Diagnostic instrument with movable electrode mounting member and methods for detecting analytes Number:7,435,384 from the United States Patent and Trademark Office (PTO) owispatent An instrument (A) detects in a sample the presence of an analyte. The sample includes electrically readable particles (126) with an agent attached thereto that binds with the analyte or is an analog of the analyte. The instrument includes a port for the sample and a pair of electrodes (25, 30), one of which has a surface portion (30a) with an agent thereat that binds with the analyte or is an analog of the analyte. The electrodes have a first position where they are separated a sufficient distance apart to enable the sample to move between the electrodes and a second position where the electrodes are in close proximity. A detection circuit, including the electrodes, has a first state when the analyte is absent and a second state when the analyte is present. A signaling device provides an indication of the state of the detection circuit.<H instrument, Diagnostic, Diagnostic, inst, 7435384.html, Patent, pto, 7435384

Title: Diagnostic instrument with movable electrode mounting member and methods for detecting analytes

Abstract: An instrument (A) detects in a sample the presence of an analyte. The sample includes electrically readable particles (126) with an agent attached thereto that binds with the analyte or is an analog of the analyte. The instrument includes a port for the sample and a pair of electrodes (25, 30), one of which has a surface portion (30a) with an agent thereat that binds with the analyte or is an analog of the analyte. The electrodes have a first position where they are separated a sufficient distance apart to enable the sample to move between the electrodes and a second position where the electrodes are in close proximity. A detection circuit, including the electrodes, has a first state when the analyte is absent and a second state when the analyte is present. A signaling device provides an indication of the state of the detection circuit.

Patent Number: 7,435,384 Issued on 10/14/2008 to Fish

Inventors: Fish; Leonard (Newport Beach, CA)
Appl. No.: 10/250,754

Filed: January 8, 2002

PCT Filed: January 08, 2002

PCT No.: PCT/US02/00461

371(c)(1),(2),(4) Date: February 06, 2004

PCT Pub. No.: WO02/054052

PCT Pub. Date: July 11, 2002

Current U.S. Class: 422/82.02 ; 422/81; 422/82.01; 422/82.03; 436/525; 436/86; 436/87; 436/94

Current International Class: G01N 27/00 (20060101); G01N 33/50 (20060101)

Field of Search: 422/81,82.01-82.04 436/86-87,94,151,525-526,537,540

References Cited [Referenced By]

U.S. Patent Documents


3799742	March 1974	Coleman
4021117	May 1977	Gohde
4054646	October 1977	Giaever
4072576	February 1978	Arwin et al.
4218298	August 1980	Shimada et al.
4219335	August 1980	Ebersole
4225410	September 1980	Pace
4233144	November 1980	Pace et al.
4287300	September 1981	Gibbons et al.
4313929	February 1982	Morita
4440638	April 1984	Judy
4444892	April 1984	Malmros
4627445	December 1986	Garcia et al.
4632901	December 1986	Valkirs et al.
4753776	June 1988	Hillman
4822566	April 1989	Newman
4840893	June 1989	Hill et al.
4859306	August 1989	Siddiqi et al.
4859421	August 1989	Apicella
4918025	April 1990	Grenner
4920047	April 1990	Giaever et al.
4945045	July 1990	Forrest et al.
4963245	October 1990	Weetall
4965187	October 1990	Tonelli
4995402	February 1991	Smith et al.
5053197	October 1991	Bowen
5063081	November 1991	Cozzette et al.
5080865	January 1992	Leiner et al.
5114674	May 1992	Stanbro et al.
5114859	May 1992	Kagenow
5133937	July 1992	Frackleton et al.
5141868	August 1992	Shanks et al.
5156810	October 1992	Ribi
5167922	December 1992	Long
5187096	February 1993	Giaever et al.
5198368	March 1993	Khalil et al.
5217905	June 1993	Marchand et al.
5223219	June 1993	Subramanian et al.
5248303	September 1993	Margolin
5281539	January 1994	Schramm
5284748	February 1994	Mroczkowski et al.
5334538	August 1994	Parker et al.
5359038	October 1994	Padron
5399486	March 1995	Cathey et al.
5401378	March 1995	King et al.
5427915	June 1995	Ribi et al.
5478526	December 1995	Sakai et al.
5503985	April 1996	Cathey et al.
5532128	July 1996	Eggers et al.
5542915	August 1996	Edwards et al.
5554339	September 1996	Cozzette et al.
5569406	October 1996	Barnhorst
5580794	December 1996	Allen
5607646	March 1997	Okano et al.
5616467	April 1997	Olsen et al.
5653939	August 1997	Hollis et al.
5660993	August 1997	Cathey et al.
5698406	December 1997	Cathey et al.
5714390	February 1998	Hallowitz et al.
5730940	March 1998	Nakagawa
5744358	April 1998	Jackowski
5776713	July 1998	Garner et al.
5798215	August 1998	Cathey et al.
5865804	February 1999	Bachynsky
5898005	April 1999	Singh et al.
5900481	May 1999	Lough et al.
5955377	September 1999	Maul et al.
5981203	November 1999	Meyerhoff et al.
6013170	January 2000	Meade
6013459	January 2000	Meade
6024883	February 2000	Jewell
6060023	May 2000	Maracas
6103538	August 2000	Kotsugai
6130037	October 2000	Lennox et al.
6133046	October 2000	Clerc
6143164	November 2000	Heller et al.
6300141	October 2001	Segal et al.
6333200	December 2001	Kaler et al.
6548311	April 2003	Knoll

