JP2003508730A - Biosensor using charge neutral conjugated polymer - Google Patents

Abstract

SUMMARY OF THE INVENTION The present invention provides a charge-neutral conjugated polymer or copolymer having a functional group for binding a biomolecular probe, as well as a biomolecular probe in electrical contact with such a polymer covalently bound. Electrodes and electrode arrangements. The present invention includes a biosensor using the electrode coated with the conjugated polymer, wherein the biosensor detects binding to a biomolecular probe by electric means such as AC impedance.

Description

Detailed Description of the Invention

TECHNICAL FIELD This application claims the priority of provisional application No. 60 / 138,437 (filed June 10, 1999). The present invention relates to the field of detector electrode arrays on a chip for performing analysis of biological substances such as DNA.

BACKGROUND ART Devices for detecting biomolecules usually consist of a support matrix for binding or capturing probe molecules, on which the detection probes are arranged. When exposed to complementary biomolecular targets (or analytes), the biodetection device produces a detectable change in radioactive, optical or electrical signal, confirming the presence of the particular biomolecular target. Usually, the biomolecular target to be detected is labeled with a marker such as ³² P, fluorescent dye or redox (depending on whether the detection means is autoradiography, fluorescence microscopy or electrical means).
Or reporter).

Another biodetection device includes a second reporter molecule. This second reporter molecule is introduced after the probe molecule interacts with its complementary biomolecular target. Like the probe molecule, this second reporter molecule also interacts with the biomolecular target, either by binding to the target or by forming a complex.

Lavache et al [Analytical Biochemistry 258 , 188-194 (1998)] describe a sequence of oligonucleotides constructed on a silicon chip with a matrix of addressable microelectrodes. Each electrode is coated with a polypyrrole copolymer, where some of the pyrrole in the copolymer has the oligonucleotide bound to the pyrrole. This polymer is prepared by an electrochemical method. The copolymer is coated on the microelectrode. Probe DNA on electrode to test sample DNA PCR amplified to contain fluorescent marker groups
The genotype of hepatitis C was detected by the hybridization of H.

International Patent Application Publication WO 95/29199 describes functionalized polypyrrole copolymers whose functional groups are designed to bind biological molecules such as DNA or polypeptides.

US Pat. No. 5,837,859 assigned to Cis Bio International describes the preparation of electrically conductive pyrrole / nucleotide / derivatized / pyrrole copolymers useful in nucleic acid synthesis, sequencing and hybridization. There is. This copolymer is produced electrochemically and coated onto microelectrodes for DNA analysis.

US Pat. No. 5,202,261 describes conductivity sensors and their use in diagnostic assays.

US Pat. No. 5,403,451 describes the detection of target analytes using polymers in combination with a periodic alternating voltage.

In typical prior art, the target DNA is usually labeled with a marker (or reporter), eg ³² P, fluorescent dye or redox. A radioactive, fluorescent or electrical signal is detected when the labeled target is exposed to a complementary probe on a conducting polymer or copolymer. Fluorescence or redox labeling is usually preferred because of the stringent experimental conditions required for radioactive labeling. However, fluorescent dyes around conductive polymers or copolymers are subject to signal quenching. On the other hand, conductive polymers or copolymers, when used for the detection of redox labeled targets,
It causes a large background noise.

DISCLOSURE OF THE INVENTION It is an object of the present invention to provide a conductive matrix when used as a support matrix for probe binding or capture for biomolecule detection and as a biodetection device for performing such detection. The elimination of inactivation of the signal derived from the functional polymer.

Another object of the invention is to reduce detection noise from conductive polymers when used as a support matrix for probe binding or capture for biomolecule detection.

Yet another object of the invention is to provide a simplified method for biomolecule detection and a biodetection device for performing such detection.

The present invention uses DN with the aid of a neutralized conjugated polymer or copolymer on the electrode.
It relates to a method for detecting biological molecules (biomolecules) such as A, RNA and polypeptides. Compared to the prior art, the present invention uses a functionalized polymer or copolymer in the neutral state instead of the conductive state as a support matrix for binding or capturing biomolecular probes in biomolecular detection devices.

