismatch. More effective transport and disconnection reduction is achieved by using stepped, preferably monotonically, stepped size reductions.

In another embodiment of the device, a biomanipulating electronic device includes a support substrate, a microposition array disposed in an area including a first side and an opposite side on the substrate, a first collection electrode disposed near the array on the substrate, And a second collection electrode disposed near the array on the substrate, wherein the first and second collection electrodes are at least partially disposed on opposite sides of the region. In a preferred embodiment, the collecting electrode has an area of at least 80% of the area of the area of the array. In this way, the sample can be collected in a relatively large area near the region containing the microlocation, from which DNA or other charged biomaterial can be provided.

In the method of using the device, the collecting electrode can be arranged to first collect the material and then repel the collected material, so that the material can be swept towards the area comprising the array. The material is conveyed wavewise over the array to allow interaction with the passive or active array. Alternatively, the material can be moved over an area of the array and effectively held in place by the application of an AC field. This embodiment has demonstrated that iterative hybridization can be performed, where the material moves to and interacts with the array, which then moves to the array for possibly another second interaction.

In another embodiment of the device design, a concentric ring design is used. An active bioelectronic device includes a support substrate, a microposition array disposed in a ring region on the substrate, a first counter electrode disposed to enclose the array on the substrate, and a collection electrode disposed inside the array on the substrate. do. In a preferred embodiment, the first opposing or return electrode is segmented and optionally has a passageway that causes segmentation to act as a passageway for electrical connection to the array. In another variation of this embodiment, multiple rings are provided around the array.

In another embodiment of the present invention, a reduced component number, preferably a five component system, is implemented in a flip chip structure to provide active biopsy. The apparatus includes a first substrate and a first substrate having at least one of the first and second surfaces supporting the electrical trace, and vias, passages or holes disposed between the first and second surfaces to allow fluid flow through the substrate, the first substrate Connected to at least one of the first and second surfaces of the substrate and connected to a microlocation array suitable for receiving fluid through the vias, passages or holes and at least a first surface disposed near, for example, a via. A second substrate comprising conductive traces, conductive wiring interconnecting the electrical traces on the second surface of the support substrate and the electrical traces on the first surface of the second substrate, such as bumps, of the first and second substrates of the support substrate. A sealant disposed between the first surface to provide a seal around and between the first substrate and the second substrate, and optionally a flow cell disposed on the first surface of the first substrate. It should. Preferably, the structure uses a flip chip structure in which flip chips are disposed in an operation direction under the support substrate. This design is particularly beneficial for reducing the number of parts in the device and improving manufacturing reliability.

In another embodiment, an active biological manipulation electronic device includes a support substrate having a first and a first surface and vias between the first and second surfaces to allow fluid flow, the second surface of the first substrate. A second substrate comprising at least a first surface suitable for facing, and a microposition array suitable for receiving the fluid, a seal disposed between the second surface of the support substrate and the first surface of the second substrate And a waveguide having a light source and an input suitable for receiving light from the light source and an output for directing the light to the array, the waveguide being substantially parallel to the support substrate, the light from the waveguide Illuminates the array. In a preferred embodiment, the light source is a laser such as a laser bar. Such devices may use support substrates that are flex circuits or circuit boards.

New and advantageous manufacturing methods may be used for some or all of the embodiments. This method is particularly beneficial for the manufacture of flip chip designs. In this structure, the chip is disposed adjacent to the substrate, the substrate comprises a via, and the structure is also adapted to receive fluid to be placed on the substrate and to flow through the via to the chip, wherein at least a portion of the chip is a sealant overcoat. Includes no area. The choice of sealant viscosity and material can effectively achieve effective coverage, good thermal contact between the substrate and the chip, and fluid sealing. In the most preferred embodiment, the method may use a photocurable sealant that is cured by light during application. Specifically, light is exposed downwardly through the vias over the substrate to the chip in the device. A photocurable insertable sealant is then applied to the interface between the substrate and the chip. The light at least partially cures the sealant as a result of exposure, whereby the sealant excludes flow to the area without the sealant. Finally, if desired, curing of the sealant may be completed by heat treatment.

In yet another embodiment, the system or chip comprises a multi-position array having electrically repeating unit cell positions. Arrays are generally organized in rows and columns, more typically in equal numbers of rows and columns. Individual unit cells of a unit cell array are selected by selectors such as one or more row selectors and one or more column selectors. The selector may be a memory, such as a shift register memory, or a decoder or a combination thereof. Input for address information may use an on-chip address generator, but usually receives an address from off-chip. In a preferred embodiment, the row selector comprises a shift register of a wider structure, such as a 1x1 structure, or a 1x4 structure. In operation, the select register is successively loaded with a value indicating selection or non-selection of a unit cell and optionally an output value (or an indicator of a value) for that cell. Optionally, memory may be provided to hold these values and continue output from the unit cell.

The system or chip optionally provides current and voltage in an active biomatrix device suitable for containing a conductive solution comprising a charged biomaterial. In one aspect, one unit cell array is provided. Each unit cell typically includes a row contact and a column contact. The row lines are disposed in the array and connected to the row contacts of the unit cell. The row selector selectively provides a row select signal to the row line. The unit cell is coupled to the power supply voltage and the electrode, and the row select signal and the column select signal serve to select the variable current output from the electrode of the unit cell. The return electrode is coupled to the potential and is suitable for contacting the conductive solution. In operation, selective driving of one or more unit cells results in the supply of current in the conductive solution.

In a preferred embodiment of the unit cell, a symmetrical structure is used. The first column selection unit, preferably the transistor and the first row selection unit, preferably the transistor, are arranged in series between a first source, such as a voltage and / or current source and a node, usually a current output node. In a preferred embodiment, the column select transistor can be precisely controlled under the application of a gate voltage from the column shift register memory. Preferably, the selection units may differ from each other in terms of controllability such that the channel length of the control transistor changes. The channel length is chosen such that the gain or other desired characteristic matches between the row and column transistors. In addition, the long channel length provides the ability to control small currents with appropriate control signals. Thus, current is output from the unit cell due to the potential application to the control gate by the potential application from the row selector and the column selector.

The unit cell circuit is preferably connected in series between a second column selection unit, preferably a transistor and a second row selection unit, and also preferably a second source, such as a voltage and / or current source and a node, usually the aforementioned current output node. It further includes a transistor. In a preferred embodiment, the first source is the power supply potential Vcc and the second source is a reference potential such as ground. Preferably the nodes are identical and there is a serial connection between Vcc and ground of the first column selection unit and the second row selection unit, the node, and the second row selection unit and the second column selection unit. Optionally, the return electrode is biased at a potential between the potential of the second source and the second source, such as Vcc / 2.

In another feature of this embodiment, a test circuit is included. The inspection circuit can be used to ensure circuit continuity by allowing inspection before application of the fluid. The first check transistor is provided between the first column select and the first row select transistor. In addition, a second check transistor is provided between the second column select transistor and the second row select transistor. Select drive ensures circuit continuity. Alternatively, the test circuit function may be performed by special programming of the row and column transistors, such as first and second row selection and turn on of the first and second column selection transistors.

In another feature of the invention, the supply current for the test position is variable. Examples of current change over time may include static direct current (ie, direct current that does not change over time), square wave, sine wave, sawtooth wave, or any waveform that changes over time. In one embodiment, the current is supplied to the column selection circuit, whether or not changing over time, and optionally provided in a digital manner in the column line for coupling with the selected electrode. Synthetic analog and digital technologies allow significant control of the waveform and the value of current supplied to the individual electrodes. Waveforms, such as current waveforms, can be generated in an on chip or off chip manner. In addition, the control or manipulation of the entire circuit, and / or the generation of signals such as current waveforms, may be achieved through the use of digital / analog converters (DACs), central processing units (CPUs), and the use of local memory for storage of values. It can be generated through the use of a clock generator for timing and control of waveforms, and through the use of a digital signal processor (DSP).

In one aspect of the invention, a system based on current control of the first current is used to effect control of the second current. Preferably, a current mirror structure is used. The current supply provides a variable current value used in the voltage generator circuit. In a preferred embodiment, multiple current sources are used and their outputs are summed up under selective control of the memory for selective inclusion. Preferably, a variable voltage is generated at the node using a voltage divider circuit that receives a variable current output. The variable voltage of the node is coupled to the control element of the unit cell, and the control element preferably provides a variable resistor between the first voltage and the output node. The variable control element thus provides a variable current output. In this way, a relatively high value of the first current can be used to control a relatively small value of the second current, which is supplied to the conductive solution applied to the active electronic device for molecular biological analysis and diagnosis. In one embodiment, current reduction by a factor of 32 allows for easy generation and provision of current to the controlled device, and generates a current of the magnitude required for effective operation of the active biological device.

In another aspect of the invention, various devices may be packaged or covered by various capture sequences. This capture sequence can be relatively short, such as when the collecting electrode is a complexity reducing electrode. In addition, relatively long capture sequences can be used where additional specificity or selectivity is required. Such a capture sequence can preferably be included on the collection electrode or between the transfer electrodes.

Accordingly, it is an object of the present invention to provide an active biological device capable of reducing manufacturing costs and achieving small size micro-locations.

Another object of the present invention is to provide a device with improved functionality.

It is still another object of the present invention to provide an apparatus capable of achieving high functionality and operability while having fewer parts than conventionally known techniques.

Another object of the present invention is to provide an apparatus which is easy to manufacture compared to the prior art.

It is yet another object of the present invention to provide a circuit and system that can eliminate or reduce the pin limit or pin out limit.

It is a further object of the present invention to provide a system that provides accurate current control in active electronic devices that is suitable for molecular biological analysis and diagnostics and can interface with larger currents generated by the control system.

TECHNICAL FIELD The present invention relates to a device useful for performing active biological manipulation and a method of manufacturing the same, and more particularly, to a device having an active electrode particularly suitable for the electrophoretic movement of nucleic acids, and a method of manufacturing the same.

1A and 1B are cross-sectional views (FIG. 1A) and a perspective view (FIG. 1B) of an active, programmable electronic matrix device (APEX).

2 is a plan view of one embodiment of the present invention using variable size electrode regions and focusing electrodes, also referred to as bug chips.

3 is a plan view of one embodiment of the present invention using a focused electrode and a pair return electrode, which is particularly useful in a method of effectively transferring charged biomaterials in a wave or sweeping manner across a microposition.

4 is a plan view of one embodiment of the present invention using a substantially circular structure with a substantially centered focusing electrode.

