JP2002536962A - Integrated portable biological detection system - Google Patents

Abstract

(57) [Summary] The present inventors have separated bacteria and cancer cells from human peripheral blood in a microfabricated electronic chip by dielectrophoresis. The isolated cells were labeled with a fluorescent dye in the nucleus and subsequently examined by laser-induced fluorescence imaging. We have released DNA and RNA electronically from isolated cells, detected specific marker sequences by DNA amplification, and then immobilized the capture probe by electronic hybridization. We demonstrate our efforts to build a "laboratory-on-a-chip", including the selection of DNA probes, dyes, and reagents, and the production of prototypes of fully integrated portable devices.

Description

DETAILED DESCRIPTION OF THE INVENTION

GOVERNMENT ASSISTANCE The government has a fund number ATP: 70NA awarded by Advanced Technology Program.
Has rights in the present invention according to NB7H3001.

FIELD OF THE INVENTION [0002] The present invention relates to devices and methods for performing active, multi-step sample preparation of biological material and molecular diagnostic analysis. More particularly, the present invention relates to an integrated, compact, portable device for causing a sample contained therein to respond to a system. In particular, the invention relates to devices and methods for performing multi-stage sample preparation and assays on either two or even a single microchip.
Applications for this collection system include food and / or quality monitoring, infectious diseases and cancer, bone marrow plastesis (eg, stem cell isolation and analysis)
Diagnosis, as well as genetics-based identification of individuals for forensic purposes.

Related Application The present application discloses “Fluorescent Imaging of Cells and Nucleic Acids in Bioelectro
Provisional Patent Application No. 60/1 filed on December 23, 1998 under the title of "nic Chips"
Claiming the benefit of issue 13,730, “Apparatus and Methods for Active Biological Sa
A simple continuation of copending application Ser. No. 08 / 709,358, filed Sep. 6, 1996, entitled "Methods for Electronic Stringen,"
cy Control for Molecular Biological Analysis and Diagnostics ", filed on July 7, 1994 and now patented application No. 08 / 271,882
Of the Automated Molecular Biological Diagnostic Syst
filed on September 9, 1994 under the title "em", and is now US Pat. No. 5,632,957.
"Appar, a continuation-in-part of application no. 08 / 304,657, issued as
atus and Methods for Active Programmable Matrix Devices ”, which was filed on September 27, 1995 and is a continuation-in-part of application number 08 / 534,454, now issued as U.S. Patent No. 5,849,486. “Self-Addressable Self-A
ssembling Microelectronic Integrated Systems, Component Devices, Mechani
sms, Methods, and Procedures for Molecular Biological Analysis and Diagn
co-pending application Ser. No. 08/986, filed Dec. 5, 1997, entitled "Ostics"
“Channel-Less Separation of Bioparticles on
a Bioelectronic Chip by Dielectrophoresis ”January 30, 1998
It is a continuation-in-part of co-pending application Ser. These specifications are hereby incorporated by reference.

BACKGROUND OF THE INVENTION The following description provides a summary of information relevant to the invention. Any information provided herein will be prior art to the claimed invention, or any publication specifically or implicitly referred to will be prior art to the present invention. Nothing is allowed. Generally, the analysis of biologically derived sample material cannot occur until the sample has been processed through many pre-analytical steps. Often, the preparation process is time consuming and cumbersome. For example, many immunological and molecular-biological diagnostic assays on clinical samples, such as blood and tissue cells, rupture or lyse the cells to remove such molecules, including proteins and nucleic acids of interest (ie, DNA and RNA). It is necessary to separate the molecule of interest from the crude sample by release and subsequent purification of such proteins and / or nucleic acids. Only after performing the processing steps can the analysis of the molecule of interest begin. In addition, the protocols used for actual analysis of the sample require a huge number of steps before obtaining useful data.

[0005] For example, charged and uncharged fine particles in a solution (such as a crude extract of cellular material or its proteins or nucleic acids) can be separated by dielectrophoresis. At the microscale, dielectrophoresis uses a glass slide-based device with exposed, ie, bare, interdigitated electrodes plated on the surface of the slide and having a flow chamber with a volume of several hundred microliters. Can be done.
With such a device, cells, proteins and nucleic acids can be separated based on their individual dielectric properties by using a separation buffer with an appropriate dielectric constant and an AC signal with suitable amplitude and frequency. However, such devices have several problems, including the non-specific binding of both isolated and non-separated cells to the glass surface and exposed portions of the electrodes. Such a device has the disadvantage that the flow chamber volume (several hundred microliters) is large enough to disturb thermal convection and remove material such as cells and large molecules initially attracted to and retained by the electrode from the electrode. There is also a problem. In addition, undesired cells and molecules do not disturb and liberate the cells of interest, since such cells and molecules can interfere with fluid flow and thus block flow during the washing step.
Not easily washed off the surface.

[0006] Conventional methods of ruptured whole cells to release proteins and nucleic acids have utilized the use of a series of high-voltage DC pulses in macroscopic devices, as opposed to microchip-based devices. . Such conventional electrolysis techniques have several problems. For example, some commercial macroscopic devices have high molecular weight (20
Dissolution conditions are used that do not release nucleic acids (greater than Kb). This is because high molecular weight molecules cannot be fitted through the pores created in the cell membrane using such methods. In addition, released nucleic acids are often lost due to their non-specific binding to the surface of the lysis chamber. Such a loss of material, especially when the molecule of interest is present at low concentrations, can cause the dielectrophoretic cell separation macroscopic device system to transfer from one device to another as the sample preparation is transported forward. This is exacerbated by the fact that it is an isolated system that allows for sample loss in material transfer.

[0007] Processing of crude lysates often requires chemical reactions to remove unwanted cellular components from specifically desired cellular components. These reactions typically involve subjecting the lysate to an enzymatic reaction such as proteinase K and a restriction enzyme or nuclease. Processing consists of strand displacement amplification (SDA
) Or by performing an amplification reaction, such as the polymerase chain reaction (PCR) method, to enhance the presence of the molecule of interest, particularly a nucleic acid. These reactions are also performed in a stand-alone process. Assays for data retrieval can be started only after the preparation and processing steps of these samples. Due to the vast number of steps between sample collection and assay, many techniques are limited in their application by lack of sensitivity, specificity or reproducibility.

[0008] Attempts have been made to use dielectrophoresis to separate and identify whole cells. For example, U.S. Pat. No. 4,326,934 to Pohl discloses a method and apparatus for cell sorting by continuous dielectrophoresis. Using such a method, cells are separated by using both positive and negative dielectrophoretic movements of cell particles. The separated cells
Characterization and / or classification is allowed by considering the characteristic deflection distance of cell movement through the positive and negative electrodes.

[0009] In another example, US Pat. No. 5,344,535 to Belts et al. Discloses a method and apparatus for the characterization of microorganisms and other particles by dielectrophoresis. In this system, cells are characterized by matching their characteristic dielectrophoretic collection rates.

[0010] In yet another example, US Pat. No. 5,569,367 to Belts et al. Disturbs the flow of cells through the device and reduces cell type differences into fractions by applying a non-uniform alternating field. A method and apparatus for separating a mixture of cells using a pair of energized interdigitated electrodes consisting of a mixed grid-like structure arranged to produce is disclosed.

In addition, other attempts have been made to combine certain processing or sub-steps together. For example, various automated systems have been proposed to prepare arrays of DNA probes on a support material. November 1992, “The 1992 San D
In the iego Conference: Genetic Recognition, Beattie et al.
Discloses the use of a micro-automated instrument system to deposit micro-droplets containing an A-array into individual micro-machined sample wells on a glass substrate.

Various other attempts have been made to describe an integrated system formed on a single chip or substrate, which may include multiple stages of sample preparation and diagnostic systems throughout. . A. Manz et al., “Miniaturized Total Chemical Analysis System
: A Novel Concept For Chemical Sensing ”, Sensers And Actuators, B1 (199
0), pp. 244-248, describes a TAS including a module adjustment construction of a miniaturized “all chemical analysis system” (TAS). In that system, sample transport, chemical reactions, chromatographic separation and detection were performed automatically.