In one aspect of the invention, aromatic monomers and functionalized aromatic monomers are electrochemically polymerized and coated on the electrode surface to produce a functionalized polymer or copolymer. The coated conjugated polymer or copolymer is in a charged, electrically conductive state. In the present invention, this charged functionalized polymer or copolymer is electrochemically reduced to a neutral state to produce a (charge neutral conjugated polymer), which is then used in any biomolecule detection. .

Charge neutral functionalized polymers or copolymers have a low electrical background when used in the electrical detection of biomolecules. Also, it does not quench the fluorescent signal when used in fluorescent detection of biomolecules. In both cases, the resulting device has a significantly improved signal /
It has a noise ratio, thus increasing the sensitivity of biomolecule detection.

That is, the present invention includes a charge-neutral conjugated polymer having a functional group for attaching a biomolecular probe to the polymer. The present invention includes electrodes in electrical communication with such polymers, arrays of such electrodes. The present invention provides a biosensor in which a biomolecule probe is covalently bound to a functional group of a charge-neutral conjugated polymer on an electrode, and the binding of a biomolecule to be detected is measured by an electrical detection means (for example, AC impedance). Includes.

BEST MODE FOR CARRYING OUT THE INVENTION The present invention relates to a method of detecting biological molecules with the aid of charge-neutral conjugated polymers on electrodes. A charge-neutral conjugated polymer means a polymer that has zero charge (negative or positive) in its backbone and still has delocalized pi electrons in its backbone. Conjugated polymers are characterized in that their backbone is accompanied by regular changes in single and double chemical bonds. Examples of conjugated polymers include polypyrrole, polyphenylene, polyacetylene, polydiacetylene, polythiophene, polyfuran, polyaniline, polycarbazole, poly (phenylene vinylene). More specifically, the invention includes charge-neutral conjugated polymers containing one or more functional groups capable of binding probe molecules. The charge-neutral conjugated polymer is coated on the electrode surface by electrochemical copolymerization of aromatic and functionalized monomers known in the art. When coated, the conjugated polymer or copolymer is electrically conductive, usually in its charged state, its charge being balanced by counterions from the polymerization solution. This charged state causes signal deactivation for nearby fluorescent markers, as in the case of fluorescence detection. It also causes noise for electrical detection.

In order to overcome these potential problems, the electrode-coated polymer or copolymer used in the present invention is first coated on the surface electrode and then coated by reverse biasing right. The charge state at that time is reduced to the charge neutral state. Polymers or copolymers in the neutral state are insulators or semiconductors, which do not quench the fluorescence of fluorescent markers in their immediate vicinity in fluorescent detection and also have limited background in electrical detection of biomolecular targets. Generates only ground noise.

Functional groups used in the present invention include, but are not limited to, amines, hydrazines, esters, amides, carboxylates, halides, hydroxyls, vinyls, vinyl carboxylates, thiols, phosphates, silicon-containing organic compounds, and these. And derivatives thereof are included. Using functional groups, biomolecular probes (e.g.,
DNA, RNA, peptides, polypeptides, proteins, antibodies, antigens and hormones) are attached to polymers or copolymers on electrodes. For example, the target DN
An oligonucleotide that is partially complementary to A is covalently attached to the neutral polypyrrole copolymer via the amine functionality.

The electrodes used in the present invention are prepared from at least one of the following materials:
Metals such as gold, silver, platinum, copper and alloys; conductive metal oxides such as indium oxide, indium oxide-tin, zinc oxide; other conductive materials such as carbon black, conductive epoxies and combinations thereof.

