KR102381031B1 - Electrochemical Biosensing Platform for Detection of Rheumatoid Arthritis Biomarker - Google Patents

Abstract

According to the present disclosure, in performing early diagnosis of rheumatoid arthritis through the detection of anti-cyclic citrullinated peptide (aCCP) antibody, which is a biomarker of rheumatoid arthritis, an autoimmune disease, disclosed is a biosensing platform for self-diagnosis capable of simply detecting or diagnosing the aCCP antibody in a sample without the help of an expert.

Description

Electrochemical Biosensing Platform for Detection of Rheumatoid Arthritis Biomarker

The present disclosure relates to an electrochemical biosensing platform for biomarker detection of rheumatoid arthritis. More specifically, the present disclosure provides an early diagnosis of rheumatoid arthritis through detection of aCCP (anti-cyclic citrullinated peptide) antibody, a biomarker of rheumatoid arthritis, an autoimmune disease. It relates to a biosensing platform for self-diagnosis that can easily detect or diagnose.

Rheumatoid Arthritis is a chronic autoimmune disease that occurs due to inflammation of the tissues surrounding the joints and is estimated to account for about 1% of the total population in Korea. It causes severe chronic pain, irreversible joint damage, swelling and stiffness. Smoking, postpartum or menopause, and other physical/mental stress are known causes of rheumatoid arthritis. If the onset of rheumatism is not detected early and left unattended, the joint is progressively destroyed and joint deformation occurs within 90% of onset patients within one year, eventually resulting in a disability that makes daily life difficult, and sometimes autoimmune abnormalities Complications may result in death of the patient.

As such, although early diagnosis of rheumatoid arthritis is necessary, the diagnosis is primarily dependent on clinical symptoms, and in many cases, diagnosis is made only after the joint damage has already progressed considerably. In addition, since the subjective symptoms are insignificant due to lethargy, muscle pain, etc., in the case of the general public, it is difficult to distinguish from other arthritis and tends to leave it unattended, so it is difficult to diagnose and cope with it early. If rheumatoid arthritis disease is not treated early, it is virtually impossible to cure after joint deformation is induced, and only simple inflammation treatment is possible, and the quality of life is reduced due to pain, fatigue, and depression. In particular, the incidence rate is highest among those aged 25 to 55, causing enormous social and economic loss.

In this regard, although the exact cause of the production of autoimmune antibodies has not been revealed, it is determined that the autoimmune mechanism induced by genetic and environmental factors attacks one's own cells. Currently, rheumatic diseases are generally diagnosed using complete blood count (CBC), increase in ESR, and C-reactive protein (CRP). Recently, rheumatoid factor (RF), Diagnosis is being made by measuring changes in inflammation levels through an increase in platelet count, an immune test (ANA: antinuclear antibody test), and joint ultrasound. Among the above-mentioned diagnostic methods, a method of diagnosing using IgM rheumatoid-like factor (RF) as a biomarker is known as a promising method. do. However, according to international diagnostic standards, RF is negative throughout the course of the disease in 20% of rheumatoid arthritis patients, indicating low sensitivity.

On the other hand, it is known that the autoantibody, anti-citrullinated protein (aCCP) antibody is produced by promoting the citrullination process in the joint of a rheumatoid arthritis patient and increasing the antigenicity of the protein. It has been reported that about 70% of patients with serum aCCP antibody positive have a chance of being diagnosed with rheumatoid arthritis within 5 years. As such, the effectiveness of treatment for rheumatoid arthritis can be evaluated by measuring the level of aCCP antibody together with RF. For example, a normal aCCP level can be considered to be <20 IU/mL, and the cut-off value of an aCCP antibody to rheumatoid arthritis is 20 UI/mL. In this regard, citrullination refers to a process in which arginine among amino acid residues is deiminated by peptidyl arginine deiminase and converted into citrulline in the post-translational modification of a protein. It is known that the citrulline process in the joints of rheumatoid arthritis patients is accelerated and the antigenicity of the protein is increased to induce the production of an autoantibody, aCCP antibody, and the aCCP antibody can be used as a biomarker for diagnosing rheumatoid arthritis.

In this regard, a method for diagnosing a citrulline-related disease such as rheumatoid arthritis using a monoclonal antibody that specifically binds to a citrullated protein has been developed (Korean Patent No. 1237002, etc.).

In the above technique, antigen-antibody binding is quantitatively diagnosed, that is, by immunoassay, Western blot, ELISA, surface-enhanced Raman scattering, radioimmunoassay, radioimmunodiffusion, immunofluorescence, and immunoblot. use etc. However, in the case of ELISA among the above methods, the incubation time is long, a large amount of samples are used, and the number of intermediate sample preparation steps is large. In addition, the detection method based on surface-increased Raman scattering is expensive and time-consuming. Therefore, there is a need for an electrochemical biosensing platform that has a small sample volume, high sensitivity, inexpensive and does not require special technology when detecting aCCP antibody.

In one embodiment according to the present disclosure, to solve the problems of the prior art and provide a high-sensitivity biosensing platform capable of quantitatively/qualitatively detecting or diagnosing aCCP antibody, a biomarker of rheumatoid arthritis, without the help of a simple and expert. do.

Another embodiment according to the present disclosure provides a method for detecting aCCP antibody in a sample using a novel platform for detection having high sensitivity for aCCP antibody.

According to the first aspect of the present disclosure,

a base matrix comprising an electrode modified with a transition metal dichalcogenide and a conductive polymer;

CCP fixed to the base matrix and having binding ability to aCCP antibody; and

At least one selected from the group consisting of (i) a conductive polymer and (ii) gold (Au), silver (Ag), palladium (Pd), copper (Cu) and platinum (Pt) having a capture ability for aCCP antibody Nanomatrix containing metal nanoparticles of;

including,

When the aCCP antibody is contained in the sample, the aCCP antibody bound to the nanomatrix is quantitatively and/or qualitatively detecting the aCCP antibody in the sample based on the electrochemical response characteristics of binding to the CCP immobilized on the base matrix. A chemical sensing platform is provided.

According to a second aspect of the present disclosure,

a) providing a base matrix to which CCP having binding ability to aCCP antibody is immobilized, wherein the base matrix includes an electrode modified with a transition metal dichalcogenide and a conductive polymer;

b) having a capture ability for aCCP antibody, (i) a conductive polymer and (ii) gold (Au), silver (Ag), palladium (Pd), copper (Cu) and platinum (Pt) selected from the group consisting of providing a liquid sample to which a nanomatrix including at least one metal nanoparticle is added; and

c) measuring the electrochemical response characteristics by bringing the CCP-fixed base matrix into contact with the liquid sample to which the nano-matrix is added;

A method for quantitatively and/or qualitatively detecting aCCP antibody in a liquid sample is provided, comprising:

Compared to the conventional method, the biosensing platform provided according to an embodiment of the present disclosure qualitatively detects aCCP antibody in a sample (liquid sample) with high sensitivity in a short time without requiring specialized skills by electrochemical sensing. and/or quantitatively detects or diagnoses. In particular, reliable detection sensitivity can be achieved even with respect to aCCP antibody present at a low concentration in a trace amount of a sample, and it has a lower detection limit compared to the conventional method, and can be implemented at low detection or diagnostic cost, making it suitable for commercialization Do. Therefore, it is expected to be widely used in the future.

