KR101755230B1

KR101755230B1 - Sensor for detecting multidrug resistance cancer cell and detecting method of multidrug resistance cancer cell using the same

Info

Publication number: KR101755230B1
Application number: KR1020150127584A
Authority: KR
Inventors: 심윤보; 프란잘 산드라
Original assignee: 부산대학교 산학협력단
Priority date: 2015-09-09
Filing date: 2015-09-09
Publication date: 2017-07-11
Also published as: KR20170030686A

Abstract

The present invention relates to a biosensor for detecting multidrug-resistant cancer cells, a method for producing the same, and an electrochemical detection method. The biosensor is a sandwich type electrochemical immunosensor, which is expressed on the cell membrane of cancer cells, By using a specific monoclonal antibody against glycoprotein (P-gp) as a detection probe and using a non-enzymatic catalyst as a reporter probe, the sensitivity to multi-drug resistant cancer cells can be improved, And it is confirmed that the stability is maintained even after long-term use. Therefore, it can be used as a biosensor for detecting multidrug-resistant cancer cells for chemotherapy of cancer patients.

Description

TECHNICAL FIELD The present invention relates to a multidrug resistant cancer cell detecting sensor and a multidrug resistant cancer cell detecting method using the same,

The present invention relates to a biosensor for detecting multidrug-resistant cancer cells, a method for producing the same, and an electrochemical detection method.

Multidrug resistance (MDR) is one of the major causes of cancer chemotherapy failure, and several mechanisms have been reported to play an important role in the development of multidrug resistance in cancer cells (Krishna and Mayer, 2000).

Among them, Permeability glycoprotein (P-gp), which is a cell membrane transport encoded by the MDR1 gene in human cells, is one of the main causes.

The permeable glycoprotein (P-gp), an energy-dependent pump, causes the cancer drug to be released from the cancer cells to maintain a concentration lower than the effective drug concentration of the cancer drug in the cancer cells, and this phenomenon causes drug resistance of cancer cells (Bellamy, 1996; Gottesman et al., 2002).

Early detection of multidrug resistance (MDR) in cancer cells can overcome cancer death by applying appropriate chemotherapy regimens for cancer patients.

Therefore, it is very important to detect MDRcc in early stage for an effective treatment strategy for cancer patients.

Up to now, expression of P-gp for the diagnosis of MDR in cancer cells has been detected by polymerase chain reaction (Murphyetal., 1990), immunohistochemistry (Chan et al., 1990) Biological methods such as flow cytometry (Lu-descher et al., 1992) and microarray (Gill et al., 2004) have been used.

However, these methods require specialized training compared to low sensitivity to MDRCC, and there is a problem in miniaturization of equipment for diagnostic field applications. As an alternative method to solve these problems, research on MDRcc detection method using a biosensor has been carried out, but the biosensor methods reported so far have been reported to have problems such as indirect, non-selective, and low sensitivity.

Therefore, there is a need to develop a biosensor for detecting MDRcc that is sensitive to MDRcc for effective chemotherapy of cancer patients and stable for long-term use.

Korean Patent Publication No. 2014-0108810 (published on September 15, 2014)

The present invention provides a detection sensor capable of selectively detecting multidrug-resistant cancer cells by enhancing sensitivity to multidrug-resistant cancer cells, and a multidrug-resistant cancer cell detection method using the same.

The present invention relates to a method for manufacturing a nanoparticle-containing nanoparticle layer, comprising: an electrode, a nanoparticle layer formed on the electrode, an electrically conductive polymer layer formed on the nanoparticle layer, and a permeable glycoprotein antibody layer formed on the electrically conductive polymer layer,

And a complex comprising a multi-walled carbon nanotube, a hydrazine and a boronic acid derivative, wherein the complex comprises a cancer cell detection unit which binds to the captured cancer cell and performs a catalytic reaction with hydrogen peroxide. For example.

The present invention relates to a method for manufacturing an electroconductive polymer layer, comprising: (a) forming an electrically conductive polymer layer by electrostatically bonding an electroconductive monomer to a surface of an electrode on which nanoparticles are electrodeposited; Fixing a permeable glycoprotein antibody to the electrically conductive polymer layer (second step); Treating the sample with the electrode on which the antibody is immobilized (step 3); Preparing a complex comprising a multi-walled carbon nanotube, a hydrazine and a boronic acid derivative (Step 4); Treating the bonded body of the fourth step on the electrode to which the sample is treated (fifth step); And a step of immersing the treated electrode in a buffer solution containing hydrogen peroxide to carry out a catalytic reaction (step 6).

The present invention relates to a method for manufacturing an electroconductive polymer layer, comprising: (a) forming an electrically conductive polymer layer by electrostatically bonding an electroconductive monomer to a surface of an electrode on which nanoparticles are electrodeposited; Fixing a permeable glycoprotein antibody to the electrically conductive polymer layer (second step); Treating the sample with the electrode on which the antibody is immobilized (step 3); Preparing a complex comprising a multi-walled carbon nanotube, a hydrazine and a boronic acid derivative (Step 4); Treating the bonded body of the fourth step on the electrode to which the sample is treated (fifth step); (Step 6) of carrying out a catalytic reaction by immersing the electrode treated with the coupling agent in a buffer solution containing hydrogen peroxide (step 6) and analyzing the reduction current by the catalytic reaction (step 7) A method for detecting a cancerous cancer cell.