5A, 5B and 5C are perspective views of a flip chip system, and FIG. 5D is a cross sectional view, FIG. 5A shows a lower side of the system, FIG. 5B shows a perspective view of a top of a flip chip structure including a sample chamber, and FIG. 5C shows Top perspective view of the via, and FIG. 5D is a cross sectional view of the flow cell.

6A and 6B are perspective and cross-sectional views of the flip chip system in one embodiment.

7A and 7B are side and top views of an edge lighting system in one embodiment of the present invention.

8 is a micrograph of the barrier for a Norland 83H dam using a 1300 J / s fiber bundle source (flex polyimide removed) masked with a flex circuit.

9 is a block diagram of a plurality of unit cell array systems.

10A is a circuit diagram of a functionalized unit cell for use with the system of FIG. 9.

10B is a voltage / timing diagram of the circuit of FIGS. 9 and 10A;

10C is a current diagram over time of the circuits of FIGS. 9 and 10A;

FIG. 11 is a component level circuit diagram of a unit cell for use with the system of FIG.

FIG. 12 is a component level circuit diagram of a unit cell including additional test circuitry for use with the system of FIG.

13 is a schematic diagram of a circuit providing current control in an active electronic device.

14 is a component level circuit diagram of a current mirror.

15 is a component level circuit diagram of a column select circuit.

16 is a component level schematic of a row selection circuit;

17 is a plan view of a physical layout of a unit cell.

18 is a plan view of a layout of a part of the 20 × 20 inspection position unit;

19 is a block diagram of an overall control and inspection system in one aspect of the present invention.

20 is a schematic block diagram of wiring between an input system and a probe card for connection with an active biometric matrix system.

21 is a graph of hybridization as a function of specific and non-specific hybridization with and without field shaping.

FIG. 22 is a graph of average MFI / s at various focusing of the Example of FIG. 2 in various focused RCA5 BTR reporters at 50 mM histidine, showing specific / non-specific binding after washing. FIG.

FIG. 23 is a current linearity graph showing the electrode current output of nanoampere as a function of current n of microamperes. FIG.

1A and 1B show a simple version of an active programmable electronic matrix (APEX) hybridization system used in the present invention. FIG. 1B is a perspective view, and FIG. 1A is a sectional view taken along the line AA ′ of FIG. 1B. In general, the substrate supports a matrix or array of electrically addressable micropositions 12. For the convenience of explanation, various micro locations are indicated by 12A, 12B, 12C and 12D. A transmissive layer 14 is disposed over the individual electrodes 12. The permeable layer allows for the transfer of relatively small charges, but reduces or restricts the flowability of large charges, such as DNA, to prevent large charges from directly contacting the electrode 12 during testing. The permeable layer 14 reduces the electrochemical degradation occurring in the DNA by direct contact with the electrode 12, in part due to the extreme pH generated by the electrolysis reaction. It also minimizes strong, nonspecific DNA adsorption to the electrode. Attachment region 16 is disposed over permeable layer 14 to provide a specific bonding location for the target material. Attachment area 16 is indicated by 16A, 16B, 16C and 16D, respectively, corresponding to the display of electrodes 12A-D. Attachment region 16 may be effectively integrated or integrated with a transmissive layer (eg, 12A) by directly including the adherent material in the transmissive material.

In operation, the reservoir 18 includes a space above the attachment area 16 containing the desired material as well as the unwanted material for detection, analysis or use. Charged 20, such as charged DNA, is located in reservoir 18. In one aspect of the invention, an active programmable matrix system includes a method for transferring charged material 20 to any particular microlocation 12. In operation, the microposition 12 generates electroless electrophoretic transfer of any charged function specific assembly 20 towards the electrode 12. For example, if the electrode 12A is positive and the electrode 12D is negative, a force electric movement line 22 runs between the electrode 12A and the electrode 12D. Electrophoretic force line 22 causes the transfer of charged conjugate 20 with pure negative charge towards positive electrode 12A. The charged material 20 with pure positive charge moves by the electrophoretic force toward the negatively charged electrode 12D. When the functionalized pure negatively charged binder 20 contacts the adhesion layer 16A as a result of the movement by the electrophoretic force, the specific functionalized binder 20 is covalently attached to the adhesion layer 16A. Optionally, electrode 24 may be disposed outside of the array. Electrode 24 may optionally be a return electrode, versus It can act as an electrode, a batch (dump) electrode, or another electrode. Optionally, a flow cell can be provided adjacent to the device for receiving fluid.

2 is a plan view of one embodiment of the present invention using focusing electrodes 42,44 and optionally transfer electrodes 50,52,54. Apparatus 20 includes a substrate 32 composed of a sufficiently rigid nonconductive material and capable of supporting components formed therein. Substrate 32 may be a flex circuit (such as polyimide, polyester, ABS, or other material such as DuPont Kapton), a printed circuit board, or preferably a semiconductor material with insulation overcoating. Connector 34 is coupled to trace 36, which is coupled to other electrical components of the system. This part can be any conductor, such as copper or gold, or other conductor known in the art. Those skilled in the art will appreciate that not all connectors 34 can be used as in a system suitable for coupling with an edge connector system. In addition, the traces 36 may vary in width depending on the demands on the traces, particularly the current demands. Thus, some traces 36 may be wider, such as coupled to focusing electrodes 42 and 44 than traces 36 coupled to micro-locations within array 38. Array 38 is preferably in the form described in connection with FIGS. 1A and 1B.

The first collection electrode 40 and the counter electrode 46 are disposed on the substrate 32. These parts generally fit within the trace of the flow cell 58 (shown in dashed lines) and comprise a relatively large proportion, preferably at least 40%, more preferably 50% and most preferably 60%. The counter electrode 46 (often functioning as a return electrode) and the collecting electrode 40 are preferably disposed around the flow cell trace 58 and can surround substantially the trace, for example up to 80%.

Usually, collection electrode 40 and counter electrode 46 are disposed on substrate 32 such that the electrophoretic lines of force extend to almost all of the flow cell volume, such as 80% or more. For example, focus 40 and counter electrode 46 may be disposed near the periphery of trace 58 of the flow cell. In yet another embodiment, they may be disposed at opposite ends of the flow cell trace 58 (see eg FIG. 3). In yet another embodiment, the counter electrode almost surrounds the trace of the flow cell with the centered collection electrode (see eg FIG. 4). The relatively large proportion of coverage of the flow cell traces and their location aids in requesting efficient electrophoretic querying of the flow cell contents.

Referring to FIG. 2, focusing electrodes 42, 44 are disposed on a substrate 32 to focus material collected on collection electrode 40 into array 38. The focusing electrodes 42, 44 are preferably arranged in a mirror image Y or V-shaped pattern with an opening that at least partially surrounds the collection electrode 40. As shown, there are two symmetrical focusing electrodes 42, 44. One focusing electrode may be used, or two or more focusing electrodes may be used. As shown, the focusing electrodes 42, 44 are substantially parallel portions (adjacent to the array) and inclined portions extending in a symmetrical manner surrounding the transfer electrodes 50, 52, 54 (transfer electrodes, optionally collecting). Adjacent electrode 40). In other words, there are first and second electrodes disposed at least partially adjacent to the microposition array, and the spacing between the first and second electrodes adjacent to the array is between the first and second electrodes in other regions away from the array. Smaller than the interval The focusing electrodes 42 and 44 optionally include portions disposed on the opposite side of the array 38 from the collection electrode 40. The focusing electrodes 42 and 44 are preferably coupled to a lead 36 that is relatively larger than the lead 36 coupled to the array 38 to allow effective current and potential transfer.

Transfer electrodes 50, 52, 54 are optionally included. Closer to the array are shown electrodes of a monotonically decreasing size. The first transfer electrode 50 is relatively smaller than the collection electrode 40, the second transfer electrode 52 is smaller than the first transfer electrode 50, and the third transfer electrode 54 is smaller. Other sizes reduce current density mismatches between locations, reducing or eliminating burnouts that occur when too large current density mismatches exist. The transfer is effectively maximized. The size ratio between the large and small electrodes is preferably 2: 1, more preferably 3: 1, and may be larger, such as 4: 1 or more.

One field shaping protocol is as follows.

Negative Bias Positive Bias Current Bias Time

Counter electrode 46 First collection electrode 40 75 μm 30 seconds

Focusing electrodes 42, 44 First transfer electrode 50 25μA 90 seconds

(-0.2μA)

First collection electrode 40

Focusing electrodes 42 and 44 Second transfer electrode 52 5 μA 180 seconds

(-0.2μA)

First transfer electrode 50

Focusing electrodes 42 and 44 Second transfer electrode 54 3μA 420 seconds

(-0.2μA)

First transfer electrode 50

Second transfer electrode 52

Focusing electrodes 42, 44 rows 3 1.5 μA 120 seconds

(-0.2μA) (500nA / pad)

Second transfer electrode 52

Third transfer electrode 54

Focusing electrodes 42, 44 rows 2 1.5 μA 120 seconds

(-0.2μA) (500nA / pad)

Second transfer electrode 52

Third transfer electrode 54

Focusing electrodes 42, 44 rows 1 1.5 μA 120 seconds

(-0.2μA)

Second transfer electrode 52

Third transfer electrode 54

Step 7 of the field shaping protocol effectively sends a response command signal to the sample volume and collects material on the array 38 for analysis. In the first step, the response command signal of the sample volume is transmitted through the negative bias of the counter electrode 46 and the positive bias of the first collecting electrode 40. The placement of the counter electrode 46 and collection electrode 40 around the trace of the flow cell 58 allows for a fast and effective response command signal to the sample volume. In the second step, material is collected adjacent to the collection electrode 40, the collection electrode 40 becomes negative (rebounded) with respect to the associated material, and the first transfer electrode 50 becomes positive (gravity). Repulsion and attraction force the transfer of material from the collection electrode 40 to the first transfer electrode 50. In addition, the focusing electrodes 42 and 44 are negatively biased. This negative (repulsion) bias provides a force that can be laterally in the conveying direction, so that the material in the solution is more concentrated. In a third step, material is collected in the first transfer electrode 50, the first transfer electrode is negatively biased (rebound), and the second transfer electrode 52 is positively biased. Collecting electrodes 42 and 44 may be negatively biased to provide repulsive force to the charged material to provide transverse components in their direction of movement to collect the material into smaller physical regions or spaces. In a fourth step, the second transfer electrode 52 can be negatively biased, with a positive biased third transfer electrode 54 at these electrodes with optional biasing of the first transfer electrode 50. The transfer can be carried out. Again, focusing electrodes 42 and 44 can maintain their negative bias. The next three steps can then be selectively separated as described above to transfer material to various rows or regions of the array.

