KR102316080B1 - Label-free electrochemical impedimetric immunosensor for sensitive detection of IgM rheumatoid factor in human serum - Google Patents

Abstract

The present specification discloses a method for manufacturing an immunosensor based on electrochemical impedance measurement for IgM-RF by performing simple immobilization of an IgG-Fc fragment antibody on a TA-SAM/IDWμE array. The formation of TA-SAM in the IDWμE array can be confirmed via AFM and EIS. TA-SAM provides a biocompatible environment for immobilizing a target IgG-Fc fragment while maintaining the biological activity of the immobilized IgG-Fc fragment. The covalent binding of the IgG-Fc fragment on the gold surface provides significant stability to the bioelectrode. The biosensor according to an embodiment of the present invention exhibited a detection limit ^{of 0.60 IU mL -1} and a dynamic range of ^{1-200 IU mL -1 .}

Description

Label-free electrochemical impedance measuring immunosensor for sensitive detection of IgM rheumatoid factor in serum and method for manufacturing the same

The present invention relates to a method, an apparatus, a computer program, and a computer-readable recording medium for detecting a rheumatoid factor.

Rheumatoid arthritis (RA) is a progressive and chronic inflammatory autoimmune disease that causes joint and pain in the elderly population, and is the most common autoimmune disease affecting nearly 1% of the global population.

Rheumatoid arthritis occurs as a result of immune dysfunction, in which altered self-epitopes themselves act as targets for autoantibodies (AAbs) produced by the host immune system. Some of the causal pathways and target proteins leading to AAb production in rheumatoid arthritis are being elucidated. AAbs, which are normally detected in the serum of rheumatoid arthritis patients, are composed of rheumatoid factors (RF) and anti-citrullinated peptide antibodies (ACPA). RF consists mainly of IgM, IgA and IgG antibodies to Fc fragments within the patient's own IgG antibody molecule. The baseline level of rheumatoid factor in human serum is 15 to 20 IU mL ⁻¹ . The IgM isotype (IgM RF) has the highest prevalence in the diagnosis of rheumatoid arthritis due to its polyvalency and is more effective for agglutination. Careful detection of IgM-RF is required for diagnosis, progression and early prediction of rheumatoid arthritis. Methods such as enzyme-linked immunosorbent assay (ELISA), agglutination test (eg, latex agglutination test (LAT) and hemagglutination test) are used as methods for detecting RF.

Aggregation reactions are simple and rapid diagnostic assays, but show significant variability, have high detection limits, and do not provide quantitative results. On the other hand, ELISA is expensive and is known to give false positive or false negative results. Despite these obstacles, efforts continue to improve the sensitivity, dynamic range, reliability, cost, and measurement speed of these analytical methods.

Detection performance is improving with the introduction of nano/micro electrodes and miniature sensing transducers in the manufacture of electrochemical sensors. Furthermore, the micro-manufacturing industry is showing tremendous ability to fabricate extremely delicate microelectrodes with low detection limits and significant dynamic range.

In this regard, in the present specification, RF antigens on an interdigitated wave type microelectrode array (IDWμE) using a thioctic acid-functionalized self-assembled monolayer (TA-SAM) It provides a method of manufacturing an electrochemical impedance immunosensor by fixing it.

A bio-electrode according to an embodiment for solving the above problems is formed on an electrode array and includes a sensing region to which an antibody is bound, and the sensing region has an interval of micrometers and is patterned in a wave shape.

A self-assembled monolayer (SAM) functionalized with thioctic acid (TA) may be immobilized on the sensing region. In the sensing region, a human IgG-Fc fragment may be covalently bound to the self-assembled monolayer (SAM). In the sensing region, bovine serum albumin (BSA) may be fixed to the self-assembled monolayer (SAM). The interval between the wave-shaped patterns in the sensing region may be 1 μm to 20 μm, and preferably, the interval between the wave-shaped patterns in the sensing region may be 7 μm.

In addition, the biosensor according to an embodiment for solving the above problems may be manufactured including the electrode described above.

In addition, a bio-electrode according to an embodiment for solving the above problem may include: (a) manufacturing an electrode array; (b) immobilizing a self-assembled monolayer (SAM) functionalized with thioctic acid (TA) on the electrode array; and (c) immobilizing a human IgG-Fc fragment to the SAM.

The fixing of the TA-functionalized SAM may include: immersing the electrode array in a TA solution to fix the TA-functionalized SAM to the electrode array; and washing the electrode array to remove thiol moieties not bound to the electrode array.

The fixing of the TA-functionalized SAM may include: rinsing the electrode array in ethanol and sonicating it; rinsing the electrode again with ethanol and deionized water; and drying the electrode array with N ₂ gas.

The immobilizing of the human IgG-Fc fragment to the SAM may include: providing a human IgG-Fc fragment solution to the electrode array; and maintaining the electrode array in a humid chamber to prevent the surface of the electrode array from drying out.

In the step of immobilizing the human IgG-Fc fragment to the SAM, the electrode array in which the human IgG-Fc fragment is immobilized to the SAM is washed with PBS (Phosphate-buffered saline), and the IgG-Fc fragment not bound to the electrode array is washed. It may further include the step of removing

Also, the electrode manufacturing method according to an embodiment may further include (d) fixing bovine serum albumin (BSA) to the SAM.

