KR20140108810A - Biosensor for Detecting Cancer Cell Treated Anti-cancer Drug and Preparation Method Thereof - Google Patents

Abstract

The present invention relates to a biosensor for detecting a cancer cell treated with an anti-cancer agent. The biosensor includes: an electrode on which an Au nanoparticle layer is electro-deposited; an electrically conductive polymer layer formed on the top of the Au nanoparticle layer; and an aptamer layer fixated on the top of the electrically conductive polymer layer. According to the present invention, the biosensor has an excellent function of specifically detecting cancer cells treated with the anti-cancer agent. As the number of cancer cells increases, fluorescent intensity and a value of impedance change of the biosensor also increase. Therefore, the biosensor can be used for quantitatively and qualitatively detecting cancer cells.

Description

TECHNICAL FIELD [0001] The present invention relates to a biosensor for detecting cancer cells treated with an anticancer agent and a method for producing the same,

The present invention relates to a biosensor that specifically and quantitatively detects cancer cells treated with an anticancer agent and a method for producing the same.

Cancer is one of the deadliest threats to human health. In the United States, nearly 1,300,000 new patients each year are cancerous, with heart disease followed by about one in four deaths, the second leading cause of death. In addition, cancer is predicted to be the primary cause of death within five years, surpassing cardiovascular disease. Solid rock accounts for most of these deaths. Although there has been considerable progress in the medical treatment of certain cancers, the overall 5-year survival rate for all cancers has improved only about 10% over the last 20 years. Cancer or malignant tumors are metastasized and grow rapidly in an uncontrolled manner, making it very difficult to find and treat in a timely manner. Also, cancer can arise from almost all tissues in the body through malignant transformation of one or several normal cells in the tissue, and they are different for each type of cancer with a specific tissue origin.

Tumor cells differ in antigenicity from normal cells due to the presence of "tumor antigens ". They may be specific for tumor cells, or are recognized as "tumor-associated antigens" (TAA) by being differentially expressed or overexpressed. Tumor cells are characterized by their primary structure, that is, their amino acid sequences are different from normal cells (derived from genomic sequence changes), and the changes in post-translational modifications, such as glycosylation and phosphorylation, The structure may also differ from normal cells, which in turn changes the antigenicity of the protein and also identifies it as a tumor-associated antigen. One typical example is protein mucin from the mammary gland, which itself is not a change in tumor cells, but autoantibodies against it are found in breast cancer patients (sometimes even in ovarian cancer). Probably because the protein is highly glycosylated in normal cells and is not exposed at all due to the dense and thick coating of the carbohydrate chains. If glycosylation is poor in tumor cells, the protein fragments acting as antigenic determinants are exposed to the immune system.

Therefore, there is a growing interest in research using an antibody targeting the tumor cell-specific antigen as a specific and more effective cancer treatment method and development of a cancer therapeutic agent for inducing apoptosis of cancer cells through the antibody. In connection with the above technology, Korean Patent Publication No. 10-2010-0045903 discloses an antibody that targets anti-DR5, a cell death receptor for cancer cells.

However, there is a need for a cancer cell detection method that is more non-invasive, simple, and universally applicable to various cancer cells because it is difficult to find antigens that are differentially expressed in cancer cells and specifically expressed in cancer cells only.

Accordingly, the present invention provides a biosensor for detecting cancer cells which is more non-invasive, simple, and universally applicable to various cancer cells and a method for producing the same.

In order to solve the above problems, the present invention provides an electrode comprising an Au nanoparticle layer electrodeposited; An electroconductive polymer layer formed on the Au nanoparticle layer; And an aptamer layer fixed on an upper portion of the electrically conductive polymer layer. The present invention also provides a biosensor for detecting cancer cells treated with an anticancer agent.

The present invention also provides a method of manufacturing a semiconductor device, comprising: electrodepositing an Au nanoparticle layer on an electrode surface; Coating an electroconductive polymer layer having a carboxyl group on the surface of the Au nanoparticle layer; And covalently bonding the carboxyl group of the electrically conductive polymer layer and the platemer to immobilize the platemer. The present invention also provides a method of manufacturing a biosensor for cancer cell treatment.

