KR100838323B1 - Impedimetric immunosensor for detecting bisphenol a and detecting method using the same - Google Patents

Abstract

An impedimetric immunosensor is provided to detect a trace amount of bisphenol A existing in a human serum sample without a marker directly, simply and sensitively, thereby being very usefully used for diagnosing diseases caused by the bisphenol A. An impedimetric immunosensor for detecting bisphenol A is characterized in that a polyclonal antibody isolated from a mouse immuno-reacted with a conjugate of a bisphenol A analog and a protein is fixed on an electrode and the electrode is coated by a nano-particle layer consisting of a conductive polymer. A method for detecting the bisphenol A comprises the steps of: (b) preparing the conjugate of the bisphenol A analog and the protein; (b) preparing the polyclonal antibody from the mouse immuno-reacted with the conjugate; (c) coating the electrode with the nano-particle layer containing the conductive polymer; (d) fixing the polyclonal antibody on the nano-particle layer; (e) removing absorption of non-specific polyclonal antibody from the nano-particle layer; and (f) subjecting the bisphenol A and the polyclonal antibody to antigen-antibody interaction. Further, the conductive polymer is selected from a group consisting of 5,4':5',2"-terthiophene-3'-carboxylic acid, diamino terthiophen, terthiophen salene analogues and formyl terthiophen.

Description

Impedimetric immunosensor for detecting bisphenol A and detecting method using the same}

1 shows a schematic diagram of an impedance immune sensor according to the present invention,

Figure 2 shows the results of QCM analysis (a: when a polyclonal antibody is fixed, b: a polyclonal antibody-antigen-immune response, c: non-specific polyclonal antibody is removed after the immune response between polyclonal antibody-bisphenol A, d: upon immune reaction between polyclonal antibody-antigen prior to blocking of the electrode surface),

Figure 3 shows the impedance spectrum before and after fixing the polyclonal antibody,

In Figure 4, A is a Bode plot of the impedance change measured while increasing the concentration of bisphenol A, B is a plot showing the correlation between the impedance change and frequency, C is a calibration plot for bisphenol A detection,

5 shows a calibration plot obtained from the standard addition method by applying the impedance immunosensor according to the present invention to a human serum sample.

<Drawing symbol>

101: glass carbon electrode 103: nanoparticle layer

105: polyclonal antibody layer 107: antigen or bisphenol A

The present invention relates to an impedance immunosensor for detecting bisphenol A that can directly and simply detect a trace amount of bisphenol A present in human serum samples and the like without a separate marker, and a detection method using the same.

Recently, since endocrine-disrupting compounds (EDCs) such as bisphenol A are released from polycarbonate plastics or epoxy resins in food cans, endocrine interest is increasing.

Bisphenol A has similar effects to endocrine hormones, estrogens and androgens, thus affecting the reproductive system as well as the central nervous system in many animals. Many studies on the endocrine activity of such bisphenol A is in progress.

Bisphenol A binds to both estrogen receptor alpha and beta to induce proliferation of estrogen receptors that express MCF-7 cells similar to estrogens. In addition, bisphenol A is expected to cause various cancer diseases such as genital disorders including a decrease in sperm count, congenital defect due to fetal exposure, prostate cancer, testicular cancer, and breast cancer, and exhibits pleiotropic activity in the brain and cardiovascular system. Therefore, the development of a simple, selective and sensitive analysis of small amounts of bisphenol A in the environment is very important.

The most commonly used methods for the detection of bisphenol A include gas chromatography combined with mass spectrometry, liquid chromatography combined with electrochemical detection, and liquid chromatography combined with mass spectrometry.

These methods show high sensitivity and specificity, but do not allow for rapid processing of many samples because they require a time-consuming pretreatment step and cannot be used for complex, expensive and immediate field measurements.

Recently, inexpensive and rapid bioassay methods have been developed as an alternative to the chromatographic method, which is a traditional method of bisphenol A detection. Typically, there is an immunoassay which is a simple and accurate detection method.

Competitive immunoassays have been reported based on immunoassay systems or surface plasmon resonance (SPR) using polyclonal antibodies chemically bound to bacterial magnetic particles (BMPs).