Field shaping protocols include current and bias time. In this embodiment, there is an inverse relationship between the size of the electrode and the amount of current supplied thereto. In addition, for the collection electrode 40 and the transfer electrodes 50, 52, 54, there is an inverse relationship between the electrode size and the bias time. In other words, the smaller the electrode, the longer the bias time. Through this protocol, the current densities in the various devices remain relatively more uniform, optionally almost similarly. In addition, as the current from a given electrode decreases (relative to larger electrodes), a relatively longer bias time is required to provide an effective amount of transfer of charged material between the various electrodes. In other words, a relatively longer bias time may be required to carry a given amount of material at a lower current for a given amount of charged material.

3 is a plan view showing another embodiment of the present invention. As shown in FIG. 2, the device 60 includes a substrate 62, a connector 64, a trace 66, and a microposition array 68. 2 and the previous description apply to the corresponding structure in the other figures. Also, the trace 66 leading to the upper left of the array 68 has been cut for simplicity of the drawing. The same applies to the structure corresponding to that shown in the lower right of the drawing. Trace 66 is the same as a relatively wider trace 66 can be used for greater current carrying capability (eg, trace 66 for first collection electrode 70 and second collection electrode 72). It can have a width or a variable width.

3 shows at least a portion of the first transfer collection electrode 70 adjacent to the array 68 starting from FIG. 2. In the embodiment of FIG. 3, the first collection electrode 70 is trapezoidal, preferably shorter upper side 70t and lower side (near than the long side 70b and bottom side 70b adjacent and parallel to one side of the array 68). It has the inclined side edge 70s tapered widely toward 70b). The second collection electrode 72 is disposed on the other side of the array 68 and has a similar shape and size (not necessarily the same). The upper side 72t is preferably shorter than the lower side 72b, so that the side edges 72s are not parallel but inclined to each other and move toward the array 68. Optionally, the electrodes 70, 72 may have other sizes such that the area of the first collection electrode 70 is almost 10% smaller (optionally nearly 20%) than the second collection electrode 72. Inlet electrode 74 and outlet electrode 76 are optionally included in traces of flow cell 78 on substrate 62. Inlet electrode 74 and outlet electrode 76 are the same size or different sizes.

In operation, the contents of the flow cell receive a response command signal by placing or biasing one of the first and second collection electrodes 70, 72 to pull (usually in positive amounts) the material to be collected. The collected material may be transferred towards the array 68 at the first collection electrode 70. The material can be effectively held on the array 68 by application of an AC field, such as, for example, a frequency in the range of 0.01 to 10 ⁶ Hz. The material may then be transferred to other electrodes 70, 72, or may be reacted repeatedly by moving the material from array 68 to electrodes 70, 72. Optionally, the micropositions of the array 68 can be electrically active or passive.

4 is a top view of a concentric ring electrode embodiment. The device 80, the substrate 82, the connector 84 trace 86 and the array 86 are as described above except that the array 86 is arranged concentrically. Preferably, the rounded concentric return electrode 90 and the central focusing electrode 92 interact to focus the material on the electrode 92 and then move to the array 92. 2 and 3 described above, the traces are shown in a cut manner.

2, 3, and 4, a capture sequence or probe can be placed on the device. Preferably they are located at least on the collecting or focusing electrode. Optionally, another sequence is disposed on the other electrode, such as the transfer electrodes 50, 52, 54 of FIG. For example, each sequence may be more specific when access to the array is made.

5A, 5B, 5C, and 5D are views showing the bottom, top, top and side exposed vias 109 taken through line AA ′ of FIG. 5B of a flip chip system. Apparatus 100 includes a first surface 104 (optionally an upper surface) and a second surface 106 (optionally) of a material suitable for the function of support and conduction, such as materials such as flex circuits, printed circuit boards, semiconductor materials, and the like. And a support substrate 102 having a bottom surface thereof. Contact 108 is connected to trace 110, which is connected to second substrate 112. The second substrate 112 may be referred to as flip chip. The second substrate 112 may be a chip, system or support optionally provided with analytical or other diagnostic material. Bump contacts such as solder bumps, indium solder bumps, conductive polymers or silver filled epoxy provide electrical contact between the traces 110 and the chip or substrate 112. An encapsulant is provided between the second surface 106 of the support substrate 102 and the first surface 114 of the second substrate 112. Generally, the opposing sides of the support substrate 102 and the second substrate 112 are those arranged in a fluid barrier contact manner through the inclusion of a sealant. Inlet 120 may be conductive to sample chamber 122 and may also be connected to assay chamber and outlet 126. 5C shows a perspective view of the support and the vias 128 formed therethrough. As shown, the side width of the via 128 is smaller than the side width of the second substrate 112. The second substrate 112 is shown in dashed lines, which is disposed below the substrate 102 in the diagram of FIG. 5C.

In a preferred embodiment, the device 100 is composed of a minimum number of components to reduce costs, reduce manufacturing simplicity, reliability, and the like. One embodiment is obtained with five parts. The device can be manufactured in five parts, while the addition of parts is possible without departing from or changing the inventive concept. These parts are as follows. First, there is a support substrate having a first surface 104 and a second surface 106 supporting electrical traces and vias 128 disposed between the first and second surfaces to allow fluid flow. Second, electrical traces connected to at least a first surface disposed to face the second surface 106 of the first substrate, and to a microposition array suitable for receiving fluid through the vias 128 (see FIGS. 1A and 1B). There is a second substrate 112 comprising a. Third, there is a conductive bump 128 that interconnects the electrical trace on the second surface of the support substrate and the electrical trace on the first surface 106 of the second substrate. Fourth, a fluid seal between the first substrate 102 and the second substrate 112 is disposed between the second surface 106 of the support substrate 102 and the first surface 114 of the second substrate 112. There is a sealant 130 to provide. Fifth, the flow cell is selectively disposed on the first surface 104 of the first substrate 102. While the number of elements varies, an advantage can be obtained from the selection of these five elements.

In operation, a sample is provided to the inlet 120 and moved to the sample chamber 122. The sample chamber 122 houses various sample processing functions, including but not limited to cell separation, cell analysis, cell component separation, complexity reduction, amplification (eg, PCR, LCR enzyme technology), and / or disruption. Can be used. The sample then moves to analysis chamber 124. The solution containing the sample moves down through the vias 128 (this can be shown in FIGS. 5C and 5D but is obscure in FIG. 5B by the analysis chamber 124). A space containing via 128 is formed and bounded below by a second substrate, wherein a sealant or adhesive 130 is formed on the second surface 106 of the support substrate 102 and the second substrate 112. 1 A barrier is formed between the interfaces of the surface 114.

In a preferred manufacturing method, a photocurable sealant is inserted or provided at the interface between the second surface 106 of the support substrate 102 and the first surface 114 of the second substrate. Light is provided through the vias 128. A dam is formed to prevent the progress of the sealant, thus maintaining the array with almost no sealant or adhesive (FIG. 8 showing the sealant free area of the array, the tip of the cured dam and the sealant on the exterior of the device). Fine picture). By suiting the size of the side widths of the vias 128, the vias 128 act as a blocking mask from incident light to cure the sealant. In addition, the sealant may be supplied at a viscosity such that an amount does not flow onto the array 18 at the interface between the second surface 106 of the support substrate 102 and the first surface 114 of the second substrate 112. Can be. Additional or final curing of the sealant may be carried out as required, for example by heat.

6A is a perspective view of a flip chip system according to an embodiment of the present invention, and FIG. 6B is a sectional view. 6A and 6B include an edge illumination member 140, a unique sample chamber 134 design and a butterfly input and output chamber design compared to FIGS. 5A, 5B and 5C. The chip or substrate 130 has a first surface 130t and a second surface 130b, at least the first surface 130t including an electrical region or trace 130 thereon or in it. Although the embodiment shown in cross-sectional view in FIG. 6B shows a trace 132 disposed on the top surface 132 of the chip or substrate 130, the electrical region may be the chip or substrate 130, for example, through the provision of a semiconductor region. It may be included in whole or in part. This semiconductor region may be controlled in an active manner to provide selective connectivity within the chip or substrate 130. Typically, the first surface 130t is the surface on which active biological interaction occurs. Optionally, the edge illumination member 140 may be disposed on approximately the same plane adjacent the first surface 130t of the chip or substrate 130. Illumination sheet 140 preferably includes holes, vias or passages 144 to allow passage of electrical connections. As shown, the illumination sheet 140 may be disposed directly on the conductive trace 132 or may be fixed directly to the adjacent support substrate sheet 150. Electrical traces 132 may be included on the first surface 130t of the substrate or chip 130. Conductive elements 136, such as solder connections, indium bumps, conductive polymers, and the like, couple conductive paths 132 on substrate 130 to conductive portions of contact traces 154. The contact trace is preferably contacted by a conductive member 156, such as a wire whisker wire or other contact for connection to the rest of the circuit. The sealant 180 is preferably disposed between the substrate or chip 130 and the next layer 150, such as a flex support layer.

In FIG. 6B, the conductive member 136 is shown on the left side and the sealant 180 is shown on the right side. Other conductive members not included in the cut plane are included to provide additional mechanical support between the substrate 130 and the trace support layer 150. The edge illumination layer 140 also includes a terminal edge 142 disposed toward the top surface 130t of the substrate or chip 130. The edge illumination layer 140 may terminate outside or inside the sealant 180. An adhesive layer 160 is disposed adjacent to the trace support layer 150 to provide an adhesion contact with the top layer 170. Top layer 170 may optionally include passages, indentations, or other cutouts as shown for inlet 176 and outlet 176 ′. As shown, the adhesive layer 160 may optionally be a die cuttable adhesive material, such as including release paper on the top and bottom surfaces prior to assembly. Suppliers of such materials include 3M or DuPont. As shown in FIG. 6A, the die cuttable adhesive material 160 may be cut to form all or part of the walls 162, 164 of the chamber. As shown, the structure can be made in any desired shape or flow cell structure.