In addition, the method for detecting a rheumatism factor according to an embodiment for solving the above problems comprises the steps of diluting a human serum sample to prepare a diluent; applying the diluent to the bio-electrode; culturing the bio-electrode in a humidity chamber; and determining the concentration of IgM-RF (rheumatoid factors) in the human serum sample based on an impedance signal generated by analyzing the cultured bio-electrode by electrochemical impedance spectroscopy (EIS). A self-assembled monolayer (SAM) functionalized with thioctic acid (TA) is fixed to the sensing region of the bioelectrode, and a human IgG-Fc fragment may be covalently bound to the self-assembled monolayer (SAM).

According to the technical details described herein, a user can use an interdigitated wave type microelectrode array (IDWμE) using a thioctic acid-functionalized self-assembled monolayer (TA-SAM). ), it is possible to manufacture an electrochemical impedance immunosensor by fixing the RF antigen.

The SAM on the surface of the TA-modified microelectrode according to the present invention provides an appropriate sensing platform for IgM detection and provides the effect of properly preserving the biological activity of immobilized IgG-Fc.

In addition, the sensor according to the present invention is reduced (3.5 x 14 mm) and can be integrated to develop a portable immune sensing system to detect IgM-RF for POC applications, making it possible to fabricate a miniaturized and portable biosensor. provides an effect.

1 is a conceptual diagram showing an image of IDWμE and a method of manufacturing the same.
2 is a diagram showing the XPS spectrum of thioxane in the IDWμE electrode array.
3 shows bare IDWμE (a), TA-SAM/IDWμE (b), IgG-Fc/EDC/NHS/TA-SAM/IDWμE (c) and IgM-RF/BSA/IgG-Fc/TA-SAM/IDWμE It is a figure showing the AFM image of (d).
4 is a diagram illustrating a CV (Cyclic voltammograms) curve according to a change in an electrode.
5 is a diagram showing a Bode diagram of the impedance magnitude and phase at different stages of deformation of the IDWμE array surface.
6 is a diagram showing the impedance response of the BSA/IgG-Fc/TA-SAM/IDWμE array and the measured values and R _ct linear calibration plots with increasing concentration of IgM-RF diluted with 1X PBS.
7 is a diagram illustrating parameters of a randle circuit model according to an exemplary embodiment.
8 is a diagram showing the selectivity and storage stability of an electrochemical IgM-RF immune sensor.
9 shows a linear calibration plot of _{the impedance response and R ct} of the BSA/IgG-Fc/TA-SAM/IDWμE arrays measured in association with increasing concentrations of diluted IgM-RF (HS-IgM-RF) in human serum. is a drawing that
10 is a block diagram of an electrode manufacturing apparatus according to an embodiment of the present invention.
11 is a block diagram of an apparatus for detecting a rheumatism factor according to an embodiment of the present invention.

The following is merely illustrative of the principles of the invention. Therefore, those skilled in the art can devise various devices that, although not explicitly described or shown herein, embody the principles of the invention and are included in the spirit and scope of the invention. In addition, it should be understood that all conditional terms and examples listed herein are, in principle, expressly intended only for the purpose of understanding the inventive concept and are not limited to the specifically enumerated embodiments and states as such. .

The above-described objects, features and advantages will become more apparent through the following detailed description in relation to the accompanying drawings, and accordingly, those of ordinary skill in the art to which the invention pertains will be able to easily practice the technical idea of the invention. .

In the specification and claims, terms such as "first," "second," "third," and "fourth," are used, if any, to distinguish between like elements, and are not necessarily used in a specific sequence. or to describe the order of occurrence. It will be understood that the terms so used are interchangeable under appropriate circumstances to enable the embodiments of the invention described herein to operate, for example, in sequences other than those shown or described herein. Likewise, where methods are described herein as comprising a series of steps, the order of those steps presented herein is not necessarily the order in which those steps may be executed, and any described steps may be omitted and/or Any other steps not described may be added to the method.

Also, terms such as "left", "right", "front", "behind", "top", "bottom", "above", "below" in the specification and claims are used for descriptive purposes, It is not necessarily intended to describe an invariant relative position. It will be understood that the terms so used are interchangeable under appropriate circumstances to enable the embodiments of the invention described herein to operate otherwise than, for example, as shown or described herein. As used herein, the term “connected” is defined as being directly or indirectly connected in an electrical or non-electrical manner. Objects described herein as being "adjacent" to one another may be in physical contact with one another, in proximity to one another, or in the same general scope or area as appropriate for the context in which the phrase is used. The presence of the phrase “in one embodiment” herein refers to the same, but not necessarily, embodiment.

In addition, in the specification and claims, references to 'connected', 'connecting', 'fastened', 'fastening', 'coupled', 'coupled', etc., and various variations of these expressions, refer to other elements directly It is used in the sense of being connected or indirectly connected through other components.

In addition, the suffixes "module" and "part" for the components used in this specification are given or mixed in consideration of the ease of writing the specification, and do not have distinct meanings or roles by themselves.

In addition, the terms used herein are for the purpose of describing the embodiments and are not intended to limit the present invention. In this specification, the singular also includes the plural unless specifically stated otherwise in the phrase. As used herein, 'comprise' and/or 'comprising' means that a referenced component, step, operation and/or element is the presence of one or more other components, steps, operations and/or elements. or addition is not excluded.