The biosensor of the present invention is a sandwich-type electrochemical immunosensor that uses a specific monoclonal antibody to a permeable glycoprotein (P-gp), which is expressed on the cell membrane of cancer cells and causes multidrug resistance, as a detection probe, It was confirmed that the use of a catalyst as a reporter probe improves the sensitivity to multidrug-resistant cancer cells and that the multidrug-resistant cancer cells can be selectively detected even when a biological sample such as serum is directly used, . Therefore, the biosensor of the present invention can be effectively used as a biosensor for the detection of multidrug-resistant cancer cells for chemotherapy of cancer patients.

1 is a schematic diagram of an immunosensor GCE / AuNPs / pTTBA / AntiP-gp / MDR _cc / APBA-MWCNT-Hyd sensor.
FIG. 2A shows the AFM image of the tapping mode (image size 500.0 nm × 500.0 nm), and FIG. 2B shows the result of checking AFM images of the tapping mode. FIG. 2A shows the result of checking the characteristics of the GCE / AuNPs / pTTBA / AntiP-gp / MDR _cc / APBA-MWCNT- (a) is an AFM image of HOPG / AuNPs film, (b) is an AFM image of HOPG / pTTBA film, (c) is an AFM image of HOPG / AuNPs / pTTBA film, (B) is S2 p and (c) is N1s, and Fig. 2C is a graph showing the XPS spectra results of the Au-coated pTTBA electrode surface (Au), AuNPs / pTTBA and (ii) GCE / AuNPs / pTTBA / AntiP- FIG. 2D shows the result of confirming the change in the frequency during immobilization of AntiP-gp in GCE obtained in 0.1 M PBS (pH 7.4) containing 4.0 mM Fe [(CN) ₆ ] 3 ^- / 4 ^- /0.3 M NaClO _4. (black), GCE / AuNPs (red), GCE / pTTBA (magenta), and GCE / AuNPs / pTTBA (blue line) electrodes are Nyquist plots.
3 is a cyclic voltammetry of the cancer cell concentration; as a result, make the (cyclic voltammetry CV) reaction, Figure 3A is 50.0mV / s, 5000 MDR _CC / mL concentrations of the cancer cells GCE / AuNPs / pTTBA / AntiP- gp / MDR _{CC / APBA (black line),} GCE / AuNPs / pTTBA / AntiP-gp / MDR CC / MWCNT-Hyd (blue line), GCE / AuNPs / pTTBA / AntiP-gp / MDR CC / APBA-MWCNT-Hyd (redline) and processing and checking the cyclic voltammetry results in electrodes, Fig. 3B is a MDR _CC As a result of the cyclic voltammetry of the GCE / AuNPs / pTTBA / AntiP-gp / MDR _CC / APBA-MWCNT-Hyd sensor reducing 4.0 mM H ₂ O ₂ , a (blank), b 1000), c (1500), d (2000), e (2500), f (3000) and g (3500) MDR _CC / mL.
Fig. 4A shows the results of checking the sensor performance. Fig. 4A is a graph showing the relationship between cancer cell growth [a (blank), b (1000), c (1500), d (2000), e (2500), f (3000) according to the MDR _CC / mL] MDR _CC FIG. 4B shows the calibration curve based on the signal obtained from the amperogram, and the result of enlarging the value between 50 and 10,000 MDR _CC / mL.
Figure 5 is the result confirming the selective detection level of MDR _CC as a result of checking the MDR _CC captured by AntiP-gp probe, Figure 5A in which the cancer cells of 30,000 MDR _CC / mL concentration treatment biosensor, 5B are MDR _CC (Ac) treated with ITO / pTTBA / AntiP-gp probe (d) and MDR _CC captured in ITO / pTTBA chip.
FIG. 6 shows the relative current responses of APBA-MWCNT-Hyd and reporter AntiP-gp-MWCNT-Hyd complexes at 25,000, 50,000 and 100,000 MDR _CC / mL cell concentrations, where the Y axis represents the current signal.

The present invention relates to a method for manufacturing a nanoparticle-containing nanoparticle layer, comprising: an electrode, a nanoparticle layer formed on the electrode, an electrically conductive polymer layer formed on the nanoparticle layer, and a permeable glycoprotein antibody layer formed on the electrically conductive polymer layer, And a complex comprising a multi-walled carbon nanotube, a hydrazine and a boronic acid derivative, wherein the complex comprises a cancer cell detection unit which binds to the captured cancer cell and performs a catalytic reaction with hydrogen peroxide. For example.

The nanoparticle layer may be a nanoparticle layer composed of gold nanoparticles or silver nanoparticles.