1 is a diagram showing a schematic sequence of detecting aCCP in a sample using (a) PANI-Au-aCCP/BSA/CCP/PANI/MoS ₂ /SPE in an exemplary embodiment, (b) CCP and Figures exemplarily showing the interaction between aCCP-bound PANI-Au nanomatrices (X is citrulline), and (c) Fe(CN) ₆ ^3- / ⁴ as redox probe or mediator ^- It is a diagram showing the aCCP immunosensing mechanism to detect aCCP using;
2 is a diagram showing the process of immobilizing CCP through EDC-NHS surface chemistry on PANI/MoS ₂ /SPE;
3 is a SEM photograph of (a) bare SPE, (b) MoS ₂ /SPE, (c) PANI/MoS ₂ /SPE, and (d) PANI-Au nanomatrix, (e) TEM photograph of MoS ₂ , (f) lattice fringe photograph of MoS ₂ (inset shows SAED pattern of MoS ₂ ), (g) TEM photograph of PANI-Au, (h) HR-TEM photograph of Au in PANI nanowire, (i) A graph showing the CV response of the PANI electrodeposition layer when CV 10 cycles were performed at a scan rate of 50 mV/s in the presence of 0.1 M aniline dissolved in 1 M HCl, and (j) MoS ₂ , PANI, PANI/ XRD patterns of MoS ₂ and PANI-Au, respectively;
4 is a cross-sectional SEM photograph of (a) SPE and (b) PANI/MoS ₂ /SPE;
5 shows (a) EDX spectrum of PANI/MoS ₂ /SPE, and elemental mapping of (b) carbon, (c) nitrogen, (d) chlorine, (e) molybdenum and (f) sulfur in PANI/MoS ₂ /SPE. ego;
6 is (a) EDX spectrum of PANI-Au nanomatrix, and elemental mapping of (b) carbon, (c) nitrogen, (d) chlorine, and (e) gold in PANI-Au nanomatrix;
7 shows (a) the full spectrum spectrum of PANI/MoS ₂ and PANI-Au, (b) Mo 3d, (c) S 2p and (d) N 1s, and PANI- in the deconvolved spectrum of PANI/MoS ₂ (e) Au 4f and (f) N 1s in the deconvolved spectrum of Au;
8 is (a) the deconvolved C 1s spectrum of PANI/MoS ₂ , and (b) the deconvolved C 1s spectrum of PANI-Au;
9 shows (a) different modified layers of SPE (bare SPE, MoS ₂ /SPE, PANI/SPE, PANI/MoS ₂ ) in the presence of 5 mM Fe(CN) ₆ ^3- / ^4- at a scanning rate of 50 mV/s. CV responses of /SPE, CCP/PANI/MoS ₂ /SPE, BSA/CCP/PANI/MoS ₂ /SPE, and PANI-Au-aCCP/BSA/CCP/PANI/MoS ₂ /SPE), (b) different modifications Bar chart of I _pa in aCCP immunosensor with layers and corresponding CV response, (c) frequencies from 1 Hz to 10 MHz in 0.1 M KCl containing 5 mM Fe(CN) ₆ ^3- / ^4- Nyquist plots (bare SPE, MoS ₂ /SPE, PANI/SPE, PANI/MoS ₂ /SPE, CCP/PANI/MoS ₂ /SPE, BSA/CCP/PANI/ MoS ₂ /SPE, and PANI-Au-aCCP/BSA/CCP/PANI/MoS ₂ /SPE), and (d) bar charts of individual layers of aCCP immunosensors and their corresponding R _ct values;
Figure 10 shows the effect of (a) CCP concentration, (b) pH, (c) PANI loading volume and (d) incubation time on the catalytic current response of aCCP immunosensor in the presence of 50 IU/mL of aCCP antibody. It is a graph showing;
11 shows (a) the detection of different concentrations of aCCP (0.25 IU/mL, 5.0 IU/mL. 25.0 IU/mL, 50.0 IU/mL, 250.0 IU/mL, 500.0 IU/mL, and 150.0 IU/mL). For this purpose, SWV response of BSA/CCP/PANI/MoS ₂ /SPE immunosensor using PANI-Au nanomatrix, (b) linear relationship between log concentration versus current response of aCCP obtained from SWV, (c) human serum albumin (HSA) ; 10 mg/mL), immunoglobulin M (IgM), RF (500 ng/mL) and CRP (1,000 ng/mL), and BSA/CCP/BSA/CCP/ using Au-PANI as nanoprobes, such as CRP (1,000 ng/mL). SWV response of PANI/MoS ₂ /SPE immunosensor to 50.0 IU/mL aCCP, and (d) bar chart showing changes in current response in the presence and absence of interfering substances;
12 is (a) a graph showing the results of 4 weeks storage (4° C.) stability measurement of aCCP immunosensor in the presence of 50.0 IU/mL aCCP, (b) 50.0 IU/mL aCCP and 5 mM Fe (CN) as a redox probe ) a graph showing the SWV response of 5 different PANI-Au-aCCP/BSA/CCP/PANI/MoS ₂ /SPE immunosensors in the presence of ₆ ^3- / ⁴ , and (c) SWV current response of 5 different electrodes is a bar chart (error bars mean standard deviation of three independent measurements); And
13 shows (a) human serum spiked with aCCP (0.25 IU/mL, 5.0 IU/mL. 25.0 IU/mL, 50.0 IU/mL, 250.0 IU/mL, 500.0 IU/mL, and 150.0 IU/mL). SWV response of the BSA/CCP/PANI/MoS ₂ /SPE immunosensor using PANI-Au as a nanoprobe for the detection of , and (b) a graph showing the linear relationship between log C _HS-aCCP versus current response change.

The present invention can all be achieved by the following description. It is to be understood that the following description describes preferred embodiments of the present invention, and the present invention is not necessarily limited thereto. In addition, the accompanying drawings are provided to aid understanding, and the present invention is not limited thereto, and details regarding individual configurations may be properly understood by the specific purpose of the related description to be described later.

Terms used herein may be defined as follows.

"Immune sensor" may refer to a sensor using an antibody that specifically binds to a target molecule in order to detect and quantify a target molecule (antigen), wherein the antibody can form a complex with the corresponding antigen. there is.

"Transition metal dichalcogenide" may refer to the general formula MX ₂ (M is any transition metal and X is a chalcogen element), wherein a single molecular layer of MX ₂ is an atomic trilayer As it is formed by, it is composed of a layer of two adjacent chalcogen atoms (X) and a transition metal atom (M) covalently bonded thereto to form an XMX layer structure.

"Chalcogen" may refer to a bar that may contain six elements, oxygen (O), sulfur (S), selenium (Se), tellurium (Te), polonium (Po), and libermorium (Lv). there is.

"Molybdenum disulfide (MoS ₂ )" belongs to a transition metal dichalcogenide with a two-dimensional structure, and MoS ₂ in bulk is composed of a single layer of S-Mo-S in the form of a sandwich bonded by weak van der Waals interaction. . In particular, there are two typical MoS ₂ phases according to the arrangement of 6 sulfur (S) atoms bonded to Mo atoms, 2H phase having sulfur atoms coordinated in the form of a prism of a 3-sided structure, and an 8-sided structure It may include a coordinated 1T phase.

A “nanosheet” may resemble a two-dimensional material, which may have a single or several single layer thicknesses, and may have transverse dimensions of hundreds of nanometers to tens of micrometers.

"Conductive polymer" may mean a polymer exhibiting conductivity. The conductive polymer may be, for example, a single monomer or a polymer of two or more monomers comprising, for example, aniline, aminophenol, diaminophenol, pyrrole, thiophene, paraphenylene, fluorene, furan, acetylene, or derivatives thereof. can

"Interfacial polymerization" may refer to a polymerization reaction that occurs at or near the interface of two immiscible solutions.

“Binding” may mean binding or linking to a surface in a covalent or non-covalent manner.

"Immobilization" may mean that any substance or bioactive agent is bound, either covalently or non-covalently, to a substrate in a direct or indirect manner.

"Polyaniline" is a polymeric cation having selective anion permeability, and is protonated in an oxidized state.

"Sample" or "liquid sample" is not limited to a specific type or form as long as it can contain the target pathogen to be detected. Illustratively, the sample may be a biological sample, for example, a biological fluid or biological tissue. Examples of biological fluids include urine, blood, plasma, serum, saliva, semen, feces, sputum, cerebrospinal fluid, tears, mucus, amniotic fluid, and the like. A biological tissue is a collection of cells, usually a specific kind of collection of intracellular substances that form one of the structural substances of human, animal, plant, bacterial, fungal or viral structures, such as connective tissue, epithelial tissue, muscle tissue, and nerve. This may be an organization or the like. Examples of biological tissue may also include organs, tumors, lymph nodes, arteries, and individual cell(s). In addition, the sample may include an environmental sample containing the biomaterial in a low concentration, for example, may include various types and types such as drinking water and food.

"Antibody" may refer to a protein molecule serving as a receptor that specifically recognizes an antigen, including immunoglobulin molecules having immunological reactivity with a specific antigen, monoclonal antibody, polyclonal antibody, full-length antibody -length antibody), antibody fragments, and recombinant antibodies can be understood as concepts including all of them.

"Diagnosing" may mean confirming the presence or characteristics of a pathological condition, and in the present disclosure, it may refer to a process of confirming whether rheumatoid arthritis develops or whether a disease has progressed or deepened.

A “biomarker” may refer to a substance that indicates that rheumatoid arthritis can be distinguished from a non-rheumatoid arthritis condition.

A “working electrode” may mean an electrode in an electrochemical system in which a specific (interesting) reaction occurs, and a “reference electrode” may mean an electrode having a stable and known electrode potential.

It may be understood that the expressions “on” and “on” are used to refer to the concept of relative position. Accordingly, not only the case where other components or layers are directly present in the mentioned layers, but also at least one other layer (intermediate layer or intervening layer) may be present between them, or additional components may be interposed or present. Similarly, the expressions “under”, “under” and “below” and “between” may also be understood as relative concepts with respect to location. Also, the expression “sequentially” may be understood as a concept of relative position.

In the case of the term "contacting", although narrowly it means direct contact between two objects, it can be understood that any additional element may be interposed in a broad sense.

In the present specification, when it is described that any component or member is “connected” or “communicated” with another component or member, unless otherwise stated, it is directly connected or communicated with the other component or member. It can be understood that not only the case where there is, but also the case where it is connected or communicated under the intervening of other components or members.

According to one embodiment of the present disclosure, a biosensing platform capable of performing early diagnosis of rheumatoid arthritis by detecting aCCP antibody, which is a biomarker of rheumatoid arthritis, in a sample is provided.

To this end, in the base matrix, the electrode surface is modified with a transition metal chalcogenide and a conductive polymer, and CCP capable of binding (selective binding) to the aCCP antibody, a target biomaterial, is fixed thereon. Separately, a nanomatrix of conductive polymer-metal nanoparticles is used as a nanomatrix probe for capturing the aCCP antibody present in the sample. As such, when the aCCP antibody is present in the sample and the sample is in contact with the CCP-immobilized base matrix, the aCCP antibody bound to the nanomatrix (in particular, the metal nanoparticles in the nanomatrix) in the sample is transferred onto the base matrix. It binds to the fixed CCP. As a result, an immunocomplex is formed between the CCP-aCCP antibodies, which changes the electrochemical signal generated from the electrode of the base matrix. By monitoring or observing the change in the electrochemical signal, it is possible to qualitatively and/or quantitatively detect or diagnose the aCCP antibody in the sample.