In addition, in the description of the invention, if it is determined that a detailed description of a known technology related to the invention may unnecessarily obscure the gist of the invention, the detailed description thereof will be omitted. Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings.

In the present specification, rheumatoid factor-Immunoglobulin M (IgM-RF) based on an impedimetric-interdigitated wave type microelectrode array (IDWμE) is non-label- free) direct detection method is disclosed. IDWμE can be functionalized as a self-assembled monolayer (SAM) of thioctic acid (TA) for antigen immobilization. IDWμE functionalized with SAM can be characterized by atomic force microscopy, energy dispersive X-ray spectroscopy and X-ray photoelectron spectroscopy.

Self-assembled monolayers provide the simplest method for generating reproducible, ultrathin, ordered and oriented monolayers capable of maintaining the activity of biomaterials. The monolayers of the present invention, made naturally from sulfur-containing compounds in contact with metallic surfaces of gold or silver, provide a suitable platform for biomolecular immobilization. Carbodiimide activation with succinimide can be used to attach proteins/antibodies to the carboxylic acid end functional groups of SAM-forming compounds.

The covalent immobilization of IgG-Fc on the SAM-modified electrode array was morphologically characterized by atomic force microscopy (AFM) and electrochemically characterized by cyclic voltammetry and electrochemical impedance spectroscopy. became In the human IgG-Fc fragment, an Fc (fragment, crystallizable) fragment is one of the functional fragments of an antibody molecule. For the gene encoding the human IgG-Fc fragment, a sequence known in the art may be used.

Impedance measurements in the presence of redox probes (potassium ferrocyanide and potassium ferricyanide) showed significant changes in both impedance spectra and charge transfer resistance upon IgM-RF binding. _{It is proposed to use hexacyanoferrate [Fe(CN) 6} ^4-/3- ] as a redox probe due to the fast movement of TA induced by the hydrophilicity of SAM.

Impedance measurements were target specific and linear for redox probes and human serum, respectively, at increasing IgM-RF concentrations between 1 and 200 IU mL ^{−1 .}

The sensor exhibited a detection limit ^{of 0.6 IU mL -1} in the presence of a redox probe and a detection limit of 0.22 IU mL ^{-1 in human serum.} The biosensor showed good relative standard deviation (RSD, 4.96%) and repeatability (RSD, 2.31%) with acceptable selectivity for IgM-RF detection. The sensor also showed good stability for 3 weeks in 1X PBS and 4 °C. Therefore, a sensitive and stable immune sensor exhibiting IDWμE properties can be integrated with a miniature potentiostat to develop a sensing system to detect IgM-RF in point-of-care applications.

Chemicals and reagents

Rheumatoid factor antibody (IgM; ≥ 95 %, 1428 IU mL ^-1 ) and rheumatoid factor antigen (Fc fragment of human IgG, IgG-Fc; 90 %, 1.760 mg mL ^-1 ) were prepared with EastCoast Bio (North Berwick, USA) It was purchased from Arotec Diagnostics (Wellington, New Zealand). Thioctic acid 99.9% was purchased from EDQM (Strasbourg, France). Potassium ferrocyanide (K ₄ [Fe(CN) ₆ ]·3H ₂ O), potassium ferricyanide (K ₃ ([Fe(CN) ₆ ]) and human serum were purchased from Sigma-Aldrich (USA).

Phosphate-buffered saline (PBS) containing 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na ₂ HPO ₄ and 1.4 mM KH ₂ PO ₄ , PBS pH 7.4 with 0.05% Tween-20 and 0.5% BSA ( PBS containing Bovine Serum Albumin) was procured from Tech and Innovation (Gangwon, Korea). The buffer was prepared using deionized water (DI water, 18.2 MΩ cm) provided by Purescience (Jungwon, Korea). All other chemicals are analytical reagent grade and were used without further purification.

1 shows an image of an IDWμE fabricated with a size of 3.5 x 14 mm. Fig. 1c shows the sensing mechanism of the impedance immune sensor based _{on R ct} measured from the high-frequency semicircle of the impedance spectrum after fixing TA-SAM, IgG-Fc fragment, BSA and IgM-RF _{, C dl} is the double layer capacitance , R _ct is the charge transfer resistance, and R _s is the solution resistance.

Hereinafter, a method of manufacturing a bioelectrode according to an embodiment of the present invention will be described with reference to FIG. 1 .

IDWμE Array Fabrication

For the impedance measurement of IgM-RF, a gold-based IDWμE array on a glass slide substrate (3.5 x 14 mm) was fabricated using a microfabrication technique. Conductive Ti and Au (with a thickness of 25 and 50 nm, respectively) layers were deposited via electron beam evaporation. The sensing region of the IDWμE array was patterned through photolithography and chemical wet etching process. As the sensing area is patterned in a wave shape, the measurement range is improved by overcoming the edge effect compared to the conventional rectangular sensing area. The sensing region of the IDWμE array may have a spacing and a width of 1 μm to 20 μm, preferably 7 μm. In addition, a 96-well polystyrene cell culture plate with a well capacity of 400 μL was used as an electrolyte reservoir for impedance spectral measurement, and further as an adapter for connecting a conductive IDWμE array pad to a potentiostat.