Preferably, upper member 170 is provided. As shown, the upper member 170 may extend almost throughout the rest of the device. Optionally, the upper member 170 may form a window 172 or other receiving surface on top of the flow cell chamber. Preferably the upper member is formed of polycarbonate or polystyrene. In the structure of FIGS. 6A and 6B, usually an array of inspection positions is selectively accessed through the upper member 172, so it would be desirable to form the upper member with a material that is nearly transparent to excitation and radiation.

6A and 6B show the structure of the flow cell. In this butterfly structure the inlet 176 is connected to the first extension region 174, where the side wall of the chamber begins with the first size d and preferably monotonously and more preferably extends linearly to form a flow cell chamber ( Size D at a point close to 134). The region of the flow cell chamber 134 is characterized by substantially parallel sidewalls 166. Preferably, a first reduced width region is provided between the flow cell region and the output. Most preferably, the reduction zone begins with width D ', most preferably D' = D, and decreases with width d ', preferably d' = d. As shown in FIGS. 6A and 6B, the height of the inlet chamber 174 is reduced from the inlet of the height H to the lower height h at the inlet to the flow cell chamber. Preferably the reduction is monotonous and most preferably linear.

In a preferred embodiment, the height h and width w of the inlet chamber are selected such that a substantially constant flow area is provided, ie the product of height h and width w (h × w) is approximately constant. Thus, as shown in the coupling view of FIGS. 6A and 6B in the portion of the inlet chamber adjacent to the inlet, the height h is relatively large and the width w is relatively small. Thus, when proceeding through the inlet chamber to the flow cell chamber, the height h increases as the width w increases. Preferably, the withdrawal chamber has a substantially identical structure, and preferably the same flow cell area is constant.

7A and 7B are cross-sectional and top views of an edge illuminated flip chip system according to one embodiment of the invention. Wherever possible, the same numbers as the elements in FIGS. 6A and 6B are used. The support substrate 150 is generally planar and includes a first surface 150t and a second surface 150b. Via 126 (shown in broken line in cross section) allows flow of fluid or solution from support substrate 150 to second substrate 130, in particular to first surface 130t of second substrate 130. do. The sealant 180 is provided between the second surface 150b of the support substrate 150 and the second substrate 130. The sealant 180 preferably provides fluid sealing to allow fluid flow to the array on the second substrate 130. A light source 190, such as a laser bar, illuminates the array on the second substrate 130. Preferably the system includes a waveguide 140 having an input 146 suitable for receiving light from the light source 190 and providing illumination through the output 142. Waveguide 140 is preferably coplanar with support substrate 150 and can be secured to it by being attached to second surface 150b of support substrate 150. An electronic device 192 for controlling the system may be included. Optionally, a surface mount electronic component 130 may be included on the substrate. A fluid engineering system 194 is provided with the system to help provide a sample to the second substrate 130.

9 is a block diagram of a plurality of unit cell arrays. In a preferred embodiment, the system or chip comprises a multi-position array with electrical repeat position cell positions. Usually, an array consists of rows and columns, more generally the same number of rows and columns, and most commonly a diagonal structure of rows and columns. For example, arrays of 10 × 10, 20 × 20 or more can be formed by this technique. The individual unit cells 212 of the array of unit cells 210 are selected by the action of selectors such as row selector 220 and column selector 230. The selectors 220, 230 may be a memory, such as a shift register memory, or a decoder, or a combination thereof. Input for address information may be used on-chip address generators but usually receive addresses from off-chip. In a preferred embodiment, row selector 220 includes shift registers in a wider structure, such as a 1x1 structure or a 1x4 structure. In operation, the selection register is sequentially loaded with a value indicating selection or non-selection of the unit cell 212 and optionally an output value for that cell. Optionally, a memory may be provided to maintain these values to continue output from the unit cell.

9, the array 210 includes a plurality of unit cells 212. In a preferred embodiment, the unit cells 212 are arranged in rows and columns, in which the display rows represent a horizontal structure for the text and the columns are shown a vertical structure for the text. You will know). Display rows and columns may also be referred to as groups or subsets of unit cells 212, such as portions of rows or columns, or groups or sets of unit cells 212 that are not linearly continuous. In general, there are unit cells 212 of m rows and n columns, where m = n and m = 2, 3, 4,... For example, the 5 × 5 matrix unit cell 212, the 10 × 10 matrix unit cell 212, and the 20 × 20 matrix unit cell 212 provide a total of 25, 100, and 400 unit cell numbers.

In FIG. 9, various levels of complexity of the unit cell 212 are shown. The unit cell 212 shown at the top is shown as a single block unit, while the unit cell 212 shown in the center of the figure is more complex, such as the structure described in greater detail in FIG. Such alternatives are shown for convenience and variety of description, and in an exemplary embodiment it will be appreciated that the structure of the individual unit cells 212 is the same for a given device.

The unit cell 212 is addressed by the action of at least one row selector 220 and at least one column selector 230. This detailed description begins with the case of the single row selector 22 and the column selector 230, and later describes the use of additional selectors 220 ', 230'. The row selector 220 receives the input information 222 and outputs a row select signal 294 on one or more row lines 224 (see FIG. 10). The select signal on row line 224 is supplied to unit cell 212 and interacts with it via, for example, row contact 226. As shown, a portion of the row line 224 is shown to the left and a portion to the right of the unit cell 212 centered on the array 210. In an exemplary embodiment, row line 224 may be of any material combination, but is electrically continuous. For example, the row line may be one continuous conductive line formed, for example, of conductive polysilicon, and may be a combination structure, such as when the conductive segments are electrically connected through a higher conductive material of a metal such as aluminum.

Column selector 230 receives an input 232 for determining row selection, or, in a preferred embodiment, the value (or correction value) of the output at unit cell 212. The column selector 230 is coupled to a column line 234 that provides a column select signal 296a-d to the unit cell 212. In a preferred embodiment the column selector 234 is coupled to the unit cell 212 via, for example, a thermal contact 236. In a preferred embodiment the thermal contact may be the control gate of a transistor, for example a field effect transistor (see FIGS. 11 and 12).

If necessary for driving the unit cell 212, a second row selector 222 ′, an input line 222 ′, a second row line 224 ′, and a column contact 226 ′ may be included. In addition, a column selector 230 ′ with an input 232 ′ may be added and coupled to the second or auxiliary column line 234 ′ coupled to the second column contact 236 ′.

As shown, row selector 220, 220 ′ and column selector 230, 230 ′ optionally include enable inputs 228, 228 ′ or chip select 238, 238 ′. One of the functions of such a signal is to allow the input of input information 222, 222 ', 232, 232' without driving row lines 224, 224 'or column lines 234, 234'. In addition, some or all of the row selector 220, 220 ′ and column selector 220, 220 ′ and column selector 230, 230 ′ may be used for the output of information, such as outputs 229, 229 ′, 239, 239 '). In one application, the output value may be a signal or bit indicating that the input data has been successfully loaded into the row selector 220, 220 'or column selector 230, 230', such as the most significant bit of the series. Optionally, this output information can be used to trigger the enable or chip select signals 228, 228 ', 238, 238'.

Within the level of detail of FIG. 9, row selectors 220, 220 ′ and column selectors 230, 230 ′ receive and use row and column input information 222, 222 ′, 232, 232 ′ and use one or more unit cells. 212, and optionally provide a signal value indicating the current (potential) level to be provided from the unit cell 212. The selectors 220, 220 ', 230, 230' may be in the form of a memory as in the form of a shift register memory or in the form of a decoder circuit, such as when the desired address is provided as input information and the output is in a decoded relationship with it. . Many circuits are known for performing this function.

Current source 240, such as a current mirror, optionally receives current source 242 and control signal 244 (VCASP). Connections 246 and 246 'couple the current from source 240 to column selector 230 and, if present, column selector 230'. As shown, the join lines 246, 246 'are separate lines (indicated by "a" to indicate the same number of wires as a). In addition, one or more current sources 242 may be provided. As described below with respect to FIG. 10C, the current value may be static and may change over time (such as in applications of pulse waveforms, sinusoids, square waves, sawtooth waves, etc.). In general, any desired variable waveform can be used.

Using the structure shown in FIG. 9, each of the unit cells 212 can be driven at a given time. Alternatively, certain unit cells 212 may be driven and other unit cells remain inactive. For example, if a given column is selected as the first value, each of the unit cells in the column associated with one or more selected rows selected by row selector 220 is driven to a value corresponding to the voltage level on the column. Other unit cells in the same column can be brought to the same or different levels by being coupled to the second column selector 230 ', where one or more row lines associated with the unit cell are driven by the second row selector 220'. Thus, in one column of unit cells, each unit cell 212 is driven with a value corresponding to a signal on column 234 associated with column selector 230 or a value on a column associated with second column selector 230 '. Can be driven, undriven, disconnected, floating, or in high impedance. In the same way, other columns can be set to the desired output level. In this way, the entire array of unit cells can be placed in a desired state or set of states. The use of the term output level and desired state includes a signal that changes as a function of time. In addition, more values within a given column 234, 234 'may be added, for example, through the addition of additional column selectors and column lines coupled to the selected unit cell 212.

10A illustrates a block diagram of the unit cell 212 and the return electrode 250. As far as possible, the number in FIG. 10 corresponds to that adopted in FIG. 9. Variable current control element 260 includes an input 262, an output 264 and a control element 266. Control element 266 is coupled to the same line as column line 234 coupled to column select 230. Selector switch 270 includes an input 272, an output 274 and a control element 276. Control element 276 is coupled to a control line, such as row line 224. The output 264 of the variable current control element 260 is coupled to the input 272 of the select switch 270. Output 274 of select switch 270 is coupled to a node providing an output current 282 (Iout). A second potential 284, such as Vcc, is provided at the input 262 of the variable current control element 260.

In operation, the application of a signal on the row line 224 to the input 276 of the selector switch 270 provides a conductive path between the node 280 and the output 264 of the variable current control element 260. . The signal value applied to the column line 234 coupled to the input 266 of the variable current control element 260 is a variable current control element 260 connected in series between the first potential node 284 and the output node 280. And a variable amount of current flowing through the selection switch 270. Although the return electrode may be another unit cell 212, the return electrode 250 completes the circuit.