The preferred detection method in this embodiment is an electrical or electrochemical method.
After exposure to the target molecule, the biosensor detects changes in the electrical signal and records the changes by means of reading (eg, display, printout). Electrical and / or electrochemical methods include, but are not limited to, AC impedance,
Select from cyclic voltammetry (CV), pulse voltammetry, square wave voltammetry, AC voltammetry (ACV), hydrodynamic modulation voltammetry, potential step method, potentiometric measurement, amperometric measurement, current step method, and combinations thereof. You can

It is more advantageous to detect biomolecular targets without the need for target labeling. The present invention provides a sensitive method for detecting biomolecular targets without the need for target labeling.

Certain biomolecules are electrically active and can give rise to unwanted background noise when detection is performed by passing a charge through these biomolecules. For example, guanine and adenine are about 0.75V each
And can oxidize at 1.05 V [Analtica Chimica Acta 319 (1996) 3
47-352]. Therefore, it is more desirable to use the impedance method for label-free biomolecule detection.

The present invention includes a method for measuring an analyte in a test sample, the method comprising the steps of: (a) aromatizing a solution in positive bias with a supporting electrolyte in solution. A polymer or copolymer coating is deposited on the electrode by electrochemically polymerizing a group monomer and a monomer having a functional group; (b) reverse biasing the electrode to neutralize the polymerized polymer. (C) covalently attaching the biomolecular probe to the neutral copolymer via the functional monomer; (d) contacting the electrode with a test sample containing an electrolyte; and (e) the analyte being a neutral polymer or copolymer. Changes in the electrical or electrochemical properties of the electrode are measured by electrical and / or electrochemical methods when bound to the above biomolecular probe.

The biosensor can include an array of electrodes in electrical contact with a matrix of charge-neutral conjugated polymers with different detection probes for detecting multiple biomolecular targets. It is also within the scope of the invention to assemble a high density biosensor with longitudinally and laterally positionable electrodes coated with thousands of detection probes for screening applications. In the case of high density arrays, it is more practical to use robotic means to place various biomolecular probes on each electrode.

[0026]   Neutral polypyrrole using the present invention in combination with an AC impedance detection method
A description will be given by using a polymer electrode array. How to create an array like this
, Schematically shown in FIG. Tip10The silicon support11Micro electrical engineering on
Prepare by the method. Probe sequence15And electrodes16Such as gold or platinum
Prepare from an inert metal. Polypyrrole12(DNA binding groupThirteen0)
.1M pyrrole + 5 μM 3-acetate N-hydroxysuccinimide pyrrole
+ 0.1M LiClO_Four/ Probe sequence in acetonitrile (0/5% water)14Up
Electrochemically coated. Then, the polypyrrole film is electrochemically neutralized 17 To Using a nanofluidic partitioning means, each probe is labeled with a different oligonucleoside.
Chid18It can be bound to ODN1 and ODN2 sequentially. Target OD
N2 solution19After hybridizing the probe sequences in
Analyzer20Using a specific DNA sequence21For impedance changes
Put out.

As a modification of the above aspect, the biomolecule probe can also be attached to an aromatic functional monomer, which can then be electrochemically polymerized with the aromatic monomer to yield a conjugated polymer.

EXAMPLES The invention is illustrated in more detail by the following examples, which use polypyrrole as the conjugated copolymer and DNA as the detection target. These examples are intended to illustrate particular embodiments of the invention and are not intended to limit the invention in its spirit or scope.

Example 1 To illustrate the present invention, a 2 mm diameter platinum electrode was used for the electrochemical coating of polypyrrole. The electrode surface was sequentially polished with 0.3 and 0.005 μm gamma alumina powder (CH Instruments, Inc.) and then washed with deionized water. After polishing, the electrodes were immersed in 1M H _S SO ₄ for 20 minutes and then washed vigorously with DI water. A CH660 potentiometer was used for polypyrrole coating. Platinum wire and Ag / AgCl were used as counter and reference electrodes, respectively. A solution containing 0.1M pyrrole + 5 μM 3-acetate N-hydroxysuccinimide pyrrole + 0.1M LiClO ₄ / acetonitrile (0.5% water) was added to
It was prepared as an electrolytic solution. Cyclic voltammetry (CV) was used for electrochemical coating. The potential difference range of CV is 0.2 to 1.3 V vs Ag for the first cycle.
/ AgCl, then -0.1 to 1.0 vs Ag / for the other 5 cycles.
Change to AgCl. Electrochemical oxidation of pyrrole gave polypyrrole as shown in FIG.