The electrically conductive polymer layer may be formed of at least one selected from the group consisting of 2,2 ': 5', 5 "-tetiophene-3'-p-benzoic acid (TTBA), 5,2 ' , And 2,5-di- (2-thienyl) -1H-pyrrole-P-benzoic acid (DTPBA), and more preferably 2,2 ' , 5 "-terthiophene-3'-p-benzoic acid (TTBA).

The boronic acid derivative may be selected from the group consisting of amino-phenylboronic acid (APBA), phenylboronic acid (PBA), thienylboronic acid (TBA) and methylboronic acid (MBA).

Permeability glycoprotein (P-gp) may be expressed in the cell membrane of the cancer cells.

The present invention relates to a method for manufacturing an electroconductive polymer layer, comprising: (a) forming an electrically conductive polymer layer by electrostatically bonding an electroconductive monomer to a surface of an electrode on which nanoparticles are electrodeposited; Fixing a permeable glycoprotein antibody to the electrically conductive polymer layer (second step); Treating the sample with the electrode on which the antibody is immobilized (step 3); Preparing a complex comprising a multi-walled carbon nanotube, a hydrazine and a boronic acid derivative (Step 4); Treating the bonded body of the fourth step on the electrode to which the sample is treated (fifth step); And immersing the treated electrode in a buffer solution containing hydrogen peroxide to carry out a catalytic reaction (step 6). The present invention also provides a method for manufacturing a multidrug-resistant cancer cell detection sensor.

According to one embodiment of the present invention, the MDR _CC of the multi-drug resistant cancer cell detecting sensor The detection limit was 23 ± 2 cells / mL (RSD <4.4%), and the detection limit was calculated using MDR _CC impedance biosensor (Zhangetal, 2014), gold nanoparticle / poly- (Chen et. Al., 2014) and QCM (Shaolian et al., 2014) biosensors for detection of the biosensor.

In addition, the relative currents of the APBA-MWCNT-Hyd and AntiP-gp-MWCNT-Hyd complexes at 25,000, 50,000 and 100,000 MDR _CC / A signal appeared. In addition, the MDR _CC detection limit of the AntiP-gp-MWCNT-Hyd conjugate was determined to be 23 ± 2 cells (n = 5) at the detection limit of APBA-MWCNT-Hyd conjugate of the present invention at 158 ± 8 cells / / mL).

Based on the above results, the multi-drug resistant cancer cell detection sensor of the present invention has a sensitivity four times higher than that of the previously reported multi-drug resistant cancer cell detection sensor, and is superior to an antibody-based immune sensor such as AntiP-gp-MWCNT- Resistant cancer cell sensitivity.

According to another aspect of the present invention, there is provided a method of manufacturing a semiconductor device, comprising: (a) forming an electrically conductive polymer layer by electrostatically bonding an electroconductive monomer to a surface of an electrode on which nanoparticles are electrodeposited; Fixing a permeable glycoprotein antibody to the electrically conductive polymer layer (second step); Treating the sample with the electrode on which the antibody is immobilized (step 3); Preparing a complex comprising a multi-walled carbon nanotube, a hydrazine and a boronic acid derivative (Step 4); Treating the bonded body of the fourth step on the electrode to which the sample is treated (fifth step); (6) carrying out the catalytic reaction by immersing the electrode treated with the complex in a buffer solution containing hydrogen peroxide (step 6) and analyzing the reduction current by the catalytic reaction (step 7) Can be provided.

The sample may be selected from the group consisting of blood, plasma, serum and urine.

The concentration of hydrogen peroxide in the buffer solution may be 2.0 to 6.0 mM, more preferably 4.0 mM, but is not limited thereto.

The reduction current may be analyzed by cyclic voltammetry (CV) or chronoamperometry, but is not limited thereto.

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.

&Lt; Referential Example 1 >

2,2 ': 5', 2 "-, 5'-, 2" -terthiophene-3'-p-benzoic acid [2,2 ' terthiophene-3 ' (p-benzoic acid); TTBA] was synthesized.

Tetrabutylammonium perchlorate (TBAP, electrochemical grade) was purchased from Fluka (USA) and purified by a conventional method, followed by drying under vacuum at 1.33 × 10 ^-3 Pa (Noh et al., 2012).

1-ethyl-3- (3- (dimethylamino) propyl) carbodiimide [1-ethyl-3- (3- (dimethylamino) propyl) carbodiimide; EDC], N-hydroxysuccinimide; NHS], dichloromethane (99.8%, anhydrous), trisodium citrate, sodium tetrahydridoborate, HAuCl ₄ .3H ₂ O, bovine serum albumin BSA) and hydrazine sulfate were purchased from Sigma Aldrich (USA).

Monoclonal p-GP antibodies, amino-phenylboronic acid (APBA), indium tin oxide (ITO) glass and H ₂ O ₂ were purchased from Sigma Aldrich (USA). MWCNTs (4-6 nm diameter, 95%) were obtained from Iljin Nanotech (South Korea).

Phosphate buffer saline (PBS) was prepared with 0.1 M disodium hydrogen phosphate, 0.1 M sodium dihydrogenphosphate and 0.9% sodium chloride.