In a preferred embodiment, the variable current control element 260 is a field effect transistor, in particular a transistor such as a MOSFET. The select switch 270 is preferably a transistor, more preferably a field effect transistor, most preferably a MOSFET. Various types of implementations may be used, such as CMOS, NMOS, CMOS, bipolar, gallium arsenide, or other structures, so long as they match the functional requirements of the system. Also in a preferred embodiment, the matching structure of the second variable current control element 260 'and the second select switch 270' is coupled between the second potential 284 'and the output node. Optionally, the channel lengths of the various devices can be arranged to achieve a symmetrical structure. For example, in a CMOS implementation, the P channel select device may have a shorter channel length than the N channel device to compensate for other electron / hole mobility (eg, 80 μ vs. 126 μ channel length). Similar numbers can be adopted for adding primes. The above description of the circuit applies to a circuit including a second variable current control element 260 ′ and a select switch 270 ′.

10B illustrates a signal that is a function of time for an exemplary control signal of unit cell 212. Generation of output signal 290 indicates completion of data input to selectors 220, 220 ', 230, 230'. Output signal 290 may be used to trigger or drive enable signal 292. The enable signal 292 may allow the selection signals for the row selection 294 and the column selection signals 296A, 296B, 296C, and 296D to be transmitted to the unit cell 212. As shown, a single row select signal 292 is provided, which is preferably supplied to select circuits 270 and 270 'having two-phase operation. The column select signals 296A, 296B, 296C, and 296D may be other values, preferably two or more values, in a preferred embodiment including at least four values, which are variable current control elements 260 and 260 '. Are provided at inputs 266 and 266 '. As described below, these values can be static or dynamic.

10C shows exemplary values of current (or voltage) as a function of time that can be supplied to the electrode. In one embodiment, a static direct current (source or sink) is provided that does not change over time. Although the value of the current is static, it will be understood that the choice of whether to allow this current to drive the electrode, usually digital selection, is used so that the electrode is selectively driven as a function of time. The second waveform of FIG. 10C represents a square wave. The square wave may be for unit direction current or bidirectional current. Offset bias can be used as needed. As shown in the third waveform of FIG. 10C, the waveform may have periodicity with subcomponent waveforms contained therein. The fourth waveform in Fig. 10C shows a general sine waveform. The fifth waveform of FIG. 10C is a sawtooth waveform. It will be appreciated that any waveform consistent with the object of the present invention may be used in connection with the devices and methods disclosed therein. A high degree of flexibility and control can be achieved by supplying waveforms that are selectively controllable by digital selection (eg, via the action of row selector 220 of FIG. 9), more specifically current waveforms. In addition, the waveforms provided at various test positions in the apparatus need not be the same. For example, a static DC waveform may be applied to the first column, a square waveform may be applied to the second column, and a sinusoidal waveform may be applied to a minute position in the column selected by the row selector.

The current waveform can be generated on or off chip. In practical implementations, the waveforms are all optionally controlled under the control of a central processing unit (CPU) or other version of microprocessor control, digital / analog converters (DACs), digital signal processors (DSPs), variable current waveform generators, Can be created through the use of on-chip memory.

11 and 12 are circuit diagrams of a unit cell driving circuit of one embodiment of the present invention. 12 includes a test circuit, such as a test transistor, but does not include FIG. The commonalities of these figures are described together.

In a preferred embodiment of the unit cell 212 a symmetrical structure is used. The first column selection unit 260, preferably the transistor, and the first row selection unit 270, preferably the transistor, are in series between a first source, such as a voltage and / or current source and a node, usually a current output node. Is connected to. In a preferred embodiment, the column select transistor 300 can be precisely controlled under the application of a gate voltage, for example from a column shift register memory (see FIG. 15). Preferably, the selection units 260, 260 'can vary their controllability, for example by varying the channel length in the control transistor. Thus, the application of the potential to the control gates 302, 312 by the application of the potential from the row selector 220, 220 ′ and the column selector 230, 230 ′ generates an output 282 of current in the unit cell. Let's do it.

The unit cell circuit 212 is a second column selection unit 270 connected in series between a second source 284 ', for example a voltage and / or current source and a node, usually a node 280 described above, i.e. a current output node. '), Preferably transistor 300' and second row select unit 270 ', preferably transistor 310'. In a preferred embodiment, the first source 284 is a power supply potential Vcc and the second source 284 'is a reference potential such as ground. Preferably, the nodes are the first column selection unit 260, 260 ′ and the second row selection unit 270, the node 280, the second row selection unit 270 ′ and the second column selection unit 260 ′. Is the same node 280 connected in series between Vcc 284 and ground 284 '.

In another form of operation of the circuit, alternatively in another mode of operation of the circuit shown in FIG. 11, the circuit drives each of the first and second row and column select transistors 260, 270, 260 ', 270' simultaneously. Continuity can be checked. In this way, the source 284 and sink 284 'are directly conductively connected.

In another aspect of the preferred embodiment a test circuit is included. 12 shows a schematic of such a system. The first test transistor 320 is between the first column select transistor 260 and the first row select transistor 270. Also, the second test transistor 330 is between the second column select transistor 260 'and the second row select transistor 270'. Selective driving ensures continuity of the circuit.

The circuit described herein may be implemented in any known technique suitable for achieving the desired functionality of the present system, but one preferred implementation mode is through a CMOS circuit. In one embodiment of the circuit of Figures 11 and 12, row select elements 260, 260 'include a relatively long channel length. These relatively large field effect transistors provide more accurate current control. For example, one embodiment of this circuit is a transistor with a channel length of 6 μm. The upper column selection unit 260 (controlled by VI_P_CSEL) has a channel length of 80 μm, and the lower column selection unit 260 '(controlled by signal VIN_N_CSEL) has a channel length of 126 μm. The difference in channel length represents the difference in mobility of electrons and holes and is measured to balance these two devices. In comparison, the remaining elements of FIGS. 11 and 12 have a channel length of 6 μm and a channel length of 4 μm. Other implementations of unit cells include series connection of transistors including dedicated series select transistors for row and column selection, and additional transistors for output level selection (current or voltage). More broadly, any circuit that receives location selection information (such as row and column selection) and value and / or polarity information and generates an output of the desired current or potential can be used.

13 is a schematic diagram of a current control system useful in the present invention disclosed therein. As far as possible, the numbering is consistent with the other figures. The circuit is selectively controllable to generate a voltage at node 342 coupled to line 234 (shown as column line 234) coupled to control element 266 of variable current control element 260. Receive current 340. The output currents from the variable current control element 260, the row select switch 270, the first power supply voltage 284 and the node 280 are as described above. Similarly, current control circuits can be used to control symmetric circuits (eg, elements 260 ', 270' of FIG. 10).

The input current 340 is supplied to the control element 344. Each current of a given subscript is fed to the control element 344 of the same subscript. The control element 344 selectively supplies current to the output 346. As shown, the current output 346 is summed so that the current at node 348 can vary based on the state of the switch or control elements 344a-d. A voltage divider is provided and a potential 350 is supplied to a resistor 352 connected to the node 342. A variable voltage is provided to node 342 by supplying current at node 348 to node 342 via resistor 352. Optionally, resistor 342 may be a device such as a transistor that conducts only when current is supplied to node 348. Thus, when each switch 344a-d remains off, this circuit does not include a resistor 352 in any conducting state (see Figure 15 for a detailed implementation).

14 is a detailed circuit of a portion of the current mirror used in the present system. Four identical circuits are shown in FIG. 14, and the description of one circuit applies equally to all circuits. Current node 400 is coupled to the output of first transistor 402 and second series connected transistor 404, which are coupled to first potential 406 (Vdd). Optionally, transistors 402 and 404 are biased or connected to power supply voltage 406. The control gate 408 of the second series connected transistor 404 is connected to the current node 400. Current node 400 is also connected to the output (source or drain) of first transistor 402. The control gate 410 of the first control transistor 402 is controlled by the signal 412. Signal 412 acts as a selection signal for the current mirror. The select signal 412 is supplied to the current mirror to generate a selective provision of current from the current node 400 to the column select circuit.

15 is a detailed circuit diagram of a column selector circuit (see, for example, column selector 230 of FIG. 9). A shift register is provided by a series of flip-flops 420, the first flip-flop receiving input information Q (0) that is optionally inverted by inverter 422 as input. As described in connection with FIG. 9, an optional output 229 may be provided from a selector such as a shift register 230. As shown, two stages, each containing four bits, are shown for the shift register. In an implementation, a 20x20 matrix or unit cell array requires 80 bits in the shift register if 4 bits are allocated to each column. The output 430 is supplied as a control signal to the current control circuit 432. As shown, the current control circuit is a first of opposite conductivity type with a control gate coupled to a signal supplied directly to the first transistor 434 and through the inverter 438 to the second transistor 436. The parallel arrangement of the transistor 434 and the second transistor 436. In operation, current supplied to node 440 is selectively supplied to output node 442 under control of signal 430. Current at the output node 442 is summed with currents from three different control circuits for the column position, which summation occurs at or before node 496.

The summed current at node 496 is sent to node 492 which acts as a voltage tap for column line 434. Logic 440, shown here as a NAND gate, receives the output of inverter 438 as an input. The output of the inverter is supplied to a NAND gate 440 which logically acts as an OR for the various inputs. Thus, selection of any one of a variety of current sources drives the gate transistor controlled by logic element 440.

Shift register 126 includes a plurality of serially connected flip-flops 420. The value signal is provided as an input to inverter 422 and then to the D input of flip-flop 420. The clock signal CM and the chip select signal CS are supplied. The output of the final flip-flop 420 (top right of FIG. 15) is fed to an inverter that provides an output bit to node 424.

16 shows a component level schematic for the shift register 450. Flip-flop 452 receives an input 454 (Q (0)) that is sent to flip-flop 452 through the Q output of one flip-flop to the D input of next flip-flop 452. Optionally, output 456 provides an indication of the most significant bit (or other load indicator) from the shift register. The enable signal 460 is provided to logic 462 (shown as a NAND gate) that receives as input the output (Q pin) of the associated flip-flop 452. The output of logic 462 controls pass circuit 464 that selectively passes signal 466 constituting row select signal 468 when it passes through it. The circuit repeats for the number of stages in the shift register 450, and this description applies to other stages as well.

17 shows a layout of an example of implementation of a unit cell. Column lines 234 and 234 'are shown vertically and are coupled to a column selector (see Figure 9). Row lines 224 and 224 'are shown horizontally. The power supply voltage VDD 500 and the second voltage 202 (VSS, eg, ground) are arranged parallel to the column lines 234 and 234 '. The optional test control lines 504 and 504 'supply control signals for the n test and p test circuits, respectively. Row line 224 is connected through conductive member 506 to gate 508 located above the underlying channel region. Row line 224 'is also coupled to gate 508' positioned over the channel region of the select transistor. Column lines 234 and 234 'are coupled to gates 510 and 510' located above the channel region, which are coupled to a first power source (VDD) 500 and a second power source voltage (VSS) 502, respectively. do. The channel lengths located under the gates 510 and 510 'are different and the difference in length is chosen such that the operating elements have similar suitable characteristics. The output of the switching transistor controlled by the gates 508, 508 ′ is supplied to the electrode 514 through the conductive member 512.