Biosensing Platform

- base matrix

According to one embodiment, the base matrix includes an electrode modified with a transition metal dichalcogenide and a conductive polymer. Illustratively, a transition metal dichalcogenide layer is formed on the electrode, and a conductive polymer layer may be sequentially formed thereon. In this regard, the electrode may use a type known in the art, specifically, it may be advantageous to use a screen-printed electrode (SPE). SPE is preferable in terms of miniaturization of the electrochemical sensor, and it is possible to reduce the amount of sample required, thereby reducing the overall size of the diagnostic system. In addition, the surface of SPE is easy to modify so that various samples can be analyzed, and common problems of conventional solid-state electrodes such as memory effect and cumbersome cleaning process can be avoided. Furthermore, when SPE is applied to an immune sensor, SPE is an electrochemical transducer with high mechanical integrity, and has the advantage of integrating a working electrode and a reference electrode in the same chip.

According to an exemplary embodiment, as a screen-printed electrode (SPE), at least one selected from carbon (C), gold (Au), platinum (Pt), silver (Ag), copper (Cu), nickel (Ni), and the like. It may be a single material. SPE is generally formed by layer-by-layer deposition of ink on a solid substrate using a screen or mesh, and can function as a working electrode. In certain embodiments, the SPE may be composed of a carbonaceous material, for example alone from graphite, activated carbon, carbon black, acetylene black, furnace black, carbon fibers, carbon nanotubes (CNT), and the like. Or they can be used in combination. In addition, the SPE may have a disk shape, a ring shape, or a comb shape of a three-electrode and two-electrode combination.

According to an exemplary embodiment, the transition metal dichalcogenide in the base matrix is a component that enhances the electrocatalytic activity of the sensor by high conductivity and intrinsic electrochemical properties, wherein the transition metal is molybdenum (Mo), tungsten (W) , titanium (Ti), tantalum (Ta), and may be at least one selected from the group consisting of zirconium (Zr). In addition, the chalcogen element in the transition metal dichalcogenide may be sulfur (S). As an example, the transition metal dichalcogenite may be selected from molybdenum disulfide (MoS ₂ ), tungsten disulfide (WS ₂ ), titanium disulfide (TiS ₂ ), tantalum disulfide (TaS ₂ ), zirconium disulfide (ZrS ₂ ), etc. , can be used alone or in combination. According to a specific embodiment, the transition metal dichalcogenide may be molybdenum disulfide (MoS ₂ ). Such a material may be in the form of a two-dimensional nanosheet having a layered structure, and has particularly high solubility with a conductive polymer. It is advantageous because the combination can provide excellent conductivity and functionality.

According to an exemplary embodiment, a coating method known in the art may be used to form a transition metal dichalcogenide layer on the electrode, for example, ACS Appl. Bio Mater. 2018, 1, 4, 1184, etc. These documents are incorporated herein by reference. Specifically, a transition metal dichalcogenide-containing ink is prepared by adding a transition metal dichalcogenide to a solvent (dispersion medium). And, it can be coated by dropping it on the electrode surface. In this case, the solvent may be an alcohol-based solvent, an ether-based solvent, an amide-based solvent, a ketone-based solvent, an aromatic solvent, an ester-based solvent, etc. For example, as an alcohol-based solvent, methanol, ethanol, 1-propanol, 2-propanol , 2-butanol, t-butanol, ethylene glycol, propylene glycol, 1,6-hexanediol, cyclohexanediol, sorbitol, xylitol, 2-methyl-2,4-pentanediol, 1,3-butanediol, 1,4 -It can be used individually or in combination from butanediol etc. Further, examples of the amide solvent include N,N-dimethylformamide, 1-methyl-2-pyrrolidone, 2-pyrrolidone, 1,3-dimethyl-2-imidazolidinone, ε-caprolactam. , formamide, N-methylformamide, acetamide, N-methylacetamide, N,N-dimethylacetamide, N-methylpropanamide and the like can be used alone or in combination. In order to enhance the adhesion to the electrode surface in the coating solution (coating dispersion), optionally, Nafion, polytetrafluoroethylene (PTFE), etc. may be used singly or in combination.

According to an exemplary embodiment, the loading amount of the transition metal dichalcogenide formed on the electrode (specifically SPE) is, for example, about 250 to 500 μg/cm 2 , specifically about 300 to 450 μg/cm 2 , more specifically It can be adjusted in the range of about 350 to 420 μg/cm 2 . Since the loading amount of transition metal dichalcogenide affects the optimization of surface modification in a limited electrode area, it may be advantageous to appropriately control it within the above-mentioned range. In addition, the transition metal dichalcogenide layer may be formed of a nanosheet, specifically, a nanosheet, and may exhibit a negative charge (specifically, in the case of MoS ₂ nanosheet).

On the other hand, according to an exemplary embodiment, the modified composite of the electrode is formed in such a way that the conductive polymer layer is positioned on the electrode on which the transition metal dichalcogenide layer is formed. At this time, the conductive polymer layer improves the electrochemical performance (conductivity, etc.) of the underlying transition metal dichalcogenide/electrode, and also induces aggregation to inhibit the leaching or leaching of the electrode material, In addition, it is possible to provide a stable chemical binding ability with a molecule capable of capturing a detection target.

As an example, the conductive polymer may be selected from a conductive polymer that typically contains an amine group (in particular, may contain a conjugated bond in the main chain), for example, polyaniline (PANI), polyethylenediamine, polypyrrole, poly It may be at least one selected from the group consisting of aminophenol and polydiaminophenol. According to an exemplary embodiment, the molecular weight (M _w ) of the conductive polymer may be, for example, about 1,000 to 100,000, specifically about 3,000 to 50,000, and more specifically about 5,000 to 20,000. Since the molecular weight of the conductive polymer affects the properties of the sensor surface complex, it may be advantageous to appropriately control it within the above-mentioned range.

According to a specific embodiment, the conductive polymer may be polyaniline (PANI), which is advantageous for forming a complex of nanostructures because polyaniline can be smoothly combined with nanoparticles and has structural durability. Polyaniline is synthesized by oxidative polymerization of aniline. Forming a conductive polymer layer through an electrochemical polymerization method among chemical polymerization and electrochemical polymerization, which is an oxidation method of aniline as a monomer, has desired conductivity. It can be advantageous for producing polyaniline.

Among the conductive polymers applicable as a conductive layer in an exemplary embodiment, polyaniline is a generic term for compounds that can exist in various compositions according to the substitution of various functional groups at the positions of rings and nitrogen and control of oxidation states, and the basic represented by the following general formula 1 have a structure

[General formula 1]

In addition, polyaniline is leucoemeraldine (1-y=0; fully reduced form), emeraldine (1-y=0.5; intermediate oxidation form), emerldine salt and perniganiline (1-y=1; completely oxidized), and is represented by the following general formula (2).

[General formula 2]

In particular, the electrochemical polymerization reaction mechanism of polyaniline can be represented as in Scheme 1 below.

[Scheme 1]

In the case of using polyaniline as a conductive polymer, the response mechanism depends on the transition from the emeraldine salt (partially oxidized and protonated to have conductivity) to the emeraldine base. In consideration of the above, in an exemplary embodiment, an electrochemical polymerization reaction, ie, electrodeposition, may be performed to form a conductive polymer layer. In the electrochemical polymerization method, the monomer in the electrolyte solution forms radicals in an electric field and polymerizes while moving to one electrode. suitable for forming In particular, it has the advantage that a uniform surface can be obtained and voltage can be easily controlled, and a dense thin film can be formed simultaneously with polymerization.

The electrochemical polymerization reaction can usually be carried out in an aqueous electrolyte solution of a strong acid, for example, various acid components such as hydrochloric acid, sulfuric acid, nitric acid, p-toluenesulfonic acid, perchloric acid, etc. can be used alone or in combination, More specifically, sulfuric acid may be used. The concentration of the strong acid in the aqueous electrolyte solution may be, for example, in the range of about 0.2 to 1.8 M, specifically about 0.3 to 1.5 M, and more specifically about 0.5 to 1.2 M. When polyaniline is used as the conductive polymer, the aniline monomer may be substituted (eg, p-CH ₃ , p-OCH ₃ , o-CF ₃ , m-CF ₃ , p-COOH, o-NH ₂ , p- NH ₂ etc.), unsubstituted aniline, specifically unsubstituted aniline, and in an aqueous electrolyte, for example, about 0.05 to 0.5 M, specifically about 0.07 to 0.4 M, more specifically about 0.08 to about 0.08 M It may be contained in a concentration of 0.2 M. In this case, the molar ratio of the strong acid/aniline monomer in the aqueous electrolyte solution may be adjusted, for example, in the range of about 3 to 20, specifically about 5 to 15, and more specifically about 8 to 12. Also, the pH range of the aqueous electrolyte solution may be, for example, about 0.1 to 1.5, specifically about 0.5 to 1.3, and more specifically about 0.7 to 1.1. The polymerization reaction conditions of polyaniline described above are exemplary, and the present invention is not necessarily limited thereto.