Foreign Patent Documents


9244055	Sep., 1997	JP
10-239240	Sep., 1998	JP
99/27367	Jun., 1999	WO

Other References

Mosbach, M. et al, Sensors and Actuators B 2000, 70, 145-152. cited by examiner .
Wong et al, Convalently-Functionalized Single Walled Carbon Nanotube Probe Tips For Chemical Force Microscopy, May 22, 1998, 2 pages. cited by other .
Goldin et al, The Great Out Of The Small, Jan. 3, 2001, 24 pages. cited by other.

Primary Examiner: Soderquist; Arlen
Attorney, Agent or Firm: Connors; John J. Connors & Assoc. Inc.

Claims

The invention claimed is:

1. A portable, single use instrument for collecting a blood sample from a host subject and testing for the presence or absence of an analyte in the collected blood sample, said instrument including a housing having a size that allows the instrument to be hand held by a user, said housing including an orifice in a portion of the housing providing access to an interior of the housing, a testing chamber within the interior of the housing, said chamber including a detection circuit that provides an indication of the presence or absence of the analyte in a blood sample, within the interior of the housing in advance of the testing chamber a supply of electrically readable particles with an agent attached thereto that binds with the analyte, said electrically readable particles being introduced into a blood sample fed to the testing chamber, said detection circuit including a pair of electrodes, said agent attaching to one of the electrodes, and at least one of said electrodes being moveable between a first position where the detection circuit has a first state and a second position where the detection circuit has a second state when the analyte is present in the sample and attaches to the agent attached to the one electrode to alter an electrical property of the detection circuit, a moveable mounting member within the interior of the housing carrying said pair of electrodes, and a needle in advance of the testing chamber mounted to the mounting member so said needle can be extended through the orifice after said housing portion makes contact with a host subject to withdraw a blood sample from the subject and then be retracted within the interior of the housing to feed the blood sample into the testing chamber, a vacuum being created in the testing chamber upon movement of the mounting member so the sample flows through the needle into the testing chamber.

2. The instrument of claim 1 including a receptacle along the passageway in advance of the testing chamber holding the supply of electrically readable particles.

3. The instrument of claim 2 where the receptacle includes a solid member comprising a matrix material holding the particles, said matrix material dissolving in the blood sample as said sample flows past said solid member to release the particles.

4. A portable, single use instrument for collecting a blood sample from a host subject and testing for the presence or absence of an analyte in the collected blood sample, said instrument including a housing having a size that allows the instrument to be hand held by a user, said housing including a needle, a cavity into which a collected blood sample flows, an orifice in a portion of the housing providing access to the cavity, a detection circuit that provides an indication of the presence or absence of the analyte in a collected blood sample flowing into the cavity, a moveable mounting member so that upon movement of the mounting member a vacuum is formed within the cavity, said needle being mounted on the mounting member so upon the mounting member being moved said needle is moved from a retracted position to a sample collection position with the needle extending through the orifice into the host subject, with a blood sample from the subject flowing through the needle into the cavity due to the vacuum.

5. The instrument of claim 4 where the detection circuit includes a pair of electrodes, one being moveable.

6. A portable, single use instrument for collecting a blood sample from a host subject and testing for the presence or absence of an analyte in the collected blood sample, said instrument including a housing having a size that allows the instrument to be hand held by a user, said housing having an orifice therein and containing a needle, a testing chamber into which a collected blood sample flows, a detection circuit in communication with the testing chamber that provides an indication of the presence or absence of the analyte in a collected blood sample flowing into the cavity, said detection circuit including a pair of electrodes, said agent attaching to one of the electrodes, and at least one of said electrodes being moveable between a first position where the detection circuit has a first state and a second position where the detection circuit has a second state when the analyte is present in the sample and attaches to the agent attached to the one electrode to alter an electrical property of the detection circuit, a supply of electrically readable particles with an agent attached thereto that binds with the analyte, said electrically readable particles being introduced into a blood sample fed to the testing chamber, a moveable mounting member so that upon movement of the mounting member a vacuum is formed within the testing chamber, said needle being mounted on the mounting member so upon the mounting member being moved said needle is moved from a retracted position to a sample collection position to draw from a host subject due to the vacuum a blood sample into the testing chamber, and one of the electrodes of the pair being mounted to move from the first position to the second position after the blood sample fills the test chamber, said agent binding with any analyte in the blood sample to change the electrical properties of the detection circuit upon the one electrode being moved from the first position to the second position.