Cell culture medium, fetal bovine serum (FBS), trypsin-EDTA, penicillin / streptomycin, and hank's balance salt (HBS) solutions were purchased from Sigma-Aldrich (USA).

All other chemicals were used for further analysis without further purification. All aqueous solutions were purified with ultrapure water using a Milli-Q water purifying system (18M Ω cm).

&Lt; Referential Example 2 >

All electrochemical experiments were performed with a three electrode cell.

A working electrode, a reference electrode, and a counter electrode were each coated with a modified glassy carbon electrode (GCE: diameter 3.0 mm), Ag / AgCl (in saturated KCl) And used as an electrode.

Voltammograms and chronoamperograms were recorded with potentiostat / galvanostat, Kosentech, model KSTP-2 (South Korea).

Conditional images were obtained using a multi-mode AFM device (Veeco Metrology) equipped with a Nanoscope IV controller (Veeco).

QCM experiments were performed using SEIKO EG & G model QCA 917 and PAR model 263A potentiostat / galvanostat (USA) and gold (Au) coated working electrode (area: 0.196 cm2; 9 MHz; AT-cutquartzcrystal).

The impedance spectra were obtained at a sampling rate of 5 points per 10 (AC amplitude: 10.0mV) with EG & G Princeton Applied Research PARSTAT 2263 using an open-circuit voltage from 100.0 kHz to 100.0 mHz.

X-ray photoelectron spectroscopy (XPS) was performed using a VG Scientific XPSLAB 250 XPS spectrometer and monochromatic AlK as a charge compensation source. SEM images were obtained with a Cambridge Stereoscan 240 and TEM images were obtained by applying an acceleration voltage of 200 kV to an AJEOLJEM-2010 electronmicroscope (Jeol High-Tech Co., Japan).

< Experimental Example 1> APBA - MWCNT - Hyd Combination preparation

First, multi-wall carbon nanotubes (MWCNTs) were functionalized according to the previously reported method (Goldman and Lellouche, 2010; Piran et al., 2009).

Briefly, 100 mg of MWCNT was treated with a mixture of concentrated 12.0 M HNO ₃ and 36.0 MH ₂ SO ₄ at 90 ° C for 2 hours and washed with distilled water until no acid was detected.

Thereafter, the resultant was dried at 80 DEG C under a vacuum condition overnight to obtain a black powder. MWCNT (3.0 mg) was then dispersed in 1.0 mL PBS (pH 7.0) containing 10.0 mM EDC / NHS solution and incubated at room temperature for 6 hours to activate -COOH of MWCNTs.

The mixture was centrifuged and the precipitate washed three times.

Meanwhile, 1.0 mg / mL hydrazine sulfate solution optimized for PBS and 10.0 mM amino-phenylbromonic acid (APBA) were prepared and activated MWCNTs were mixed with hydrazine sulfate (Hyd) and APBA solution.

The mixture was stored at 4 < 0 > C overnight and then centrifuged.

After centrifugation, the sediments were washed five times with PBS to prevent APBA and Hyd from remaining.

The compound obtained in the above procedure was dispersed in 1.0 mL of PBS and stored at 4 ° C in a refrigerator until used.

< Experimental Example 2> Immune sensor Probe Produce

A sensor was fabricated as shown in Fig. A 1.0 mM TTBA monomer solution was electro-polymerized on the surface of GCE / AuNPs to form a polymer layer of TTBA monomer and immobilized AntiP-gp on the surface of the polymer layer.

< Experimental Example 3> Sample preparation of cancer cells

P-gp overexpressed uterine sarcoma MDR _cc (ATCCs Number: CRL-1977 ™ and ATCCs Number: CRL-2274 ™) were obtained from the American Type Culture Collection (ATCC, Manassas, USA). Control experiments and interference studies were performed with SKBr-3, HeLa, OSE and HEK-293 cell lines, which were obtained from Korean cell line bank.

The cells were cultured in a suitable medium containing 10% inactivated fetal bovine serum (FBS) and 100 units / mL penicillin / streptomycin at 37 ° C and 5% CO ₂ .

MDR _cc Were cultured in McCoy's 5A medium and maintained in media containing 4.0 μg / mL adriamycin. Before each experiment, cells were suspended in sterile PBS and reconstituted with disposable C-Chip (South Korea) under an optical microscope (Olympus).

< Experimental Example 4> Sensor The probe MDR cancer cell detection using

GCE / AuNPs / pTTBA / AntiP-gp sensor probes were incubated with the MDR _cc (CRL-1977 ™) for 30 minutes and then washed with the same buffer to remove unbound cells.

Next, the GCE / AuNPs / pTTBA / AntiP-gp / MDR _cc / APBA-MWCNT-Hyd probes were formed by incubating the GCE / AuNPs / pTTBA / AntiP-gp / MDR _cc electrodes with APBA-MWCNT- And washed with the same buffer. The final GCE / AuNPs / pTTBA / AntiP-gp / MDR _cc / APBA-MWCNT-Hyd probes were tested at a scan rate of 50.0 mV / s with potential cycles between +0.6 and -0.7 V.