Formation of self-assembled monolayers

The electrode array was cleaned via sonication for 2 minutes with successive solutions of acetone, ethanol and water, rinsed with DI water, and impurities removed under a low stream of N2 gas. To immobilize the TA-SAM, the electrode array was immersed in 10 mM TA ethanol (99.5%) for 12 h. After SAM modification, TA/IDWμE was extensively washed with 70% ethanol and deionized water to remove all unbound thiol moieties. To remove H-bonded thiol, the substrate was placed in fresh absolute ethanol and sonicated for 2 minutes to perform additional rinsing. Immediately after sonication, the electrodes were again rinsed extensively with 70% ethanol and ultrapure water and dried with _{N 2 gas.}

Atomic force microscopy (AFM) and Energy Dispersive X-ray Spectroscopy (EDX)

AFM was performed using an ambient air scanning probe microscope (XE-100 Park ystems, Korea). Images were recorded in normal non-contact mode using XEP software. Elemental analysis was performed using a scanning electron microscope (Japan Electron HITACHI S-4700) equipped with a HORIBA EX 250.

X-ray photoelectron spectroscopy

X-ray photoelectron spectroscopy (XPS) measurements were performed using a Thermo Electron Corporation (Waltham, Massachusetts, US) spectrometer with a _{monochromatic Al K α X-ray source.} Survey spectra were first recorded and then area scans were performed over the S(2p) and C(1s) optoelectronic binding energy regions. A 200 eV pass energy, 1 eV step size, 50 ms dwell time, and 400 μm x 400 μm X-ray spot size were used for the survey scans (range=1200 to -5 eV).

Area scans (Au 4f, C 1s, S 2p, O1s) revealed typical bandwidths of 15-20 eV, 50 eV pass energies, 1 eV step size and 50 ms dwell time. All XPS spectra were analyzed using a standard Gaussian curve fit with Shirley background subtraction applied.

Preparation of IgM-RF bioelectrodes

To covalently immobilize human IgG-Fc fragment (50 μgmL ⁻¹ ) on the TA-activated surface, 10 μL (50 μg) of human IgG-Fc fragment solution was dropped onto the electrode chip surface, and the binding process was performed. In order to prevent the electrode surface from drying during the period, it was kept in a humid chamber for 1 hour. The human IgG-Fc fragment was immobilized by a covalent coupling reaction between the TA-SAM reactive succinimidyl group on the electrode surface and the amino group of the IgG-Fc fragment. The formed electrodes (IgG-Fc/TA-SAM/IDWμEs) were thoroughly washed with 1X PBS to remove unbound IgG-Fc fragments. Finally, to block non-specific binding, the electrodes were thoroughly washed with IX PBS containing 0.05% tween-20 and 0.5% BSA for 2 min to form BSA/IgG-Fc/TA-SAM/IDWμEs. According to the World Health Organization (WHO) standard RF serum (serum), IgM-RF concentration can be expressed as ^{IU mL -1.} Next, different amounts of IgM-RF were mixed with 10 mM PBS (pH 7.4) or human serum to obtain final concentrations of ^{1, 10, 50, 100 and 200 IU mL -1 .} A fresh solution was prepared for each experiment.

Cyclic voltammetry

1 , electrochemical measurements were performed using an IVIUM CompactStat potentiostat (Eindhoven, Netherlands) with a modified bare IDWμE array comprising a pair of gold conductive pads acting as actuation/sensing electrodes and counter/reference electrodes, respectively. became Cyclic voltammetry using an IVIUM CompactStat potentiostat (Eindhoven, Netherlands) in a background solution _{of 5 mM K 3} Fe(CN) ₆ /K ₄ Fe(CN) ₆ (1:1) in 1X PBS (10 mM, pH 7.4) (CV) was performed. CV scans were measured in a potential window of +0.5 V to -0.5 V and a scan rate of ^{50 mVs −1 .} A three-electrode system was used with a platinum coil as the counter electrode, Ag/AgCl (saturated NaCl) as the reference electrode, and an exposed or modified IDWμE array as the working electrode.

Electrochemical impedance spectroscopy

Impedance analysis of the modified electrode was performed using an input potential of 50 mV amplitude scanned over the 1-100,000 Hz frequency range in which the frequency ratio was increased by 5 steps in 10 divisions, 5 mM K ₃ Fe(CN) ₆ /K _{4 in 1X PBS.} Fe(CN) ₆ (1:1) background solution was performed. Before experiments, _{all electrolyte solutions were deoxygenated by bubbling with pure N 2} gas, and all experiments were performed at room temperature (25° C.). Impedance fitting was performed by an equivalent circuit model on the measured impedance spectrum using nonlinear curve fitting software (ZView; Scribner Associates Inc., Southern Pines, USA). For all electrochemical reaction studies of immune sensors, pH 7.4, the recommended pH for biomolecules to initiate higher biological activity, was chosen. The process of immobilization of TA-SAM, immobilization of IgG-Fc to an electrode array and development of an immune sensor is shown in FIG. 1 along with the basic operating principle. Fig. 1a shows the wave type pattern of the sensor region, Fig. 1b shows a configuration in which TA-SAM is formed on an Au-IDWμE electrode array, and an IgG-Fc fragment and BSA are coupled to TA-SAM is conceptually show Hereinafter, experimental results of the sensor according to an embodiment of the present invention will be described.