In addition, various enzyme-linked immunosorbent assays (ELISA) have been used for the detection of bisphenol A. However, the general ELISA method is a complicated multistage process, has a problem of being expensive and difficult to use in the field. As an alternative to the general immunoassay process, SPR-based photoimmune sensors and total internal reflection fluorescence (TIRF) are being used as new methods. However, photo-immune sensors are time-consuming multi-step methods and require large and expensive equipment, and thus are not effective compared to other technologies.

Therefore, there is a need for the development of an impedance immunosensor capable of detecting bisphenol A in a field sensitive, selective and low cost. In this respect, the impedance immunosensor for detecting electrochemical bisphenol A is a potential alternative to the photoimmune sensor. In particular, there have been no reports on the impedance immunosensor for detecting electrochemical bisphenol A.

Electrochemical immunosensors can provide real-time immediate monitoring of bisphenol A and can specifically detect trace amounts of bisphenol A in biological fluids.

In relation to the development of such an immunosensor, the development of electrochemical techniques based on potentiometric, amperometric, and conductivity measurements has been investigated. Amperometric immunosensors are widely used for indirect measurement of analytes such as enzymes labeled with antigens. . However, enzyme labeling procedures are time consuming and complex and require multiple processes.

On the other hand, a direct electrochemical immunosensor without a label is an attractive approach for measuring affinity by observing changes in electronic or interfacial properties of the immunocomplex formed on the electrode surface. Thus, a direct detection approach at the electrochemical immunosensor simplifies the sensing protocol.

For example, there are cases where electrochemical impedance spectroscopy (EIS) has been applied in biometric processes, and DNA hybridization measurements, histidine tag protein measurements, other immune sensors and DNA-protein interactions using EIS. Has been studying. However, no direct detection using bisphenol A markers using impedance immunosensors has yet been reported.

Accordingly, the present inventors immobilized a polyclonal antibody isolated from a mouse immunized with a combination of a bisphenol A analog and a protein to an electrode, and the electrode has an impedance immunosensor for detecting bisphenol A coated with a nanoparticle layer composed of a conductive polymer. The present invention has been completed by the development.

Accordingly, an object of the present invention is to produce an impedance immune sensor that does not use a marker, and to detect the trace bisphenol A contained in human serum samples directly, simply and with high sensitivity. It is to provide a detection method used.

In order to achieve the above object, the present invention is to fix the polyclonal antibody isolated from the mouse immunized with a combination of bisphenol A analog and protein to the electrode, characterized in that the electrode is coated with a nanoparticle layer consisting of a conductive polymer It provides a impedance immunosensor for detecting bisphenol A.

The impedance immunosensor for detecting bisphenol A may include an electrode selected from a gold electrode or a glass carbon electrode; A nanoparticle layer made of a conductive polymer coupled to the surface of the electrode through an electrolytic polymerization reaction; And a polyclonal antibody layer fixed by a carboxyl group of the conductive polymer by a covalent bond.

When bisphenol A is used alone as an antigen for producing polyclonal antibodies, an immune response cannot be initiated. Therefore, a conjugate with a protein must be used. However, there is a problem in that the structure of bisphenol A is changed upon binding between one of the hydroxyl groups of bisphenol A and the amino group of the protein, resulting in low selectivity.

Thus, in the present invention, a bisphenol A analog that is not bisphenol A, for example, is bound to a protein by using a carboxyl group at the end of hydroxyphenylvaleric acid, and thus obtained polyclonal antibody is used as an antigen. Production solves the problem of using bisphenol A.

As the bisphenol A analogs, those selected from the group consisting of hydroxyphenylvaleric acid, 2,4-dinitrophenol, p -nitrophenol, o -nitrophenol, 2,6-dimethylphenol and 2,4-dichlorophenol are used. do.

In addition, bovine serum albumin (BSA), hemocyanin, ovalbumin, and the like are used as the protein, and preferably bovine serum albumin is used.

The conductive polymer may be 5,2 ': 5', 2 "-terthiophene-3'-carboxylic acid (5,2 ': 5', 2" -terthiophene-3'-carboxylic acid) or diaminoterthiophene. (diamino-terthiophen), terthiophen salene analogues, or formyl terthiophen, preferably 5,2 ': 5', 2 "-terthiophene-3'-carboxylic acid. do.