On the other hand, the member corresponding to the working electrode in the electrochemical polymerization reaction may be an electrode modified with the transition metal dichalcogenide prepared above, and the reference electrode is Hg ₂ SO ₄ , Ag/AgCl, Ag/Ag ⁺ , Hg/Hg ₂ SO ₄ , RE-6H, Hg/HgO, KCl saturated calomel half cell (SCE), platinum salt bridge platinum filament reference electrode, and the like. In addition, when using a three-electrode system, as a counter electrode, for example, a conductive metal material such as a platinum electrode (platinum filament electrode, platinum ring electrode, etc.), a gold electrode, a silver electrode (or silver net electrode), or electrodes made of a combination thereof (platinum/gold electrodes); glass, quartz, plastic film, mica and aluminum plates coated with conductive particles; titanium electrode; A silver/mercury film electrode or the like can be used.

An exemplary electrochemical reaction may use cyclic voltammetry or amperometry, and the basic principle of each of the methods is known in the art. In the former case, the thickness of the conductive polymer layer to be polymerized increases as the cycle increases, while in the latter case, the thickness of the conductive polymer layer to be polymerized increases as time increases. In addition, when the conductive polymer layer is formed through an electrochemical reaction, the applied voltage is, for example, about -1 to 1.5 V, specifically about -0.5 to 1.3 V, and more specifically about -0.4 V to 1.2 V range. can be selected from In the case of the cyclic voltage potential method, not only the applied voltage but also the intensity of the current can be adjusted, for example, it can be selected within the range of about 10 ^-6 to 10 ^-1 A. In addition, when a voltage is applied in the cyclic voltage potential method, the scanning speed can be appropriately adjusted in the range of about 5 to 500 mV/s, and the polarization phenomenon and electrical conduction resistance through the oxidation and reduction peaks in the oxidation-reduction reaction are reduced. It can be observed and it can be confirmed that a polymer layer is formed. The loading amount of polyaniline as a conductive polymer can be controlled according to the number of cycles in the cyclic voltammetry and polymerization time in the amperometric method.

The conductive polymer layer thus formed may have a nanofiber or nanowire structure, wherein the diameter of the nanofiber or nanowire is, for example, about 1 to 100 nm, specifically about 3 to 50 nm, more specifically about 5 to 20 nm. In addition, the conductive polymer layer exhibits porosity and may provide a higher adhesive tendency compared to an unmodified electrode. Moreover, the amine group-containing conductive polymer, specifically the polyaniline layer, can exhibit a positive charge and thus electrostatically interact with the underlying transition metal dichalcogenide layer, specifically the negatively charged MoS ₂ layer, resulting in a strong interaction can induce

In particular, the transition metal dichalcogenide has good conductivity, and when it is functionalized with a conductive polymer, its solubility can be improved. Furthermore, the electrochemical activity of the electrode (base matrix) may be significantly increased by the interaction between the conductive polymer layer and the transition metal dichalcogenide layer.

- Fixation of CCP

According to one embodiment, when the sample containing the aCCP antibody is subsequently contacted, CCP that can be selectively bound to the aCCP antibody is immobilized on the base matrix. For immobilization of CCP, a surface chemical route known in the art, for example, a coupling immobilization of a covalent bond (specifically immobilization using a crosslinker) may be applied. As an example, the CCP may be immobilized on the conductive polymer/transition metal dichalcogenide/electrode through bonding of the carboxyl group and the amine group by the EDC/NHS reaction. In this case, the carboxyl group of CCP reacts with EDC to form an unstable amine-reactive ester intermediate, and NHS or sulfo-NHS is attached to the carboxyl group of CCP to form a metastable active ester intermediate, and then interacts with the amine group in the conductive polymer It forms a stable amide bond with CCP.

On the other hand, in addition to the EDC / NHS route described above, glutaraldehyde, N,N'-dichlorohexyl carbodiimide, dithiol-bis-succinimidyl propionate (Dithiobis) Succinimidyl Propionate), diisopropylcarbodiimide, or 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide and N,N'-carbonyldiimidazole may be used.

According to an exemplary embodiment, the CCP may be immobilized on the base matrix by contacting the base matrix with a solution containing CCP. In this case, the concentration of the CCP-containing solution is, for example, about 10 to 200 μg/ml, specifically about 60 to 140 μg/ml, more specifically about 80 to 120, in consideration of the electrode area and measurement sensitivity optimization of the sensor. It can be adjusted in the μg/ml range.

- Nanomatrix

According to one embodiment, apart from the above-described base matrix, a nanomatrix including a conductive polymer and metal nanoparticles as a probe (nanoprobe) for capturing aCCP antibody in a liquid sample is provided separately.

In this regard, the conductive polymer may be selected from the range of the conductive polymers previously included in the base matrix, specifically, the conductive polymers containing an amine group, and may be the same as or different from the conductive polymer in the base matrix. According to a specific embodiment, polyaniline can be used as a conductive polymer in the nanomatrix. Since polyaniline can be smoothly combined with nanoparticles and has good structural durability, it has advantageous properties for forming a nanostructured complex. may be advantageous for the capture of

In addition, the metal nanoparticles may be at least one material selected from the group consisting of gold (Au), silver (Ag), palladium (Pd), copper (Cu) and platinum (Pt), and more specifically, gold (Au). Gold (Au) has high conductivity and biocompatibility, and in particular has advantageous properties for signal amplification, so it may be advantageous to use it as a metal component in a nanomatrix.

On the other hand, the conductive polymer, specifically polyaniline, can be prepared by a method different from the electropolymerization method that can be adopted when forming the above-described base matrix, for example, by an interfacial polymerization method, and metal nanoparticles are also synthesized during interfacial polymerization to synthesize the conductive polymer- Nanocomposites containing metal nanoparticles can be prepared. As an example, when the conductive polymer is polyaniline, the aniline monomer is dissolved in an organic solvent, for example, at least one selected from benzene, toluene, xylene, and the like, while an aqueous medium (specifically an acid solution, more specifically HCl, etc.) The polymerization reaction can be performed by dissolving the metal precursor in a strong acid solution of Illustratively, the molecular weight (M _w ) of the conductive polymer for a nanomatrix may be, for example, about 1,000 to 100,000, specifically about 3,000 to 50,000, and more specifically about 5,000 to 20,000, as the molecular weight is in the range of the sensor surface. Considering that it affects the properties of the composite, it may be advantageous to appropriately control it within the above-mentioned range.

When synthesizing metal nanoparticles in a nanomatrix, a compound containing typically Au(III) ions may be used as a metal precursor, and gold(III) chloride may be more typically used. As an example, the gold (Au) precursor may be 1 or 2 or more selected from the group consisting of HAuCl ₄ , AuCl, AuCl ₂ , AuCl ₃ , Na ₂ Au ₂ Cl ₈ , NaAuCl ₂ , and hydrates thereof, more specifically As such, it may be HAuCl ₄ . In an exemplary embodiment, the concentration of the metal precursor in the aqueous medium may range from about 1 to 10 mM, specifically about 2 to 5 mM, and the concentration of the monomer (particularly aniline) of the conductive polymer in the organic solvent is, for example, For example, it may be in the range of about 0.1 to 1 M, specifically, about 0.3 to 0.7 M, but this may be understood as an example.

According to an exemplary embodiment, the conductive polymer in the prepared nanomatrix may be in the form of nanofibers or nanowires, and may be in the form in which metal nanoparticles are attached or embedded in the nanofiber or nanowire matrix. In this case, the size (diameter) of the metal nanoparticles may be, for example, about 3 to 20 nm, specifically about 4 to 18 nm, more specifically about 6 to 14 nm. As the size of the metal nanoparticles affects the binding to the conductive polymer and/or protein, it may be advantageous to be adjusted in the above-described range in consideration of this. In addition, the metal nanoparticles (specifically Au nanoparticles) in the nanomatrix, for example, about 0.1 to 0.4 nm, specifically about 0.15 to 0.3 nm, more specifically about 0.2 to 0.25 nm of a lattice fringe (lattice fringe) can have In addition, in the case of a conductive polymer in the form of nanofibers or nanowires in the nanomatrix, the diameter thereof may be, for example, about 1 to 100 nm, specifically about 3 to 50 nm, more specifically about 5 to 20 nm. there is.

As described above, the nanomatrix containing the conductive polymer-metal nanoparticles is added to or contacted with the liquid sample. Specifically, it may be bound to the metal nanoparticles in the nanomatrix. Specifically, it is possible to provide a high loading capacity for the aCCP antibody by physical adsorption and electrostatic interaction between the metal nanoparticles and the aCCP antibody.

The content of conductive polymers and metal nanoparticles in the nanomatrix varies according to the types of conductive polymers and metals used, so it is difficult to uniformly specify them. However, when the content of metal nanoparticles is too small, the amount of capture for the aCCP antibody in the sample decreases, thereby lowering the detection sensitivity, whereas when the content of metal nanoparticles is too high, the sensor response characteristics (e.g., change in current) To the extent that this reduced problem can be induced, it can be determined in consideration of the advantages.

In an exemplary embodiment, the nanomatrix and the liquid sample may be contacted under stirring or shaking conditions. In this case, the liquid sample may be a body fluid or blood. In this way, after the nanomatrix-aCCP antibody is formed, for the purpose of suppressing a phenomenon such as contamination of the remaining active site of the nanomatrix by an external component, it can be optionally blocked by BSA or the like. there is.