The electrodes were purged with nitrogen gas during all steps of the electrochemical coating. The coated polypyrrole coating with associative functional groups was uniform and its color was blue. This polypyrrole film is in an oxidized form (charged and conductive state). To prepare the neutralized polypyrrole, the electrode was placed back into the electrolyte and -0.
It was cycled over a potentiometric range of 2-0.3 vs Ag / AgCl, which range is the reduction region for this electrochemical system. Neutralization of the polypyrrole coating is shown in FIG. Electrodes coated with a neutralized polypyrrole film were washed vigorously for probe oligonucleotide binding.

5′-amino substitution was then carried out by direct displacement of the leaving N-hydroxysuccinimide group in dimethylformamide containing 10% phosphate buffer pH = 8.0 for 16 hours at room temperature. The oligonucleotide was attached onto the neutral polypyrrole film. Oligonucleotides with a terminal amino group at the 5'-phosphorylated position:
CCC TCA AGC AGA was used. For comparison, the oxidized polypyrrole film was modified with oligonucleotides by the same procedure as above.

DNA-bound and non-DNA-bound, oxidized and neutralized polypyrrole coated electrodes were tested in deionized water on a Solartro 1260 impedance analyzer.
A platinum sheet with an area of 10 cm ² was used as the counter electrode. A frequency sweep method using a bias of 500 mV was performed over the frequency range of 100 mHz to 1 MHz. Since the double-layer capacitance is proportional to the area of the electrode surface, the relationship between the capacitance Cc of the counter electrode surface and the capacitance Cp of the probe electrode is Cc.
>> Cp. Therefore, if the total capacitance of this detection system is Ct, then Ct =
1 (1 / Cp + 1 / Cc) = Cp Cc / (Cp + Cc) = Cp. Furthermore, the solution resistance of a disc-shaped ultramicroelectrode can be expressed as: Ru = 1 / (4k
r) (1) [where r is the radius or side length of the electrode and k is the conductivity of the solution]. Ru due to the small probe is much larger than the counter electrode. The results obtained from the AC impedance can be representative of changes originating from the probe. This is because the surface area of the probe is much smaller than the surface area of the counter electrode.

The results of the experiment are shown in FIGS. 4, 5 and 6. FIG. 4 shows the capacitance change vs. electrode surface vs. frequency, where an electrode surface based on oxidized polypyrrole with oligonucleotide binding has a greater capacitance response in the low frequency range than a surface without oligonucleotide binding. It is shown that.
However, the signal / noise ratio is not large. Figure 5 shows that the capacitance of the electrode surface based on neutralized polypyrrole with oligonucleotide binding is significantly higher than the capacitance of the surface without oligonucleotide binding. FIG. 6 shows that the capacitance at the electrode surface based on neutralized polypyrrole with oligonucleotide binding is about 4 times greater than the capacitance on the surface based on oxidized polypyrrole.

Hybridization of an oligonucleotide probe on neutral polypyrrole with its complementary strand shows a significantly improved signal / noise ratio compared to the probe on charged polypyrrole.

Example 2 Electrodes coated with a neutralized polypyrrole coating were washed vigorously for DNA binding. Electrode coated with polypyrrole, 80 μL DMF and 20 μL 15 nM
It was placed in a mixture of 5i-amino-3i-fluorescein-labeled 15bp oligonucleotides for 4 hours at room temperature. At the end, the electrodes were washed with TBE buffer, thoroughly washed with deionized water and dried in air at room temperature. The conditions were not optimized.