In addition, the nanoparticle layer is coated with a conductive polymer on the electrode through the electrolytic polymerization according to a known method ( Anal . Chem . , 73, 5629-5632, 2001; Anal . Chem . , 77, 4854-4860, 2005). It is produced, the particle diameter of the nanoparticles grown at a scanning speed of 1 V / s is 5 to 40 nm, the thickness of the nanoparticle layer is 20 to 300 nm, preferably 200 nm. Since the nanoparticles of the nanoparticle layer coated with the conductive polymer have a maximum surface area, the polyclonal antibody may be fixed to the nanoparticle layer.

In the impedance immunosensor according to the present invention, the immobilization of the polyclonal antibody to the nanoparticle layer and the interaction between the polyclonal antibody and the bisphenol A are carried out by the quartz crystal microbalance (QCM) and the electrochemical impedance spectroscopic technique. ; EIS) technology.

Bisphenol A can be measured using impedance changes according to specific immune interactions observed on the surface of the impedance immune sensor, wherein the polyclonal antibody content is 200-300 ng, pH is 5-9, and immune response measurement. It is preferable that the temperature is 15-50 ° C. because bisphenol A can be easily measured with high sensitivity and high selectivity.

In particular, the impedance immunosensor according to the present invention has a very high sensitivity that can effectively detect even bisphenol A of 0.3 ng / ml in the field, and a high performance liquid using a generally expensive and complicated device to detect bisphenol A. Detection limits similar to chromatography are shown.

In addition, the present invention comprises the steps of preparing a conjugate of a bisphenol A analog and a protein; Preparing a polyclonal antibody from a mouse immunized with the conjugate; Coating a nanoparticle layer containing conductive polymer on the electrode; Fixing the polyclonal antibody to the nanoparticle layer; Removing adsorption of non-specific polyclonal antibodies remaining in the nanoparticle layer; And it provides a method for detecting bisphenol A, characterized in that comprising the step of performing an antigen antibody reaction between bisphenol A and polyclonal antibody.

The conjugate is prepared by activating a carboxyl group of a bisphenol A analog and covalently bonding an amine group of a protein with a carboxyl group of the activated bisphenol A analog, wherein activation is carried out at a carbodiimide or N-hydroxysuccinimide. Performed by one or more compounds selected.

The nanoparticle layer is prepared by coating a conductive polymer on the electrode through an electrolytic polymerization according to a known method ( Anal . Chem . , 73, 5629-5632, 2001; Anal. Chem . , 77, 4854-4860, 2005). The particle diameter of the nanoparticles grown at a scanning speed of 1 V / s is 5 to 40 nm, and the thickness of the nanoparticle layer is 20 to 300 nm, preferably 200 nm.

The step of fixing the polyclonal antibody to the nanoparticle layer is to activate the carboxyl group of the conductive polymer present in the nanoparticle layer using one or more compounds selected from carbodiimide or N-hydroxysuccinimide, the activated carboxyl group and polyclonal antibody The polyclonal antibody is immobilized on the nanoparticle layer through covalent bonds between amine groups.

In order to remove the adsorption of the nonspecific polyclonal antibody remaining in the nanoparticle layer, it is first washed with a phosphate buffer solution to remove the weakly adsorbed antibody, and reacted in a phosphate buffer solution (PBSTG) and bovine serum albumin solution containing tween 20 and gelatin. Remove nonspecifically adsorbed antibodies.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the present invention is not limited by these examples.

Example 1 Impedance Immune Sensor

1) Preparation of BHPVA-BSA Binder

4,4-bis (4-hydroxyphenyl) valeric acid [4,4-bis (4-hydroxyphenyl) valeric acid; BHPVA] was combined with bovine serum albumin (BSA) and used for mouse immunization.

In order to bind BHPVA and BSA, the carboxyl group of BHPVA was first activated using NHS (N-hydroxysuccinimide) and EDC [1-ethyl-3- (3-dimethylamino-propyl) carbodiimide]. That is, 7.0 mg of BHPVA, 3.5 mg of NHS, and 5.9 mg of EDC were weighed and dissolved in 1.0 ml of dimethylsulfoxide (DMSO), followed by stirring for 2 hours.