Still another proposed integration system according to Stapleton, US Pat. No. 5,451,500, describes a system for automated detection of target nucleic acid sequences. In this system, multiple biological samples are individually placed in a two-dimensional format into a matrix containing a carrier. Various multi-electrode systems are also disclosed, which are intended to perform multiple aspects of biological sample preparation or analysis. “Silicon Semiconductor Wafer for Analyzin
g Micronic Biological Samples ", Pace, US Pat. No. 4,908,112
No. 1 describes an analytical separation device, wherein a channel in a semiconductor device forms a capillary-sized conduit, and the electrodes are located in the channel to activate the movement of liquid through the conduit. . In addition, “Method and Device for
Moving Molecules by the Application of a Plurality of Electrical Fields
In US Patent No. 5,126,022 entitled "Invention," Soane et al., For many purposes, such as various transformations that are either physical or chemical in existence,
The application of an electric potential causes the material to move through the groove to the electrode, where the selected component is
An antigen that is reactive with a given charged particle that is moved in a medium or is brought into contact with a complementary component, dye, fluorescent tag, radiolabel, enzyme-specific tag or other type of chemical. A system is described that can guide into various grooves filled with antibodies. Further, Clark et al., US Pat. No. 5,194,133 entitled "Sensor Devices"
In this article, an elongated microfabricated channel containing a material such as starch, agarose, alginate, carrageenan or a polyacrylamide polymer was formed to cause separation of the sample fluid as the fluid passed along the channel. A sensor device for analyzing a sample fluid, including a substrate having a surface, is disclosed. Biological materials can include, for example, binding proteins, antibodies, lectins, enzymes, a range of enzymes, or lipids.

Various devices are known for eluting DNA from various surfaces. For example, Shukla, entitled "Apparatus for Electroelution", U.S. Pat.
No. 340,449 describes a system and method for eluting macromolecules such as proteins and nucleic acids from solid matrix materials such as membranes such as polyacrylamide, agarose and PVDF in an electric field. The material is eluted from the solid phase to a volume partially defined by the molecular weight exclusion membrane. Okano et al., “Separation
of Polynucleotides Using Supports Having a Plurality of Electrode-Contai
US Patent No. 5,434,049 entitled "ning cells" discloses a method for detecting a plurality of target polynucleotides in a sample, the method comprising the steps of eluting the captured target polynucleotides. Applying the potential of the individual chambers to act on the eluted material, where the eluted material is available for collection.

[0015] Other devices for performing nucleic acid diagnostics have been designed, where R. Lip
shutz et al., “Integrated Nucleic Acid Diagnostic Device” (US Pat. No. 5,856,
No. 174) and R. Anderson et al., “Integrated Nucleic Acid Diag.
Nostic Device "(US Pat. No. 5,922,591) requires at least two reaction chambers for sample preparation and analysis.

[0016] P. Wilding et al., "Integrated cell isolation and PCR analysis using silic
on microfilter-chambers ”, Anal. Biochem. 257, pp. 95-100, 1998;
CH Li and DJ Harrison, “Transport, manipulation, and reaction o
f biological cells on-chip using electrokinetic effects ”, Anal. Chem.,
69, pp. 1564-1568, 1997, there are still other achievements aimed at the partial integration of complete sample handling systems.

MA Burns et al., “An integrated nanoliter DNA analysis device”, Scienc
e, 282, pp. 484-487, 1998; SC Jacobson and JM Ramsey, "Integrate
d microdevice for DNA restriction fragment analysis ”, Anal. Chem., 68,
pp. 720-723, 1996; LC Waters et al., “Microchip device for cell lysis, mu.
ltiplex PCR amplification, and electrophoretic sizing ”, Anal. Chem., 70
pp. 158-162, 1998; and AT Woolley et al., "Functional integration of
PCR amplification and capillary electrophoresis in a microfabricated DNA
Other attempts have been made to integrate chemical reactions and detections, such as the analysis device ", Anal. Chem., 68, pp. 4081-4086, 1996.

In general, as can be understood from the above examples, systems and methods have been described which are sufficient for fully integrated self-contained samples responsive to systems using electronically active microchips. Not offered to. A further vast system has been described as
It is extremely labor intensive and time intensive, requiring multiple steps and human intervention either during or between processes, which together are not optimal and result in sample loss, contamination and operator intervention. Allow errors. In addition, the use of multiple processing steps using multiple machines or complex automated equipment systems to perform individual processes is impeded except for the largest laboratories, both in terms of spending and physical space requirements. May be used. For the reasons mentioned above,
These techniques are limited and lacking. They can be easily combined into a fully self-contained integrated diagnostic assay, on a single electronically addressable microchip,
In particular, it does not form a system that can perform assays for data retrieval for nucleic acid and protein-derived information. Despite the long recognized need for such integrated systems without complex fluidics and unsuitable valve systems, no satisfactory solution has been proposed before. Therefore, there is a continuing need for methods and devices that lead to improved dielectrophoretic separation of biological cells and improved biological stability of the separated cells, and furthermore, the preparation of cells without complex fluidics. There is a continuing need for methods and devices that can improve analysis and integrate cell separation, preparation, purification and analysis into a single self-contained system.

SUMMARY OF THE INVENTION Accordingly, provided herein is an active integrated multi-step sample preparation of biological samples and molecular diagnostic analysis using a minimum number of electronically addressable microchips. An integrated portable system and device for performing. In a preferred embodiment, the systems and devices have a single flow cell element for separating, preparing and analyzing pre-selected eukaryotic and / or prokaryotic cells from a mixed population of cells. The single flow cell element includes an electronically addressable microchip having a grid of individual electrodes therein. The grid may include a number of individual electrodes and may be fabricated on a silicon wafer, as is well understood in electronics technology. Generally, such a grid may include from 4 to 25 to as many as 100 or 10,000 or more individual electrodes. An example of this single flow cell is that the flow cells, including the electronic grid, can perform simple fluid manipulations of the cells for their respective separation, lysis, cleaning of material from the cell of interest, and analysis of such material. Intended. The preselected eukaryotic or prokaryotic cell may be of any type (eg, blood cells, cancer cells, stem cells, bacteria, etc.) and of any origin (eg, animals, biopsies, blood, organs, Forensic samples, stool, water, etc.). For example, specific examples of cells that can be separated from whole blood include Escherichia coli, Salmonella typhimurium,
ella typhimurium, Staphylococuus epidermas
dermus), Micrococcus lysodeikticus
) And cultured cervical carcinoma cells.

In another embodiment, the device of the present invention may include at least one additional flow chamber for performing either various sample preparation steps or analysis of the prepared sample. For example, in one alternative embodiment, the system includes first and second flow cell chambers each having an electronically addressable microchip. In this alternative embodiment, the first chamber may be used to separate and lyse cells and perform a sample preparation step, while using the second chamber,
Sample material can be analyzed directly on the second flow cell microchip.

In another alternative embodiment, the method includes at least two flow cells, wherein the first flow cell does not have an electronically addressable grid but has at least a heating element coupled thereto, Can be used to adjust the temperature of the material in the cell. The second flow cell is intended to have an electronically addressable microarray used to perform the analysis of the sample material.

Regardless of the embodiment utilized, the system of the present invention comprises: (1) separating eukaryotic cell types from each other and eukaryotic cell types from prokaryotic cell types; Processing the sample material directly from the state to a more purified state, and (
3) Provides the ability to analyze sample material directly on a microchip grid. Such an ability would cause a new use of an electronic bias at one level of voltage in the form of a dielectric current to cause dielectrophoresis of the cells, then increase the voltage to lyse the captured cells, and then Changing the bias mode from AC mode to DC mode to address specific electrodes on the array of flow cell (s), for capture / hybridization to probes previously bound to the electrode array This is possible by causing the transport of the molecule. The system of the present invention further provides that tubing and a simplified arrangement of solenoid operated valves and pumps can transport other suitable sample preparation reagents to and away from the flow cell (s). Intend. In addition, in embodiments having a first flow chamber without a microchip, the system directly lyses cells in a sample and analyzes the material of interest without having to separate cell types. Intended to be able to. In such embodiments, the flow cell has a heating element that can be used to raise the temperature to directly lyse the cells in the sample. Following such lysis, a sample preparation step, such as protease treatment or nucleic acid amplification, may be performed, followed by transport of the amplified species to a second flow cell containing an electronically addressable microchip.