7. The instrument of claim 6 including a spring member interactive with the needle that forces the needle through the orifice and into the subject when the needle is in the sample collection position, with a blood sample from the subject flowing through the needle into the testing chamber due to the vacuum.

Description

RELATED PATENT APPLICATIONS

This application is utility patent application based on a PCT application international application number PCT/US02/00461, international filing date Jan. 8, 2002, which in turn is based on U.S. provisional patent application Ser. No. 60/260,250, entitled "Diagnostic Instruments And Methods For Detecting Analytes," filed Jan. 8, 2001. These related applications are incorporated herein by reference and made a part of this application.

DEFINITIONS

As used herein:

"analyte" means any molecule that is in a sample and is being assayed (analyzed);

"analog of an analyte" means a chemically modified analyte, such as, for example, an analyte molecule connected to a linker molecule and will not bind with the analyte;

"binding agent" means any molecule or group of molecules that that are able to interact with an analyte;

"class of analyte" means a group of analytes that are detected b y closely similar methods: There are five main classes of clinically relevant analytes: Class I proteins, Class II nucleotides such as, for example, RNA and DNA, Class III small molecules, Class IV electrolytes, and Class V cell detection;

"electrically readable particle or particles" means a particle or particles whose physical state, or presence or absence, can be determined through the use of an electronic circuit.

"electrolyte or electrolytes" means a compound or compounds that when dissolved in an aqueous medium dissociate into ions that make the medium conductive.

"small molecule or molecules" means a molecule or molecules that do not have binding sites and have, or may be modified to have, associated therewith an electrically detectable characteristic.

BACKGROUND OF THE INVENTION

A myriad of different clinical analysis methods for detecting the presence or absence of various classes of molecules (analytes) have been developed. These analysis methods are used widely in the biological field in detecting the presence of such molecules as proteins and other biomolecules. Proteins have been mainly analyzed by immunoassays such as RIA or ELISA format, DNA by gel or capillary electrophoresis after PCR amplification, small molecules such as glucose and cholesterol, by various color reactions, either chemical or enzymatic, and electrolytes such as sodium or chloride by ion sensitive electrodes. Recently, biochip or biodisc arrays have been developed for protein and DNA analysis. Instrumentation is widely different depending on the application.

In clinical laboratories and large hospitals hundreds of samples are processed with expensive and large automated analyzers. Smaller analyzers, such as microtiter well plate readers, are used in medium or small laboratories. The fastest growing market is the point-of-care (POC) market. Glucose and HCG (pregnancy test) are examples of well established tests in which, typically, strips or dipsticks are used. Although a glucose test by necessity is quantitative, strip tests are qualitative or semi-quantitative at best with very limited dynamic range. The current technology does not allow quantitative assays in vivo, except for oxygen and possibly for glucose.

In all tests in which analyzers are used, the sample is taken first in a separate container such as a syringe or test tube or placed on a strip. Before actual assay, an aliquot of a sample or the strip is transferred into an analyzer. Transfer adds an inconvenient and potentially harmful step, because laboratory personnel can be exposed to pathogens. Although this problem has partially been solved by a cassette and applicator instrument designed for an optical disc based assay, even then a cassette must be inserted into an optical disc.

It would be highly preferable if a sampling device, the syringe or cassette, would be able to perform the actual assay immediately without any further transfer of a sample. Furthermore, it would be desirable to perform tens, or even hundreds, of different assays for various classes of analytes from the same sample. The instrument may be disposable, and of low cost.

SUMMARY OF THE INVENTION

This invention has several features. Without limiting the scope of this invention as expressed by the claims that follow, its more prominent features will now be discussed briefly. After considering this discussion, and particularly after reading the section entitled, "DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS," one will understand how the features of this invention provide its benefits, which include, but are not limited to, quick and accurate detection of analytes, either quantitatively or qualitatively, or both, elimination of exposure of health care workers to infection, rapid analysis of samples containing multiple analytes, and provision of low cost disposable instruments or modules. In addition to application in the medical field, this invention may be used in connection with food and water safety measurements, military, and even anti-terrorist purposes.

