KR100580620B1 - Novel electrically conductive polymer, sensor using the same and a method for detecting a target molecule using the same - Google Patents

Abstract

The present invention relates to a novel conductive polymer, specifically, to a conductive polymer represented by the following general formula (I).

(I)

(I)

Wherein m is 1, 2, or 3, and R is hydroxysuccinimidyl, hydroxyphthalimidyl or pentafluorophenolyl group. The conductive polymer of the present invention can be effectively used in sensors and methods for detecting target molecules by hybridization.

Thiophene, conductive polymer, nucleic acid, protein, hybridization

Description

Novel conductive conductive polymer, sensor using the same and a method for detecting a target molecule using the same

1 is a schematic diagram showing an example of a sensor using a conductive polymer of the present invention.

Figure 2 shows an example of a sensor using a conductive polymer of the present invention consisting of three electrodes.

3 is a diagram showing an example of polymerizing the conductive polymer of the present invention by using a constant current (chronopotentiometry).

4 is an electron micrograph of the conductive polymer membrane of the present invention polymerized by the constant current method.

5 is a cyclic voltammogram obtained by repeating five times the conductive polymer membrane of the present invention polymerized by the constant current method.

FIG. 6A is a cyclic voltammogram obtained at three different scan rates for the conductive polymer film of the present invention polymerized by the constant current method, and FIG. 6B is a scan rate and peak current obtained from the cyclic voltammogram of FIG. 6A. Correlation graph of.

Figure 7 is a schematic diagram showing the hybridization process of the immobilized probe DNA and the target DNA complementary thereto.

FIG. 8 is a result of measuring cyclic voltamograms for two spots each including a probe DNA immobilized on an electrode coated with a conductive polymer of the present invention and a target DNA hybridized with the probe DNA.

Figure 9 shows the correlation between the concentration of the target DNA and the peak current obtained by hybridizing by adding the target DNA of various concentrations to the probe DNA immobilized on the electrode coated with the conductive polymer of the present invention, and measuring the cyclic voltamogram Drawing.

10 is a view showing the difference in peak current according to the single base difference in the target DNA.

The present invention relates to a new conductive polymer, a sensor including the electrode coated with the conductive polymer, and a target material detection method using the conductive polymer.

Currently, a lot of research is being conducted on the development of a sensor for detecting a biological material using the principles of electrochemistry. Many studies have been made on ion sensors, gas sensors, and biosensors because of the advantages of using the principle of electrochemistry. Among biological materials, monitoring information on the presence or absence of DNA hybridization or changes in protein tertiary structure is very important in the fields of genomics and proteomics. To this end, there has been the development of sensors using organic materials (such as intercalators) and electroconductive polymers with electrochemical activity. In the case of a sensor using an intercalator, much research has been conducted so that it is currently on the market.

Meanwhile, in the case of the conductive polymer, only a few representative monomers to be polymerized in the electrode should be used, and research has been relatively slow due to difficulty in controlling physical properties of the polymerized polymer itself. Pyrrole, thiophene, aniline, etc. are used as a representative conductive polymer sensor, but aniline is effective in acidic conditions, so pyrrole and thiophene are the main research targets. Has been.

Among them, pyrrole is not easy to use for a long time due to low oxidation potential (see US Patent No. 6,201,086). Thiophene maintains a higher oxidation potential than pyrrole but is more hydrophobic, which makes it unsuitable for systems employing biomolecules using water as a basic solvent (Bauerle P. and Emge, A., Adv. Mater., 3: 324 (1998).

It is an object of the present invention to provide a novel conductive polymer.

It is also an object of the present invention to provide a novel monomer compound which can be used for the polymerization of the conductive polymer.

It is also an object of the present invention to provide an electrode coated with the conductive polymer.

Another object of the present invention to provide a sensor comprising an electrode coated with the conductive polymer.

Still another object of the present invention is to provide a method for detecting a target material using the conductive polymer.