25 μL of dimethylformamide containing 20% phosphate buffer, pH = 8.0
300 μM concentration of 5'-amino substituted oligonucleotide in
It was attached to the neutral polypyrrole coating on the microelectrode by direct displacement of the leaving N-hydroxysuccinimide group. An oligonucleotide with a terminal amino group at the 5'-phosphorylated position: CCC TCA AGC AGA was used as an example. After the reaction, the microelectrode was thoroughly washed with DI water, then baseline A
The C impedance was measured. For hybridization, the probe bound to polypyrrole on the microelectrode was added to different concentrations (μ) in 1 × SSC buffer.
M-aM) of the target molecule. Hybridization was carried out in a water bath at 37 ° C. for 24-48 hours in a closed conical tube. The microelectrode was then washed at room temperature with sufficient 1xSSC solution and then the AC impedance was measured.

Solartron Impedance Frequency An with Electrochemical Interface 1287
Impedance was measured before and after hybridization of polypyrrole microelectrodes using alyzer 1260. The counter and reference electrodes were platinum and Ag / AgCl, respectively. The measurement is an open circuit voltage (OCV) in 1M LiClO ₄ solution.
I went there. The measured complex impedance vs. frequency is shown in FIG. 8 for single and hybridized DNA, which shows a large difference in impedance before and after hybridization.

In this experiment, an electrode of this kind is able to detect 0.1 amol of target DNA in solution due to the neutralized form of the polypyrrole film.

Example 3 Experiments were conducted on the specificity of electrodes based on polypyrrole. Eight probe-coupled electrodes were hybridized in buffer containing 2 pM and 2 fM of perfectly correct and 3-base mismatched target 15mer DNA, respectively. The results show a significant difference between fully hybridized DNA and mismatched hybridized DNA. In addition, the electrodes were placed in 1 × SSC buffer at 37 ° C. and 38 ° C.
Washing was performed for a minute. AC impedance measurements show that the AC impedance for improper hybridization approaches the baseline of single-stranded DNA as the wash temperature increases, but the AC impedance for perfect hybridization remains nearly constant. Was done. These results are shown in FIG. 7 and FIG.
Shown in. FIG. 9 is a plot from FIG. 8 and shows that the resistance of the improper DNA system gradually decreases with increasing washing temperature and returns to the baseline of single-stranded DNA.

The present invention can be used in any solution containing a metal or polymerizing cation that is capable of reacting with DNA and is ionically conductive.

The above examples are intended to be illustrative of the present invention and are not intended to limit the spirit or scope of the present invention.

[Brief description of drawings]

FIG. 1 is a schematic diagram for preparation of an array of polypyrrole coated electrodes and for detection by AC impedance.

FIG. 2 is a structural diagram of a polypyrrole copolymer.

FIG. 3 is a structural diagram of an electrochemically reduced neutral polypyrrole copolymer.

FIG. 4 is a graph showing the relationship between capacitance and frequency in an electrode based on oxidized polypyrrole with and without DNA binding.

FIG. 5 is a graph showing the relationship between capacitance and frequency in an electrode based on neutral polypyrrole with and without DNA binding.

FIG. 6 is a graph showing a comparison of capacitance and frequency response between oxidized and neutralized polypyrrole-based electrodes with DNA binding.

FIG. 7 is a graph showing an AC impedance plane measured in a perfectly properly hybridized DNA and a single-stranded DNA system.

FIG. 8: Frequency-complex diagram (Freq) obtained from neutralized polypyrrole electrode
uency Complex diagram).

FIG. 9 is a graph showing impedance planes measured in a DNA hybridized improperly with 3 bases and in a single-stranded DNA system.

FIG. 10 is a graph in which resistance and ω ^−1/2 are plotted with respect to AC impedance measured in a DNA hybridized inappropriately with 3 bases and a single-stranded DNA system.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12M 1/34 C12Q 1/68 A 4J043 C12N 15/09 ZNA G01N 27/02 D 4J100 C12Q 1/68 27 / 30A G01N 27/02 27/48 Z 27/30 27/46 336M 27/48 386Z C12N 15/00 ZNAA (81) Designated countries 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, K E, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, K , KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK , DM, 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, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM , TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Si Son United States 84044 East Gold Poppy Way 4521 (72), Phoenix, Arizona Inventor Bien Chohn United States 85282 Arizona Tempe, Apartment B111, South Mill Ave 3730 (72) Inventor George Maracas United States 85048-9512 East Bighorn Ave 2613F Term, Phoenix, Arizona 2G060 AA06 AC10 AF06 AF20 4B024 AA11 AA19 AA20 CA09 HA12 4B029 AA07 AA23 AA27 CC03 FA02 4B063 QA01 QA18 QQ42 QQ52 QR32. JA45