The first feature is that the present invention solves most, if not all, of the problems associated with the prior art by a novel electronic/mechanical approach. It provides instruments and methods to detect the presence of a selected analyte in a sample including electrically readable particles having an agent attached thereto that binds with the selected analyte (Case I). In some embodiments of this invention, however, electrically readable particles need not be employed. These embodiments take advantage of a change in electrical characteristics between a pair of movable electrodes due to changes in their displacement with respect to each other (Case II). The electrodes may be formed by a photo-resist etching process or a plating process.

When electrically readable particles are used, they have binding agents at their surfaces. These binding agents bind with the analyte. They can be antibodies, oligonucleotides, or any other type of recognition molecule. The binding interaction between the analyte and the agent precedes detection of an electrical property, or a change in the electrical. Either the absence or the attendance of this electrical property, or a change in the electrical property, is sensed by a detection circuit which determines the presence or absence of the analyte. The electrical property may be current, resistance, conductance, inductance, capacitance, voltage, magnetic flux, or a phase shift. In accordance with the present invention, an electrically readable particle is bound to an electrode by an affinity binding in order to induce a significant change in the electrical properties of the detection circuit. The effect can be much greater than is obtained by binding an electro-active moiety onto a similar electrode, even if this moiety is catalytic. Moreover, the binding of an electrically readable particle is universal and independent of the chemical nature of the binding pair.

Small molecules and electrolytes can advantageously be detected electrochemically by the present invention. Electrically readable particles are not used in assaying for small molecules and electrolytes. The embodiment of this invention that does this type of assay for either a specific small molecule or a specific electrolyte detects the absence or present of an ion current using a pair of electrodes. One electrode has a surface portion that collects thereat the specific small molecule or electrolyte, as the case may be. This one electrode is at a first polarity. The other electrode has a surface portion that collects thereat small molecules or electrolytes other than said specific small molecule or electrolyte in question. This other electrode is at a second polarity opposite said first polarity. The electrodes have (i) a first position where they are separated a sufficient distance apart to enable the sample to move there between and to suppress the ion current and (ii) a second position where the electrodes are sufficiently close to each other to establish an ion current. In the first position the electrodes are separated a first distance apart sufficient to enable the sample to move between said electrodes and a second position where the electrodes are in close proximity to each other. The first distance is, greater than the second distance.

There is a detection circuit, including the electrodes, that has a first state when the specific small molecule or electrolyte is absent from the sample and a second state when the specific small molecule electrolyte is present in the sample. A signaling device provides an indication when the detection circuit is in the second state.

Almost all clinical or chemical assays can be made using both Case I and Case II embodiments of this invention. Essentially all Class I through V molecules identified above may the assayed. The molecules include, but are not limited to, peptides, proteins, glycoproteins, oligonucleotides, DNA, RNA, steroids, lipids, lipoproteins, carbohydrates, cathecolamines, several drugs, oxygen, nitric oxide, nitrous oxide, carbon dioxide, nitro, nitroso, azo, heterocyclic compounds, isocyanates, phenols, amines, most sulfur containing compounds, such as disulphides, dithiocarbamates, thiobarbiturates, thioureas, thiols, sulphonates, sulphides, and sulphoxides, and also cations, anions, chelates, and organometallics.

In the Case I embodiments of this invention, the detection circuit measures the electrical property produced when an affinity binding occurs between the analyte and the binding agent, for example, antigen-antibody interaction, DNA hybridization, ligand-receptor binding, enzyme-substrate, or enzyme-inhibitor interaction. The detection circuit includes an electrode, and preferably, uses at least one pair of relatively large surface area electrodes that in one position are widely separated, although sub-micrometer electrodes are still within the scope of this invention. Nevertheless, an individual sub-micrometer sized particle, or group of such particles, is large enough to fill the gap between the electrodes when these electrodes are in a second position in close proximity with each other to produce a detectable change in an electrical characteristic of the detection circuit.

One Case I embodiment of the present invention has a flow through capillary with a movable wall. The capillary contains two or more electrodes, at least one of which is connected to the movable wall. At least one electrode is coated with an agent that binds to the analyte. In the presence of the analyte, electrically readable particles having a binding thereon, such as a gold spheres, are bound onto the surface of the electrode coated with a binding agent. When two electrodes are allowed to approach, so that both of them are in close proximity of the electrically readable particles, for example, a current is observed that is relative to the number of the electrically readable particles and the applied electric potential. Electrically readable particles will make the capacitor leaky. Thus, electrical properties can be changed by moving the electrodes in a controlled way into close proximity and measuring the electrical property change such as conductance, resistance, inductance, capacitance, or phase shift, in order to find out the modulation of these properties by an analyte. The instrument has a mechanism for bringing two electrodes into close proximity. The close proximity is obtained by providing an accurate moving mechanism and/or physical structures that prevent a direct contact between the electrodes and accurately space the electrodes apart a predetermined distance. This allows accurate electrochemistry to be performed quickly and at low cost.