Detection (diagnosis) of aCCP antibody in a sample using a biosensing platform

According to one embodiment, in the biosensing platform (or immunosensor platform) prepared above, the CCP-immobilized base matrix is a nanomatrix (if negative) or a nanomatrix to which aCCP antibody is bound (if positive) is added It comes into contact with the sample (liquid sample). According to an exemplary embodiment, prior to contact with the liquid sample, a blocking layer may be formed using a reagent known in the biosensor field in order to prevent exposure of the active site in the modified electrode (base matrix). In this regard, as the blocking layer, for example, at least one selected from the group consisting of bovine serum albumin (BSA) and gelatin (for example modified hydroyzed porcine gelatin, specifically trade name ^Prionex® ) Since the material used is available, the surface of the CCP-immobilized base matrix may be subjected to blocking treatment, and incubation may be additionally performed.

In addition, a suitable pH for the formation of an immunocomplex between the CCP immobilized on the base matrix and the aCCP antibody captured on the nanomatrix during the contacting process is, for example, about 6 to 8.5, specifically about 7 to 8, more specifically about 7.2 to 7.5 can be adjusted in the range of In addition, the contact time or the incubation time may be adjusted in the range of, for example, about 10 to 120 minutes, specifically about 20 to 100 minutes, and more specifically about 40 to 80 minutes. The above-described conditions are understood as illustrative, and may be changed according to the types of materials constituting the biosensing platform.

According to one embodiment, when the CCP-immobilized base matrix and the nanomatrix-containing liquid sample come into contact to form an immunocomplex of the CCP-aCCP antibody, the response characteristic through the electrochemical signal is measured to qualitatively evaluate the aCCP antibody in the sample. /Can be quantitatively detected or diagnosed. In this case, the electrochemical signal may mean a signal detectable by an electrochemical measuring device, specifically, a signal detectable by a working electrode (corresponding to the base matrix) that is a detection unit in the biosensor. Such signals may include currents, voltages (voltages converted from currents), and digital signals corresponding thereto. According to an exemplary embodiment, the electrochemical response characteristics are, for example, square wave voltammetry, cyclic voltammetry, linear sweep voltammetry, differential pulse voltammetry. Method (differential pulse voltammetry), electrochemical impedance spectroscopy (Electrochemical impedance spectroscopy), etc. can be measured by selecting at least one.

According to an exemplary embodiment, the electrochemical response characteristic may be measured in the presence of a redox probe. In this regard, the redox probe is a redox pair ferro/ferricyanide, 3,3',5,5'-tetramethylbenzidine (TMB), 1-hydroxy-3-methoxycarbonyl-2,2, It may be at least one selected from 5,5-tetramethylpyrrolidone (CMH), methylene blue (MB), and the like, and more specifically, a redox pair ferro/ferricyanide (Fe(CN) ₆ ^3- / ^{4 -} ) can be In particular, when the redox probe (mediator) is used together with SPE, it has the advantage of accelerating the catalytic reaction of the target. As an example, when the aCCP antibody is contained in the sample to form a nanomatrix to which the aCCP antibody is immobilized, and this is added to the CCP-immobilized base matrix, electron transfer is restricted by affinity interaction between the aCCP antibody and CCP. However, it shows a lower current response characteristic than the case in which the aCCP antibody is not present in the sample.

According to an exemplary embodiment, the redox probe may be provided in a form contained in a salt solution, wherein the salt solution may include KCl, NaCl, PBS and/or serum. Also, the concentration of the redox probe may be, for example, about 2 to 10 mM, specifically about 3 to 8 mM, more specifically about 4 to 6 mM, but is not limited thereto.

The biosensing platform (or immunosensor) provided according to this embodiment has a lower limit of detection (LOD) compared to the prior art, for example, about 1 IU/mL or less, specifically about 0.1 to 0.5 IU /mL, more specifically about 0.15 to 0.25 IU/mL, but this should be understood as illustrative. As such, the biosensing platform according to the present embodiment may exhibit a significantly lower detection limit compared to a conventional detection platform.

Hereinafter, preferred examples are presented to help the understanding of the present invention, but the following examples are provided for easier understanding of the present invention, and the present invention is not limited thereto.

Example 1

Materials and devices used in this example are as follows.

substance used

- MoS ₂ , HAuCl _{4.3H 2 O} , toluene, potassium ferricyanide ([K ₃ Fe(CN) ₆ ]), potassium ferrocyanide ([K ₄ Fe(CN) ₆ ]), KCl, aniline, N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), N-hydroxysulfosuccinimide (NHS), BSA, Nafion, isopropyl alcohol and HCl were each purchased from Sigma-Aldrich.

- 21-mer cyclic filaggrin peptide and aCCP were purchased from Peptron.

- Human serum and human serum albumin were purchased from Sigma-Aldrich.

- Immunoglobulin M (IgM) RF was purchased from EastCoast Bio.

- C-reactive protein (CRP) was purchased from Abcam.

analysis device

- The surface morphology was analyzed using SEM (Hitachi S-4700; operating acceleration voltage: 15 kV, energy dispersive X-ray) and TEM ((TEM-FEI Tecnai, voltage 300 kV).

- The crystalline phase was analyzed using SmartLab ^ㄾ X-ray diffractometer (Rigaku).

- UV spectra were recorded using a Varian Cary (1 cm path length, containing quartz cells).

- Electrochemical measurements and characterization were performed using an Ivium CompactStat ^™ potentiostat.

- Carbonaceous screen-printed electrodes (working and counter electrodes are made of carbon and reference electrodes are of Ag/AgCl) were purchased from Metrohm Dropsens.

- X-ray photoelectron spectroscopy (XPS) elemental surface analysis was performed using a PHI 5000 Versa Probe (Ulvac-PHI) spectrometer (equipped with monochromator Al Kα (1486.6 eV)).

- FTIR (Fourier transform infrared) spectroscopy analysis was performed using a Jasco-4600 FTIR spectrometer.

- To peel the MoS ₂ nanosheet, sonics VCX 130 equipped with a titanium probe was used.

-pH analysis was performed using a Thermo Fisher Scientific pH meter.

A. Biosensing platform production

In this example, in order to detect aCCP antibody in a sample, a biosensing platform was manufactured according to the steps shown in FIG. 1 .

Preparation of Au-PANI nanomatrix

A PANI-Au nanocomposite was prepared by interfacial polymerization. Interfacial polymerization was initiated by introducing an aqueous solution in which 3 mM HAuCl 4 was dissolved in 0.1 M HCl into toluene (organic phase) in which 0.5 M aniline was dissolved, and PANI nanofibers were slowly formed in the aqueous phase of the solution. At the same time, during the polymerization reaction, Au nanoparticles were also synthesized and incorporated into the PANI nanofibers. The prepared PANI-Au solution was centrifuged at room temperature, and was diffused three times with water for purification. The purified PANI-Au solution was stored at 4°C.

Preparation of PANI-Au-aCCP

To obtain a standard calibration curve, 7 different concentrations of aCCP were calibrated. Calibration standards are expressed in IU/mL. 5 μl of the prepared PANI-Au nanoprobe was mixed with aCCP under shaking conditions for 1 hour, and then 0.5% BSA was added to block the remaining active sites on the PANI-Au nanoprobe. Then, PANI-Au-aCCP was incubated at 4° C. for 1 hour.

PANI/MoS ₂ -Manufacture of modified SPE

MoS ₂ NS (5 mg) was dispersed in 1 mL of isopropyl alcohol and then added to 50 μl of Nafion. The prepared solution was sonicated using a probe at 45% amplitude for 5 minutes. 10 μl of the prepared MoS ₂ ink was dropped on the SPE to load 50 μg (about 398.1 μg/cm 2 ). To improve the electrochemical performance of the MoS ₂ /SPE electroactive matrix, a fine PANI thin film layer was electrochemically attached on the MoS ₂ /SPE. The electrochemical polymerization of aniline was carried out in 1 M HCl containing 0.1 M aniline at a potential range of -0.4 to 1.0 V over 10 cycles. The redox conversion of leucoemeraldine to perniganiline via emeraldine and emerldine was confirmed by corresponding characteristic peaks at potentials of 0.25 V, 0.54 V, and 0.92 V. The electropolymerized PANI-modified MoS ₂ /SPE was washed with running water to remove the inactivated adsorbed aniline monomer from the electrode surface. As the electrosynthesized PANI is a porous material, it strongly adhered on MoS ₂ /SPE because it has a tendency to exhibit high adhesion compared to bare SPE.

BSA/CCP/PANI/MoS ₂ /SPE manufacturing

The selection of a biological recognition mechanism is an important matter in the manufacture of a biosensor, and in this example, a citrulline-containing cyclic filaggrin peptide (21 mer) was used to recognize the aCCP antibody. As shown in Figure 2, CCP was immobilized on PANI/MoS ₂ /SPE using EDC-NHS surface chemistry. To this end, the carboxyl group of CCP (100 μg/mL, 1X PBS) was reacted with EDC (0.4 M) for 30 min under stirring to form an unstable amine-reactive O-acyl isourea ester intermediate. When 0.1 M sulfo-NHS was added to the synthesis solution, NHS was attached to the carboxy group of CCP to form a meta-stable active ester intermediate. Finally, when the prepared solution was dropped on PANI/MoS ₂ /SPE, amine groups in PANI promoted stable amide bond with CCP. To prevent exposure of active sites in PANI/MoS ₂ /SPE, 5.0 μl of 0.5% BSA was dropped onto CCP/PANI/MoS ₂ /SPE and incubated at 4°C. After incubation, the prepared BSA/CCP/PANI/MoS ₂ /SPE was washed twice with 10 μl of 1X PBS.