In another embodiment, the collection system is intended for direct processing of cells selectively separated or lysed in a first chamber to obtain any material of interest from such cells. I do. Such processing is generally directed to isolating and purifying proteins and nucleic acids from cellular contaminants. A vast array of techniques can be performed in the preparation of the molecule of interest, including, but not limited to, enzymatic treatment to remove protein material from the nucleic acid of interest using protease K, and removal of nucleic acid from protein using nucleases Enzyme treatment, digestion residue adsorption, nucleic acid amplification (eg, by PCR and SDA), in situ buffer exchange, and receptor-ligand or antibody binding or binding to the protein of interest Other protein-protein binding reactions, such as enzyme-substrates, are included.

[0024] In another embodiment, the analysis of the prepared sample material may involve a number of preselected hybridization formats. In a preferred format, the nucleic acid of interest is, as is known to those skilled in the art of electronically addressable microchips,
Selectively hybridizes via an electronically-directed process. Such hybridization formats include nucleic acids (R
NA, DNA, pNA). Other formats contemplated for use in the system include the selective capture of a protein of interest, such as by an antibody or other protein binding probe attached to an electron grid. These can include other protein-protein interactions such as receptor-ligand and enzyme-substrate binding.

In still other embodiments, the device comprises elements (eg, buffer vials, tubes, etc.) for transporting and transporting samples, buffers, enzymes and reagents to and from the flow cell (s). System, small solenoid valve and at least one pump). Still other embodiments are provided for detecting analytes and molecules of interest to be assayed. In a preferred embodiment, such elements include a battery powered diode laser (preferably 635).
nm) and a CCD camera coupled with astronomical filters and zoom lenses of the individual electrodes of the microchip grid (s) of the first and / or second flow cell chamber (s). . Still other detection methods are described in P. Ropp and H. Holden Thorp, Chemistry & Biology, 1999, Vol. 6, N
It is also contemplated that light emission using a light emitting device, such as direct electrochemical detection, well known to those skilled in the art, such as the detection described in o. 9, pp. 599-605, is not required. It is further intended that each of these electronic components be coordinated via a computer and appropriate programming software, as is well understood by those skilled in the electronics arts.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Referring now to specific embodiments of the present invention, there is provided a portable laboratory-on-a-chip system, wherein an electronically addressable microchip is provided. The sample may be processed and analyzed in at least a single flow cell containing The elements of the device are a sample preparation and analysis flow chamber, a fluid handling system, and a luminescence source (such as a battery-powered 635 nm diode laser) and detection electronics (a series of filters and a CCD camera coupled with a microchip astronomical zoom lens). Is contained in a portable casing or housing. Outside the portable casing is a computer, such as a personal computer connected to the portable housing by a cable.

Referring now to FIG. 1, a flow chart illustrates three different sample handling stages for performing a bioassay intended to use the laboratory-on-a-chip system of the present invention. . These are: (1) sample processing, (2)
Chemical reaction, and (3) analyte detection.

Sample handling in stage (1) processing generally involves the preparation of crude biological samples (eg, blood, urine, stool, mixed cell populations, etc.) in order to isolate molecules of interest such as nucleic acids and proteins. Processing cells from others). Sample handling in chemical reaction stage (2) potentially involves many types of molecular biological reactions to clean and even isolate, purify or amplify the molecule of interest, But not limited to, enzyme-based reactions of treatment with proteases, nucleases and restriction digestion, PCR and SDA-based nucleic acid amplification, in situ buffer exchange, chemical labeling such as with radioisotopes and fluorescent markers, and Includes immune-based and protein-protein reactions such as antibody-antigen reactions, ligand-receptor reactions and enzyme-substrate reactions. Sample handling in the analyte detection stage (3) can be done via a large number of formats, including optical, electrochemical and radioisotope detection of fluorescent emission. In a preferred embodiment, the detection includes hybridization using nucleic acids or capture proteins on an electronic grid, and detection using fluorescence imaging.

In FIG. 2, the system is illustrated in a schematic form showing a top-down or perspective view of the basic components. In this figure, a flow cell 11 is mounted using a support 10. On the back side of the flow cell 11 mounted on the support, there is a heating element 12. In addition, the flow cell may be connected to a port and a desalting column 13 via hollow fibers to purify, desalinate, and introduce different buffers to the flow stream. The heating element is used to heat the flow cell, either for direct thermo-induced lysis of cells in the chamber or for use in stage 2 process reactions such as temperature cycling for enzyme inhibition and nucleic acid amplification. I can do it.

As will be appreciated by those skilled in the art, the flow cell and other electronically actuated components such as solenoid operated valves and pumps, lasers and detection cameras are provided with cables 19 to housing 18 for all programming and control purposes. For example, a power supply and a computer, which are electronically interconnected with the computer 20, are coupled to the electrodes of the flow cell 11 for programming and operation. In a preferred embodiment, the electronic signals that can be generated by the computer software used with the system include a sinusoidal AC component and its use in dielectrophoresis, and a square DC component and its use in hybridization. In some cases, AC components are included. In a still further aspect, the time-varying electronic signal used in the system preferably includes an offset signal, such as when the offset signal is a DC signal.

FIG. 2 shows a light emitting source 14 that emits a laser beam 15. Preferably, the light source 14 is a diode laser. The beam 15 is preferably incident on a beam splitter 16 that directs at least a portion of the beam 15 toward a flow cell 11 microtip for sample analysis. The radiation emitted from the microtip grid electrode returns on the entrance path, at least a part of which passes through a beam splitter 16 to a detector 17. In a preferred embodiment, the detector 17 includes a charge-coupled device (CCD) detector. Other detectors may also be used. However, because it is not bulky, it is preferable to have a detector that generally has a relatively larger range of coverage relative to its depth (in the direction of emitted radiation).

FIG. 3 shows a perspective view of one design or style of the integrated portable laboratory on a chip system for overall portability. The computer 20, preferably a portable notebook personal computer, includes conventional elements such as keyboards, function keys, monitors and input devices such as trackballs and mice. The housing 18 serves to include not only a preparation, reaction and analysis platform including a flow cell, but also additional equipment utilized in conjunction with a computer 20 to operate the system. For example, the housing 18 may include a power supply, waveform generator, laser, flow cell (s), CCD camera, and other electronics, fluid systems, and reagents necessary for operation and control of the system (FIG. 4).

As an example of a flow cell design, FIG. 5 shows a cross-sectional view of such a cell. The electronically addressable microarray 23 is a 1.0 inch square pin grid array (PGA) (068 PPGA, 400 Square Cavity, Spectrum).
It is mounted on a substrate 10 containing Semiconductor Materials (San Jose, CA). The ceramic chip, heater,
Element 12 (Dawn 505, Dawn Electronics, Carson City, Nevad)
a). The heating element 12 may be joined in any of a number of ways. In a preferred embodiment, the bonding may be performed by placing a thermally conductive flexible adhesive 22 between the backside of the electronic microchip and the ceramic heater and incubating the adhesive at an appropriate temperature and time. further,
As is well understood in the art, these microchips are coated with a thin osmotic layer 25, such as a hydrogel, agarose, a polymer of acrylamide, or a sol-gel matrix. This osmotic layer 25 protects the cell and the molecule of interest (ie, biomaterial) from the electrochemistry that occurs on the electrode surface that would damage the biomaterial if it were directly exposed to the electrode.