Another Case I embodiment of the present invention comprises two opposing electrode arrays intersecting at right angles. Because the electric potential can be coupled between any pair of electrodes between two arrays, the number of high field areas, i.e., active working areas on electrodes can be much larger than the number of electrodes. A simple example is an orthogonal arrangement of two linear electrode arrays. Because two or more electrode arrays can be used, the number of tests can be quite large. The instrument in which multiple analytes are assayed simultaneously employs a microprocessor to record the electrical properties of each assay site independently, or a combination of assay sites that do not interfere with each other. This instrument is greatly simplified as compared with prior art devices employing electrode arrays and it facilitates the construction of massive, very low cost arrays.

The present invention enables, perhaps for the very first time, a disposable self-contained instrument that is able to perform hundreds, or even thousands of tests qualitatively, quantitatively, or both, and quickly from a very small amount of sample. It solves most problems associated with the prior art. The assay can be performed in the sample collection instrument. No transfer or aliquoting of the sample is required. Results are obtained fast and they are quantitative. The detection is very sensitive, because a single electrically readable particle can be detected, and only one analyte molecule is necessary for the binding of that particle. The electrode arrays may allow multiple tests to be performed from each sample. The instrument may incorporate a low cost microprocessor and display, or be inserted into an adapter for connection to a computer having a microprocessor and display.

All these advantages combined enable construction of a self-contained disposable instrument that is able to analyze quantitatively, sensitively, and quickly several different analytes simultaneously. The instrument can also be modular, so that only the sample collection and measuring unit is disposable, while processing and display units are used repeatedly, or it can be a single use, disposable hand held instrument used for specific tests, or a computer equipped with an adapter.

In a third embodiment of the instrument of this invention, disposable modules, or self-contained assay instruments for the Point-of-Care, are provided. The modules can have sample collection and processing equipment including, for example, cell separation, cell lysis, reagent storage and mixing, and analyte fractionation, such as chromatography and electrophoresis. The modules have the electrodes for actual measurement. These modules can be used in combination with a processing and display instrument that has been designed for this purpose or modules can be connected via an adapter to a computer, such as a personal computer (PC). Self-contained disposable instruments may also have processing and display capability. The information can be transferred to a permanent storage unit, such as PC hard drive, before the disposal. The measurement modules, which have a sample inlet port, are in a rack or in a conveyer belt. A pipetting robot will add samples to each module, and after a short incubation time another robot will put each module into a data collection and processing system.

This invention includes several methods. Broadly, the method of this invention detects in a sample the presence of an analyte. It includes the steps of

(a) introducing into the sample electrically readable particles with an agent attached thereto that binds with the analyte or is a n analog of the analyte,

(b) providing an instrument including

a pair of electrodes, said electrodes having a first position where they are separated a first distance apart sufficient to enable the sample to move between said electrodes and a second position where the electrodes are in close proximity to each other, said first distance being greater than said second distance,

a detection circuit, including the electrodes, that has a first state when the analyte is absent from the sample and a second state when the analyte is present in the sample, said second position determining the state of the detection circuit,

a signaling device that provides an indication of the state of the detection circuit with the electrodes in the second position,

(c) contacting the sample, including the electrically readable particles, with the electrodes while said electrodes are in the first position,

(d) removing any unbound particles from between the electrodes, and

(e) moving the electrodes to the second position.

In another method the electrically readable particles are stored on a holding electrode. These stored particles may or may not carry a binding agent. If they do not, they arrived independently at the surface of this electrode through electrostatic attraction. By reversing either the charge of the particles, or of the electrode, the non-specifically bound particles are removed, while specifically bound particles remain bound.

DESCRIPTION OF THE DRAWING

The preferred embodiments of this invention, illustrating all its features, will now be discussed in detail. These embodiments depict the novel and non-obvious instrument and methods of this invention as shown in the accompanying drawing, which is for illustrative purposes only. This drawing includes the following figures (Figs.), with like numerals indicating like parts:

FIGS. 1A and 1B are schematic illustrations of a first embodiment of the instrument of this invention depicting test sites where analytes in a sample are individual molecules with at least two spaced binding sites.

FIGS. 2A and 2B are schematic illustrations of a second embodiment of the instrument of this invention, which is similar to the first, depicting test sites where DNA specimens are the analytes in the sample.