The sensor according to the present invention is excellent in the ability to specifically detect cancer cells treated with anticancer drugs, and the fluorescence intensity and the impedance change value are also increased proportionally as the number of cancer cells is increased. As a result, the cancer cells can be detected qualitatively and quantitatively It can be used effectively as a sensor.

Fig. 1 shows a production schematic diagram and a cancer cell detection principle of the biosensor of the present invention.
2 is an XPS result graph for analyzing the composition of the sensor electrode surface.
Figure 3 shows the reaction of (a) Daunomycin with (a) GC / AuNPs / pTTBA and (b) cancer cells against GC / AuNPs / pTTBA / (D) GC / AuNPs / pTTBA / platamater electrode reacted with (c) GC / AuNPs / pTTBA / platemaker electrode and Daunomycin treated cancer cells. Insertion figure: CV with solution (dark line) and 0.01 mM daunomycin not containing daunomycin in solution (red line) using GC / AuNPs / pTTBA electrode. (B) shows the impedance result when the daunomycin is processed (⊚) and not processed (◈), and when the aptamer is not immobilized () and immobilized (▴). Interpolation: Indicates impedance signals. (C) Daunomycin reacted with the sensor showed the impedance results for the various numbers of HeLa treated. (D) The calibration curve (n = 5).
4 is a graph of the impedance results for a) cancer cells (HeLa) and b) normal cells (OSE) treated with darunomycin in 0.1 M PBS (pH 7.4) using GC / AuNPs / pTTBA /
5 (a) and 5 (b) show SEM images of the surface of the sensor electrode reacted with cancer cells treated with Daunomycin, and (c) - (f) show the SEM images of the cancer cells treated with Daunomycin Fluorescence microscope image at excitation / emission? ₄₀₀ /? ₅₉₀ is shown.

The present invention relates to an electrode comprising an Au nanoparticle layer electrodeposited; An electroconductive polymer layer formed on the Au nanoparticle layer; And an aptamer layer fixed on an upper portion of the electrically conductive polymer layer. The present invention also provides a biosensor for detecting cancer cells treated with an anticancer agent.

The inventors of the present invention have studied cancer cell detection methods that are more noninvasive, convenient, and universally applied to various cancer cells, and have found that lipid having a low pitch such as phosphatidylserine is mainly distributed on the surface of cancer cells, Attention has been paid to the fact that a large amount of anionic molecules such as salicylic acid and heparin sulfate are present, and the cancer cell has a characteristic of absorbing a cationic anticancer drug which is electrochemically active and exhibits fluorescence property, and the anticancer drug specifically absorbed into cancer cells is targeted The inventors confirmed that a cancer cell detection biosensor has excellent cancer cell specific detection ability and completed the present invention.

The Au nanoparticle layer enhances the sensitivity of the biosensor. As the electrode to which the Au nanoparticle layer is deposited, a glassy carbon electrode or an indium tin oxide electrode may be used, but the present invention is not limited thereto.

In one embodiment of the present invention, the cationic anticancer agent to be treated in cancer cells is any one or more selected from the group consisting of daunomycin, bleomycin, daunorubicin, doxorubicin, epirubicin, dirubicin and mitomycin- Daunomycin (DAN) may be used, but is not limited thereto.

In another embodiment of the present invention, the aptamer that specifically binds to the anticancer agent may be an aptamer consisting of the nucleic acid sequence of SEQ ID NO: 1, but is not limited thereto. The general contents of platamers are described in Bock LC et al., Nature 355 (6360): 5646 (1992); Hoppe-Seyler F, Butz K "Peptide aptamers: powerful new tools for molecular medicine". J Mol Med. 78 (8): 42630 (2000); Cohen BA, Colas P, Brent R. "An artificial cell-cycle inhibitor isolated from a combinatorial library". Proc Natl Acad Sci USA. 95 (24): 142727 (1998).

In another embodiment of the present invention, the electrically conductive polymer layer serves to improve electrical conductivity, which may include a terthiophene monomer, and more specifically, the terthiophene monomer is 2,2 ': 5' , 2 '' - Entity thiophene -3 (p-benzoic acid) (2,2 ': 5', 2 '' - terthiophene-3 (p -benzoic acid)), or 5,2 ': 5', 2 "- 3 '-carboxylic acid (5,2': 5 ', 2 "-terthiophene-3'-carboxylic acid), preferably 2,2': 5 ' ( p -benzoic acid) (2,2 ': 5', 2 "-terthiophene-3 ( p- benzoic acid)).