The present invention provides a conductive polymer represented by the following general formula (I).

(I)

(I)

Wherein m is 1, 2, or 3, and R is hydroxysuccinimidyl, hydroxyphthalimidyl or pentafluorophenolyl group. Where n is zero or a positive integer.

The present invention also provides a conductive polymer represented by the following general formula (II).

（II)

(II)

Wherein m is 1, 2, or 3, and R 'is hydroxysuccinimidyl, hydroxyphthalimidyl, pentafluorophenolyl, or probe group, and the conductive polymer At least one of R ′ is a probe group. Where n is zero or a positive integer.

As used herein, the term "probe" refers to a molecule capable of specifically binding to a target material. Such probes include nucleic acids and proteins, preferably DNA, RNA, PNA, antibodies, antigens, enzymes, coenzymes, substrates or coenzymes.

The conductive polymer of the present invention may be prepared by polymerizing a monomer by a method such as cyclic votammetry, chronopotentiometry, and chronoamperometry. In the present invention, the conductive polymer has a high oxidation potential.

The present invention also relates to N-hydroxyphthalimidyl 3-thiophenyl acetate represented by the following structural formula (III) or pentafluorophenolyl 3-thio represented by the structural formula (IV). Phenyl acetate (pentafluorophenolyl 3-thiophenlyl acetate).

(III)

(III)

(IV)

(IV)

The compounds may be used as the preferred monomer in synthesizing the conductive polymer of the present invention.

The present invention also provides an electrode coated with the conductive polymer. As the electrode material, a commonly used material such as platinum may be used.

In addition, the present invention provides a sensor comprising an electrode coated with the conductive polymer. The sensor is composed of the components of a conventional sensor, except that it includes an electrode coated with the conductive polymer. The sensor may include a working electrode, a counter electrode, and a standard electrode which are commonly used.

1 is a block diagram showing an example of a sensor including an electrode coated with a conductive polymer of the present invention. As shown in Fig. 1, the sensor includes an electrode including a working electrode 4, a counter electrode 12, and a standard electrode 14 coated with a conductive polymer 6 of the present invention, to which a probe 8 is covalently bonded. And a constant voltage meter 2 capable of measuring the voltage. The working electrode 4 coated with the conductive polymer of the present invention to which the probe in the sensor is covalently bonded may be manufactured by the following procedure. A thiophene-based monomer capable of binding an amine group to a probe is synthesized. It is preferable that the said monomer is capable of electropolymerization. The monomer may be polymerized on a substrate and a probe such as DNA may be coupled thereto to prepare an electrode coated with the conductive polymer of the present invention, in which the probe is covalently bonded. In addition, the probe is first covalently bonded with a thiophene-based monomer, and then polymerized on a substrate of the thiophene-based monomer to which the probe is bonded, thereby preparing an electrode to which the probe is coated with the conductive polymer of the present invention. You may. The electrode thus prepared is used as a working electrode (WE) of the sensor.

The sensor of the present invention can be used to detect the target substance 10 in a sample. When the sample is brought into contact with the probe 8 coupled to the working electrode of the sensor, the target material 10 present in the sample is hybridized with the probe, and a change in voltage or current is generated by the hybridization reaction. By measuring such a change in voltage or current, the target substance 10 in the sample can be detected (Fig. 1).

The change in voltage or current is considered to be caused by the following mechanism, but is not limited thereto. The amount of current measured at a particular oxidation / reduction potential depends on the extent to which the conductive polymer on the substrate forms a double conjugation. When the probe covalently bonded to the conductive polymer of the present invention hybridizes with the target material, the degree of formation of the double conjugated state is lower than that of the non-hybridized case. Thus, when the probe and the target material hybridize, the amount of current decreases. For example, when the DNA probe hybridizes with the target DNA to form double stranded DNA, the amount of current decreases. Therefore, the target material can be detected by measuring a decrease in the oxidation / reduction current or an increase in the oxidation / reduction potential depending on whether the probe and the target material are hybridized.