18 shows a plan view of a portion of a 20x20 array of unit cells. 18 shows a portion of the entire chip, and it can be seen that structures such as unit cells, shift registers, row and column decoders, and current mirrors are usually repeated identically throughout the chip. Multiple unit cells are included (shown in detail in FIG. 17). The opposite and return electrodes 520 are preferably disposed around the array of unit cells. The electrode 520 surrounds the unit cell array. Optionally, multiple electrodes can be used to surround the array. In a preferred embodiment, four L-shaped electrodes surround the array (shown in the corner), with each electrode surrounding almost one quarter of the array. These electrodes can be used to move unwanted materials and act as dump or batch electrodes. Row selector 220 ″ (having a number corresponding to the number in FIG. 9) is disposed on the right side of the array (electrode 520 in FIG. 18). Column selector 230 ″ is disposed outside of array and electrode 520. Current mirror circuit 240 ″ is optionally disposed at the corner of the chip. Selection and placement of chip components is made to optimize the function of the device. Inclusion of chip components, usually disposable components, increases device cost but allows for local control of functionality. Various arrangements are possible, but the structure shown in FIG. 18 is a preferred embodiment of a 20x20 chip.

19 is a schematic diagram of the entire system. The control computer 530 is coupled to the test board 532 and the probe card 534 via a bus 540. Optionally, a connector such as an RS 232 connector is used. The probe card 534 interfaces with the actual active electronics. Output from the device may be provided to a receiving system 542, which may include an analog / digital converter for providing digital data to the computer system 530 via the bus 520.

20 is an enlarged block diagram of the test board and the probe card of FIG. 19. Serial port connection 550 is coupled to a universal asynchronous receiver transmitter (UART) 552 via controller 554. Bus wiring is coupled to current source 556 and current sink 558. Various digital / analog converters are provided, such as dump DAC 560 and bias DAC. Shift register 264 is coupled to the probe card 534. Analog / digital converter 556 may receive an output signal, for example, from a probe card. If the shift register includes an output (see outputs 229, 229 ', 239, 239' and FIG. 9), a shift register loopback 568 may be provided.

21 shows a graph of electron hybridization using the chip of FIG. 2. The graph shows the fluorescence intensity at MFI / s as a function of column number. The three bar graphs, shown in columns 1, 2 and 3, show hybridization on the left bar graph compared to nonspecific hybridization on adjacent right columns using field shaping. The three pairs indicated by column 1, column 2 and column 3 above the indicator “standard” represent the same system without field shaping. The discrimination between specific versus non-specific combinations is substantially smaller than when field shaping is used. The sequence was ATA5 / ATA7 / biotin, and 10 pM RAC5 / BTR.

22 is an experimental graph performed in the system shown in FIG. The y axis represents the average MFI / second and the x axis represents the various rows of various concentrations. The first pair of paragraphs shows a 50 nM concentration of RAC5 / BTR reporter at 50 mM histidine and shows specific and nonspecific binding after washing. The first pair shows rows 1 and 2 comparing the specific binding (ATA5 / RCA5) to the nonspecific binding (ATA7 / RAC5), showing an improvement of 12: 1 and 50: 1. Middle pairs in the bar graph represent the 50 pM concentration of the RAC5BTR reporter and represent the 3.9: 1 and 4.9: 1 ratios of specific binding to nonspecific binding signal strength. The final set of paired bar graphs shows the 1 pM concentration of the RAC5BTR reporter and the 4.4: 1 and 4.0: 1 ratios of specific to nonspecific binding.

FIG. 23 is a graph of current linearity showing the electrode current output of nanoamperage as a function of the current input of microamperes. The description is provided to represent various lines on the graph.

Although the foregoing description has been given for the purpose of understanding and understanding the invention, those skilled in the art will understand that various modifications and changes can be made in light of the teachings of the invention without departing from the spirit or scope of the appended claims.

Claims (211)