The BSA solution was prepared by dissolving 45 mg of BSA in 4.0 ml of 0.1 M NaHCO ₃ solution (adjust the pH of the solution to 7.0 using HCl). BHPVA-NHS-EDC solution was added dropwise to the BSA solution, stirred for 2 hours at a temperature of 25 ℃, and then dialyzed with 0.01 M PBS for 24 hours at 4 ℃. In addition, the solution was dialyzed with pure water for 48 hours with changing water 8 times. Finally, the white crystals obtained by lyophilization of the solution were stored at -20 ° C.

2) Production and Purification of Polyclonal Antibodies

Three 4 week old Balb / C mice were immunized. Each mouse received 4 mg of BHPVA-BSA conjugate suspended in Freund's complete adjuvant (FCA) or Freund's incomplete adjuvant (FIA) on a total of four times on Days 0, 11, 23 and 34. Immunization by intraperitoneal injection over and blood was collected from tail vein at day 45.

The first immune response used 1.5 mg of BHPVA-BSA conjugate suspended in FCA as antigen, and the subsequent three immune responses used 0.5, 1.0 and 1.5 mg of BHPVA-BSA conjugate suspended in FIA as antigen, respectively.

Antititer antibody titer was measured by ELISA using BHPVA-BSA coated on a microplate. The resulting polyclonal antibody was purified by modified caprylic acid-saturated ammonium sulfate (SAS) method.

3) Preparation of Nanoparticle Layer Containing Conductive Polymer

0.1 mM 5,2 ': 5', 2 "-Ter dissolved in 0.1 M TBAP (tetrabutylammonium perchlorate, electrochemical grade; Fluka) / CH ₂ Cl ₂ (99.8%, anhydrous, sealed under N ₂ gas; Sigma) solution Offen-3'-carboxylic acid (TTCA) monomers were cyclically shifted in potential from 0 to 1.6 V over three times at 1000 mV / s in a known manner to form nanoparticle layers on glass carbon electrodes ( Biosens . Bioelectron . , 19, 1565-71, 2004; Biosens . Bioelectron . , 21, 257-65, 2005; Anal . Chem . , 77, 4854-4860, 2005).

Excess monomer used was removed by washing with CH ₂ Cl ₂ . And the TTCA monomers used were prepared by known methods ( Synth . Met . , 126, 105-110, 2002). At this time, the thickness of the nanoparticle layer was 200 nm.

QCM (quartz crystal microbalance; SEIKO EG & G model QCA 917 and PAR model 263A Potentiostat / Galvanostat) analysis was performed to measure the amount of nanoparticles formed on the glass carbon electrode surface.

As a result, the frequency change (Δf) was 0.96 ± 0.04 kHz, and the amount of nanoparticles formed after three cycles calculated using a known formula (Anal. Chem., 73, 5629-5632, 2001) was 1.05 ± 0.17. Μg, which corresponds to 3.6 ± 0.6 × 10 ⁻⁹ mol / cm ² .

4) Fixation of Polyclonal Antibody

The nanoparticle layer coated on the glass carbon electrode was immersed in 0.01 M phosphate buffer solution (pH 7.0) containing 10 mM EDC for 6 hours to activate the carboxyl group of the nanoparticle layer. The glass carbon electrode coated with the nanoparticle layer treated with EDC was washed with phosphate buffer solution, and then reacted at 4 ° C., 0.01 M PBS (pH 7.0), 1: 500 solution of polyclonal antibody for 12 hours.

The polyclonal antibody was immobilized on the nanoparticle layer through a covalent bond between the carboxyl group of the nanoparticle layer and the amine group of the polyclonal antibody. The nanoparticle layer modified with a polyclonal antibody coated on the glass carbon electrode was washed with PBS to remove the weakly adsorbed antibody, and then 0.1 hour in a PBSTG solution (PBS solution containing 0.05% Tween20 and 0.8% gelatin) for 0.1 hour. Non-specific adsorption was removed by reaction for 1 hour in% BSA solution.

This electrode was washed three times with PBS and stored at 4 ° C. and used for the measurement of bisphenol A. A schematic diagram of the impedance immunosensor for detecting bisphenol A is shown in FIG. 1.

Hereinafter, a glass carbon electrode (area = 0.07 cm 2), Ag / AgCl (in saturated KCl), platinum wire coated with a nanoparticle layer modified with a polyclonal antibody prepared above were used as a working electrode, a reference electrode, and a counter electrode, respectively. Cyclic voltammograms were measured using potentiostat / galvanostat and Kosentech model KST-P2 (South Korea).