Figure 2 is a block diagram showing another example of a sensor including an electrode coated with a conductive polymer of the present invention. The sensor consists of a working electrode 4, a counter electrode 12, and a reference electrode 14. The conductive polymer of the present invention is coated on the working electrode.

The present invention also provides a method for detecting a target substance comprising the following steps.

(a) electrically synthesizing the thiophene-based conductive polymer of the present invention on a conductive substrate;

(b) coupling a probe to a surface of the conductive polymer film;

(c) contacting a target material that specifically reacts with the probe; And,

(d) detecting the target material by measuring a difference in voltage or current between the conductive polymer in which the target material is coupled to the probe and the conductive polymer in which the target material is not coupled or inconsistent with the probe.

The present invention also provides a method for detecting a target substance comprising the following steps.

(a) electrically synthesizing a thiophene-based conductive polymer to which the probe of the present invention is bound on a conductive substrate;

(b) contacting a target material that specifically reacts with a probe in the conductive polymer; And,

(c) detecting the target material by measuring a difference in voltage or current of the conductive polymer to which the target material is coupled with the probe and the conductive polymer to which the target material is not coupled or inconsistent with the probe.

The conductive substrate is preferably a gold or platinum electrode. The probe is preferably DNA, RNA, PNA, antibody, antigen, enzyme, substrate or coenzyme. In addition, the target material includes any one capable of specifically binding to the probe, but is preferably a nucleic acid or a protein.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited by these examples.

Example

Example 1: N-hydroxyphthalimidyl 3-thiophenyl acetate

N-hydroxyphthalimidyl 3-thiophenyl acetate, which is one of the monomers used to prepare the conductive polymer of the present invention, was synthesized by the following procedure. First, 1 equivalent of 3-thiophenyl acetic acid and 1 equivalent of N-hydroxyl phthalimide are reacted under DCC under normal temperature and chloroform solvent conditions, and the structure is obtained by recrystallization. N-hydroxyphthalimidyl 3-thiophenyl acetate was obtained as a pure compound of III).

The NMR measurement values of the N-hydroxyphthalimyl 3-thiophenyl acetate are as follows.

¹ H-NMR (CDCl ₃ ): 7.87 (m, C ₆ H ₄ , 4H), 7.31 (m, C ₄ H ₃ SCH ₂ , 1H), 7.24 (s, C ₄ H ₃ SCH ₂ , 1H), 7.11 (m, C ₄ H ₃ SCH ₂ , 1H), 4.01 (s, C ₄ H ₃ SCH ₂ , 2H)

Example 2 Synthesis of Pentafluorophenolyl 3-thiophenyl Acetate

Pentafluorophenolyl 3-thiophenlyl acetate, one of the monomers used in the preparation of the conductive polymer of the present invention, was synthesized as follows.

First, 1 equivalent of 3-thiophenyl acetic acid and 1 equivalent of pentafluorophenol are reacted under DCC under normal temperature and dichloromethane solvent conditions, and the structure (IV) is obtained through silica gel column chromatography. Pentafluorophenolyl 3-thiophenyl acetate was obtained as a pure compound.

The NMR value of the obtained pentafluorophenolyl 3-thiophenyl acetate is as follows.

¹ H-NMR (CDCl ₃ ): 7.82 (2H), 7.72 (2H), 7.28 (1H), 7.26 (1H), 7.07 (1H), 4.0 (2H)

Example 3 Polymer Polymerization on Electrodes

In a solution of 0.1 M dopant TBAHFP (tetrabutylamine hexafluorophosphate) dissolved in acetonitrile, 0.1 M of N-hydroxyphthalimidyl 3-thiophenyl acetate (Formula III) synthesized in Example 1 was dissolved. The monomer was polymerized on the platinum electrode using the method (FIG. 3). That is, on the platinum electrode placed on a 0.5 mm thick glass substrate, a current was fixed at 0.4 mA and the polymer was polymerized for 40 seconds to form a uniform conductive polymer film.