In order to obtain the catalytic reaction, GCE / AuNPs / pTTBA / AntiP-gp / MDRCC / APBA-MWCNT-Hyd probes were placed in 0.1 M PBS containing 4.0 mM H ₂ O ₂ and a -0.45 V potential vs. Ag / A chronoamperometric experiment was performed.

< Example 1> Characteristics and shape of electrode surface

In order to characterize the electrode surface, AuNPs were electrodeposited on GCE using a potential step method and AuNPs electrodeposition was confirmed by linear sweep voltammetry (LSV).

As a result, the maximum instantaneous current increase with increase of the sweep number was observed while confirming the electrodeposition of AuNPs on the GCE. From the above results, it was confirmed that the AuNPs electrodeposition and the GCE / AuNPs surface conductivity were increased.

Thereafter, a 1.0 mM TTBA monomer solution was electro-polymerized to form a pTTBA film on the surface of GCE / AuNPs. Monomer oxidation peaks were observed at +1.2 V during an anodic scan from 0.0 to +1.4 V using a 1.0 mM monomer solution.

In addition, a positive reduction peak was observed at + 0.8 V through a reverse anodic scan at + 1.4 V and the reduction peak was due to the reduction of the oxidized polymer film formed on the GCE / AuNPs surface.

In order to identify the fabricated sensor probes, modified steps were characterized using SEM, AFM, XPS, QCM and EIS.

SEM images of the GCE / AuNPs layer confirmed that the AuNPs particle size was 20.0 ± 1.5 nm, and the SEM image of the GCE / AuNPs / pTTBA surface confirmed the pTTBA film over the AuNPs.

The TEM image also confirmed that the AuNPs size was 20.0 ± 1.5 nm.

After the electropolymerization, the morphology of the polymer layer was confirmed by AFM.

As a result, it was confirmed that (a) HOPG / AuNPs, (b) HOPG / pTTBA and (c) HOPG / AuNPs / pTTBA layers were formed on the surface of pyrolytic graphite (HOPG) .

It was confirmed that AuNPs / polymer film composed of the same constituent of small particles was present on the surface of the polymer AuNPs-coated HOPG electrode, and it was confirmed that the nanoparticles were successfully produced according to the above results.

The particle size of the AuNPs / pTTBA film was 70.5 ± 15.5 nm. The root mean-square roughness of AuNPs, pTTBA and AuNPs / pTTBA films was 3.56, 1.61 and 2.67 nm, respectively. Respectively.

Next, the characteristics of the electrode surface were confirmed using XPS.

After 50 seconds of Ar ion gas etching, all XPS spectra were obtained and the C1s peak at 284.6 eV was calibrated as the internal standard.

As a result, XPS spectra were obtained on the surfaces of (i) GCE / AuNPs / pTTBA and (ii) GCE / AuNPs / pTTBA / AntiP-gp as shown in FIG. 2B. Referring to FIG. 2B, two peaks were observed at 284.6 and 286.5 eV positions by pTTBA in the C1s spectrum of GCE / AuNPs / pTTBA (i), and these peaks were identified as C = C, CC, And C = O, CO bonds.

A new CN bond formed between the -COOH group of pTTBA and the -NH ₂ group of AntiP-gp was confirmed at 285.1 eV after immobilizing AntiP-gp antibody as in GCE / AuNPs / pTTBA / AntiP-gp (ii) The C = O bond migrated to positive energy and was identified at 286.7 eV.

Referring to FIG. 2B (b), in the polymer-coated film, the S2p peak was found at 163.7 eV, and the peak coincided with the C-S bond. This bond is due to the sulfur component of pTTBA, and no peak was observed at the 163.7 eV position on the GCE / AuNPs surface.

2B (c), the peak in the N1s spectrum was observed at 399.7 and 399.1 eV after AntiP-gp fixation, and the peak was found between the -NH ₂ group of AntiP-gp and the -COOH group of pTTBA The formation of CN bond by covalent bonding confirmed that AntiP-gp was successfully immobilized through the peak.

On the other hand, in GCE / AuNPs / pTTBA surface (i), peaks were not observed at 399.7 and 399.1 eV.

From the above results, it was confirmed that AntiP-gp successfully binds to the nanocomposite.

In addition, immobilization was confirmed using a quartz crystal microbalance (QCM), and the amount of AntiP-gp immobilized on the polymer film was confirmed by changing the frequency.

As a result, in AntiP-gp immobilization as shown in Fig. 2 (C), the frequency reduction with the total frequency change (f) of 287 Hz reached a complete steady state after 95 minutes. The result corresponds to a mass change (? M) of 315.8 ± 12.2 ng.

From the above results, it was confirmed that AntiP-gp was successfully immobilized on the pTTBA layer.

Impedance spectrometry was performed to identify the characteristics of the probe layer at each modification step.