In an electronic device for active biological manipulation, Support substrates; A microposition array disposed in a first region on the substrate; A first collection electrode disposed on the substrate; First and second focusing electrodes disposed on the substrate, the first and second electrodes having a proximal end and a distal end, wherein the proximal end is at least partially disposed adjacent to the microposition array and adjacent to the array; And a spacing between proximal ends of a second electrode is less than a spacing between distal ends of the first and second electrodes; And At least one counter electrode disposed on the substrate Electronic device comprising a. The electronic device of claim 1, further comprising at least one transfer electrode, wherein the transfer electrode is disposed between the first collection electrode and the array on the substrate. The electronic device of claim 2 having at least two transfer electrodes. The electronic device of claim 3, wherein the transfer electrodes have different sizes. The electronic device of claim 4 wherein the ratio of large electrodes to small electrodes is at least 2: 1. The electronic device of claim 4 wherein the ratio of large electrodes to small electrodes is at least 3: 1. The electronic device of claim 4 wherein the ratio of large electrodes to small electrodes is at least 4: 1. The electronic device of claim 4, wherein the transfer electrode decreases in size closer to the array. The electronic device of claim 8, wherein the reduction in size is monotonous. The electronic device of claim 2, wherein the at least one transfer electrode is smaller than the first collection electrode. The electronic device of claim 10, wherein an area ratio of at least one transport electrode of the collection electrode and the transport electrode is at least 4: 1. The electronic device of claim 1 further comprising a capture sequence. The electronic device of claim 12, wherein the capture sequence is disposed adjacent to the collection electrode. The electronic device of claim 12, wherein the collection electrode is a complexity reducing electrode. The electronic device of claim 1, further comprising a second power source for connection with the focusing electrode. The electronic device of claim 1, wherein the focusing electrode is negatively biased. The electronic device of claim 1, wherein the focusing electrode is actively biased to zero. The electronic device of claim 1 further comprising a flow cell. The electronic device according to claim 1 or 2, wherein the counter electrode and the first collection electrode are arranged to send a response command signal to a substantial portion of the volume of the flow cell. 20. The electronic device of claim 19, wherein the portion is at least 50%. 20. The electronic device of claim 19, wherein the portion is at least 75%. 20. The electronic device of claim 19, wherein the counter electrode and the collection electrode are disposed along substantially the entirety of the periphery of the trace of the flow cell. 23. The electronic device of claim 22, wherein the counter electrode and the collection electrode are disposed along at least 80% of the periphery of the prow cell trace. 20. The electronic device of claim 19, wherein the counter electrode and the collection electrode are disposed at substantially opposite ends of the flow cell trace. 19. The electronic device of claim 18, wherein an area of the collection electrode and the counter electrode proportional to the trace of the flow cell is at least 40%. 19. The electronic device of claim 18, wherein an area of the collection electrode and the counter electrode proportional to the trace of the flow cell is at least 50%. 19. The electronic device of claim 18, wherein an area of the collection electrode and the counter electrode proportional to the trace of the flow cell is at least 60%. 19. The electronic device of claim 18, wherein the flow cell comprises an inlet. 19. The electronic device of claim 18, wherein the flow cell comprises an outlet. A method of analyzing a biological sample using the electronic device for active biological manipulation of claim 1, comprising: Providing the sample to the device; Placing the first collection electrode to attract the desired charged biomaterial to focus the desired charged biomaterial on the collection electrode; Applying an electric potential to the focusing electrode to provide a component of a force across a line disposed between the collection electrode and the micropositional array; And Transferring material from the collection electrode to the microposition array How to include. 31. The method of claim 30, wherein the focusing electrode is actively biased. The method of claim 31, wherein the active bias of the focusing electrode is zero. The method of claim 31, wherein the bias is negative. 33. The method of claim 30, further comprising providing an attractive force bias to attract said desired sample to said transfer electrode. 31. The method of claim 30, wherein biasing the transfer electrode to attract the desired sample material after providing the desired sample to the collection electrode. 33. The method of claim 30, wherein the biasing is sequentially applied to the transfer electrodes. 31. The method of claim 30, wherein at a time after the desired material is collected at the collection electrode, the collection electrode is biased to repel the desired sample. 31. The method of claim 30, further comprising actively biasing at least a predetermined microposition in the micropositional array to draw the desired biomaterial. An electronic device for performing active biological manipulation on a solution containing a charged biomaterial, Support substrates; A microposition array disposed in an area on the substrate; A first transfer electrode disposed on the substrate; A second transfer electrode disposed on the substrate and having a different size from the first transfer electrode; And A control system coupled to the first transfer electrode and the second transfer electrode to allow selective activation of the electrodes such that transfer of the charged biomaterial from the first transfer electrode to the second transfer electrode occurs; Electronic device comprising a. 40. The electronic device of claim 39, wherein an area ratio between the first transfer electrode and the second transfer electrode is at least 2: 1. 40. The electronic device of claim 39, wherein an area ratio between the first transfer electrode and the second transfer electrode is at least 3: 1. 40. The electronic device of claim 39, wherein an area ratio between the first transfer electrode and the second transfer electrode is at least 4: 1. 40. The electronic device of claim 39, wherein the second transfer electrode is closer to the micro array than the first transfer electrode, and the second transfer electrode has an area smaller than the first transfer electrode. 40. The electronic device of claim 39, comprising at least a third transfer electrode, wherein the third transfer electrode is different in size from both the first and second transfer electrodes. 45. The electronic device of claim 44, wherein the first transfer electrode, the second transfer electrode, and the third transfer electrode are arranged to monotonously decrease in size as they approach the array. 40. The electronic device of claim 39, further comprising first and second focusing electrodes disposed on the support substrate, wherein the first and second electrodes are not parallel. 40. The electronic device of claim 39, further comprising a flow cell. 40. The electronic device of claim 39, further comprising at least a first counter electrode and a second quartz electrode arranged to send a response command signal to a substantial portion of the volume of the flow cell. In an active biological manipulation electronic device, Support substrates; A microposition array disposed on the substrate; A first collection electrode disposed on the substrate; First and second focusing electrodes disposed on the substrate, wherein the first and second electrodes are at least partially disposed adjacent to the microposition array, and a gap between the first and second electrodes adjacent to the array is Less than a gap between the first and second electrodes in another area away from the array; A first transfer electrode disposed on the substrate; A second transfer electrode disposed on the substrate and having a different size from the first transfer electrode; A control system coupled to the first transfer electrode and the second transfer electrode, the control system allowing selective activation of the electrodes to effect transfer of the charged biomaterial from the first transfer electrode to the second transfer electrode; And At least one counter electrode disposed on the substrate Electronic device comprising a. In an active biological manipulation electronic device, Support substrates; A microposition array disposed on the substrate; A first collection electrode disposed on the substrate; First and second focusing electrodes disposed on the substrate, wherein the first and second electrodes are at least partially disposed adjacent to the microposition array, and a gap between the first and second electrodes adjacent to the array is Less than a gap between the first and second electrodes in another area away from the array; A first transfer electrode disposed on the substrate; A second transfer electrode disposed on the substrate and having a different size from the first transfer electrode; A control system coupled to the first transfer electrode and the second transfer electrode, the control system allowing selective activation of the electrodes to effect transfer of the charged biomaterial from the first transfer electrode to the second transfer electrode; At least one counter electrode disposed on the substrate; And A flow cell disposed adjacent to the support substrate and surrounding the collection electrode and the counter electrode, wherein the counter electrode and the control electrode are disposed adjacent to the periphery of the flow cell; Electronic device comprising a. 51. The electronic device of claim 50, wherein the first transfer electrode and the second transfer electrode are disposed between the first and second focusing electrodes. 51. The electronic device of claim 50, wherein the first transfer electrode is disposed farther from the microposition array than the second transfer electrode, and the first transfer electrode is larger than the second transfer electrode. In an electronic device for biological manipulation, Support substrates; A microposition array disposed in an area on the substrate; A first collection electrode disposed adjacent said array on said substrate; And A second collection electrode disposed on the substrate adjacent the array and at least partially opposite the region; Electronic device comprising a. 54. The electronic device of claim 53, wherein the first collection electrode and the second collection electrode are disposed at substantially opposite ends. 54. The electronic device of claim 53, further comprising a flow cell, wherein the flow cell is supported on the substrate and suitable for defining traces of the flow cell. 56. The electronic device of claim 55, wherein the first collection electrode and the second collection electrode are disposed at substantially opposite ends of the trace of the flow cell. 54. The electronic device of claim 53, wherein the first collection electrode has at least 80% of the area of the region. 54. The electronic device of claim 53, wherein the first collection electrode has at least 100% of the area of the region. 54. The electronic device of claim 53, wherein the first collection electrode has at least 120% of the area of the region. 54. The electronic device of claim 53, further comprising a capture sequence. 61. The electronic device of claim 60, wherein the capture sequence is disposed adjacent to the collection electrode. 55. The electronic device of claim 53 wherein the collection electrode is a complexity reducing electrode. 54. The electronic device of claim 53 further comprising a focusing electrode. 56. The electronic device of claim 55, wherein an area of the first collection electrode and the second collection electrode proportional to the trace of the flow cell is at least 40%. 56. The electronic device of claim 55, wherein an area of the first collection electrode and the second collection electrode proportional to the trace of the flow cell is at least 50%. 56. The electronic device of claim 55, wherein an area of the first collection electrode and the second collection electrode proportional to the trace of the flow cell is at least 60%. 56. The electronic device of claim 55, wherein the flow cell comprises an inlet. 56. The electronic device of claim 55, wherein the flow cell comprises an outlet. 54. The electronic device of claim 53, wherein the first collection electrode and the second collection electrode are the same size. 54. The electronic device of claim 53, wherein the first collection electrode and the second collection electrode are different in size. 71. The electronic device of claim 70, wherein the first collection electrode is smaller than the area of the second collection electrode. 71. The electronic device of claim 70, wherein the first collection electrode is at least substantially 10% smaller than the area of the second collection electrode. 54. The electronic device of claim 53, wherein the first collection electrode is trapezoidal. 54. The electronic device of claim 53, wherein the second collection electrode is trapezoidal. 74. The electronic device of claim 73, wherein the ladder has a bottom side adjacent to the array and a top side away from the array, wherein the bottom side is longer than the top side. 54. A method of analyzing a biological sample using an electronic device for performing the active biological manipulation of claim 53, Providing a sample to the device; Placing the first collection electrode to attract the desired charged biomaterial to focus the desired charged biomaterial on the collection electrode; At least a portion of the micropositional array disposed on the substrate disposed from the first collection electrode toward the second collection electrode by positioning the second collection electrode to attract the desired charged biomaterial with respect to the first collection electrode. Transferring the charged biomaterial onto the substrate such that interaction occurs between the charged biomaterial and the array How to include. 77. The method of claim 76, using electronics for performing active biological manipulations, wherein the array is electrically passively maintained. 77. The method of claim 76, using electronics for performing active biological manipulations, wherein the array is electrically active to facilitate interaction between the array and the charged biomaterial. 77. The method of claim 76, using electronics for performing active biological manipulations, wherein the charged biomaterial moves like waves on the array. 77. The method of claim 76, using electronics for performing active biological manipulations, wherein the charged biomaterial moves on the array. 77. The method of claim 76, using electronics for performing active biological manipulations, wherein the charged biomaterial moves on the array and is held on its side with respect to the substrate. In an active biological manipulation electronic device, Support substrates; A microposition array disposed on the substrate; A first collection electrode disposed to surround the array on the substrate; And Opposing electrodes disposed in the array on the substrate Electronic device comprising a. 83. The electronic device of claim 82, wherein the first collection electrode is segmented. 83. The electronic device of claim 82, wherein a plurality of rings are provided surrounding the array. 83. The electronic device of claim 82, further comprising a capture sequence. 86. The electronic device of claim 85, wherein the capture sequence is disposed adjacent to the collection electrode. 86. The electronic device of claim 85, wherein the collection electrode is a complexity reducing electrode. 83. The electronic device of claim 82, wherein the microposition is disposed in a circular shape. In an active biological manipulation electronic device, Board; A flow cell suitable for containing a fluid containing a biomaterial, the flow cell being supported by the substrate and defining traces on the substrate; A plurality of electrodes disposed on the substrate, the electrodes comprising at least a return electrode and a collection electrode, wherein the return electrode and the collection electrode are disposed around the flow cell trace to direct response to almost all of the volume of the flow cell Send a signal- Electronic device comprising a. In an active biological manipulation electronic device, Support substrates having first and second surfaces, the support substrates having vias between the first and second substrates to allow fluid to flow through the substrates, the second surfaces supporting electrical traces. -; A second substrate comprising at least a first surface, the first surface being adapted to face the second surface of the first substrate, the second substrate comprising conductive traces connected to the microposition array, The array is adapted to receive the fluid through the via; Electrical wiring disposed between an electrical trace on a second surface of the support substrate and an electrical trace on a first surface of the second substrate; A sealant disposed between the second surface of the support substrate and the first surface of the second substrate, the sealant providing fluid sealing between and between the first substrate and the second substrate; And A flow cell disposed on a first surface of the first substrate Electronic device comprising a. 91. The electronic device of claim 90, wherein the support substrate is a stretch circuit. 91. The electronic device of claim 90, wherein said support substrate is a circuit board. 91. The electronic device of claim 90, wherein the second substrate is a semiconductor substrate. 95. The electronic device of claim 90, wherein the electrical wiring is conductive bumps. 95. The electronic device of claim 94, wherein the conductive bumps are solder. 