B. Results and Discussion

PANI/MoS ₂ and characterization of PANI-Au.

Structural properties of the surface-modified SPE were confirmed using SEM, and differences between individual layers thereof were analyzed. First, the bare SPE has a high surface roughness due to the intrinsic property of carbon (FIG. 3a). After the MoS ₂ ink was dropped on the SPE, it was confirmed that MoS ₂ nanosheets were formed as well as the sheet-like properties of MoS ₂ ( FIG. 3b ). It can be seen that the PANI layer electropolymerized on MoS ₂ /SPE has a nanowire structure (Fig. 3c). Cross-sectional SEM pictures of PANI/MoS ₂ /SPE were distinguished from individual MoS ₂ nanosheets and electrodeposited PANI layers ( FIGS. 4a and 4b ).

Referring to FIG. 3D , it can be seen that Au nanoparticles (NP) are embedded in the PANI nanowire matrix. As a result of elemental dispersion X-ray and elemental mapping analysis, the presence of individual elements such as C, N, Cl and Au in the PANI/MoS ₂ sample ( FIGS. 5A to 5F ) and C, N, Cl in the PANI-Au nanomatrix and the presence of individual elements such as Au ( FIGS. 6a to 6e ). Morphological and structural analyzes of MoS ₂ nanosheets and PANI-Au nanomatrices were performed using TEM. Referring to FIG. 3e , it can be seen that MoS ₂ nanosheets having a 2D structure having a thin sheet shape and good crystallinity are formed. The hexagonal lattice and characteristic lattice fringes (0.62 nm) in the inset indicate the presence of the (002) plane of MoS ₂ (Fig. 3f). According to Fig. 3g, a large amount of Au NP-loaded PANI nanowires were observed, and the size range of Au NPs dispersed in the PANI nanowires was approximately 6-14 nm. The presence of large amounts of Au NPs in PANI can facilitate the capture of large amounts of aCCP antibodies through electrostatic interactions. The presence of Au NPs with a lattice fringe of 0.24 nm can be confirmed from the high-resolution TEM photograph (FIG. 3h).

The crystal phases of MoS ₂ , PANI, PANI/MoS ₂ , and PANI-Au layers were analyzed using XRD ( FIG. 3j ). The presence of a strong characteristic peak at 14.4° for MoS ₂ nanosheets supports that MoS ₂ nanosheets have good crystallinity. In the case of the electropolymerized PANI sample, a broad peak was observed at 24°, indicating that PANI has amorphous properties. The PANI/MoS ₂ XRD pattern showed a characteristic peak of MoS ₂ (14.4°) and a characteristic peak of PANI (24.0°). From this, the crystal phase does not change during the electropolymerization of PANI on the MoS ₂ layer, and it can be confirmed that PANI and MoS ₂ are present in the PANI/MoS ₂ nanocomposite layer. The interfacially polymerized PANI-Au sample showed a broad amorphous characteristic peak at 24.9°C. Au (111), Au (200), Au (220), and Au (311) characteristic peaks corresponding to Au nanoparticles were observed at 38.2°, 44.4°, 64.7°, and 77.6°, respectively, which is a bar of Au NPs. Indicating a face-centered cubic structure, especially the highest peak intensity was observed in the Au (111) plane. Elemental compositions of PANI/MoS ₂ and PANI-Au nanocomposites were analyzed using XPS. The full survey spectrum shows the coexistence of Mo, S, C, O, and N elements in PANI/MoS ₂ and Au, C, O, and N elements in the PANI-Au nanocomposite ( FIG. 7a ). PANI/MoS ₂ exhibited Mo 3d, S 2p, C 1s, O 1s, and N 1s spectra, and the extended Mo 3d spectral region contains S 2s, Mo 3d _5/2 , and Mo 3d _3/2 . (Fig. 7b). The S 2s spectrum was captured at 226.7 eV, and the deconvolved Mo 3d _5/2 peak and Mo 3d _3/2 peak were recorded at 229.4 eV and 232.5 eV, respectively. The S 2p spectrum contains S 2p _3/2 , S 2p _1/2 , and S2 peaks at 162.1 eV, 163.3 eV and 164.2 eV, respectively ( FIG. 7c ). Due to the presence of PANI in PANI/MoS ₂ , the C 1s deconvolved region showed a CC/CH peak and a CN peak at 284.8 eV and 286.3 eV, respectively ( FIG. 8a ), and the N 1s deconvolved spectrum was Presence of quinoid amine structure (-N=) (398.7 eV), benzenoid amine structure (-NH-) (399.6 eV), and nitrogen cation radicals (-N ⁺ -) (401.5 eV) was shown (Fig. 7d). The PANI-Au nanocomposite contains Au 4f, C 1s, O 1s, and N 1s electron oxidation states. In Fig. 7e, the Au 4f deconvolution spectra are Au 4f7/2 (84.7 eV) and Au 4f5/2 ( 86.7 eV). The C 1s spectrum of PANI-Au showed a CC/CH peak and a CN peak at 285.0 eV and 286.8 eV, respectively ( FIG. 8b ). The deconvolved N 1s peaks of PANI-Au are three nitrogens corresponding to (-N=) (398.6 eV), (-NH-) (399.6 eV), and (-N ⁺ -) (401.6 eV). from (Fig. 7f).

PANI-Au-aCCP/BSA/CCP/PANI/MoS ₂ Electrochemical characterization of /SPE immunosensors

- Cyclic potential method (CV)

Prior to aCCP detection, the electrochemical properties of individual layers of modified SPE were analyzed using CV in the presence of 5 mM Fe(CN) ₆ ^3- / ^4- in 0.1 M KCl. As observed from Figure 9a, bare SPE produced a low current response (I _pa =123.6 μA, I _pc =104.1 μA) due to undesirable electrochemical behavior. Consumption of the electrochemical current response (I _pa =41.6 μA, I _pc =34.0 μA) was observed even after the electrode surface was modified with MoS ₂ ink. This results from the formation of an electron-blocking layer between MoS ₂ /SPE and Fe(CN) ₆ ^3- / ⁴ ions at the electrode interface.

The PANI-modified electrode showed a higher current response (I _pa =703.8 μA, I _pc =609.1 μA) due to the strong interaction between the amine group of PANI and Fe(CN) ₆ ^3- / ^4- . Since PANI was electropolymerized on MoS ₂ /SPE, the electronegativity of MoS ₂ /SPE was completely neutralized, and in the case of PANI/MoS ₂ /SPE, a high current response (I _pa = 950.2 μA, I _pc =838.5 μA) was obtained. was captured As such, it can be concluded that the electrochemical activity of PANI/MoS ₂ /SPE increases due to the interaction between MoS ₂ and PANI. When CCP was immobilized on PANI/MoS ₂ /SPE, an insulating layer was formed to limit electron transport, thereby induced a sharp decrease in the redox peak current (I _pa =320.4 μA, I _pc =208.6 μA). The remaining non-adsorbed sites of CCP/PANI/MoS ₂ /SPE were blocked upon fixation with 0.5% BSA.

The CV response of BSA/CCP/PANI/MoS ₂ /SPE supports the reduced current response (I _pa =256.8 μA, I _pc =198.9 μA). Finally, upon addition of PANI-Au-aCCP, limited electron transfer (resulting from the affinity interaction between aCCP and CCP) produced a low current response (I _pa =216.9 μA, I _pc =151.7 μA) (Fig. 1c).

The importance of capture of aCCP in PANI-Au was characterized by the CV response of aCCP/BSA/CCP/PANI/MoS ₂ /SPE in the presence and absence of PANI-Au nanomatrices. At this time, the consumed current response (I _pa =95.8 μA, I _pc =100.3 μA) was observed in the absence of PANI-Au. On the other hand, when the PANI-Au nanocomposite in which the aCCP antibody was captured was added, an enhanced current response (I _pa =216.9 μA, I _pc =151.7 μA) was observed, indicating that the high conductivity of the PANI-Au nanocomposite was SWV This is because it facilitates the detection of aCCP through The bar chart in FIG. 9b shows the change in I _pa at individual levels constituting the modification of SPE. The above results suggest that the aCCP immunosensor was successfully implemented using the BSA/CCP/PANI/MoS ₂ /SPE immunodetection platform and the PANI-Au nanomatrix for capturing the aCCP antibody.