The polymer layer may form a covalent bond with an amine group attached to the 5'end of the extruder by activating the carboxyl group with carbodiimide or N-hydroxysuccinimide.

According to another aspect of the present invention, Coating an electroconductive polymer layer having a carboxyl group on the surface of the Au nanoparticle layer; And covalently bonding the carboxyl group of the electrically conductive polymer layer and the platemer to immobilize the platemer. The present invention also provides a method of manufacturing a biosensor for cancer cell treatment.

In one embodiment of the present invention, the aptamer is composed of the nucleic acid sequence of SEQ ID NO: 1, and a covalent bond is formed between the amine group attached to the 5-terminal end of the nucleic acid sequence of SEQ ID NO: 1 and the carboxyl group of the electrically conductive polymer layer.

According to the present invention, the biosensor according to the present invention is excellent in the ability to distinguish cancer cells treated with daunomycin from non-cancerous cells, and has an impedance increase value (ΔR _ct ) in proportion to the cell number in the range of 80 to 10000 cell / The biosensor of the present invention is advantageous in qualitatively and quantitatively detecting cancer cells without additional dye or coloring agent.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

< Experimental Example >

The following experimental examples are intended to provide experimental examples that are commonly applied to the respective embodiments according to the present invention.

1. Reagents

The electroconductive polymer 2,2 ': 5', 2 "-terthiophene-3 ( p- benzoic acid) (TTBA) was synthesized by Paal-Knorr pyrrole condensation. Tetrabutylammonium perchlorate (TBAP, electrochemical grade) was purchased from Fluka (USA) and vacuum dried at 1.33 x ^10-3 Pa after stagnation. (EDC), N-hydroxysuccinimide (NHS), dichloromethane (99.8%, anhydrous and N ₂ packaged), and trisodium The citrate was purchased from Sigma Co. (USA). Sodium tetraborate hydridoborates and HAuCl ₄ · 3H ₂ O was purchased from Aldrich (USA). The SELEX-derived aptamer of the present invention was used with an amine group attached at 5 '(NH ₂ -5'-GGGAATTCGAGCTCGGTACCATCTGTGTAAGGGGTAAGGGGTGGGGGTGGGTACGTCTAGCTGCAGGCATGCAAGCTTGG-3', SEQ ID NO: 1 ). PAGE purified aptamers were purchased from Bioneer (S. Korea) and used. HPLC grade DAN was purchased from Sigma-Aldrich (USA) and stored at 4 占 폚. The stock solution was stored at -20 ° C in sterile Tris EDTA buffer (10.0 mM Tris-HCl and 1.0 mM EDTA) at pH 8.0.

 RPMI 1640 medium, Dulbecco's Modified Eagle's medium (DMEM), human serum, fetal serum (FBS), trypsin-EDTA, penicillin / streptomycin, Hank? The balance salt (HBS) solution was purchased from Sigma-Aldrich. All aqueous solutions were prepared with secondary distilled water purchased from Milli-Q water purifying system (18 MΩ cm).

2. Biosensor manufacturing

FIG. 1 shows a production diagram of the biosensor of the present invention. AuNP was electrodeposited on a glassy carbon electrode (GCE) before the polymerization reaction of TTBA. Electrodeposition was carried out by using a linear sweep voltammetry (LSV) from 1.5 to 0.4 V in a 0.5MH ₂ SO ₄ solution containing 0.001% HAuCl _{4. The} electrodeposition time was 60 seconds, the electrodeposition potential was -1.0 V, / s, and the potential cycle was 3 cycles.

PTTBA on GC / AuNP was prepared by electropolymerizing 1.0 mM TTBA monomers present in 0.1 M TBAP / CH ₂ Cl ₂ at a scan rate of 0.1 V / s at a rate of 0.0-1.4 V (vs. Ag / AgCl) Lt; / RTI &gt; After the polymerization, the carboxylic acid functional groups of the pTTBA layer were activated with EDC / NHS and the aptamers were fixed to form a GC / AuNP / pTTBA / plastomer layer. Thereafter, the unbound functional groups were removed by washing with 0.1 M mercaptoethanol for 1 minute, and the formation of each layer was confirmed by XPS and the fixation by quartz crystal microbalance (QCM).