The microchip can then be bonded to a molded or machined medical grade plastic cell 27 such that the electronic microarray 23 constitutes the inside bottom surface of the well in the flow chamber. The flow cell 27 provides a compartment 29 for containing a biological sample material and a buffer that is layered on top of the microchip. The flow cell may be further designed to have at least two to four ports 30 for sample distribution and extraction. The flow cell 27 may further be configured to interface with an external fluid distribution and removal system and to receive a tubing for desalination. In addition, the flow cell compartment is covered and sealed by a fused quartz window or lid 28 for visual access to the microarray analysis site or capture pad. The window 28 can include any thickness of quartz, but is typically about 0.015 inches thick.

Window 28 may be coupled to the flow cell in any of a number of ways,
And the flow cell can be coupled to the chip. The preferred method uses an ultraviolet (UV) curable adhesive 26 developed for the assembly of optomechanical components. The coupling of the flow cell to the chip can be either directly to the exposed surface of the chip or to a permeable layer overlaying the microarray, also using an adhesive 26. The tubing is coupled to the input and output ports using various methods, depending on the size, sizing and tubing base material of the tubing.

In another embodiment, the integration system may use two flow cells, one for sample preparation and a second for analysis. FIG. 6 shows such a specific example in a schematic form. The first flow chamber 31 is coupled to the heating element 12 and may or may not have an electronically addressable microchip 31a, if desired (31b). . In either format, ie, with or without an electronic microchip, flow cell 31 is used to perform stepwise cell separation and / or cell lysis, and to clean and lyse cells. Further preparation of the recovered target molecule is performed. Further preparation of the molecule of interest can include isolation of the specific protein of interest, isolation of the nucleic acid of interest, such as removing protein material from the nucleic acid by treatment with a protease, and amplification of the nucleic acid of interest, such as by PCR and SDA. It is intended to include many of the chemical reactions and steps described above.

The second flow cell 32 is used for sample analysis. In a preferred embodiment, both flow cells 31 and 32 are located on the same side of support 10. However, these flow cells can be arranged in other arrangements, including back contact or stacked configurations. Furthermore, these two flow cells can be connected via hollow fibers to purify, desalinate and introduce different buffers into the flow system. Further, each flow cell can be designed to have independent temperature control elements, if desired.
As will be appreciated by those skilled in the art, each electronically addressable microchip in the flow cell and other electronically actuated components such as solenoid operated valves and pumps, as in a single flow cell arrangement, It is electronically interconnected with the computer 20 by cables for all programming and control purposes. FIG. 6 also shows a light emitting source 14 that emits a laser beam 15 as described above and operates in the same manner. A camera 17 with an astronomical zoom lens on the flow cell electronic grid is also integrated on the movable track 33 so that both the first and second flow cell electronic microarray grids can be examined.

FIG. 7 is a schematic diagram of another system showing a support 10 including a sample preparation 31 a (shown as a flow cell including electrodes) and an analysis 32 (shown as 25 electrode grids) flow cells. Each flow cell preferably has an electrically operated three-way valve 45.
And a plurality of ports 42 that connect to a tubing or hollow fiber 44 that leads to the. With respect to the preparation flow cell 31a, the cell is intended to have at least two ports 42, optionally at least three ports, and in a preferred embodiment four ports. For the analysis flow cell 32, this cell preferably has two ports. These ports 42 and their individual tubing 44 may be used for various functions at different times. For example, the three-way valve 45 and the pump 4
Based on the direction of flow entering or exiting flow cells 31 and 32 directed by the directional setting of 6, at one point the port may be considered as an input port, while at another point it may be May include ports. Flow in the chamber "pushes" and "pulls" the liquid in various orders through the ports
By mixing. Such mixing can be advantageous, for example, during the amplification operation.

FIG. 7 also illustrates other components of the self-contained system. In one embodiment, reservoirs 21a-e are provided for carrying samples and reagents, such as for washing, amplification, hybridization, and disposal. In another embodiment, the support 10 has a small desalting column or hollow fiber ion exchange unit 13 attached thereto for desalting small sample volumes. Prior to attempting to electronically address the amplified nucleic acids to the specific capture pad of the microarray, it may be necessary to perform a desalting step to reduce the ionic strength of the sample. This is because the reagents used in the amplification step have an ionic strength that prevents movement of the molecule of interest under an electronically addressed protocol. Once the amplified sample has been desalted, it can be directed to the flow cell 32 for analysis on the microarray.

Regardless of whether a single or multiple flow cell integrated system of the present invention is used, cell separation is performed by dielectrophoresis. In such methods, polarizable particles, including those with no net charge (eg, eukaryotic and prokaryotic cells) are subjected to a "dielectric" force of a non-uniform electric field. As long as the effective polarizability of the particles is different under the surrounding medium, the cells will remain subjected to the dielectric force. The directions of migration of the different cell types are: (1) the surface charge of the cell wall or cell lipid bilayer membrane, (2) the conductivity and permittivity of such cell membranes and cell walls, and (3) cell morphology and structure. Configuration. Escherichia coli (Escherichia coli) can be obtained from a mixed cell population (eg, blood cells) using dielectrophoresis as practiced in the latest invention.
coli, Salmonella typhimurium, Micrococcus lysodeikticus and Staphylococcus epidermidis, as well as cancer cells such as cultured cervical carcinoma cells. Selectively isolate.

In a preferred embodiment, a flow cell useful for separating cells, as practiced in the present invention, has at least 100 individually biasable so that a dielectric force pattern can be generated across the electronic grid of the microchip. May be designed to include electronically addressable microchips having individually addressable microelectrodes. For example, FIG. 8 shows a pattern of two applied forces. In FIG. 8A, a square wall pattern is shown, while 8B shows a checkerboard pattern. Each of these patterns provides unique strong and weak ionic fields that provide sufficient dielectric force to separate different cell types. In addition, each of the force patterns
The square wall and checkerboard are also illustrated in the computer generated field pattern shown in FIGS.

In addition, the electrodes of the microarray in the flow cell (s) may be biased all together as a single array for performing cell separation, lysis and analysis, or may be programmed to perform the separation, lysis and analysis steps. The sub-array can be formed in any of a number of patterns to perform. In other words, one subset may be biased to separate cells, another set to bias cell lysis, and yet another set used for analysis.

In the example of cell separation, FIG. 11 shows the separation of Micrococcus lysodeikticus from whole blood. Bacteria are concentrated above the electrodes, while unwanted blood cells, both red and white blood cells, are present in the dark areas between the electrodes. Cells were dissociated using a bias protocol that included a 10 V peak-to-peak sinusoidal signal at 10 kHz. Although this signal value was used in this example, in general, cell separation can be performed using a sinusoidal signal, where the volts are 2-20 peak-peak, while the frequency is 5-50 kHz.

Undesired cells are washed from the flow chamber, while cells of interest are retained. This is done by maintaining an attractive bias for the cells of interest and creating a flow of wash buffer through the flow chamber. Once the cells of interest have been isolated in the flow chamber, they can be processed in any of a number of ways for further processing. In one embodiment, J. Cheng et al., "Preparat
ion and hybridization analysis of DNA / RNA from E. coli on microfabricate
d bioelectronic chips ", Nature Biotechnol., 16, pp. 541-546, 1998, lysing the cells by applying a high voltage pulse of up to 450 volts with a pulse width of 10 μs to 50 μs. In another example, E. coli cells were separated from blood cells, as shown in Figures 14-17, using a square wall dielectric force pattern in Figure 14, while using a checkerboard in Figure 15. For example, the separation for the checkerboard pattern used a biased form of a 10 kHz 10V peak-to-peak sinusoidal signal at 10 kHz, Figures 16 and 17 show the results after washing the square wall and checkerboard pattern separation. Shows how neatly the bacterial cells were isolated.