FIGS. 2C through 2G are schematic illustrations of the second embodiment of the instrument of this invention depicting using nano tubes as the conductive particles, wherein

FIG. 2C shows a pair of spaced apart electrodes with nano-tubes in a sample that is between the spaced apart electrodes,

FIG. 2D shows the electrodes of FIG. 2C moved into close proximity with each other and the analyte attached to the left handed assay site,

FIG. 2E is a side elevational view of a nano-tube.

FIG. 2F is an illustration of an end of the nano-tube being chemically treated to form thereat a binding site,

FIG. 2G is a cross-sectional view taken along line 2G-2G of FIG. 2E.

FIGS. 3A and 3B are schematic illustrations of a third embodiment of the instrument of this invention including an electrode that has at least one electrical property in a detection circuit that (a) does no t change because the analyte attaches to the agent on the electrically readable particles and (b) that changes in the absence of the analyte due to the agent on the electrically readable particle binding to the analog of the analyte on the electrode.

FIGS. 3C and 3D are schematic illustrations of another version of the third embodiment of the instrument of this invention where an analog of the analyte is bound to the particle.

FIG. 4 is a schematic illustration of the fourth embodiment of the invention showing an assay site for detecting electrolytes.

FIG. 5 is a schematic illustration of another version of the fourth embodiment of the invention showing an assay site for detecting small molecules such as glucose.

FIG. 6A is a schematic illustration of a fifth embodiment of the instrument of this invention shown in a plan view.

FIG. 6B is a cross-sectional view taken along line 6B-6B of FIG. 6A showing a capillary structure in an open position.

FIG. 6C is an enlarged fragmentary view taken along line 6C of FIG. 6B.

FIG. 7A is a cross-sectional view similar to that shown in FIG. 6B but with the capillary structure in a closed position.

FIG. 7B is an enlarged fragmentary view taken along line 7A of FIG. 7.

FIG. 8A shows cross-sections of different capillary structures.

FIG. 8B is a cross-sectional view of a capillary structure with two separate walls, one being moveable relative to the other.

FIG. 9 is a plan view of a sixth embodiment of this invention employing a grid of overlying orthogonally oriented electrodes creating at the overlying intersections of the electrodes an array of test sites.

FIG. 10 is a perspective view of one of the test sites created the overlying orthogonally oriented electrodes shown in FIG. 9.

FIGS. 11A through 11G are schematic illustrations depicting the steps employed in making the grid shown in FIG. 9.

FIG. 12 is a plan view of an array of assay sites having a holding electrode in advance of the array.

FIG. 13A is a cross-sectional view of a seventh embodiment of this invention schematically illustrating an instrument employing electrodes coated with a thin layer of soft material and the electrodes in an open position.

FIG. 13B is a cross-sectional view of the seventh embodiment of this invention schematically illustrating the instrument shown in FIG. 13A with the electrodes in a closed position.

FIG. 14A is cross-sectional view of an eighth embodiment of this invention schematically illustrating the use of a flexible, resilient electrode.

FIG. 14B is a plan view taken along line 14B-14B of FIG. 14A.

FIGS. 15A through 15C are cross-sectional views of a ninth embodiment of this invention schematically illustrating an instrument employing a roller moving past different assay sites.

FIGS. 16A through 16D are cross-sectional views of a tenth embodiment of this invention schematically illustrating a hand held, single use, portable, disposable instrument with a needle that is extended to collect a sample of blood from a subject and retracted after collecting the sample.

FIG. 16E is a plan view of the instrument shown in FIGS. 16A through 16D.

FIG. 16F is a plan view of a display on the instrument shown in FIGS. 16A through 16D providing a qualitative readout that a sample is positive for E. Coli in the sample and a quantitative readout indicating the amount present in the sample.

FIG. 17 is a schematic illustration of a gold particle coated with a resistive material.

FIGS. 18A through 18D show a eleventh embodiment of this invention employing a disposable module used with the portable, reusable, computer adapter shown in FIGS. 19A and 19B and is used with the computer shown in FIG. 20, wherein

FIG. 18A is a cross-sectional taken along line 18A-18A of FIG. 18B,

FIG. 18B is a cross-sectional taken along line 18B-18B of FIG. 18A,

FIG. 18C is a cross-sectional taken along line 18C-18C of FIG. 18B,

FIG. 18D is a cross-sectional taken along line 18D-18D of FIG. 18B.

FIG. 19A is a cross-section view of a computer adapter to be inserted into a USB port of the computer shown in FIG. 20, showing the disposable module of FIGS. 18A through 18D about to be inserted into the computer adapter.