3. Cell sample preparation

MCF-10A and HEK-293 (human breast cancer), HeLa (human cervical cancer), MCF-7 (human breast adenocarcinoma), HT-29 Cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA). HeLa and MCF-7 cell lines were cultured in DMEM medium, and HT-29 and SK-BR-3 cell lines were cultured in RPMI 1640 medium. All non-cancer cells were cultured in DMEM medium. Cells were cultured in each medium containing 10% heat-inactivated fetal bovine serum, 100.0 unit / mL penicillin and 100.0 unit / mL streptomycin at 37 ° C under 5% CO ₂ . The medium was replaced every two days. Then, all cancerous / non-cancerous cells were washed with HBS to remove the medium and treated with 1.0 μM DAN in PBS (pH 7.4) for 20 minutes at 4 ° C in an ice bath to eliminate DAN internalization. To remove DAN from unabsorbed cell surface, cells were centrifuged at 1000 rpm. Experimental groups not treated with DAN were used as positive controls, and experimental and control cells were incubated with the biosensor. In addition to the biosensor of the present invention, a GC / AuNP / pTTBA probe with an aptamer removed was also used for comparative experiments. The surface of the probe was immediately measured by electrochemical impedance spectroscopy (EIS) after reaction with the cells.

4. Analysis

The electrochemical cell for EIS analysis used a Ag / AgCl reference electrode on a saturating KCl solution, a platinum counter electrode, and a three electrode cell using GC / AuNP / pTTBA / platemer of the present invention as a working electrode. The Faraday impedance spectrum was recorded using PARSTAT 2263 (USA) with an open circuit potential of 100.0 kHz to 100.0 MHz. All electrochemical impedance measurements were performed in PBS (pH 7.4) buffer, and the impedance spectra were collected in the form of Nyquist plot.

< Example  1 > of the manufactured biosensor XPS  And QCM  analysis

XPS analysis was performed using a VG Scientific XPSLAB 250 XPS spectrometer to analyze the formation of each layer of the GC / AuNP / pTTBA / platemater biosensor. The XPS spectrum used the C1 peak at 284.6 eV as the internal standard. The results are shown in Fig. (a) is a surface of a GC / AuNP / pTTBA sensor, and (b) is a surface analysis result of GC / AuNP / pTTBA / The TTBA coated electrode showed a S2p peak corresponding to the CS bond at 163.8 eV, which is attributed to the sulfur content of pTTBA. N1 peaks of the zipper fixed to the surface were confirmed at 398.8 and 399.8 eV positions, respectively, corresponding to the CN, -NH and O = C-NH- bonds, which were formed between the NH ₂ group of the extruder and the COOH group of pTTBA Covalent bonds. The platemaker-fixed electrode exhibits a very distinct P2p peak at 131.9 eV, which corresponds to the phosphate, the main structural element of the platameric backbone, suggesting that the aptamer is successfully bound to the surface.

Using a gold-coated working electrode (area: 0.196 cm 2; 9.0 MHz; AT-cut quartz crystal), the degree of fixation was determined based on frequency variation with quartz crystal microbalance (QCM) for pTTBA film. When the aptamer was stationary, the decrease in frequency reached a steady state through a 0.11 kHz change (Δ f ) after one hour, corresponding to a mass change (Δ m ) of 132.8 ± 7.2 ng. Therefore, the number of plumder molecules on the electrode surface was found to be 1.7 x 10 ^-11 mol / cm ² . The results suggest that the aptamer is effectively immobilized on the pTTBA layer of the inventive sensor.

< Example  &Lt; 2 &gt; Detectability  Review

CV (cyclic voltammetry) and electrochemical impedance spectroscopy (EIS) of the platemater biosensor were analyzed to examine the binding between DAN and cancer cells. The results are shown in Fig. 3 and Fig.

One. CV (Cyclic voltage current)

First, the electrochemical behavior of the DAN solution was analyzed with an electrode with only TTBA (GC / AuNP / pTTBA) without an electrode, and the redox peak was -0.65 / -0.55 V vs. Ag / AgCl (see also FIG. 3A internal view). The CV peak of DAN is due to the following reaction: DAN (ox) + 2e ^- + 2H ⁺ → DAN (red).