Characterization of TA self-assembled monolayers

EDX measurement was performed to confirm the immobilization of the SAM layer on the electrode surface by studying the elemental composition of the electrode after SAM treatment. A detailed description of the EDX spectrum will be omitted. The efficiency and thiol-gold interaction properties of SAM immobilization were further verified using XPS. S 2p and C 1s XPS bands focusing on the contributions of sulfur and carbon atoms, which characterize the SAM-Au interaction, are shown in Fig. 2 b and c, respectively.

Morphological Characteristics of Fabricated Bioelectrodes

AFM topological images were recorded to study the morphological properties of the electrode surface after each major IDWμE fabrication step. 3 shows bare IDWμE (a), TA-SAM/IDWμE (b), IgG-Fc/EDC/NHS/TA-SAM/IDWμE (c) and IgM-RF/BSA/IgG-Fc/TA-SAM/IDWμE (d) shows the AFM image. Hereinafter, it will be described with reference to FIG. 3 .

Figure 3a shows the polycrystalline surface of bare gold IDWμE with an average roughness of 1.95 nm. The electrode surface after TA-SAM fixation was transformed into a uniform and highly densely ordered structure with a smooth topography and an average roughness of 1.06 nm when compared with bare gold IDWμE (Fig. 3b). The surface of the electrode modified with the IgG-Fc/TA-SAM film was changed to a particle structure, indicating successful immobilization of the protein antibody on the surface and showing an increase of 3.72 nm in average roughness (Fig. 3c). After binding of IgM-RF to the BSA/IgG-Fc/TA-SAM/IDWμE surface, a clear protruding topography was generated over the entire electrode surface due to stable immune complex formation with an increase in surface roughness of 5.03 nm (Fig. 3d). .

Hereinafter, electrochemical properties of the bioelectrode manufactured according to an embodiment of the present invention will be described.

Cyclic voltammetry

Cyclic voltammetry (CV) of bare Au-IDWμE arrays and modified Au-IDWμE arrays was performed in _{a 1x PBS solution of 5 mM K 3} Fe(CN) ₆ /K ₄ Fe(CN) ₆ (1:1). This was used to record the changes that occurred in the electrochemical behavior introduced at different stages of electrode change (Figure 4). The CV of bare IDWμE _{shows the electrochemical response to Fe(CN) 6} ^4- / ^3- with oxidation and reduction peaks (Fig. 4, curve i). After the electrodes were functionalized with TA-SAM (TA-SAM/IDWμE), the CV curve changed sharply and the peak current of TA-SAM/IDWμE decreased dramatically when compared to IDWμE (Fig. 4, curve ii). The effect may be increased due to the formation of very high levels of the SAM insulating layer, which results in a hindrance of _{[Fe(CN) 6} ] ^{3-/4- ion transport towards the electrode.} In addition, the CV of EDC-NHS/TA-SAM/IDWμE (Fig. 4, curve iii) shows clear oxidation and reduction peaks compared to the TA-SAM layer due to the formation of a neutral amine bond between the terminal carboxyl groups of SAM and EDC/NHS. indicates Thus, ions can migrate to the modified EDC-NHS/TA-SAM electrode.

Moreover, the IgG-Fc/EDC-NHS/TA-SAM/IDWμE electrode (Fig. 4, curve iv) shows a reduced ferricyanide response, and an enlarged peak-to-peak separation, indicating that the This suggests that the insulating biomolecule layer is fixed on the electrode surface.

Electrochemical impedance spectroscopy (EIS)

EIS was used to characterize the interfacial properties of electrode surfaces during electrode fabrication and electrode reaction studies using different IgM-RF concentrations. Figure 5 shows the IDWμE array surface at different transformation steps for detection of IgM-RF in 1X PBS solution (pH 7.4) of _{5 mM K 3} Fe(CN) ₆ /K ₄ Fe(CN) _{6 (1:1).} Bode diagrams of impedance magnitude (|Z|) (a) and phase (-Φ) (b) are shown. Hereinafter, it will be described with reference to FIG. 5 .

5 a and b are impedance spectra shown as Bode diagrams (Z vs -Φ) in electrodes changed differently using a _{5 mM K 3} Fe(CN) ₆ /K4Fe(CN) _{6 (1:1) solution in 1X PBS.} shows In the curve i of the Bode diagram in Fig. 5a, the bare IDWμE array mainly shows three distinct sections in the impedance magnitude (|Z|), which is the double layer capacitance (mid frequency region), and the charge transfer with the highest |Z| value. Corresponds to resistance (low frequency region), and solution resistance (high frequency region). It is assumed that the equivalent circuit model (Randall circuit) is also fitted with the impedance spectrum of the electrode shown in Fig. 5A. At frequencies higher than 10 kHz, the total impedance of the system is governed by the ohmic resistance and parasitic impedance of the _{electrolyte R S .} In the measured frequency range, the electrode interface impedance was parallel to _{R ct .}

In addition, the capacitive electrode interface impedance is expressed as a constant phase element (CPE) of the measured impedance, and can be expressed as 1/[CPE-T·(iω) ^CPE-P ] (where i and ω is the imaginary unit and the angular frequency of the sine wave signal). The parameters of the Randall circuit model determined by fitting the measured impedance spectrum are shown in FIG. 7 .