The poly (N-hydroxyphthalimidyl 3-thiophenyl acetate) conductive film formed as described above was observed through a scanning electron microscope, and the results are shown in FIG. 4.

In addition, in order to determine the reproducibility of the poly (N-hydroxyphthalimidyl 3-thiophenyl acetate) conductive membrane, the cyclic voltamo was repeated five times by applying voltage from the platinum electrode at the same scan rate under acetonitrile / TBAHFP medium. Gram was measured. The results are shown in FIG. As shown in Fig. 5, the conductive polymer of the present invention exhibited a substantially reversible oxidation wavelength at 1.0 V, which showed high electrical reactivity and high reproducibility.

In addition, in order to determine the conductive properties of the poly (N-hydroxyphthalimidyl 3-thiophenyl acetate) conductive film, voltage was applied through a platinum electrode at three scan rates and a cyclic voltamogram was measured. The results are shown in Figs. 6A and 6B. FIG. 6A is a cyclic voltammogram and FIG. 6B is a graph showing a correlation between the scan speed and the peak current calculated from the cyclic voltammogram. As can be seen in Figure 6b, the scan rate and the peak current for the conductive polymer of the present invention shows a linear relationship, it can be seen that it has a good conductivity characteristics.

Example 4 Immobilization of DNA Probes and Confirmation of Hybridization with Target DNA

1. Immobilization of DNA Probes

The DNA probe was immobilized on the conductive polymer film polymerized in Example 3. 100 μM probe DNA (5′-NH ₂ -GTTCTTC T CATCATC-3 ′: SEQ ID NO: 1) having an amine group at 5 ′ was added to the conductive polymer membrane and reacted for about 12 hours. The immobilization of the DNA probe is performed by replacing the hydroxyphthalimide group in the conductive polymer of the present invention with an amine group at the 5 'end of the DNA probe. In addition, the immobilization of the protein probe can be accomplished by substituting hydroxyphthalimide in the conductive polymer membrane using an amine group of lysine in the amino acid residue.

2. Hybridization with Target DNA

100 μM of completely complementary target DNA (5′-NH ₂ -GATGATG A GAAGAAC-3 ′: SEQ ID NO: 2) was added to the single-stranded DNA probe immobilized on the conductive polymer of the present invention, and hybridized at 40 ° C. for 3 hours. . 7 is a diagram schematically illustrating a process of hybridizing a target DNA to a single-stranded DNA probe. The same hybridization in the absence of the target DNA as a control. After the hybridization reaction, the peak current was measured using a cyclic current method to confirm hybridization. The results are shown in FIG.

8 is a result of measuring a cyclic voltammogram by applying a voltage to determine whether the hybridization of the two spots of the probe DNA is immobilized on the electrode coated with the conductive polymer of the present invention.

As shown in FIG. 8, it can be seen that the current amount at the same voltage is significantly reduced (about 1/2) at the time of DNA hybridization (ds DNA), due to the large functional group attached to the side of the conductive polymer due to the hybridization of DNA. It shows a decrease in the amount of current during oxidation / reduction of the conductive polymer.

In addition, in order to determine the sensitivity of the electrode to which the probe DNA is immobilized on the electrode coated with the conductive polymer of the present invention, the hybridization is performed as described above by varying the concentration of the target DNA completely complementary to the probe DNA. A mogram was obtained. From the results, a correlation graph of the peak current amount according to the concentration of the target DNA was obtained (Fig. 9).

As a result, the sensitivity of the electrode containing the conductive polymer of the present invention was obtained from the slope at the origin of the graph. The sensitivity was 0.62㎂ / nmole, which means that the sensor including the electrode containing the conductive polymer of the present invention can detect a target DNA in solution at about 1 nmole.