HeLa cervical cancer and ovarian surface epithelial (OSE) non-cancer cells were counted (2.5 × 10 ² cells mL ^-1 ) using a disposable hemocytometer in the presence of an optical microscope and cultured with DAN. To remove DAN from unabsorbed cell surface, cells were centrifuged at 1000 rpm and cancerous and non-cancerous cells were named CC _D and NC _D , respectively. The cells were then reacted with the biosensor of the present invention for 15 minutes and after washing, the CV was recorded in the range of -0.30 to -1.0 V at a scanning rate of 50.0 mV / s in 0.1 M deoxygenated PBS. The pair of oxidizing source peaks due to DAN being absorbed into the cancer cell membrane and detected by the biosensor (n = 5, RSD <3.9%) is about -0.61 / -0.58V vs. Ag / AgCl (Figure 3A, blue line d)). DAN peak potential shift compared to CV in DNA solution suggests DAN binding to the cancer surface. The DNA peak current of the present invention was proportional to the scan rate by the electrochemical reaction of DAN localized on the surface, suggesting that DAN reacted with cancer cells was absorbed by the biosensor. However, the redox peak was not observed in cancer cells that were not treated with DAN (see Figure 3A line), NC _D (non-cancer cells) showed weak peak absorption (see the green line in Figure 3A) Suggesting specific binding to the surface of cancer cells as compared to cancer cells.

2. EIS  Spectrum Review

The EIS spectrum of the Nyquist plot was recorded on PBS before and after the interaction of CC _D with the biosensor and the results are shown in Figure 4 (a) cancer cells treated with DAN, and b) normal cells. Referring to FIG. 4 a, a rapid increase in R _ct value (ΔR _ct ) was observed (2902.5 ± 112.6 Ω) after interaction between CC _D (1.0 μM) and the biosensor. However, referring to FIG. 4 b, when the biosensor of the present invention interacted with NC _D under the same conditions, a 14-fold lower ΔR _ct was identified (223.2 ± 13.5 Ω).

In comparative experiments, EIS was measured after interaction between DAN (without cancer cells) and probe, and ΔR _ct (~ 188.4 ± 12.3 Ω), which is similar to the result of interaction between NC _D and biosensor. However, when CC _D interacted with the same probe, the impedance increased significantly. The results show that the combination of the DAN-absorbed cancer cells and the biosensor is ΔR _ct Suggesting that it is associated with an increase in value.

When the concentration of DAN is 0.01 μM, CC _D Lt; RTI ID = 0.0 _&gt; While the value increased to 2186.5 ± 84.2Ω, NC _D of ΔR _ct The value did not increase almost to 32.5 ± 3.5 Ω, which is 68.3 times lower than the value of CC _D (n = 5, RSD <4.2%, see Fig. 4).

3. Impedance change review

In order to check whether the impedance change is specific to the interaction between the biosensor and the CC _D , a comparative experiment was conducted according to whether or not the platemaker was fixed and the DAN treatment was performed. The result is shown in FIG. 3B. FIG. 3B shows the impedance results of the case where the daunomycin is processed (⊚) and not processed (◈), and the case where the aptamer is not immobilized () and immobilized (▴). First, the interaction between DAN-treated cancer cells and a sensor without aptamer (GC / AuNPs / pTTBA) was measured (1-◈ ★) and GC / AuNPs / pTTBA, which is negatively charged due to carboxyl groups on the surface, due to the electrostatic repulsion between the negatively charged ΔR _ct The value did not increase. This clearly shows that DAN untreated cancer cells do not have any affinity for the GC / AuNPs / pTTBA surface. In another comparative experiment, the interaction between DAN-untreated cancer cells and an aptamer with a fixed electrode (GC / AuNPs / pTTBA / aptamer) was measured (2-¬), and the biosensor It was found that the ΔR _ct value was not increased due to the negative charge due to the deprotonation of the phosphate functional group and the electrostatic repulsion between the negative charge and the negative charge on the cancer cell surface. The interaction between the DAN-treated CC _D and the GC / AuNPs / pTTBA electrode was also measured (3- ◎ ★), and CC _D There is no functional group with affinity for ΔR _ct The value did not increase. Thus, ΔR _ct Value increased only when DAN was absorbed into cancer cell membrane and detected by biosensor (4- ▲).