The change in the measured impedance magnitude (|Z|) after the deformation process at the IDWμE array surface _{affects the R ct} and C _dl of the electrode. Electrodes incubated with TA showed larger |Z|. This indicates a larger-scale SAM functionalization at the electrode surface _{, resulting in blocking of [Fe(CN) 6} ] ^3- / ⁴ ion transport towards the electrode surface (Fig. 5a, curve ii). After EDC-NHS activation, the measured |Z| The value decreased, which resulted in the formation of neutral amine bonds that promote ion migration to the EDC/NHS modified electrode (Fig. 5a, curve iii). In addition, immobilization of IgG-Fc fragments (50 μg mL ⁻¹ ) and BSA (0.5%) to EDC/NHS activated SAM electrodes leads to an increase in |Z| values at low frequencies. (Fig. 5a, curve iv and curve v). In particular, cross-linking of biomolecules onto the electrode surface is suspected to interfere with the tunneling of redox pairs towards the sensor platform.

Electrochemical Reaction Characterization of IgM-RF/BSA/IgG-Fc/TA-SAM/IDWμE Bioelectrodes

The IgM-RF electrochemical impedance immune sensor according to an embodiment of the present invention is well expressed as _{R ct indicating resistance at low frequencies.} These results demonstrate that the R _ct variation on the modified electrode can be a detection parameter for the delicate measurement of IgM-RF. To this end, this resistance was measured over a wide range of IgM concentrations (1 to 200 IU mL ^{−1 ).} To evaluate the response to IgM-RF, immune sensors were incubated with different concentrations of IgM-RF for 10 min. Then, EIS measurements were performed at 5 mM [Fe(CN) ₆ ^-3 / ^{-4 ].}

Figure 6a shows the impedance response of the BSA/IgG-Fc/TA-SAM/IDWμE array performed at 5mM [Fe(CN) ₆ ^-3 / ^{-4 ] and the increase in the concentration of IgM-RF diluted with 1X PBS.} Shows the measured value, b of FIG. 6 is from 1 to 200 IU mL ^-1 _{R ct} linear calibration plots for increasing IgM concentrations are shown. Each data point is the average of three independent measurements (n = 3). Ranges are indicated using error bars.

As shown in FIG. 6 , the R _ct value increased with increasing concentration of IgM rheumatoid factor. As shown in FIG. 6 a, _{the change in R ct} value was proportional to the linear increase in the concentration of IgM-RF in the range of 1-200 IU mL ^{-1 .} As shown in Fig. 6b, the linear regression equation is expressed as y = 4415115.5 + 2477.7 * x with a correlation coefficient of 0.9983.

Also, the calculated limit of detection was 0.6 IU mL ^-1 (S/N = 3). Limit-of-detection (LOD) was determined using (3*SD/sensitivity), where SD represents the standard deviation of the measured impedance signal with respect to blank and sensitivity represents the slope of the linear curve. Response studies _{clearly showed that R ct} increased linearly with increasing IgM-RF concentration. This phenomenon can be explained by the formation of a kinetic barrier to electron movement due to the increase of IgM-RF molecules bound to the immobilized IgG-Fc fragment on the electrode array.

The change in R _ct of the deformed electrode was used as a quantification parameter for the delicate measurement of IgM-RF. Therefore, R _ct was used as a physical signal in the sensor to detect and quantify IgM-RF through specific immune responses promoted on the electrode surface.

In this way, the fabricated immune sensor established a proof-of-concept for use in the direct detection of IgM-RF without further amplification of electrical signals. Sensors are other RF biosensors already reported in the literature and commercially available IgM-RF quantitative ELISA kits, Biomatic (15.6-1000 mIU mL ^-1 ), IgM RF (Immco, 6-50 IU mL ^-1 ), Sigma (0- 50 IU mL ^-1 ), Diamedix (100 IU mL ^-1 ), and Inova (6.25-100 IUmL ^-1 ) showed significant dynamic range and detection limit.

Interference studies

IgM-RF (10 IU) such as human chorionic gonadotrophin (HCG; 10 IUmL ^-1 ), insulin (10 IUmL ^-1 ) or tumor necrosis factor-α (TNF-α; 10 IUmL-1) mL-1) The effect of some agents interfering with measurement was tested in the BSA/IgG-Fc/EDC-NHS/TA-SAM/IDWμE bioelectrode reaction.

Figure 8 shows the selectivity (a) and storage stability (a) of the electrochemical IgM-RF immunosensor at an IgM-RF concentration of 10 IU mL ^-1 (100 μL of 5 mM [Fe(CN) ₆ ^{-3/-4 ] in PSB).} b) is shown. The inset in the graph represents the change in _{R ct} as a function of the interference agent ( FIG. 8 a ) and time ( FIG. 8 b ). Each data point represents the average of three values (n = 3). Ranges are indicated using error bars.

With these agents is mixed with 1X PBS were separately exposed to the bio-electrode, each of the EIS spectra as shown in Figure 8 is a ^{_{[Fe (CN) 6 3- /}} 4-] 1-100,000 Hz frequency region in solution was obtained over The test results showed that the response of the bioelectrode was not significantly affected even in the presence of these interfering agents compared to the control bioelectrode (BSA/IgG-Fc/EDC-NHS/TA-SAM/IDWμE), and the prepared bioelectrode The electrode showed high selectivity for IgM-RF measurement.