FIG. 19B is a cross-section view similar to that of FIG. 19A showing the disposable module of FIGS. 18A through 18D inserted into the computer adapter.

FIG. 20 is a schematic illustration of a computer network with the assembly of the disposable module and computer adapter shown in FIG. 19B being connected to the USB port in a computer.

FIGS. 21A through 21C depict schematically the twelfth embodiment of this invention showing a sample carrier to be used with the automatic testing instrument shown in FIG. 21 wherein

FIG. 21A is a plan view of the sample carrier comprising the top plate shown in FIG. 21B and the bottom plate shown in FIG. 21C,

FIG. 21B is a plan view of the top plate of the sample carrier, and

FIG. 21C is a plan view of the bottom plate of the sample carrier.

FIG. 22 is a plan view of an automatic detection system for testing numerous samples on a sustained basis over a prolonged period of time.

FIG. 23 is a detection circuit employed in this invention where changes in inductance, resistance and/or capacitance are detected.

FIG. 24 is a detection circuit employed in this invention where changes in voltage are detected.

FIG. 25 is a detection circuit employed in this invention including a multiplexer.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

First Embodiment

FIG. 1 depicts an instrument A including a pair of conductive electrodes 25 and 30, respectively mounted on insulator base support members 21 and 20. The electrodes 25 and 30 have substantially flat, planar surfaces 25a and 30a facing each other and each lying in different parallel planes. These electrodes 25 and 30 are directly opposed to each other vertically, and each have an area that exceeds about 0.5 square micrometer, and typically, each have an area of from about 10 square micrometers to about 10 square millimeters. The electrodes 25 and 30 are shown in a spaced apart position in FIG. 1A and, after introducing a sample and then removing unbound material from between the electrodes 25 and 30, moved to the dotted line position shown in FIG. 1B. As shown in FIG. 1B schematically, the electrodes 25 and 30 are brought into close proximity, almost but no t quite touching. Typically, the distance between the electrodes 25 and 30, when in the position shown in FIG. 1A, is from about 5 micrometers to about 5 millimeters and, when in the position shown in FIG. 1B, is from about 10 nanometers to about 5 micrometers. A third electrode 31, spaced from the electrode 30, and also mounted on the base support 20, provides a second assay site. Binding agents 116 and 117, are respectively on the surfaces of the electrodes 30 and 31. As discussed further in connection with the embodiments shown in FIGS. 9 through 12, a plurality of pairs of directly opposed electrodes form numerous assay sites that enable an instrument according to this invention to detect several different analytes in any sample.

The base support members 21 and 20 are enclosed within a housing 21a having an inlet port 21b providing an opening in the housing through which a sample is introduced into the instrument A and an outlet port 21c through which sample flows from the housing. The port 21a may have a cover 21d that is moveable between open and closed positions. As illustrated, the cover 21d is shown in an open position. The sample includes electrically readable particles 126 and 127 and binding agents, the antibodies 114 and 115, are bound respectively to the surfaces of these electrically readable particles 126 and 127. The electrically readable particles are introduced into the sample prior to, during, or after the sample is fed into the instrument A. In this illustration, the sample also includes an analyte 118, for example, insulin. This analyte 118, having at least two spaced apart binding sites, only reacts with the binding agent 116 on the surface of the electrode 30. Upon introduction of the sample, including the particles 126 and 127 and the analyte 118, into the instrument A between the electrodes 25 and 30, the analyte attaches to the particles 126 and to the binding agent 116 on the surface of the electrode 30. Thus, on the left side, the first assay site, the electrically readable particle 126 on the electrode surface forms an antigen conductor. No affinity binding occurs on the right side, the second assay site, with the particle 127, because there are no analytes present that bind with both the binding agents 115 and 117.

In accordance with this invention, the directly opposed pairs of electrodes, electrodes 25 and 30 and electrodes 25 and 31, are moved from the position shown in FIG. 1A to the position shown in dotted line in FIG. 1B. A motor M having its output shaft (not shown) is connected to a drive mechanism D that, upon energizing the motor, moves the support member 21 downward towards the other electrodes 30 and 31 on the support 20. In the position shown in FIG. 1A, the pair of electrodes 25 and 30 is separated a sufficient distance to allow the sample to flow between this pair of electrodes and out the outlet port 21c. After an adequate time period has elapsed to allow binding to occur if the analyte 118 is present, all the assay sites are washed, for example with distilled water, to remove unbound material. An alternately mechanism such as depicted in FIG. 12 may be used. This mechanism shown in FIG. 12 will be discussed subsequently in greater detail. After washing, the motor M is energized to move the electrode to the dotted line position shown in FIG. 1B. Preferably, pressure is applied to the particle 126 bound to the electrode 30. This is especially important when the detection circuit responds to changes in conductivity. The pressure is controlled. It must not be so great as to cause a short circuit between the electrodes 25 and 30, yet sufficient to the break surface tension. The application of pressure is carefully controlled so that the electrodes pairs of an assay site do not touch, but will insure electrical contact between the particles 126 bound to the electrode 30 and also between these bound particles and the electrode 25. As discussed subsequently, a spacer device may be employed to insure that the electrodes do not touch and short out the detection circuit. When the detection circuit responds to changes in inductance, capacitance, or phase shift, the application of pressure is not critical.