Example 4: Comparison of the degree of hybridization due to base mismatch

In the conductive polymer membrane polymerized in Example 3, the same probe DNA (5′-NH ₂ -GTTCTTC T CATCATC-3 ′: SEQ ID NO: 1) was immobilized on an electrode, and different target DNAs were hybridized to each electrode. DNA probes and target DNA roneun full complementarity of DNA (5'-GATGATG A GAAGAAC- 3 ': SEQ ID NO: 2) (hereinafter referred to as "PM" is also called) and 5'-G GATGATG GAAGAAC a DNA containing a base Bo emergency One base, using a sequence having a sequence of -3 '(SEQ ID NO: 3) (hereinafter also referred to as "TG") and 5'-GATGATG C GAAGAAC-3' (SEQ ID NO: 4) (hereinafter also referred to as "TC") The electrode according to the difference measured the selectivity. The selectivity was obtained for circulating voltammogram for each sample hybridized with probe DNA and target DNA. Controls were hybridized without target DNA and circulating voltamograms were measured. Next, the ratio of each peak current to the peak current of the control was determined from the cyclic voltammogram.

The results are shown in FIG. In FIG. 10, TC, TG and PM show the ratio of peak currents (Ip (ds) / Ip (ss)) of the samples hybridized with each of these target DNAs to the peak currents for the control.

As shown in FIG. 10, the Ip (ds) / Ip (ss) value for the fully complementary target DNA is 52%, and the Ip (ds) / Ip (ss) value of the TG and TC probe DNAs with one base mismatch. Are 29.3% and 24.3%, respectively. These results indicate that by hybridizing the probe DNA and the target DNA on the conductive polymer of the present invention, it is possible to distinguish the target DNA having a single base difference from the fully complementary target DNA.

According to the monomer of the present invention, it can be used to synthesize a polymer having good conductivity.

The conductive polymer of the present invention can be effectively used in a sensor for detecting a target molecule by hybridization.

According to the sensor of the present invention, by including the electrode coated with the conductive polymer of the present invention having a high oxidation potential, it is possible to detect whether the probe and the target material are hybridized with high sensitivity.                     

According to the method for detecting the target substance of the present invention, the target substance can be detected at high speed and high sensitivity.

<110> Samsung Electronics Co. Ltd. <120> Conductive polymer, sensor using the same and method for          detecting a target molecule using the same <130> SI004142 <150> KR10-2002-0012729 <151> 2002-03-09 <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> probe DNA <400> 1 gttcttctca tcatc 15 <210> 2 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> target DNA which is completely complementary to the probe DNA (SEQ          ID No. One) <400> 2 gatgatgaga agaac 15 <210> 3 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> a target DNA which is complementary to the prove DNA (SEQ ID No.          1) except 1 base <400> 3 gatgatggga agaac 15 <210> 4 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> a target DNA which is completely complementary to the prove DNA          (SEQ ID. 1) except 1 base <400> 4 gatgatgcga agaac 15

Claims (12)

Conductive polymer represented by the following general formula (I):

(I) Where m is 1, 2, or 3, R is hydroxysuccinimidyl, hydroxyphthalimidyl or pentafluorophenolyl group, and n is 0 or a positive integer. Conductive polymer represented by the following general formula (II):

(II) Wherein m is 1, 2, or 3, R 'is hydroxysuccinimidyl, hydroxyphthalimidyl, pentafluorophenolyl, or probe group, and at least one of R' of the conductive polymer is a probe group N is 0 or a positive integer. The conductive polymer of claim 2, wherein the probe is a nucleic acid or a protein. The conductive polymer of claim 3, wherein the probe is DNA, RNA, PNA, antibody, antigen, enzyme, substrate, or coenzyme. N-hydroxyphthalimidyl 3-thiophenyl acetate or pentafluorophenolyl 3-thiophenyl acetate. An electrode coated with the conductive polymer of claim 1. The sensor comprising an electrode coated with the conductive polymer of claim 1. delete delete delete delete delete

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