In another example, mammalian cells are separated on an electronic grid in a flow chamber. Figures 12, 13A and 13B show the separation of cervical carcinoma cells. FIG.
Shows the first separation, where the cells move on the electrodes of the grid, where the field is at its highest value by subjecting the cells to a positive dielectrophoretic force. A mixture of normal human leukocytes and erythrocytes mixed with carcinoma cells is extruded into the space between the electrodes, where the field is at a minimum as reflected by the pink diamond shaped spot. After washing, the carcinoma cells are retained by the electrodes (FIG. 13A), while all normal blood cells are washed out. FIG. 13B shows cells after staining with propidium iodide.
For example, mammalian cells can be separated using a checkerboard format and run at 30 kHz
A V peak-to-peak sinusoidal signal may be applied for 3 minutes to separate. J. Cheng et al., “Is
olation of Cultured Cervical Carcinoma Cells Mixed With Peripheral Blood
Cells on a Bioelectronic Chip ”, Anal. Chem. V. 70, pp2321-26, 1998.

After lysis, both cellular proteins and nucleic acids can be retained for analysis. An example showing that nucleic acids can be retained from lysed cells is provided in FIG. There, E. coli DNA was run on a PAGE gel, chromosomal DNA and plasmid DN.
A indicates that both are held. If nucleic acids are desired for analysis, further cleaning and purification may include treatment with a protease such as proteinase K. Following such treatment, the lysate may be either analyzed immediately or amplified and subsequently analyzed. If another embodiment is used, the sample may be further processed in a flow chamber or transported to a second chamber for such protease treatment, amplification and analysis. In general, protease treatment can be performed in a suitable buffer at 60 ° C. for 15 minutes. Temperature control can be achieved by using a ceramic heater element bonded to the backside of the microchip. Inactivation of proteinase K
This can be done by heating the sample at 95 ° C. for 2 minutes. Overall, the process of cell separation, lysis, and protease treatment can take 15-25 minutes.

If proteins are desired, nucleic acids can be removed by treating the sample with restriction enzymes and nucleases. In addition, proteins can be treated with various enzymes and partial protease treatments to release specific proteins of interest from cell membranes and other cellular components. Following the sample processing steps described above, further processing can be performed including amplification (eg, by PCR and SDA) and labeling with radioisotopes or fluorescent markers.

In an example of processing of a chemical reaction stage, a particular nucleic acid sequence (ie,
Salmonella enterica invA and spaQ genes were amplified using SDA. SDA is preferred for this portable system because amplification can be performed under isothermal conditions of 50-60 ° C., thereby eliminating the high temperature denaturation cycle associated with PCR. However, in one embodiment, the portable system is:
If the flow chamber is equipped with the above-described heating element capable of performing repeated high temperature cycles, PCR amplification can be performed.

As shown in FIGS. 19 and 20, SDA can be effectively performed on the device with product yields comparable to the positive control performed in conventional reaction tubes. In this example, Salmonella enterica in a volume of 50 μl was used.
erica) and blood cells were mixed and injected into a flow cell containing an electrode array at 10,000 sites. Bacterial cells can be generated using a checkerboard pattern of 10-15.
Blood cells were separated by dielectrophoresis using a 10V peak-to-peak signal bias protocol with a sinusoidal waveform having a frequency of kHz. The cell mixture is pumped into the flow cell at a rate of 31 μl per minute until the flow chamber is filled and then 12 μl until a total volume of 50 μl has passed through the chamber.
The flow rate was adjusted to 0.4 μl / min. Cells were held above the electrodes by the effect of the positive dielectrophoretic force established by the protocol. The flow cell was then washed with water by pumping the buffer in the reverse direction at a rate of 114 μl / min for 10 minutes to remove cells that were not retained by the positive bias. The fluid contents of the flow cell were then replaced with a solution of SDA reagent to amplify either the spaQ or the invA gene sequence.

200V with a square waveform of 10 ms duration and a total of 40 separate pulses
Total nucleic acids were isolated from bacterial cells by first lysing the cells by applying voltage to the electrodes with a pulsed direct current. (Alternatively, cells can be stored in 95 chambers.
Can also be dissolved by heating to 5 ° C. for 5 minutes). The mixture of concentrated SDA reagent and buffer stock was introduced into the flow cell and mixed with the denatured target nucleic acid to give the following final concentrations of SDA reaction components: 500 nM amplification primer, 50 nM
"Bumper" primer, 9.5 mM magnesium acetate, 35 mM potassium phosphate buffer, pH 7.6, 80 μg / ml bovine serum albumin and 1.4 m
M of each dATP, dGTP, TTP and alpha-thiolated dCTP. Amplification primers were designed to amplify 81 base pairs of the invA and spaQ genes and contained the following nucleic acid sequences:

[0051]

[Table 1]

The bumper primer contained the following sequence:

[0053]

[Table 2]

The released nucleic acid was then denatured by heating the chamber to 90 ° C. for 5 minutes. The chamber was then brought to 60 ° C. and 10 μl of S containing enzyme
DA reagent buffer was introduced into the chamber to initiate amplification (ie, 40 units of BsoB1 restriction endonuclease and 16 units of exo ^- Bst DNA polymerase). In the system of the present invention, amplification using SDA can be performed in 25 to 35 minutes. In this example, a reaction volume of 5 μl aliquot was added 30 min after the reaction to PA
Removed for GE analysis (FIGS. 19 and 20).

The third stage of sample handling, including detection of the molecule of interest, is preferably performed on proteins and nucleic acids of interest. Such detection may employ various forms of hybridization to probes previously bound to the microarray. For example, nucleic acids (eg, RNA, DNA and pRN) can be used to bind nucleic acid analytes (eg, amplified or unamplified target nucleic acids) from a sample by hybridization. Proteins can also be attached to capture molecules attached to the array (
Ie protein-ligand binding interaction). Such capture molecules can include proteins or other molecules, and binding interactions can include interactions such as antigen-antibody, enzyme-substrate, and receptor-ligand binding.

For nucleic acids, target species, whether amplified or not, specific microarrays in either a single (or secondary) flow chamber for capture by oligonucleotide probes tethered to it. The capture pad is electronically addressed. Preferably, the electrode array of the flow cell (ie, flow cell 11, 31a or 32 depending on the specific example and protocol used) comprises:
It has at least 25 individually addressable electrodes coated with a permeable layer (eg, an acrylamide-based hydrogel). The target nucleic acid is biased with a positive sine wave signal generated using a function generator / irregular waveform generator (33120A, Hewlett Packard, Santa Clara, CA). The capture probe-target hybrid is then detected using the fluorescence-labeled reporter probe used in the portable device shown in FIG. 2 and a CCD-based optical imaging system. Detection using this arrangement takes approximately 5 minutes to complete.

Continuing from the above example, amplification products of the spaQ and invA genes were available for capture and analysis. Prior to capture, the SDA reaction solution and amplification products are
It was passed through a desalting column with a volume of μl and the buffer was subsequently exchanged for 50 mM histidine buffer. The amplification product was then addressed to a specific pad of the electrode, which included a gene-specific probe bound to a permeable layer overlaying the electrode. This was followed by 200 mM NaCl, 10 mM Tris, pH 8.0.
Wash the chamber with 1 mM EDTA, then 200 mM NaCl, 10
A bodipy 630-labeled probe oligonucleotide specific for the amplified product at a concentration of 0.5 μM in mM Tris, pH 8.0, 1 mM EDTA and 100 μg / ml calf thymus DNA was introduced. The reporter probe solution was left in the assay cell for 10 minutes and then washed with 7-800 μl of 50 mM histidine buffer. The chamber was then visualized using a 630 nM helium-neon laser and a computer controlled CCD camera. FIG. 21 shows the results of binding of the spaQ gene amplification product to the individual pads of the electrodes in the flow chamber. Binding was generated as a control in the reaction tube and then compared to the annealing of the amplification product introduced into the flow cell chamber after addressing fractions 1 and 2 and the amplification product generated in the flow cell was captured by the capture pad. It shows that annealing was performed satisfactorily in minutes 1 and 2). The level of binding for the control amplification product was higher due to the higher level of amplification obtained in the control reaction.
We note that there is little non-specific binding of amplification products from flow cell reactions bound to capture sites with non-specific capture probes.