Storage stability of bioelectrodes

BSA/IgG-Fc/EDC-NHS/TA-SAM/IDWμE arrays were selected and R _ct change after IgM-RF addition was measured as a function of time (storage stability). The storage stability of the BSA/IgG-Fc/EDC-NHS/TA-SAM/IDWμE arrays was determined by measuring the _{R ct} in IgM-RF at ^{10 IU mL −1 over a regular interval of 3 days for approximately 21 days.} Specifically, BSA/IgG-Fc/EDC-NHS/TA-SAM/IDWμE bioelectrodes are stored at 4 °C in 1X PBS when not in use. As a result, it was confirmed that 98% of the initial response of the bioelectrode was maintained after 3 weeks as shown in FIG. 8B .

Reproducibility

Reproducibility is an important factor in the clinical application of immune sensors. The reproducibility of the immune sensor was tested via intra-assay and inter-assay relative standard deviations (RSD). The intra-assay precision of the immune sensor was tested by detecting 10 IU mL ^-1 of IgM-RF in three replicates, 200 in three independently prepared BSA/IgG-Fc/EDC-NHS/TA-SAM/IDWμE. Inter-assay accuracy was evaluated by measuring IgM-RF of IU mL ^{-1 .} The intra- and inter-assay RSDs were evaluated to be 4.96% and 2.31%, respectively, indicating acceptable precision and reproducibility with the immunosensor.

Excellent electrode stability is observed due to the covalent bonding of IgM-RF molecules with the gold surface, which prevents leakage of biomolecules from the electrode surface. In particular, impedance microelectrode-based IgM-RF detection has several advantages over other methods. The detection and analysis method as in an embodiment of the present invention (a) operates as a label, (b) is widely applicable, and (c) the Warburg diffusion phenomenon occurring in a micro-machined electrode (d) sample preparation is economical, and (e) direct signal reading in the form of electrical signals is possible.

IgM-RF measurement in human serum samples

To demonstrate the practical applicability of the proposed immune sensor, IgM-RF was detected in human serum samples using a BSA/IgG-Fc/EDC-NHS/TA-SAM/IDWμE array. To avoid matrix effects and build robust calibration curves, dilutions of human serum in IX PBS buffer (1:500) were prepared. Human serum samples containing IgM-RF were applied to the electrodes and incubated for 10 minutes in a constant humidity chamber to react with all available IgG-Fc fragments. After incubation with human serum, EIS measurements were recorded in 5-mM [Fe(CN) ₆ ^-3 / ^{-4 ] electrolyte solution.}

9 shows the impedance response (a) of the BSA/IgG-Fc/TA-SAM/IDWμE array measured in relation to the increase in the concentration of IgM-RF (HS-IgM-RF) diluted in human serum (a) and the increase in human serum. A linear calibration plot of _{R ct} versus diluted IgM-RF concentrations (1 to 200 IU mL ⁻¹ ) is shown (b). Each data point represents the average of three independent measurements (n = 3), and ranges are indicated using error bars.

Compared with blank, it was observed that the serum sample impedance signal increased with increasing IgM-RF concentration (Fig. 9a). The concentration of IgM-RF in human serum samples was determined with reference to the calibration curve shown in FIG. 9B .

The electrochemical response measured with human serum samples is dependent on the IgM-RF concentration, demonstrating the absence of matrix interference during IgM detection and suggesting the excellent specificity of the developed impedance sensor. The change in R _ct value is proportional to the linear increase in IgM-RF concentration in the range of 10-200 IU mL ^{-1 .} The linear regression equation can be expressed as y = 274,422.6 + 1435.13 * x with a correlation coefficient of 0.9941 ( FIG. 9 b ). In addition, the calculated LOD was 0.22 IU mL ^-1 (S/N = 3).

As described above, an immunosensor based on electrochemical impedance measurement for IgM-RF can be fabricated by performing simple immobilization of an IgG-Fc fragment antibody on a TA-SAM/IDWμE array. The formation of TA-SAM in the IDWμE array can be confirmed via AFM and EIS. TA-SAM provides a biocompatible environment for immobilizing a target IgG-Fc fragment while maintaining the biological activity of the immobilized IgG-Fc fragment. The covalent binding of the IgG-Fc fragment on the gold surface provides significant stability to the bioelectrode, effectively demonstrating its use as an impedance biosensor in measuring IgM-RF in serum samples. The immune sensor exhibited a detection limit of 0.60 IU mL ^-1 and a dynamic range of ^{1-200 IU mL -1 .} Also, due to this small architecture (3.5 × 14 mm) and the ability to integrate this sensor with a small potentiostat, the sensor according to an embodiment of the present invention can be used for POC applications. In addition, the sensor according to an embodiment of the present invention showed limited stability for 3 weeks. In addition, the fabricated bioelectrodes are cost-effective because they do not require expensive and sophisticated instruments and fluorescent dyes. The sensor according to an embodiment of the present invention may be used for diagnosing an individual suspected of having arthritis even if the ^{RF level is less than 1 IU mL -1 .}

The electrode manufacturing method and the rheumatism factor detection method according to the embodiment described above include an electrode manufacturing apparatus and rheumatism provided with a hardware configuration for operation and a control unit consisting of a processor, a memory, a communication unit, and a user interface (input/output device) for controlling the same. It may be implemented as a factor detection device.