The pairs of electrodes 25 and 30 are components in an electrical circuit such as, for example, the detection circuits depicted in FIGS. 23 through 25. These detection circuits each have a first state when the analyte is absent from the sample and a second state when the analyte is present in the sample, and a signaling device, for example, a liquid crystal display (LCD) that provides an indication when the detection circuit is in the second state. The signaling device provides a qualitative read-out identifying the analyte, or a quantitative read-out corresponding to the amount of analyte in the sample, or both. These detection circuits will be discussed in greater detail subsequently.

Because very small electrically readable particles 126 and 127 are used, the instrument A is very sensitive. It can even detect a single molecule of the analyte 118. For example, a cancer cell will produce a protein molecule or molecules that are indicative of the presence of cancer cells. In the early stages of the cancer, only very low levels of this marker protein molecule are in a sample of a subject's blood. Conventional testing techniques are not usually capable of detecting such low concentrations of these identifying protein molecules. The attachment of a single marker molecule to an electrically readable particle that is in the presence of an electrode at an assay site creates a change in state in the detection circuit that indicates the presence of this single molecule.

Second Embodiment

The second embodiment of this invention, the instrument B shown in FIG. 2, is similar to that shown in FIG. 1, but the housing 21a and motor M and drive mechanism D are not shown. This embodiment is a schematic representation of a DNA test. The electrodes 30 and 31 are coated with different probes, that is binding agents, respectively probes 216 and 217. The analyte or target 218 is able to bind with the probe 216 as well as with a probe 214 on the surface of the electrically readable particle 226. The probes 215 and 217 do not bind with the target 218. As discussed above in connection with FIGS. 1A and 1B, after binding of the target 218, unbound material is removed from the space between the electrodes 25 and 30 and the electrodes are moved towards each other and a detection circuit is then activated.

FIGS. 2C through 2G schematic represent another DNA test. In accordance with one aspect of this invention, as shown in FIGS. 2C through 2G, the conductive particles may be nano-tubes 26, such, for example, manufactured by Carbon Nanotechnologies, Inc. of Huston, Tex. Nano-tubes 26 are graphite-like elongated tubes having either opened or closed ends and hallow interiors with an outside diameter D of from about 1 nanometer to about 50 nanometers and a length L of from about 10 nanometers to about 10 micrometers. As shown in FIG. 2G, the nano-tubes 26 have an inside diameter D1 of from about 10 to 20 nanometers, with a wall thickness of an low as a single atom.

As illustrated in FIG. 2F, the opposed ends 26a and 26b of the nano-tubes have been reacted with such chemical reagents as a mixture of osium tetraoxide and periodic acid, bromine, chromic acid, or potassium permanganate, to form reactive groups. When the mixture of osium tetraoxide and periodic acid is used the reactive groups are oxygen sites. These oxygen sites are reacted with amino terminated oligonucleotides R to form at the opposed ends binding sites 26a and 26b. These binding sites on the nano-tubes 26, in the presence of the analytes 15a and 15b, which must both be present, bind with the analytes at the right hand assay site shown FIGS. 1C and 1D. There are binding agents 16 and 16a, respectively at the surfaces of the electrodes 20 and 21 that bind with the analytes attached to one nano-tubes 26. The analytes 18 do not bind with the binding agents 17 and 17a at the electrodes 21 and 20 at the right hand assay site shown in FIGS. 1C and 1D. To make the nano-tubes 26 soluble they are coated with a detergent such as tween-20. In accordance with one aspect of this invention, the electrodes 20 and 21 are more into closed proximity with each other as shown in FIG. 1D prior to measuring for attachment of the analytes to an assay site. A reference discussing chemical modification of the ends of the nano-yubes is discussed by S> S> Wong et al in Covalently-Functionalized Single-Walled Carbon Nanotube Probe Tips for Chemical Force Microscopy, J. Am. Chem. Soc. 1998, 120, 8557-8558.

Third Embodiment

In FIGS. 3A and 3B, a competitive or inhibitory assay is illustrated using a third embodiment of this invention, the instrum