- Electrochemical impedance spectroscopy

Bare SPE, MoS ₂ /SPE, PANI/SPE, PANI/MoS ₂ /SPE, CCP/PANI/MoS ₂ /SPE, BSA/CCP/PANI/MoS ₂ /SPE, and PANI-Au-aCCP/BSA/CCP/ The electrochemical charge transfer resistance (R _ct ) contribution of specific electrode layers, such as PANI/MoS ₂ /SPE, was characterized using EIS Nyquist plots. The frequency was set in the range of 1 Hz to 1 MHz, and the Nyquist plot was fitted using the most suitable equivalent circuit. The charge transfer properties of the electrode surface are determined by the bulk solution resistance (R _s ), the double-layer capacitance formed by ions at the electrode interface (C _dl ), and the charge transfer resistance (R _ct ), which is the ratio at the electrode-electrolyte interface. This is due to the Dox characteristic current flow and Z _w (indicating Warburg impedance). In Fig. 9c, the semicircular part of the Nyquist plot means R _ct at the electrode face, and the linear region indicates the diffusion of electrons at the electrode surface. After fitting with an appropriate equivalent circuit, the R _ct values obtained for bare SPE, MoS ₂ /SPE, PANI/SPE, and PANI/MoS ₂ /SPE were 159.1 Ω, 293.9 Ω, 20.6 Ω, and 15.6 Ω, respectively. As shown in Fig. 9c, bare SPE exhibits an undesirable electrochemical response due to low electrochemical activity, while MoS ₂ /SPE induces a higher R _ct due to the electronegative charge accumulated on the surface of MoS ₂ /SPE. did The lowest R _ct was obtained for PANI/MoS ₂ /SPE (15.6 Ω), indicating high conductivity of PANI/MoS ₂ /SPE. The fixation of CCP was confirmed by increased R _ct (342.1 Ω). The increased R _ct (582.1 Ω) in BSA/CCP/PANI/MoS ₂ /SPE means that the unbound CCP active site was blocked in CCP/PANI/MoS ₂ /SPE. Finally, the highest R _ct (928.7 Ω) of PANI-Au-aCCP/BSA/CCP/PANI/MoS ₂ /SPE supports the interaction between aCCP and CCP, which restrictively blocks electron transport at the electrode interface. The overall results (Fig. 9d) suggest that the immunosensor for aCCP was successfully fabricated.

Optimization of biosensors

First, it was tested by varying the concentration of CCP (25.0-150.0 μg/mL) with respect to 50.0 IU/mL of the aCCP-attached PANI-Au nanomatrix in the manufactured immune sensor. As the concentration of CCP was changed from 25.0 to 50.0 μg/mL, the change in catalytic current response (ΔI) gradually increased, and the highest ΔI was observed for CCP of 100.0 μg/mL. When the concentration of CCP was 125.0 to 150.0 μg/mL, ΔI started to decrease ( FIG. 10A ).

When CCP protein in excess of 100 μg/mL is loaded on the compartmentalized electrode surface, CCP may aggregate and block the citrulline antibody binding site, thereby interfering with CCP-aCCP immunocomplex formation upon addition of PANI-Au-aCCP. there is. Therefore, inhibition of the formation of CCP-aCCP immunocomplexes may be the result of weakening the electron-blocking layer (derived from the immunocomplexes) on the compartmentalized electrode surface, leading to a decrease in ΔI. Therefore, in the case of the detection system according to the present embodiment, the optimal concentration may be selected as CCP of 100.0 μg/mL.

In addition, ΔI for the aCCP immunosensor using PANI-Au in the presence of 50.0 IU/mL of aCCP antibody was measured while changing the pH level (6.0-8.2). ΔI gradually increased from pH 6.0 to 7.0, and the maximum ΔI response was obtained at pH 7.4 ( FIG. 10b ). The biological activity between CCP and aCCP antibody was maintained with high affinity near neutral pH (7.4), which may be due to the high electrocatalytic current response.

Since the aCCP antibody can be absorbed in the PANI-Au nanomatrix through the electrostatic interaction between the aCCP antibody and PANI-Au, the loading volume of PANI-Au plays an important role in the performance of the detection system. The loading volume of the PANI-Au nanocomposite varied in the range of 0 to 10 μl (Fig. 10c). SWV current responses obtained by varying the loading volumes of PANI-Au (0 µL, 0.5 µL, 2.5 µL, 5.0 µL, 7.5 µL, and 10.0 µL) can be obtained with high current responses from 0-5.0 µL of PANI-Au and , especially at 5.0 μL, the maximum current response was elicited. In addition, the current response decreased in 7.5-10.0 μL of PANI-Au, which is due to steric hindrance at the electrode interface.

To analyze the effect of incubation time on the electrochemical performance of the aCCP immunosensor, the aCCP immunosensor using PANI-Au in the presence of 50.0 IU/mL of aCCP antibody was evaluated at different incubation times (0-90 min) ( Fig. 10d). The maximum SWV peak current change was observed at 60 min.

PANI-Au-aCCP/BSA/CCP/PANI/MoS ₂ Characterization of SWV Response of the /SPE Immunosensor

Under the increased assay conditions, using the SWV electroanalysis technique, the assay performance of BSA/CCP/PANI/MoS ₂ /SPE immunosensor was investigated in the presence of 5 mM Fe(CN) ₆ ^3- / ⁴ as a redox mediator. did The entire assay was performed in the presence of 40 μl of electrolyte (1X PBS, pH = 7.4).

After continuous addition of aCCP, the peak current response at 0.37 V gradually decreased, and the dynamic detection range of aCCP was analyzed (Fig. 11a). It shows that the change in the peak current response of the sensor is linearly related to the log concentration of 0.25 to 1500 IU/mL aCCP (Fig. 11b). ΔI means a decrease in peak current corresponding to the aCCP concentration, and may be calculated by Equation 1 below.

[Equation 1]

ΔI = I _{no aCCP} -I _aCCP

In the above equation, I _{no aCCP} is the peak current of the immunosensor in the absence of aCCP, and I _aCCP corresponds to the peak current observed after addition of aCCP.

The obtained formula was ΔI(μA) = 4.4713log C _aCCP (IU/mL)+3.962 (R ² = 0.9974), and the detection limit of the immune sensor was calculated using the standard method presented by IUPAC. LOD can be expressed by Equation 2 below.

[Equation 2]

LOD = 3S _D /S

where S _D is the standard deviation of 10 blanks in the absence of aCCP, and S is the slope of the calibration curve.

As such, the aCCP immunosensor detected aCCP antibody with a log dynamic range of 0.25-1500 IU/mL, and the LOD was 0.16 IU/mL in 1X PBS (S/N=3). The results compared with the aCCP detection limit presented in the previously reported literature are shown in Table 1 below.

division dynamic range
(IU/mL) detection limit
(IU/mL) sample type note electrochemical
amperometric method 10-1000.0 2.5 PBS Analyst. 145 (2020) 4680-4687 Fluorescence Immunoassay
(BioPlex ^TM 2200)
3-300.0
0.2 HS RSC Adv. 4 (2014) 32924-32927 ELISA
(ImmunLisa ^TM CCP) 25-3200.0 1.6 HS RSC Adv. 4 (2014) 32924-32927 electrochemical SWV 0.25-1500.0 0.16
PBS
the present invention electrochemical SWV 0.25-1500.0 0.22 10% HS the present invention

.

The reason that the immunosensor according to the embodiment has a higher detection limit can be considered because the loading capacity of the aCCP antibody on the PANI-Au nanomatrix is increased. In addition, it is considered that the electrocatalytic activity of the PANI/MoS ₂ /SPE electrode (base matrix) induced a significant improvement in the electrocatalytic activity. The PANI base matrix not only improves the electrochemical performance, but also facilitates the immobilization of CCP on PANI/MoS ₂ /SPE through stable amide bond formation between CCP and PANI via EDC-NHS surface chemistry. The derived experimental results suggest that the base matrix of the PANI/MoS ₂ nanocomposite and the PANI-Au NP nanomatrix are potential candidates for providing a broad dynamic range and better detection limits.

Principle of aCCP Immune Sensor

Referring to FIG. 1B , due to the CCP immobilized on the surface-modified electrode in the aCCP immunosensor, the CCP-immobilized SPE can recognize the aCCP antibody with higher affinity. In an experiment using a redox probe of Fe(CN) ₆ ^3- / ^4- , a higher SWV response could be obtained in the absence of the aCCP antibody, whereas a decrease in the current response was observed in the presence of the aCCP antibody. In addition, when PANI-Au-aCCP/BSA/CCP/PANI/MoS ₂ /SPE was completely isolated using 0.5% BSA, the change in current response was dependent on the aCCP concentration.

Applications of biosensors

When a specific protein is present together in a serum sample, the SWV response of the immune sensor was checked in the presence of 50.0 IU/mL aCCP antibody, and a study on interfering substances was performed. Human serum albumin (HSA; 10 mg/mL), immunoglobulin M (IgM) RF (100 ng/mL), and CRP ab (inflammatory antibody) (1,000 ng/mL) were tested against 50 IU/mL aCCP. Under optimized conditions, analytes were incubated at 4° C. for 60 minutes after the addition of interfering substances, and then washed twice with 40 μl of IX PBS. Then, SWV studies were performed in the presence of 5 mM Fe(CN) ₆ ^3- / ^4- in 1X PBS (pH=7.4). 11C shows the recorded SWV response to aCCP 50.0 IU/mL on BSA/CCP/PANI/MoS ₂ /SPE using a PANI-Au nanomatrix prepared to capture aCCP together with the aforementioned interfering material. The bar chart of the SWV response ( FIG. 11D ) shows little deviation in the current response (ΔI), and the % relative standard deviation (RSD) obtained was 4.67%, which was less than 5%. Error bars mean aCCP current response in three independent measurements (n=3). As such, the aCCP immunosensor according to the embodiment can detect the aCCP antibody with high selectivity.