Example 2 QCM Review

In order to examine the immobilization of the polyclonal antibody to the nanoparticle layer and the interaction between the antigen and bisphenol A for the immobilized polyclonal antibody, QCM was performed as follows. At this time, the QCM experiment was performed using SEIKO EG & G model QCA 917 and PAR model 263A potentiostat / galvanostat (USA).

1) QCM review for immobilization of polyclonal antibodies

A nanoparticle layer was grown on one side of a quartz crystal gold electrode, and the gold electrode coated with the nanoparticle layer was washed and then immersed in a 0.01 M EDC solution dissolved in a phosphate buffer solution (pH 7.0) for 6 hours. The carboxyl group on the surface of the nanoparticle layer was activated, and the polyclonal antibody was immobilized on the gold electrode in the same manner as in Example 1.

As shown in FIG. 2, the frequency change was observed during the immobilization of the polyclonal antibody as a function of time, and as a result, the frequency gradually decreased and became stable after 2 hours.

Therefore, it is judged that the fixation of the polyclonal antibody to the nanoparticle layer is completed within 2 hours at room temperature, and the frequency change (Δf) after 2 hours is ca. It was 232 Hz, and the amount of polyclonal antibody immobilized on the nanoparticle layer was 255 ± 18 ng.

2) QCM review of the interaction of antigen and bisphenol A against polyclonal antibodies

The QCM was carried out as above, but the non-specific adsorption was removed by treating the nanoparticle layer immobilized with polyclonal antibody for 1 hour in PBSTG solution and 0.1% BSA solution, respectively, and then the specific interaction between antigen and bisphenol A was observed. Reviewed.

As a result, as shown in Fig. 2, the decrease in frequency reached a stable state after 20 minutes of binding between the polyclonal antibody-antigen and the polyclonal antibody-bisphenol A, respectively, and the frequency change (Δf) was the polyclonal antibody and antigen. The liver interactions were 138 Hz and the polyclonal antibody and bisphenol A interactions were 130 Hz, with positive changes of 151.7 ± 22 ng and 143 ± 13 ng, respectively.

In particular, if the nonspecific adsorption of the polyclonal antibody is not removed, the frequency decrease is stabilized after 30 minutes, and the frequency change is observed at 165 Hz, which is not preferable, so the nonspecific adsorption must be removed.

Example 3 Impedance Examination

Impedance analysis was measured using a EG & G PAR model 273A potentiostat / galvanostat (USA) and a lock-in amplifier (PAR EG & G model 5210) connected to a personal computer. The frequency was scanned at 100 kHz to 10 Hz or 100 Hz at an open circuit voltage to obtain 5 points per 10, and the amplitude of a sine voltage of 10 mV was used. In this case, the impedance change was measured in a buffer medium (0.01M PBS, pH 7.0) at a temperature of 25 ± 0.2 ℃.

1) Examination of impedance change for immobilization of polyclonal antibody

Impedance spectra before and after polyclonal antibody fixation in 0.01 M PBS solution (pH 7.0) are shown in FIG. 3, where A represents a Bode plot and B represents a Nyquist plot.

The electrode coated with the nanoparticle layer had an increased impedance after fixing of the polyclonal antibody, exhibited high conductivity due to the conductive polymer constituting the nanoparticle layer, and increased the resistance of the nanoparticle layer by immobilizing the polyclonal antibody on the nanoparticle layer. Therefore, impedance value increased after fixation of polyclonal antibody.

As shown in A of FIG. 3, the maximum difference before and after the fixing of the polyclonal antibody was observed at a log frequency of 3.7 Hz, and as shown in FIG. 3B, the charge transfer resistance according to the fixing of the polyclonal antibody was shown. resistance) increased.

The impedance spectrum of the present invention is shown as shown in FIG. 3B using a general equivalent circuit. At this time, R _s is the solution resistance, R _p is the electrolytic resistance, W is the Warburg factor, Q _dl is the constant phase element (constant phase element).

The R _s , R _p , W and Q _dl are obtained from experimental data and are shown in Table 1. R _s is the bulk characteristics of the electrolytic solution, and is a value that is not affected by chemical modification to fix the polyclonal antibody to the nanoparticle layer.