4. Quantification Detectability  Review

In order to confirm that the detection sensor of the present invention is applied to various cancer cell detection, experiments were similarly performed on DAN-treated MCF-7, HT-29 and SKBr-3 cancer cell lines. ΔR _ct for all the experimental cell lines The value increased rapidly. In addition, to ensure that the present invention may be applied to quantitative detection of cancer cells, various amounts of sikyeotgo DAN--treated HeLa cells (HeCC _D) a biosensor with the reaction, and the results are shown in Figure 3 C. Referring to Figure 3C, ΔR _ct Value gradually increased as the number of HeCC _D increased. Based on the HeCC _D impedance data, a calibration curve was derived and is shown in Figure 3D. The calibration curves were linear at 80 to 10000 cells / mL in the HeCC _D range and the detection limit was 43 ± 3 cell / mL (RSD <4.4%) (95% confidence level, n = 5). The linear regression equation is ΔRct (Ω) = 33.05 + 0.727 [HeCC _D ] and the correlation coefficient is 0.998.

< Example  3> on sensor surface Whether or not it  Confirm

The interaction between biosensor and CC _D was confirmed by microscopic analysis of sensor surface. After EIS spectrum measurement, the biosensor surface was analyzed by SEM and the results are shown in FIG. Referring to FIGS. 5A and 5B, it can be seen that CC _D of 5.0 to 7.0 μm is present on the biosensor.

In addition, since DAN has an intrinsic fluorescence activity, CC _D on the biosensor electrode was confirmed by fluorescent microscope image inspection on an indium tin oxide (ITO) electrode, and the results are shown in FIGS. 5C to 5F. Referring to Figures 5C-5F, the various numbers of CC _Ds detected by ITO / pTTBA / Is detected, and it is detected as the amount of CC _D to be cultured with the probe increases And the amount of CC _D was also increased. As the amount of CC _D detected increases, R _ct The value will increase, and therefore cancer cells can be qualitatively and quantitatively detected by the biosensor of the present invention without additional dyeing or coloring agents.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined in the appended claims. will be. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> Pusan National University Industry-University Cooperation Foundation <120> Biosensor for Detecting Cancer Cell Treated Anti-cancer Drug and          Preparation Method Thereof <130> ADP-2013-0038 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 80 <212> DNA <213> Artificial Sequence <220> <223> Aptamer for anticancer drug <400> 1 gggaattcga gctcggtacc atctgtgtaa ggggtaaggg gtgggggtgg gtacgtctag 60 ctgcaggcat gcaagcttgg 80

Claims (8)

An electrode electrodeposited with an Au nanoparticle layer;
An electroconductive polymer layer formed on the Au nanoparticle layer; And
And an absorber layer fixed on top of the electrically conductive polymer layer
Biosensor for detecting cancer cells treated with anticancer drugs. The method according to claim 1,
Wherein the anticancer agent is at least one selected from the group consisting of daunomycin, bleomycin, daunorubicin, doxorubicin, epirubicin, duralubicin, and mitomycin-C. The method according to claim 1,
Wherein the aptamer comprises the nucleic acid sequence of SEQ ID NO: 1. The method according to claim 1,
Wherein the electrically conductive polymer layer comprises a terthiophene monomer. 5. The method of claim 4,
The terthiophene monomer
2,2 ': 5', 2 '' - Entity thiophene -3 (p-benzoic acid) (2,2 ': 5', 2 '' - terthiophene-3 (p -benzoic acid)), or 5,2 ' : 5 ', 2 "-tetiophen-3'-carboxylic acid (5,2': 5 ', 2"-terthiophene-3'-carboxylic acid). The method according to claim 1,
Wherein the electrically conductive polymer layer is a polymer layer in which a carboxyl group is activated by carbodiimide or N-hydroxysuccinimide. Electrodepositing an Au nanoparticle layer on the electrode surface;
Coating an electroconductive polymer layer having a carboxyl group on the surface of the Au nanoparticle layer; And
And covalently bonding a carboxyl group of the electrically conductive polymer layer and an electrode pad to fix the electrode pad. 8. The method of claim 7,
The above aptamer is composed of a nucleic acid sequence of SEQ ID NO: 1, and a covalent bond is formed between an amine group attached to the 5 'end of the nucleic acid sequence of SEQ ID NO: 1 and a carboxyl group of the electrically conductive polymer layer. &Lt; / RTI &gt;

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