GCE / AuNPs, GCE / pTTBA and GCE / AuNPs / pTTBA as in FIG. 2D also in 0.1 M PBS (pH 7.4) containing 4.0 mM Fe [(CN) ₆ ] 3 ^- / 4 ^- /0.3 M NaClO ₄ The Nyquist plots of the electrodes were obtained and the results of R _S , R _P 1, R _P 2, CPE1 and CPE2 were obtained by fixing the experimental results to the equivalent circuit using Zview2 impedance software .

In the equivalent circuit, R _S represents the solution resistance, R _P 1 and R _P 2 represent the polarization resistance, and W represents the Warburg element. CPE1 and CPE2 also represent constant phase elements.

The R _P value was determined from the intersection point between the Zre axis and the extrapolation of the curve obtained from the Nyquist plot of the impedance spectroscopy.

As a result, GCE, GCE / AuNPs, GCE / pTTBA and polarization resistance (R _P) values of GCE / AuNPs / pTTBA electrodes each were identified as 636.2, 230.9, 10006.7 and 1113.1 Ω, wherein R _P value was decreased by about 9x The conductivity of polymer electrode with AuNP was increased by electrodeposition of AuNP.

< Example 2> Multidrug resistance ( Multidrug resistance; MDR) Cancer cell detection confirmation

GCE / AuNPs / pTTBA / AntiP-gp sensor was incubated with 5000 MDR _CC / mL (CRL-1977 ™) for 30 minutes. The GCE / AuNPs / pTTBA / AntiP-gp / MDR _CC probes were then incubated with the APBA-MWCNT-Hyd conjugate for 30 minutes and washed three times with the same buffer solution. Thereafter, cyclic voltammetry (CV) was performed at a scanning speed of 50.0 mV / s and a potential cycle of +0.6 and -0.7 V in 0.1 M PBS in which oxygen was removed.

As a result, the maximum redox peak was observed at -50 / -90 mV as shown in the red curve of FIG. 3A, and the result was attributed to the electrochemical characteristics of Hyd and APCA-MWCNT-Hyd and GCE / AuNPs / pTTBA / AntiP-gp / MDR Indicates that the _CC electrode has successfully interacted.

The maximum redox reaction observed at -50 / -90 mV is directly proportional to the scan rate between 10.0 and 60.0 mV / s, suggesting that this is due to the electrode reaction by the hyd on the electrode surface.

In order to confirm the above, experiments similar to those performed previously were performed except Hyd and APBA.

As a result, neither the redox curve nor the redox curve was observed in both cases, as shown in the wave and black curves of FIG. 3A, and these results were confirmed to be due to the absence of recognition molecules on the sensor surface such as Hyd, electrochemically active group and APBA.

From the above results, it was confirmed that the electrochemical signal for detecting MDR _CC was obtained by APBA and Hyd.

In addition, the ability of GCE / AuNPs / pTTBA / AntiP-gp / MDR _CC / APBA-MWCNT-Hyd probe to promote H ₂ O ₂ reduction reaction was confirmed.

The sensor probe was immersed in oxygen-depleted 0.1 M PBS containing 4.0 mM H ₂ O ₂ and subjected to cyclic voltammetry (CV).

As a result, a very distinct reduction peak was confirmed at -400 mV as shown in Fig. 3B. On the other hand, APBA and Hyd were removed and cyclic voltammetry (CV) was performed in the same manner as above. As a result, no reduction peak appeared at -400 mV.

From the above results, it was confirmed that the reduction peak was due to H ₂ O ₂ reduction by Hyd attached to the sensor probe.

Next, in order to confirm the curve caused by the immune response, MDR _CC The H ₂ O ₂ catalyst current was observed.

As a result, as shown in FIG. 3B, MDR _CC CVs were recorded as the number increased, and MDR _CC As the number increased from 1000 to 3500 cells / mL, the current response increased.

In the figure, a represents a blank, and b (1000), c (1500), d (2000), e (2500), f (3000) and g (3500) represent MDR _CC / mL.

A linear graph was obtained with a linear regression equation according to the following equation, and the correlation coefficient was 0.997.

? I (μA) = 2.43 + 0.002 [MDR _CC ]

From the above results, the biosensor of the present invention can be applied to MDR _CC Was found to be effective for accurate detection.

Next, the experimental parameters of the sensor probe were optimized by time - of - day chronoamperometry to confirm the detection limit of MDR _CC .

< Example 3> Check the performance of the sensor

The performance of GCE / AuNPs / pTTBA / AntiP-gp sensor probes according to H ₂ O ₂ reaction was confirmed by increasing the number of MDR _CC (CRL-1977 ™ cell) by APBA-MWCNT-Hyd conjugate bound to MDR _CC .

Time-of-day currents were applied to Ag / AgCl and GCE / AuNPs / pTTBA / AntiP-gp / MDR _CC / APBA-MWCNT-Hyd probes at -0.45 V in oxygen-depleted PBS containing 4.0 mM H ₂ O ₂ .

As a result, as shown in FIG. 4A, H ₂ O ₂ The reduction of catalyst signal was observed, and the MDR _CC And it was confirmed that it is directly proportional to the amount. Also, it was confirmed that the current reaction gradually increased with the increase of the number of MDR _CCs .