97. The electronic device of claim 95 wherein the conductive bumps are indium solder. 95. The electronic device of claim 94 wherein the conductive bumps are conductive polymers. 95. The electronic device of claim 94, wherein the conductive bumps are silver filled epoxy. 91. The electronic device of claim 90, wherein the sealant is a photocurable polymer. 105. The electronic device of claim 99, wherein the photocurable polymer is water based. 91. The electronic device of claim 90, further comprising an edge irradiation member disposed for irradiation of the first surface of the second substrate. In the analysis system of a solution containing a charged biomaterial, Inlet; Outlet; An inlet chamber connected to said inlet, said inlet chamber having a cross-sectional area A in a plane substantially perpendicular to the flow direction and having a variable height and width as a function of distance from said inlet; And An outlet chamber connected to said outlet, said outlet chamber having a cross-sectional area A 'in a plane substantially perpendicular to the flow direction and having a variable height and width as a function of distance from said outlet; Including; And said inlet chamber and outlet chamber are adapted for fluid flow therebetween, said inlet chamber and outlet chamber having a substantially constant cross-sectional area. A flip chip structure having a chip disposed adjacent to a substrate comprising a via, the structure being adapted to receive fluid to lie on the substrate and to flow the chip down through the via, the at least one of the chip Wherein the portion comprises an area free of sealing overcoats, Attaching the chip to the substrate; Providing a photocurable insert sealant at the interface between the substrate and the chip; Exposing light to the chip through the via to a device on the substrate; At least partially curing the sealant as a result of the exposure to prevent the sealant from flowing into the area free of the sealant; And Completing curing of the sealant How to include. 103. The method of claim 103, wherein the sealant is curable. 107. The method of claim 103, wherein said light is ultraviolet light. In an active biological manipulation electronic device, A support substrate having first and second surfaces and vias disposed between the first and second surfaces to allow fluid flow; A second substrate comprising at least a first surface, the first surface being adapted to face a second surface of the first substrate, the second substrate comprising a microposition array, the array being the fluid Suitable for accepting; A sealant disposed between the second surface of the support substrate and the first surface of the second substrate; Light source; And A waveguide having an input suitable for receiving light from the light source and an output suitable for directing the light toward the array, wherein the waveguide is substantially parallel to the support substrate and light exiting the waveguide is directed to the array. Investigated Electronic device comprising a. 107. The electronic device of claim 106, wherein said light source is a laser. 108. The electronic device of claim 107, wherein the laser is a laser bar. 107. The electronic device of claim 106, wherein the waveguide is attached to the support substrate. 109. The electronic device of claim 109, wherein the waveguide is a polymer. 107. The electronic device of claim 106, wherein said light source is a laser. 107. The electronic device of claim 106, wherein a plurality of waveguides are provided. 107. The electronic device of claim 106, wherein the support substrate is a stretch circuit. 107. The electronic device of claim 106, wherein the support substrate is a circuit board. 107. The electronic device of claim 106, further comprising a fluid engineering system disposed on the first surface of the support substrate. 107. The electronic device of claim 106, further comprising solder bumps between the support substrate and the second substrate. In an active electronic control system of biological response in a multi-location environment, An array of unit cells arranged in a matrix; A row selector operatively connected to the array external to the array to selectively address the rows of the array; A column selector operatively connected to the array external to the array to selectively address the columns of the array; An input coupled to the row selector and column selector, the input adapted to receive unit cell selection information; A current circuit for receiving an input current and coupling a signal to the unit cell for current generation; And A power connector suitable for receiving power and for supplying power to the unit cell System comprising. 118. The system of claim 117 wherein the row selector comprises a memory. 118. The system of claim 118, wherein the memory is a shift register memory. 119. The system of claim 119, wherein the shift register is a 1x1 structure. 118. The system of claim 117 wherein the row selector comprises a decoder. 118. The system of claim 117, wherein the column selector comprises a memory. 123. The system of claim 122 wherein the memory is a shift register memory. 123. The system of claim 123 wherein the shift register memory is a 1x1 structure. 123. The system of claim 123 wherein the shift register memory is a 1x4 structure. 118. The system of claim 117, wherein the column selector comprises a decoder. 118. The system of claim 117, further comprising a variable current waveform generator. 118. The system of claim 117, further comprising a current mirror system that receives a current at a first value and outputs a current at a second value. 129. The system of claim 128, wherein said second value is less than said first value. 129. The system of claim 129, wherein the second value is at least 20 times smaller than the first value. 118. The system of claim 117 further comprising a multiplexer for alternating input of matrix selection. 118. The system of claim 117, wherein the input current comprises a current waveform. 134. The system of claim 132, wherein the current waveform is a static direct current waveform. 134. The system of claim 132, wherein the current waveform is a square wave. 134. The system of claim 134, wherein the current waveform is an asymmetric square wave. 134. The system of claim 132, wherein the current waveform is a sine wave. 134. The system of claim 132, wherein the current waveform is a sawtooth wave. A circuit for controlling output current in an active biologically controlled reaction system, A first column select transistor suitable for control by a column selector; A first row select transistor suitable for control by a row selector, wherein the first select transistors are connected in series with each other between a node and a first power source; An output connected to the node; A second column select transistor suitable for control by a column selector; And A second row select transistor suitable for control by a row selector, the second select transistors being connected in series with each other between the node and a second power supply; Circuit comprising a. 138. The circuit of claim 138 wherein the output is directly connected to the node. 138. The circuit of claim 138 wherein the first row select transistor and the second row select transistor are CMOS transistors. 138. The circuit of claim 138 wherein the first and second column select transistors are CMOS transistors. 145. The circuit of claim 141, wherein the channel length of the column select transistor is longer than the channel length of the row select transistor. 138. The circuit of claim 138 further comprising a first test transistor across the first power supply and the node. 143. The circuit of claim 143 wherein the first test transistor is suitable for control by a test signal. 143. The circuit of claim 143 further comprising a second test transistor across the second power supply and the node. 145. The circuit of claim 145 wherein the first test transistor is suitable for control by a test signal. 138. The circuit of claim 138 wherein the first power source is Vcc. 138. The circuit of claim 138 wherein the second power source is ground. A circuit for supplying a current to an active biological matrix device suitable for containing a conductive solution comprising a charged biomaterial, A variable current control element comprising a control element suitable for receiving an input, an output, and a control signal; Selection switch having an input, an output and a control element, wherein the input is connected to the output of the variable current element to provide a series connection between the variable current control element and the selection switch, the control element receiving a second control signal. Suitable for receiving, one of an input of the variable current control element and an output of the selection switch is connected to a first potential, and the other is suitable for contacting the conductive solution; And A return electrode connected to a second potential to contact the conductive solution Including; And a current is supplied between the return electrode and the other of an input of the variable current control element and an output of the selection switch in the presence of the conductive solution. 149. The circuit of claim 149, wherein the variable current control element is a transistor. 151. The circuit of claim 150, wherein the transistor is a MOSFET. 151. The circuit of claim 149, wherein the select switch is a transistor. 152. The circuit of claim 152 wherein the transistor is a MOSFET. 149. The circuit of claim 149, wherein an input of the variable current control element is connected to the first potential and the output of the select switch is suitable for contacting the conductive solution. 149. The circuit of claim 149, wherein the output of the select switch is connected to the first potential and the input of the variable current control element is suitable for contact with the conductive solution. 149. The method of claim 149, wherein A second variable current control element comprising a control element suitable for receiving an input, an output, and a third control signal; And A second selection switch having an input, an output and a control element, the input being connected to an output of the second variable current element to provide a series connection between the second variable current control element and the second selection switch; The control element is suitable for receiving a fourth control signal, one of an input of the second variable current control element and an output of the second select switch is connected to a third potential, and the other is in contact with the conductive solution. Suitable for Circuit further comprising. 156. The circuit of claim 156, wherein the second variable current control element is a transistor. 162. The circuit of claim 157 wherein the transistor is a MOSFET. 156. The circuit of claim 156, wherein the first variable current control element is of a first conductivity type and the second variable current control element is of opposite conductivity type. 159. The circuit of claim 156 or 159, wherein the second variable current control element is a p-channel element. 156. The circuit of claim 156, wherein the first variable current control element and the second variable current control element are selected to have similar performance characteristics. 162. The circuit of claim 161 wherein the circuit parameter is a device gain. 156. The circuit of claim 156, wherein a series connection is provided in the order of said first potential, said variable current control element, said selection switch, said second selection switch, said second variable current control element, and said third potential. 156. The circuit of claim 156, wherein the first potential, the select switch, the variable current control element, the second variable current control element, the second select switch and the third potential are connected in series. 156. The circuit of claim 156, wherein the first potential, the variable current control element, the selection switch, the second variable current control element, the second selection switch, and the third potential are connected in series. 158. The circuit of claim 156, wherein the first potential, the select switch, the variable current control element, the second select switch, the second variable current control element and the third potential are connected in series. 156. The circuit of claim 150 or 156, wherein the potential is VDD. 167. The circuit of claim 167, wherein the potential is a power supply voltage. 168. The circuit of claim 168, wherein the power supply voltage is substantially in the range of 0-10V. 168. The circuit of claim 168 wherein the power supply voltage is substantially 5V. 156. The circuit of claim 156, wherein the second potential is substantially one half of the power supply potential. 162. The circuit of claim 156 wherein the third potential is ground. 172. The circuit of claim 172 wherein the second potential is between the power supply potential and ground. 172. The circuit of claim 173, wherein the second potential is substantially one half of the power supply potential. 149. The circuit of claim 149, wherein the control signal is a static signal. 149. The circuit of claim 149, wherein the control signal is a square wave signal. 149. The circuit of claim 149, wherein the control signal is an asymmetric square wave signal. 149. The circuit of claim 149, wherein the control signal is a sine wave. 151. The circuit of claim 149, wherein the control signal is a sawtooth wave. A circuit for supplying a current to an active biological matrix device suitable for containing a conductive solution comprising a charged biomaterial, A first variable current source having an output; A voltage divider having an input suitable for receiving a first potential, a second input coupled to the output of the first variable current source, and an output node suitable for supplying a second potential that varies as a function of the output of the first variable current source ; A variable current control element having an input, an output, and a control element coupled to the node suitable for supplying the second potential, the input coupled to a third potential, the output adapted to contact the conductive solution Coupled to the first electrode; And Return electrode connected to return potential Including; And a current is variably supplied between the return electrode and the first electrode in the presence of the conductive solution. 182. The circuit of claim 180, wherein the first variable current source comprises a switched source. 181. The circuit of claim 181 wherein the first variable current source comprises a plurality of switch current sources. 182. The circuit of claim 182 wherein the switch current source is controlled by a memory. 185. The circuit of claim 183 wherein the memory is a two phase memory. 184. The circuit of claim 184 wherein the two-phase memory is flip flop. 185. The circuit of claim 183 wherein the memory comprises a shift register. 182. The circuit of claim 180, wherein the voltage divider comprises a switchable resistor element. 187. The circuit of claim 187 wherein the switchable voltage divider comprises a transistor connected in series. 185. The circuit of claim 188 wherein the series connected transistor is controlled by logic. 185. The circuit of claim 189 wherein the logic to receive a memory status output as an input. 189. The circuit of claim 189, wherein the logic controls the inclusion of a resistor in the voltage divider when any memory is on. 185. The circuit of claim 189 wherein the logic is a NAND gate. 182. The circuit of claim 182 wherein the outputs of the switch current source are summed. 180. The circuit of claim 180, wherein the first potential is equal to the second potential. 194. The circuit of claim 194, wherein the first potential and the second potential are the power supply potential. 182. The circuit of claim 180, further comprising at least one current mirror for supplying current to the first variable current source. 182. The circuit of claim 180, further comprising a test transistor for providing a series connection with the third potential, wherein the output is coupled to a first electrode suitable for contacting the conductive solution. A system for supplying current to an active biological matrix device suitable for containing a conductive solution comprising a charged biomaterial, An array of unit cells, each unit cell comprising a row contact and a column contact, a row line coupled to the row contacts of the array, and a row selector coupled to the row line to provide a row select voltage; A thermal line coupled to the thermal contacts of the array; A column selector coupled to the column line to provide two or more column voltage states to the column line, wherein the unit cell is coupled to a power source and an electrode, wherein the row select voltage and the column voltage state are output from an electrode of the unit cell Supplying a variable current; And Return electrode coupled to the potential and suitable for contact with the conductive solution Including; And a current is supplied between the various unit cells including the return electrode in the presence of the conductive solution. 199. The system of claim 198, wherein the return electrode is a unit cell of the array. 199. The system of claim 198, wherein the row selector comprises a memory. 213. The system of claim 200, wherein the memory comprises a shift register memory. 203. The system of claim 201, wherein the shift register memory is a 1x1 structure. 199. The system of claim 198, wherein the row selector comprises a decoder. 199. The system of claim 198, wherein the column selector comprises a memory. 204. The system of claim 204, wherein the memory comprises a shift register memory. 205. The system of claim 205, wherein the shift register memory comprises a plurality of bits per column of unit cells. 206. The system of claim 206, wherein there are at least four bits per column of unit cells. 199. The system of claim 198, wherein the unit cell further comprises a second row contact and a second row line. 199. The system of claim 198, further comprising a second row selector. 199. The system of claim 198, wherein the unit cell further comprises a second column contact and a second column line. 197. The system of claim 198, further comprising a second column selector.