Stability and Accuracy

The storage stability of the aCCP immunosensor was evaluated by measuring its performance after storage at 4°C for 4 weeks. SWV responses were recorded in the presence of 50.0 IU/mL of aCCP ( FIG. 12A ). The sensor retained 92.3% of the initial peak current even after 4 weeks. Moreover, the calculated % RSD of ΔI was 2.92%, which was less than 5%. As such, the aCCP immunosensor could be stored for 3 weeks and still showed efficient results. The reproducibility of aCCP detection in the presence of 50.0 IU/mL of aCCP for the immunosensor was evaluated. Fig. 12b shows the SWV response obtained for five different electrodes, and the bar chart shows the five different electrodes and their corresponding ΔI values (Fig. 12c). The standard deviation from the three measurements was expressed as error bars (n=3), and the calculated % RSD of reproducibility was 3.32%. Reproducibility was measured through 8 experiments for the same electrode, and the calculated % RSD of the precision experiment (intra-assay) was 3.49%.

Real-time analysis in human serum samples

The sensor was applied to human serum samples spiked with aCCP antibody to evaluate the real-time applicability of the sensor under optimized conditions. Due to the complexity of human serum, serum samples were diluted to 10% (v/v) in IX PBS (pH=7.4). However, prior to analyzing the serum samples spiked with aCCP antibody, the SWV for the diluted serum was recorded and considered as a blank value. aCCP antibody-spiked serum samples (0.25 IU/mL, 1.0 IU/mL, 10.0 IU/mL) in the presence of redox probe, 5 mM Fe(CN) ₆ ^3- / ^4- in 1X PBS (pH=7.4) , 100.0 IU/mL, 500.0 IU/mL, 100.0 IU/mL, and 1500.0 IU/mL) were analyzed under optimized conditions. The SWV readings of serum samples spiked with aCCP antibody were subtracted from the blank value to eliminate the matrix effect of human serum. The recorded SWV response is plotted in FIG. 13A , and when the concentration of aCCP is increased, the current response at 0.29 V gradually decreased. Referring to FIG. 13A , the principle of the immune sensor for human serum was found to follow the same mechanism and tendency as observed in 1X PBS. However, the oxidation potential was slightly shifted in the negative direction due to the matrix effect in human serum. The obtained SWV peak current was subtracted from the peak current of 10% human serum, and the calculated value is plotted in FIG. 13B . A linear equation of logC _HS-aCCP vs ΔI may be expressed by Equation 3 below.

[Equation 3]

ΔI (μA) = 2.8695 logC _HS-aCCP (IU/mL)+2.1503 (R ² =0.9944)

In the above equation, the error bar means the standard deviation of three independent measurements.

The calculated LOD of detection of aCCP in aCCP-spiked human serum was 0.22 IU/mL, and the log dynamic linear range was 0.25-1500 IU/mL (S/N = 3). Moreover, the dynamic range of detection of 2.5-15000 IU/mL of aCCP antibody in 100% serum matched the actual concentration of aCCP antibody in human serum.

Considering the above results, the PANI-Au nanomatrix can increase the loading amount of the aCCP antibody, thereby increasing the sensitivity of the aCCP immune sensor. In applying the BSA/CCP/PANI/MoS ₂ /SPE immunosensor, when the aCCP detection was performed in 1X PBS, the LOD was 0.16 IU/mL, and when performed in 10% human serum, the LOD was 0.22 IU/ mL (log dynamic range is 0.25-1500.0 IU/mL). In addition, the aCCP immune sensor according to the embodiment was able to distinguish aCCP from other proteins in the human body, and exhibited excellent selectivity due to CCP fixation. Furthermore, the aCCP immunosensor can be stored at 4° C. and maintained storage stability for 3 weeks. In this example, the total time required for aCCP detection (including CCP fixation and PANI-Au-aCCP addition) was within 2 hours. As such, the sensor according to the embodiment can provide advantages compared to the results of conventional studies. For example, the detection procedure is simple, and the required sample volume (10 μl) is 10 times smaller than that of a commercial ELISA kit. is the amount of capacity.

Simple modifications or changes of the present invention can be easily used by those of ordinary skill in the art, and all such modifications or changes can be considered to be included in the scope of the present invention.

Claims (21)

A base matrix comprising a modified electrode in which a transition metal dichalcogenide layer and an amine group-containing conductive polymer layer are sequentially loaded on the electrode, the transition metal in the transition metal dichalcogenide is molybdenum (Mo), tungsten (W), at least one selected from the group consisting of titanium (Ti), tantalum (Ta) and zirconium (Zr);
CCP fixed to the base matrix and having binding ability to aCCP antibody; and
From the group consisting of (i) an amine group-containing conductive polymer and (ii) gold (Au), silver (Ag), palladium (Pd), copper (Cu) and platinum (Pt) having capture ability for aCCP antibody a nanomatrix comprising at least one selected metal nanoparticle;
including,
When the aCCP antibody is contained in the sample, the aCCP antibody bound to the nanomatrix is quantitatively and/or qualitatively detecting the aCCP antibody in the sample based on the electrochemical response characteristics of binding to the CCP immobilized on the base matrix. Chemical sensing platform. The electrochemical sensing platform according to claim 1, wherein the conductive polymer contained in each of the base matrix and the nanomatrix is the same or different. The electrochemical sensing platform according to claim 2, wherein the conductive polymer is at least one selected from the group consisting of polyaniline (PANI), polyethylenediamine, polypyrrole, polyaminophenol, and polydiaminophenol. According to claim 1, wherein each of the molecular weight (M _w ) of the conductive polymer contained in the base matrix and the molecular weight (M _w ) of the conductive polymer contained in the nano-matrix is determined in the range of 1,000 to 100,000 electricity, characterized in that Chemical sensing platform. The electrochemical sensing platform of claim 1, wherein the chalcogen element in the transition metal dichalcogenide is sulfur. The electrochemical sensing platform of claim 1, wherein the CCP is immobilized on the base matrix by EDC-NHS surface chemistry. The electrochemical sensing platform according to claim 1, wherein the size of the metal nanoparticles in the nanomatrix is in the range of 3 to 20 nm. The electrochemical sensing platform of claim 1, wherein the conductive polymer in the base matrix and the nanomatrix has a nanofiber or nanowire shape. The electrochemical sensing platform according to claim 1, wherein the nanomatrix is formed by interfacial polymerization, and the conductive polymer in the base matrix is formed by electropolymerization. The electrochemical sensing platform of claim 1, wherein the electrode is a screen printed electrode (SPE). 11. The method of claim 10, wherein the screen printing electrode is at least one material selected from the group consisting of carbon (C), gold (Au), platinum (Pt), silver (Ag), copper (Cu) and nickel (Ni) Electrochemical sensing platform, characterized in that consisting of. The electrochemical sensing platform according to claim 1, wherein the loading amount of the transition metal dichalcogenide is set in the range of 250 to 500 μg/cm 2 . The electrochemical sensing platform according to claim 1, further comprising a blocking layer on the surface of the base matrix to which the CCP is immobilized in order to prevent exposure of the active site on the base matrix to which the CCP is immobilized. a) providing a base matrix to which CCP having binding ability to aCCP antibody is immobilized, wherein the base matrix is a modified electrode in which a transition metal dichalcogenide layer and an amine group-containing conductive polymer layer are sequentially loaded on the electrode Including, wherein the transition metal in the transition metal dichalcogenide is at least one selected from the group consisting of molybdenum (Mo), tungsten (W), titanium (Ti), tantalum (Ta) and zirconium (Zr);
b) having a capture ability for aCCP antibody, (i) an amine group-containing conductive polymer and (ii) gold (Au), silver (Ag), palladium (Pd), copper (Cu) and platinum (Pt) consisting of providing a liquid sample to which a nanomatrix including at least one metal nanoparticle selected from the group is added; and
c) measuring the electrochemical response characteristics by bringing the CCP-fixed base matrix into contact with the liquid sample to which the nano-matrix is added;
A method for quantitatively and/or qualitatively detecting aCCP antibody in a liquid sample, comprising: 15. The method of claim 14, further comprising the step of blocking the surface of the base matrix to which the CCP is fixed prior to step c). 15. The method according to claim 14, wherein step c) is performed under conditions of pH 6 to 8.5. 15. The method of claim 14, wherein the CCP-immobilized base matrix is formed by contacting the base matrix with a CCP-containing solution, wherein the concentration of the CCP-containing solution is set in the range of 10 to 200 μg/ml. . 15. The method of claim 14, wherein the electrochemical response characteristic is square wave voltammetry, cyclic voltammetry, linear sweep voltammetry, differential pulse voltammetry (Differential pulse voltammetry) voltammetry) and electrochemical impedance spectroscopy (Electrochemical impedance spectroscopy), characterized in that the measurement by at least one means selected from the group consisting of. 15. The method of claim 14, wherein the liquid sample is a bodily fluid or blood. 15. The method of claim 14, wherein step c) is a redox pair ferro/ferricyanide, 3,3',5,5'-tetramethylbenzidine (TMB), 1-hydroxy-3-methoxycarbonyl-2 , 2,5,5-tetramethylpyrrolidone (CMH) and methylene blue (MB), characterized in that the method is carried out in the presence of a redox probe at least one selected from the group consisting of. The method according to claim 20, wherein the redox probe is provided in a form contained in a salt solution, wherein the concentration of the redox probe is set in the range of 2 to 10 mM.

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