In the figure, a represents a blank, and b (50), c (100), d (500), e (1000), f ) And j (100,000) represent the respective MDR _CC / mL.

Also, as shown in FIG. 4B, a calibration curve based on the chronoamperograms obtained by the above experiment was confirmed, and the calibration curve shows linear response with MDR _{CC in the} range of 50 to 100,000 cells / mL.

A linear graph was obtained with a linear regression equation such as the following equation, and the correlation coefficient was 0.998.

? I = 0.9351 (占 0.1181) + 0.0025 (占 0.0000067) [MDR _CC ]

Through the standard deviation of the blank measured five times, MDR _CC (RSD <4.4%). The detection limit was 23 ± 2 cells / mL.

The detection limits are based on recently reported MDR _CC impedance biosensors (Zhang et al., 2014) using gold nanoparticles / poly-aniline nanofibers, chemiluminescence for cancer cell detection (Chen et al., 2014) QCM (Shaolian et al., 2014) biosensor.

In addition, another P-gp overexpressed cell host, CRL-2274 ™, was purchased from ATCC (USA) and the same experiment was performed.

As a result, the current response was increased with CRL-2274 ™ cell increase, but it was 17% lower than that of CRL-1977 ™ cell. These results indicate that the concentration of P-gp expressed on the cell membrane of CRL-2274 It is confirmed that it is low.

< Example 4> Sensor selectivity, reproducibility, stability and actual sample analysis

In order to confirm the selectivity of the nano-biosensor according to the present invention, SKBr-3, HeLa, OSE and HEK-293 cells were used at cell concentrations of 1000, 30,000, 75,000 and 100,000 cells / Selectivity.

As a result, the current response of the cells was similar at a concentration of 30,000 cells / mL as shown in FIG. 5A.

This result is due to the absence of an immune response between AntiP-gp and P-gp on the cell surface, and it was confirmed that P-gp was not expressed or only a very small amount was expressed on the cell surface of the cells.

In addition, the selectivity of the nano-biosensor was confirmed using compounds such as albumin, fibrinogen, and glucose that are present in the actual sample matrix.

As a result, it was interesting that no current reaction appeared from the compound.

Nano biosensor of the present invention from the results of MDR _CC In the detection, it was confirmed that there is very high selectivity and no interference or positive error signal appears at all.

Next, RSD <3.5% (n = 5) was obtained as a result of the reproducibility analysis. RSD of the electrode was less than 3.2% when the same conditions were applied.

This difference is expected to be due to surface modification and / or very small changes under optimum conditions.

Finally, the stability of the sensor probe over time was confirmed. As a result, the selectivity of the immune sensor was maintained at 96% for 6 weeks, and the reactivity was decreased by about 13% after 6 weeks. .

From the above results, it was confirmed that the stability of the sensor lasted for 6 weeks. The stability of the sensor is due to strong covalent bonding of the antibody on the stable conductive polymer-nanoparticle complex.

MDR _CC detection was confirmed in human serum samples in a similar dynamic range (50-100,000 MDR _CC / mL) to confirm the biomedical application of nanobiosensors.

MDR _CC was dispersed in five-fold diluted serum samples and incubated with GCE / AuNPs / pTTBA / AntiP-gp sensor probes. After washing, the sensor probe was treated with APBA-MWCNT-Hyd conjugate and reacted with H ₂ O ₂ .

As a result, the current response increased as the number of MDR _CCs increased.

From the above results, it was confirmed that the biosensor of the present invention is effective for the detection of MDR _CC in serum, and it is confirmed that it can be used for biomedical analysis.

Calibration plots for serum samples were obtained with a linear regression equation as follows: The correlation coefficient was 0.998.

? I = 0.881 + 0.0023 [MDR _CC ]

From the above results it was confirmed that the sensor of the present invention can be effectively used to detect MDR _CC in a complex biological sample matrix.

The detection limit of MDR _CC in serum samples was 28 ± 2 cells / mL (RSD <4.8%) (95% confidence level, n = 5). The detection limit for the blood sample was 5 cells / mL higher than the detection limit for buffer solution (23 cells / mL), but this difference in detection limit is negligible due to the matrix effect of the blood constituents.

In addition, it was confirmed whether MDR _CC could be detected in the mixed cell sample of the biosensor.

For this, MDR _CC was mixed with SKBr-3, HeLa, OSE and HEK-293 cells and detected with a biosensor probe.

As a result, the detection sensitivity was 97% (n = 5), which was interestingly higher than when MDR _CC alone was detected.

Biosensors of the invention From the above results, it was confirmed that the MDR _CC is to be used very effectively in MDR _CC detected when present in combination with other cancer cells and normal cells.

< Example 5> Comparison with existing reporter antibody-based immunoassay methods

To verify the MDR _CC captured by AntiP-gp probe, ITO / pTTBA / AntiP-gp and MDR _CC sensor to the bait incubated, washed and then trapped in the surface of ITO / pTTBA / AntiP-gp MDR CC Was observed under an optical microscope, and a microscopic image of the ITO / pTTBA / AntiP-gp sensor with different concentrations of MDR _CC treated as shown in Fig. 5B was confirmed.