R _p and Q _dl are numerical values that vary depending on the dielectric constant and conductivity at the interface between the electrode and the electrolyte, and R _p values change when another substance is adsorbed on the electrode surface. Q _dl is a numerical value that varies depending on the dielectric constant of the layer separating the ion charge and the electrode surface, the surface area of the electrode, and the thickness of the separation layer.

The large decrease in Q _dl shown in the present invention means that the amount of polyclonal antibodies immobilized on the nanoparticle layer is greatly increased due to the high surface area, so that the polyclonal antibodies exhibit the maximum surface area range.

2) Examination of impedance change for interaction between antigen and bisphenol A for polyclonal antibody

After fixing the polyclonal antibody and blocking the free active site, the nanoparticle layer fixed with the polyclonal antibody was washed with PBS and stored in 0.01M PBS solution. The impedance of the polyclonal antibody-antigen and polyclonal antibody-bisphenol A interaction was performed by adding 40 ng / ml of antigen and bisphenol A dissolved in PBS solution at the open circuit voltage.

It can be seen from the Bode plot shown in FIG. 3A that the impedance is reduced by the interaction between specific polyclonal antibody-antigen and polyclonal antibody-bisphenolA.

This decrease in impedance is believed to be due to a change in conductivity or dielectric constant due to immune interaction. The maximum difference in impedance values was observed at a log frequency of 3.7 Hz. Since the decrease in impedance is proportional to the concentration of antigen or bisphenol A, the decrease in impedance is believed to be caused by specific polyclonal antibody-antigen and polyclonal antibody-bisphenol A binding.

Thus, the specific recognition of antigen and bisphenol A by polyclonal antibodies can be measured by Bode plot without other reducing probes or markers.

In addition, the Nyquist plot measured before and after antigen and bisphenol A addition showed that the charge transfer resistance was reduced due to the polyclonal antibody-antigen and polyclonal antibody-bisphenol A interaction.

The impedance spectrum of the present invention was measured using the equivalent circuit shown in the inset of FIG. Where R _s is the solution resistance, R _p is the electrolytic resistance, W is the Warburg element, Q _dl is the constant phase element, and n is the slope of the Warburg term.

The R _s , R _p , W, Q _dl and n are obtained from experimental data, and are shown in Table 1. W, Q _dl , R _p was sensitively changed by antigen or binding between bisphenol A (BPA) and polyclonal antibody (PAb), and in particular, the decrease in R _p and increase in Q _dl were prominent by this binding.

In addition, the value of Δ | Z |, which is the impedance difference obtained from the Bode plot, is obtained simply and directly, which is useful for the detection of bisphenol A.

PAb fixation After fixing PAb After antigen binding After BPA binding R _s (Ω㎠) 601.4 600 520 530 Q _dl (Fcm ^-2 s- ^(1-α) ) 1.3 × 10 ^-6 3.7 × 10 ^-9 1.27 × 10 ^-7 1.15 × 10 ^-7 R _p (Ω㎠) 3.42 × 10 ^-9 1360 900 999.7 n 0.76 0.78 0.81 0.79 W (Wcm2s ^β ) 1.2 × 10 ⁵ 120.5 56.3 50.75 Chi-squared 1.0 × 10 ^-4 5.3 × 10 ^-4 3.1 × 10 ^-4 4.4 × 10 ^-4

< Example  4> Review of interference effects

Phenol, hydroquinol, p -hydroxybenzoic acid, p -nitrophenol, o -nitrophenol, 2,4-dinitrophenol, 2,6-dimethylphenol for measuring the selectivity of the impedance immunosensor according to the invention The impedance change was measured in the presence of BHPVA and phenolic compounds such as 2,4-dichlorophenol, 3-chlorophenol, 2-chlorophenol and the like.

Before examining the interference effect, the impedance immunosensor coated with the nanoparticle layer immobilized with polyclonal antibody was first washed with PBSTG and then reacted in 0.1% BSA solution for 1 hour to remove nonspecific binding.

As a result, BHPVA, an immunogen, significantly prevented the detection of bisphenol A as expected. However, other phenolic compounds did not interfere with the detection of bisphenol A. That is, no significant change was observed in the Δ | Z | value obtained after bisphenol bonding (40 ng / ml) even when added at a concentration 100 times higher (5 µg / ml) for other phenolic compounds.