In FIG. 22, the amplified SDA product for the spaQ gene was also addressed to a specific pad on the microarray using a two alternating bias format. In one bias protocol, the amplicon was addressed using a 1 kHz 3.5 V peak-to-peak sinusoidal biased AC format. This protocol also used a DC offset voltage of + 2.3V for 5 minutes. In another protocol, a direct current (DC) format containing +2.5 V for 2 minutes was used. As will be appreciated, the DC protocol can transport the molecule of interest to the capture site with greater efficiency as the DC bias pad shows a larger binding signal. The results further demonstrate the flexibility of the system in that the processing of sample materials (cells and molecules) can be manipulated with the wide range of adjustments possible using this electronic-based laboratory-on-a-chip system. . For example, although shown in the above examples as providing lower transport mobilities for specific sequences, the use of an AC format may be used, where the voltage range is 2.0-5.0 peak-to-peak, Frequency range 1 to 10k
Hz, and the DC offset is in the range of 1-5 volts. This range of flexibility provides for the transport of molecules under changing buffer conditions.

Visual detection of amplification reaction products by probe hybridization is 635
This can be done by pointing a battery powered diode laser capable of generating at least approximately 2 mW of laser power with an emission wavelength of nm.
Using a laser, a fluorescent dye-labeled reporter probe (BODIPY-630) was used.
To excite). The wavelength of the emission filter is 670 nm. The dichroic mirror has a wavelength cutoff of 645 nm. Alternatively, as will be appreciated by those skilled in the art, direct electrochemical voltammetric detection systems may be used instead of light-based detection.

While the foregoing invention has been described in some detail by both drawings and examples for purposes of clarity and understanding, without departing from the spirit or scope of the appended claims,
It will be readily apparent to one skilled in the art in light of the teachings of the present invention that certain changes and modifications may be made thereto.

The file of this patent contains at least one drawing executed in light and dark. Copies of this patent with color drawing (s) will be provided by the Office upon request and payment of the necessary fee. The present invention will be further described with reference to the accompanying drawings.

[Sequence list]

[Brief description of the drawings]

FIG. 1 is a simplified flow chart of the system showing three basic stages of the system including sample preparation, chemical reaction and analyte detection.

FIG. 2 is a schematic diagram showing the simple overall hardware of the system, including a flow chamber 11 with a heating element 12, a small desalting column 13,
A laser 14 having an optical path 15 and a detector such as a CCD camera 17 are included.

FIG. 3 is a perspective view showing one example of an external design relating to the portable system of the present invention. The portable system includes a housing 18 that is integrated with a computer and a monitor 20 via a cable 19.

FIG. 4 shows sample and reagent vials 21a-e, heating element 12
, A CCD camera 17, a laser 14, a series of pumps 46 for each reagent vial, and various components such as the focus knob 4 and other features such as the area 5 for electronic components. is there.

FIG. 5 is a cross-sectional view of a flow cell having a microtip grid. The flow cell generally includes a microtip grid 23 on the inner bottom surface of the flow cell chamber 29. The flow cell is covered by a quartz window 28. The underside of the microchip substrate has a heating element 12 coupled thereto. The flow cell has at least two input ports 30.

FIG. 6 is a schematic diagram showing another specific example having two flow cells.
In this embodiment, the first flow cell may include either a cell 31a with an electronically addressable microchip or a cell 31b without such a microchip. In any case, the flow cell 31
a, b have a heating element 12 associated therewith. In this embodiment, second flow cell 32 has an electronically addressable microchip coupled to heating element 12. As in the preferred embodiment illustrated in FIG. 2, the integrated system is located on a movable track 33 that locates the desalination column 13, the laser 14 with the optical path 15, and the astronomical detector of any flow cell. It further includes a detector 17.

FIG. 7 is a schematic diagram showing a specific example of two flow chambers, in which a first flow cell 31a and a second flow cell 32 are arranged close to each other. Also shown is a flow system for directing buffers, reagents and analytes through the system flow cell and desalting column.

FIG. 8 illustrates a bias pattern format that can be used to create a pattern of dielectric forces separating eukaryotic or prokaryotic cells. (A) shows a dielectric pattern of a square wall. (B) shows the checkerboard force pattern.

FIG. 9 is a computer display of a dielectric field distribution corresponding to a square wall force pattern.

FIG. 10 is a computer display of a dielectric field distribution corresponding to a checkerboard force pattern.

FIG. 11 shows a photograph of an electronically addressable microarray in a flow cell during cell separation. The photo specifically illustrates bacterial species separated from whole blood using a checkerboard dielectric force pattern applied to the electrodes of the flow chamber. Bacteria are brightly colored concentrations at the field charge maximum directly above the electrodes of the array, while blood cells are present between the electrodes in the minimum area of the field charge.

FIG. 12 shows eukaryotic cells (cultured HeLa cells) separated from blood cells. The mixture of normal human white and red blood cells is pushed into the space between the electrodes, where the field is minimal as reflected by the pink diamond-shaped spot.

FIGS. 13A and B show eukaryotic cells separated from blood cells (culture He) after washing (FIG. 13A) and after staining with propidium iodide (FIG. 13B).
La cells).

FIG. 14 shows a series of photographs of separating bacterial cells from blood cells using a square wall dielectric force field pattern.

FIG. 15 shows a series of photographs of separating bacterial cells from blood cells using a checkerboard dielectric force field pattern.

FIG. 16 shows the isolated bacteria after washing the square wall pattern.

FIG. 17 shows the isolated bacteria after washing the checkerboard pattern.

FIG. 18 is a PAGE photograph showing the analysis of nucleic acids released from bacteria by electrolysis. Lanes 1 and 6 are markers; lanes 4 and 5 are RNase-treated and untreated whole bacterial DNA, respectively; lanes 2 and 3 are supercoiled and linear plasmid pCR2 released from bacteria.
.1 DNA.

FIG. 19 is a PAGE showing amplification of two S. enterica genes, spaQ and invA by SDA in a flow cell of the invention.
It is a photograph. From left to right, lanes 1 and 6 are molecular weight markers, lanes 2 and 3 are negative and positive controls, respectively, and lane 5 is the result of amplification of the invA gene-specific DNA sequence amplified using the device of the present invention. It is.

FIG. 20 is a PAGE showing amplification of two S. enterica genes, spaQ and invA by SDA in a flow cell of the invention.
It is a photograph. Lane 2 is the molecular weight marker, lanes 1 and 4 are negative and positive controls, respectively, and lane 3 is the amplification result for the spaQ gene.

FIG. 21 is a photograph showing the hybridization results for the spaQ gene. Here, two electrode pads with probes specific for the spaQ gene were tested twice for hybridization and found to be specific for the gene. A typical third pad was hybridized with the amplified material from a positive tube reaction, and a fourth pad with a non-specific probe was tested for hybridization to the amplification product generated in the flow cell.

FIG. 22 is a photograph showing an amplified spaQ gene product addressed to the capture site of a microarray electrode using two different bias conditions.
The top row is twice addressing the pad with capture probes specific for the spaQ sequence (lanes 3 and 4), while lanes 1 and 5 have non-specific probes. The upper row used a sine wave AC format, while the lower row used a direct current (DC) format. The results show that the DC protocol binds a higher degree of amplicon, so using the DC format over the AC format is slightly better at transporting nucleic acids to the electrodes.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification code FI Theme coat ゛ (Reference) G01N 33/48 G01N 33/48 M 4B063 33/53 33/53 D 4D054 M 33/566 33/566 37 / 00 101 37/00 101 103 103 ZCC ZCC // C12N 15/09 ZNA C12Q 1/68 A C12Q 1/68 C12R 1:19 G01N 27/447 1:42 (C12Q 1/68 C12R 1:45 C12R 1:19 ) 1: 285 (C12Q 1/68 G01N 27/26 331K C12R 1:42) C12N 15/00 ZNAA (C12Q 1/68 C12R 1:45) (C12Q 1/68 C12R 1: 285) (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ , LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZW (72) United States 92128 Capewood Lane 14021, San Diego, California, USA Inventor Michael Heller United States 92024 Hawk View Drive, Encinitas, CA No. 1614, 7214 Inventor Ed Sheldon United States 92131 Birch Bluff Place, San Diego, California No. 12515 (72) Inventor Jonathan Diver United States 92131 Sunshine Peak Coat, San Diego, California 11909 (72) James P. O'Connell, Inventor United States 92075 Lana Beach, Solana Point Circle, No. 166 (72) Inventor Dan Smolco United States 91935 Bee Valley Road, Jamal, California No. 20555 (72) Inventor Shira Jalari United States 92122 8510 Costa Verde Boulevard, San Diego, California David Willowby, Inventor David Willowby, 92102 San Diego, California 1312 Twenty-Force Street 1311 F-term (reference) 2G043 AA03 BA16 CA03 DA02 EA01 GA07 KA02 LA01 2G045 BB24 CB01 CB21 DA13 DA36 FA19 FB01 FB02 FB03 FB12 2G054 AA08 AB03 AB04 BB08 CA21 CA22 CA23 EA03 EA06 FA44 GA05 4B024 AA20 CA01 CA09 HA14 HA19 4B029 AA07 FA12 4B063 QA01 QA08 QA19 QA08 QA19 QA08 4D054 FA10