As a result, as the MDR _CC concentration was increased from 10 ² to 10 ⁴ cells / mL as shown in FIG. 5B (ac), the number of cells captured by the sensor was increased. From the results, it was found that the ITO / pTTBA / AntiP- MDR _CC detection was possible.

On the other hand, as shown in FIG. 5B (d), no MDR _CC was detected in the ITO / pTTBA electrode in which AntiP-gp was not immobilized. From the results, it was confirmed that AntiP-gp is essential for MDR _CC detection.

In addition, the present invention was compared with conventional immunoassays based on reporter antibodies.

First, a reporter AntiP-gp-MWCNT-Hyd conjugate was prepared by a carbodiimide coupling reaction according to an existing report of the present inventors (Zhu et al., 2010).

MWCNT-Hyd and Reporter AntiP-gp-MWCNT-Hyd complexes at 25,000, 50,000 and 100,000 MDR _CC / mL cell concentrations, as in the previous experiment.

As a result, a very high signal was obtained in the APBA-MWCNT-Hyd complex as shown in FIG.

In addition, MDR _CC detection limit of reporter AntiP-gp-MWCNT-Hyd conjugate was confirmed to be 158 ± 8 cells / mL (95% confidence level, n = 5).

The results were 7 times higher than the detection limit using APBA-MWCNT-Hyd conjugate (23 ± 2 cells / mL).

This result is due to the weak immune response between the reporter AntiP-gp-MWCNT-Hyd complex and the MDR _CC when the sensor probe is captured, and this weak immune response is due to the low concentration of P-gp on the MDR _CC membrane.

It has been previously reported that changes in the concentration of cell surface antigens (eg, P-gp) have a significant effect on antibody binding (Langmuir et al., 1991; Velders et al., 1998) As shown in FIG.

However, APBA-MWCNT-Hyd conjugates were not affected by the sensitivity of biosensors even at low P-gp concentrations because APBA-MWCNT-Hyd conjugates use glycans, which are highly expressed on the cancer cell surface, as target molecules .

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Claims

A cancer cell capturing unit configured to capture cancer cells; an electrode; a nanoparticle layer formed on the electrode; an electrically conductive polymer layer formed on the nanoparticle layer; and a permeable glycoprotein antibody layer formed on the electrically conductive polymer layer; And
And a complex comprising a multi-walled carbon nanotube, a hydrazine and a boronic acid derivative, wherein the complex comprises a cancer cell detecting part which binds to the captured cancer cell and performs a catalytic reaction with hydrogen peroxide. sensor.

The multi-drug resistant cancer cell detection sensor according to claim 1, wherein the nanoparticle layer is a nanoparticle layer composed of gold nanoparticles or silver nanoparticles.

[4] The method of claim 1, wherein the electrically conductive polymer layer comprises at least one selected from the group consisting of 2,2 ': 5', 5 "-tetiophene-3'-p-benzoic acid (TTBA) (TTCA) and 2,5-di- (2-thienyl) -1H-pyrrole-P-benzoic acid (DTPBA) are electrolytically mixed with each other to produce a multidrug-resistant cancer cell.

The boric acid derivative according to claim 1, wherein the boronic acid derivative is selected from the group consisting of amino-phenylboronic acid (APBA), phenylboronic acid (PBA), thienylboronic acid (TBA) and methylboronic acid Sensor for detecting cancer cells.

Forming an electrically conductive polymer layer on the surface of the electrodeposited electrode by electrospinning the electroconductive monomer (first step);
Fixing a permeable glycoprotein antibody to the electrically conductive polymer layer (second step);
Treating the sample with the electrode on which the antibody is immobilized (step 3);
Preparing a complex comprising a multi-walled carbon nanotube, a hydrazine and a boronic acid derivative (Step 4);
Treating the bonded body of the fourth step on the electrode to which the sample is treated (fifth step); And
Immersing the treated electrode in a buffer solution containing hydrogen peroxide to carry out a catalytic reaction (step 6).

Forming an electrically conductive polymer layer on the surface of the electrodeposited electrode by electrospinning the electroconductive monomer (first step);
Fixing a permeable glycoprotein antibody to the electrically conductive polymer layer (second step);
Treating the sample with the electrode on which the antibody is immobilized (step 3);
Preparing a complex comprising a multi-walled carbon nanotube, a hydrazine and a boronic acid derivative (Step 4);
Treating the bonded body of the fourth step on the electrode to which the sample is treated (fifth step);
(6) carrying out the catalytic reaction by immersing the electrode treated with the coupling agent in a buffer solution containing hydrogen peroxide and
And analyzing the reduction current by the catalytic reaction (Step 7).

[Claim 7] The method according to claim 6, wherein the sample is selected from the group consisting of blood, plasma, serum, and urine.

[Claim 7] The method according to claim 6, wherein the concentration of hydrogen peroxide in the buffer solution is 2.0 to 6.0 mM.

7. The method of claim 6, wherein the reduction current is analyzed by cyclic voltammetry (CV) or chronoamperometry.