Therefore, by using the impedance immunosensor of the present invention, bisphenol A can be selectively detected by distinguishing it from other phenolic compounds.

Example 5 Quantitative Detection of Bisphenol A

The dilution ratio of the polyclonal antibody was 1: 500, and the bisphenol A was quantitatively analyzed by measuring the impedance decrease while increasing the concentration of bisphenol A under an immunoreaction condition of pH 8.0 for 20 minutes at a temperature of 30 ° C.

4A shows a Bode plot for impedance analysis before and after bisphenol A interaction. Impedance measurements were performed after addition of bisphenol A at various concentrations of 1 to 300 ng / ml. The value of Δ | Z | increased with increasing concentration of bisphenol A.

4B shows the difference in log impedance values obtained from the immunosensor after interacting with bisphenol A at various concentrations. The value of Δ | Z | increased in the frequency range between 10 Hz and 100 kHz. However, the maximum value of Δ | Z | was obtained at a log frequency of 3.7 Hz. Therefore, the value of Δ | Z | was chosen as the calibration plot at a log frequency of 3.7 Hz.

4C shows a calibration plot of bisphenol A detection. The impedance immune sensor of the present invention showed a linear correlation between the value of Δ | Z | and the concentration of bisphenol A. The linear range was 1-100 ng / ml. In addition, the detection limit of the impedance immune sensor of the present invention was 0.3 ng / ml.

Example 5 Application to Human Serum Samples

In order to apply the impedance immunosensor prepared in Example 1 to the actual clinic, the trace amount of bisphenol A present in healthy human serum samples was measured. Human serum samples were purchased and used, and the bisphenol A concentration was measured using a standard addition method.

5 shows a calibration plot obtained from the standard addition method. A linear plot was extrapolated to the negative x-axis to calculate bisphenol A concentration in human serum samples. The concentration of bisphenol A in serum samples was 400 ± 27 pg / ml.

Meanwhile, when multi-electrode electrochemical detection based on known high-performance liquid chromatography was used, bisphenol A of 320 pg / ml was detected. Therefore, the impedance immunosensor of the present invention can be very useful for the detection of bisphenol A in serum samples.

As described above, the use of the impedance immunosensor according to the present invention is caused by bisphenol A because it can directly and simply detect a trace amount of bisphenol A present in human serum samples without a separate marker. It can be very useful for diagnosing diseases in vivo.

Claims (6)

A polyclonal antibody isolated from a mouse immunized with a combination of a bisphenol A analog and a protein is fixed to an electrode, and the electrode is coated with a nanoparticle layer composed of a conductive polymer. The method of claim 1, wherein the bisphenol A analog is selected from the group consisting of hydroxyphenyl valeric acid, 2,4-dinitrophenol, p -nitrophenol, o -nitrophenol, 2.6-dimethylphenol and 2.4-dichlorophenol. Impedance immunosensor for detecting bisphenol A. The method of claim 1, wherein the conductive polymer is 5,2 ': 5', 2 "-terthiophene-3'-carboxylic acid (5,2 ': 5', 2" -terthiophene-3'-carboxylic acid), dia An impedance immunosensor for detecting bisphenol A, characterized in that it is selected from the group consisting of diaminoterthiophene, terthiophen salene analogues, and formyl terthiophene. Preparing a conjugate of a bisphenol A analog and a protein; Preparing a polyclonal antibody from a mouse immunized with the conjugate; Coating a nanoparticle layer containing conductive polymer on the electrode; Fixing the polyclonal antibody to the nanoparticle layer; Removing adsorption of non-specific polyclonal antibodies remaining in the nanoparticle layer; And Performing an Antibody Antibody Response Between Bisphenol A and Polyclonal Antibody Bisphenol A detection method using the impedance immunosensor according to claim 1, characterized in that consisting of. The method of claim 4, wherein the conjugate is prepared by activating the carboxyl group of the bisphenol A analog and covalently bonding the carboxyl group of the activated bisphenol A analog to an amine group of the protein. 6. The method of claim 5, wherein said activation is performed by at least one compound selected from carbodiimide or N-hydroxysuccinimide.

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