Claims (139)

[Claims] 1. A method comprising: a. And further comprising at least one input port and at least one output port coupled to the flow cell, a plurality of individually addressable electrodes located within the flow cell, and a heating element coupled to the flow cell;
Wherein the flow cell is adapted to receive the sample via an input port, the electrode is operably coupled to a power source adapted to electronically rupture cell membranes in the sample, and wherein the electrode is An electronic system for cell separation, cell lysis, sample preparation and sample analysis, further comprising an active matrix array adapted to bind to a predetermined molecule of interest in the sample for analysis of the molecule. B. A detector operably positioned to detect a molecule bound to the active matrix array by a detectable signal; and c. An integrated system for analysis of eukaryotic and / or prokaryotic cells in a biological sample, including a power supply. 2. The integrated system according to claim 1, wherein the sample preparation further comprises a cell separation step. 3. The integrated system according to claim 2, wherein the cell separation is performed electronically. 4. The integrated system according to claim 1, further comprising a light emitting source operably positioned to provide radiation to the active matrix array. 5. The integrated system according to claim 4, wherein the light emitting source is a laser. 6. The integrated system according to claim 5, wherein the laser is a diode laser. 7. The integrated system according to claim 5, including a beam splitter. 8. The beam splitter of claim 7, wherein the beam splitter is positioned to receive the light emission, reflect at least a portion thereof to the active matrix array, and then pass at least a portion of the radiation from the active matrix array. Integrated system. 9. The integrated system according to claim 1, wherein said plurality of individually addressable electrodes comprising an active matrix array are used as a unit for each cell separation, cell lysis and sample analysis. 10. The integration of claim 1 wherein said plurality of individually addressable electrodes including an active matrix array are used independently of each other in the construction of a subset of electrodes for cell separation, cell lysis and sample analysis. system. 11. The method of claim 1, wherein the sample preparation comprises a further reaction step after electrolysis.
An integrated system as described. 12. The integrated system according to claim 11, wherein the further reaction comprises an enzymatic reaction. 13. The integrated system according to claim 11, wherein the further reaction comprises a chemical reaction. 14. The integrated system according to claim 11, wherein the further reaction comprises a protease reaction. 15. The method of claim 1, wherein the protease reaction comprises a protease-K reaction.
4. The integrated system according to 4. 16. The integrated system according to claim 1, wherein the further reaction comprises an amplification reaction. 17. The integrated system according to claim 16, wherein the amplification includes SDA. 18. The integrated system according to claim 16, wherein the amplification comprises PCR. 19. The integrated system according to claim 16, wherein the amplification comprises a linear amplification. 20. The integrated system according to claim 16, wherein the amplification comprises exponential amplification. 21. The integrated system according to claim 1, wherein the power supply supplies a time-varying signal to at least certain electrodes. 22. The integrated system according to claim 21, wherein the time-varying signal includes an AC component. 23. The integrated system according to claim 22, wherein the AC component is a sine wave. 24. The integrated system according to claim 21, wherein the time-varying signal includes an offset signal. 25. The integrated system according to claim 24, wherein the offset is a DC offset. 26. The integrated system according to claim 1, further comprising a control system. 27. The integrated system according to claim 26, wherein the control system is a computer. 28. The computer of claim 2, wherein the computer is a personal computer.
7. The integrated system according to 7. 29. The integrated system according to claim 28, wherein the personal computer is a notebook computer. 30. The integrated system according to claim 1, wherein the electronic flow cell includes at least one port in addition to the input port and the output port. 31. The integrated system according to claim 30, wherein the electronic flow cell includes at least two ports in addition to the input port and the output port. 32. The integrated system of claim 31, wherein the port is at substantially 90 ° to another port. 33. The integrated system according to claim 30, wherein at least two ports are facing each other. 34. The integrated system according to claim 1, wherein the detector is a CCD detector. 35. The integrated system according to claim 1, which is a closed system. 36. The integrated system according to claim 1, which is a self contained system. 37. The integrated system according to claim 1, further comprising a fluid system. 38. The integrated system according to claim 37, wherein the fluid system includes a pump. 39. The integrated system according to claim 37, wherein the fluid system includes a plurality of valves. 40. The method according to claim 1, wherein the sample analysis comprises hybridization and protein-
2. The integrated system according to claim 1, comprising binding of molecules to an array selected from the group consisting of ligand binding interactions. 41. The integrated system according to claim 40, wherein the hybridization is DNA-DNA hybridization. 42. The integration system according to claim 40, wherein the hybridization is DNA / RNA hybridization. 43. The integrated system according to claim 40, wherein the hybridization is pNA / nucleic acid hybridization. 44. The integration system of claim 40, wherein the protein-ligand binding is selected from the group consisting of receptor-ligand, antigen-antibody and enzyme-substrate binding. 45. a. Further, the apparatus includes at least one input port, a first flow cell coupled to the input port, a plurality of electrodes located within the flow cell, and at least one output port, wherein the first flow cell is connected via the input port. A first source for sample preparation, the electrode being operably coupled to a power source adapted to electronically rupture cell membranes in the sample. An electronic system of b. Additionally, the method includes at least one input port, a second flow cell coupled to the input port, a plurality of electrodes located within the second flow cell, and at least one output port, wherein the electrode is configured to analyze the molecule. An active matrix array adapted to bind to a predetermined molecule of interest in the sample for use with an input port of the second electronic system coupled to at least one output port of the first electronic system. A second electronic system for sample analysis, characterized in that: c. A detector operably positioned to detect a molecule bound to the active matrix array by a detectable signal; and d. An integrated system for the analysis of biological samples containing eukaryotic and / or prokaryotic cells, including a power source. 46. The collection system of claim 45, wherein the sample preparation further comprises a cell separation step. 47. The integration system according to claim 46, wherein the cell separation is performed electronically. 48. The integrated system of claim 45, further comprising a light emitting source operably positioned to provide radiation to the active matrix array. 49. The integrated system according to claim 48, wherein the light emitting source is a laser. 50. The integrated system according to claim 49, wherein the laser is a diode laser. 51. The integrated system according to claim 48, comprising a beam splitter. 52. The beam splitter of claim 51, wherein the beam splitter is positioned to receive the light and reflect at least a portion thereof to the active matrix array, and then pass at least a portion of the radiation from the active matrix array. Integrated system. 53. The integrated system of claim 45, wherein said plurality of individually addressable electrodes of said first electronic system are used as respective units of said cell separation and cell lysis. 54. The integrated system according to claim 45, wherein the plurality of individually addressable electrodes of the second electronic system are used as a unit for the analysis. 55. The integrated system of claim 45, wherein said plurality of individually addressable electrodes of said first electronic system are used independently of each other in a configuration of a subset of electrodes for cell separation and cell lysis. 56. The plurality of individually addressable electrodes of the second electronic system are used independently of one another in a configuration of the subset of electrodes for the analysis.
5. The integrated system according to 5. 57. The integrated system of claim 45, wherein sample preparation comprises a further reaction step after said electrolysis. 58. The integrated system according to claim 57, wherein the further reaction comprises an enzymatic reaction. 59. The integrated system according to claim 57, wherein the further reaction comprises a chemical reaction. 60. The integrated system of claim 57, wherein the further reaction comprises a protease reaction. 61. The protease reaction comprises a protease-K reaction.