Claims (17)

[Claims] 1. A charge-neutral conjugated polymer having a functional group for binding a biomolecular probe. 2. The charge-neutral conjugated polymer according to claim 1, which has a functional group for attaching a biomolecular probe to the charge-neutral conjugated polymer, which is polypyrrole, polyphenylene, polyacetylene, polydiacetylene, polythiophene,
A charge neutral conjugated polymer selected from the group consisting of polyfuran, polyaniline, polycarbazole, poly (phenylene vinylene) and copolymers and combinations thereof. 3. The polymer according to claim 1, which is produced by electrochemical polymerization. 4. The functional group is selected from the group consisting of amine, hydrazine, ester, amide, carboxylate, halide, hydroxyl, vinyl, vinylcarboxylate, thiol, phosphate and silicon-containing organic groups.
The polymer described in. 5. An electrode in electrical communication with the charge-neutral conjugated polymer of claim 1. 6. The electrode comprises gold, silver, platinum, copper and alloys; indium oxide, indium oxide-tin, zinc oxide; or carbon black, conductive epoxy,
Or the electrode of Claim 5 which consists of these combinations. 7. An array of electrodes according to claim 6. 8. The electrode array of claim 7, wherein the charge-neutral conjugated polymer is polypyrrole. 9. An electrode in electrical communication with a matrix of a charge-neutral conjugated polymer having a functional group to which a biomolecule probe is covalently bound, and electrically detecting the binding of the biomolecule to the biomolecule probe. A biosensor device for detecting a biomolecule, the biosensor device comprising: 10. An array of electrodes in electrical communication with a matrix of charge-neutral conjugated polymers having functional groups to which the biomolecule probe is covalently bound, as well as electrically binding the biomolecule to the biomolecule probe. A biosensor for detecting a biomolecule comprising a means for detecting. 11. The electrical detection means is AC impedance, periodic voltammetry (CV), pulse voltammetry, rectangular wave voltammetry, AC voltammetry (ACV), hydrodynamic modulation voltammetry, potential step method, potentiometric measurement, current measurement, current The biosensor according to claim 10, which is selected from step methods and combinations thereof. 12. The biosensor according to claim 11, wherein the electrical detection means is AC impedance. 13. The method of claim 12, wherein the one or more electrodes have oligonucleotides that are partially complementary to the target DNA covalently attached to a charge-neutral conjugated polymer that is in electrical communication with the electrodes. The biosensor described. 14. The biosensor of claim 13, wherein the DNA in the test sample hybridizes to an oligonucleotide covalently attached to a charge-neutral conjugated polymer on one or more electrodes in the sequence. 15. A method of measuring an analyte in a test sample comprising the steps of: (a) a charge-neutral conjugated polymer covalently bound to a binding group that directly or indirectly binds to the analyte. An electrode in electrical communication with the matrix of; (b) contacting the matrix of the charge-neutral conjugated polymer with a test sample containing the analyte; and (c) the analyte bound to the neutral conjugated polymer. Electrically detect. 16. The analyte is detected by AC impedance.
The method described in. 17. A method of making an analytical electrode comprising the steps of: (a) electrochemically polymerizing pyrrole and a functionalized pyrrole to provide an oxidized polypyrrole functionalized pyrrole copolymer; (b) on the electrode. (C) electrically reducing the copolymer to provide an electrically neutralized copolymer;
And (d) covalently attaching to the functionalized pyrrole in the copolymer a biomolecular probe that binds directly or indirectly to the analyte.