0 integrated system. 62. The integrated system according to claim 57, wherein the further reaction comprises an amplification reaction. 63. The integrated system according to claim 62, wherein the amplification comprises SDA. 64. The integrated system according to claim 62, wherein the amplification comprises PCR. 65. The integrated system according to claim 62, wherein the amplification comprises a linear amplification. 66. The integrated system of claim 62, wherein the amplification comprises exponential amplification. 67. The integrated system according to claim 45, wherein the power supply supplies a time-varying signal to at least certain electrodes. 68. The integrated system according to claim 67, wherein the time-varying signal comprises an AC component. 69. The integrated system according to claim 68, wherein the AC component is a sine wave. 70. The integrated system according to claim 67, wherein the time-varying signal includes an offset signal. 71. The integrated system according to claim 70, wherein the offset is a DC offset. 72. The integrated system according to claim 45, further comprising a control system. 73. The integrated system according to claim 72, wherein the control system is a computer. 74. The computer according to claim 7, wherein the computer is a personal computer.
3. The integrated system according to 3. 75. The integrated system according to claim 74, wherein the personal computer is a notebook computer. 76. The integrated system of claim 45, wherein the first flow cell of the first electronic system includes at least one port in addition to the input port and the output port. 77. The integrated system of claim 76, wherein the first flow cell of the first electronic system includes at least two ports in addition to the input port and the output port. 78. The integrated system of claim 77, wherein the port is at substantially 90 ° to another port. 79. The integrated system of claim 76, wherein at least two ports are present facing each other. 80. The integrated system according to claim 45, further comprising at least one heater element. 81. The integrated system according to claim 80, wherein the heater is disposed on the first electronic system. 82. The integrated system according to claim 80, wherein the heater is disposed on the second electronic system. 83. The integrated system of claim 80, wherein the heater element is in thermal contact with either the first or second electronic system. 84. The integrated system according to claim 45, wherein said detector is a CCD detector. 85. The integrated system according to claim 45, which is a closed system. 86. The integrated system according to claim 45, which is a self-contained system. 87. The integrated system according to claim 45, further comprising a fluid system. 88. The integrated system of claim 87, wherein said fluid system comprises a pump. 89. The integrated system according to claim 87, wherein the fluid system includes a plurality of valves. 90. Sample analysis comprising hybridization and protein-
46. The integrated system according to claim 45, comprising binding molecules to an array selected from the group consisting of ligand binding interactions. 91. The integration system according to claim 90, wherein the hybridization is DNA-DNA hybridization. 92. The integration system according to claim 90, wherein the hybridization is DNA / RNA hybridization. 93. The integrated system according to claim 90, wherein the hybridization is pNA / nucleic acid hybridization. 94. The protein-ligand binding may be a receptor-ligand, an antigen-
90. The integrated system of claim 90, wherein the integrated system is selected from the group consisting of an antibody and an enzyme-substrate bond. 95. a. Additionally, the flow cell includes at least one input port, a first flow cell coupled to the input port, a heater element, and at least one output port, wherein the flow cell is adapted to receive the sample via the input port. A first non-electronic system for preparing a sample, wherein the heater is coupled to heat the first flow cell to a temperature sufficient to induce rupture of a cell membrane in the sample; b. . Additionally, the apparatus includes at least one input port, a second flow cell coupled to the input port, a plurality of electrodes located within the second flow cell, and at least one output port, wherein the electrodes are for analyzing a molecule. An active matrix array adapted to bind to a predetermined molecule of interest in the sample of the first, wherein the input port of the second electronic system is coupled to at least one output port of the first non-electronic system. A second electronic system for analyzing a sample, characterized by: c. A detector operably positioned to detect a molecule bound to the active matrix array by a detectable signal; and d. An integrated system for the analysis of biological samples containing eukaryotic and / or prokaryotic cells, including a power source. 96. The integrated system of claim 95, further comprising a light emitting source operably positioned to provide radiation to the active matrix array. 97. The integrated system according to claim 96, wherein the light emitting source is a laser. 98. The integrated system according to claim 97, wherein said laser is a diode laser. 99. The integrated system according to claim 96, comprising a beam splitter. 100. The beam splitter of claim 99, wherein the beam splitter is positioned to receive the emission and reflect at least a portion thereof to the active matrix array and to pass at least a portion of the radiation from the active matrix array. Integrated system. 101. The integrated system according to claim 95, wherein said plurality of individually addressable electrodes of said electronic system are used as said analysis unit. 102. The plurality of individually addressable electrodes of the electronic system,
97. The integrated system according to claim 95, wherein the integrated systems are used independently of each other in the configuration of the electrodes of the analysis subset. 103. The integrated system of claim 95, wherein sample preparation comprises a further reaction step after said heater induced lysis. 104. An integrated system according to claim 103, wherein the further reaction comprises an enzymatic reaction. 105. The integrated system of claim 103, wherein said further reaction comprises a chemical reaction. 106. The integrated system of claim 103, wherein the additional reaction comprises a protease reaction. 107. The integrated system according to claim 106, wherein the protease reaction includes a protease-K reaction. 108. The integrated system of claim 103, wherein said further reaction comprises an amplification reaction. 109. The integrated system according to claim 108, wherein said amplification comprises SDA. 110. The integrated system of claim 108, wherein said amplification comprises PCR. 111. The integrated system of claim 108, wherein said amplification comprises linear amplification. 112. The integrated system of claim 108, wherein said amplification comprises exponential amplification. 113. The integrated system of claim 95, wherein the power supply supplies a time varying signal to at least certain electrodes. 114. The time varying signal includes an AC component.
An integrated system as described. 115. The integrated system according to claim 114, wherein the AC component is a sine wave. 116. The integrated system according to claim 113, wherein the time-varying signal includes an offset signal. 117. The integrated system according to claim 116, wherein said offset is a DC offset. 118. The integrated system according to claim 95, further comprising a control system. 119. The integrated system according to claim 118, wherein said control system is a computer. 120. The integrated system according to claim 119, wherein the computer is a personal computer. 121. The integrated system according to claim 120, wherein the personal computer is a notebook computer. 122. The integrated system of claim 95, wherein the non-electronic first flow cell includes at least one port in addition to the input port and the output port. 123. The integrated system of claim 122, wherein the non-electronic first flow cell includes at least two ports in addition to the input and output ports. 124. The integrated system of claim 123, wherein the port is at substantially 90 ° to another port. 125. The integrated system of claim 122, wherein at least two ports are facing each other. 126. The integrated system according to claim 95, further comprising at least two heater elements. 127. The integrated system according to claim 126, wherein the heater is disposed on the electronic system. 128. The integrated system of claim 126, wherein the heater element is in thermal contact with either the non-electronic or electronic system. 129. The integrated system according to claim 95, wherein said detector is a CCD detector. 130. The integrated system according to claim 95 which is a closed system. 131. The integrated system according to claim 95, which is a self-contained system. 132. The integrated system according to claim 95, further comprising a fluid system. 133. The integrated system of claim 132, wherein said fluid system comprises a pump. 134. The integrated system of claim 132, wherein the fluid system includes a plurality of valves. 135. The integrated system of claim 95, wherein the sample analysis comprises binding of the molecules to an array selected from the group consisting of hybridization and protein-ligand binding interactions. 136. The integrated system according to claim 135, wherein the hybridization is DNA-DNA hybridization. 137. The integrated system according to claim 135, wherein the hybridization is DNA / RNA hybridization. 138. The integrated system according to claim 135, wherein the hybridization is pNA / nucleic acid hybridization. 139. The integrated system of claim 135, wherein said protein-ligand binding is selected from the group consisting of receptor-ligand, antigen-antibody and enzyme-substrate binding.