10 is a block diagram of an electrode manufacturing apparatus according to an embodiment of the present invention. The electrode manufacturing apparatus 100 according to an embodiment of the present invention includes an electrode array forming unit 110 for forming an electrode array, and thioctic acid (TA) functionalized SAM (self) in the electrode array as in the electrode manufacturing method described above. -assembled monolayer) may include a SAM fixing part 120 and an antibody fixing part 130 fixing a human IgG-Fc fragment to the SAM. The electrode manufacturing apparatus may further include a BSA fixing unit 140 for fixing bovine serum albumin (BSA) to the SAM and a control unit 150 for controlling the components.

For example, the SAM fixing unit 120 immerses the electrode array in a TA solution to fix the TA-functionalized SAM to the electrode array, and washes the electrode array to remove thiol moieties not bound to the electrode array. can be removed The SAM fixing unit 120 may place the electrode array in ethanol and perform ultrasonic treatment to perform rinsing, rinsing the electrode again with ethanol and deionized water, and drying the _{electrode array with N 2 gas.}

In addition, the antibody fixing unit 130 may provide a human IgG-Fc fragment solution to the electrode array, and maintain the electrode array in a humid chamber to prevent the surface of the electrode array from drying out. The antibody fixing unit 130 washes the electrode array in which the human IgG-Fc fragment is immobilized on the SAM with phosphate-buffered saline (PBS) to remove the IgG-Fc fragment not bound to the electrode array.

11 is a block diagram of an apparatus for detecting a rheumatism factor according to an embodiment of the present invention. The rheumatoid factor detection apparatus 200 according to an embodiment of the present invention includes a diluent preparation unit 210 that prepares a diluent by diluting a human serum sample as in the rheumatoid factor detection method described above, and applies the diluent to the bio-electrode, The detection unit 220 determines the concentration of IgM-RF (rheumatoid factors) in a human serum sample based on an impedance signal generated by culturing the bio-electrode in a humidity chamber and analyzing the cultured bio-electrode by electrochemical impedance spectroscopy (EIS). and a control unit 230 for controlling the components.

In addition, the electrode manufacturing method and the rheumatism factor detection method according to the embodiment described above may be implemented in the form of a program command that can be executed through various computer means and recorded in a computer-readable medium. The computer-readable medium may include program instructions, data files, data structures, etc. alone or in combination. The program instructions recorded on the medium may be specially designed and configured according to the embodiment, or may be known and used by those skilled in the art of computer software. Examples of the computer-readable recording medium include magnetic media such as hard disks, floppy disks and magnetic tapes, optical media such as CD-ROMs and DVDs, and magnetic such as floppy disks. - includes magneto-optical media, and hardware devices specially configured to store and carry out program instructions, such as ROM, RAM, flash memory, and the like. Examples of program instructions include not only machine language codes such as those generated by a compiler, but also high-level language codes that can be executed by a computer using an interpreter or the like.

Each of the drawings referenced in the description of the previous embodiment is merely an embodiment shown for convenience of description, and items, contents, and images of information displayed on each screen may be modified and displayed in various forms.

Although the present invention has been described with reference to one embodiment shown in the drawings, this is merely exemplary, and those of ordinary skill in the art will understand that various modifications and equivalent other embodiments are possible therefrom. Accordingly, the true technical protection scope of the present invention should be determined by the technical spirit of the appended claims.

Claims (15)

Board;
an electrode array formed on the substrate, in which a plurality of fingers of a first electrode and a plurality of fingers of a second electrode are alternately disposed; and
a sensing region formed on the electrode array;
Each of the plurality of fingers of the first electrode and the plurality of fingers of the second electrode has an interval and a width in micrometers and is patterned in a regular wave shape without straight edges,
A self-assembled monolayer (SAM) functionalized with TA (Thotic Acid) is fixed to the plurality of fingers of the first electrode and the plurality of fingers of the second electrode patterned in the wave shape,
A human IgG-Fc fragment is covalently bound to the TA-functionalized SAM,
An electrode having Bovine Serum Albumin (BSA) fixed to the SAM to which the human IgG-Fc fragment is covalently bound to block non-specific binding.
delete delete delete The method of claim 1,
The distance and width of the plurality of fingers of the first electrode and the plurality of fingers of the second electrode are 1 μm to 20 μm, respectively.
6. The method of claim 5,
An electrode having an interval and a width of each of the plurality of fingers of the first electrode and the plurality of fingers of the second electrode of 7 μm.
A biosensor comprising an electrode according to any one of claims 1 to 6.
(a) manufacturing an electrode array formed on a substrate, in which a plurality of fingers of a first electrode and a plurality of fingers of a second electrode are alternately disposed;
(b) immobilizing a self-assembled monolayer (SAM) functionalized with TA (thioctic acid) on the electrode array by immersing the electrode array in 10 mM TA ethanol;
(c) 10 μL (50 μg) of a human IgG-Fc fragment solution in the TA-functionalized SAM was dropped onto the electrode array, and the electrode array was placed in a humid chamber to prevent the surface of the electrode array from drying out. ) to covalently bind a human IgG-Fc fragment to the TA-functionalized SAM; and
(d) immobilizing BSA (Bovine Serum Albumin) for blocking non-specific binding to SAM to which the human IgG-Fc fragment is covalently bound,
The plurality of fingers of the first electrode and the plurality of fingers of the second electrode each have an interval and a width of a micrometer unit, and are patterned in a regular wave shape without straight edges.
9. The method of claim 8,
After step (b),
washing the electrode array with 70% ethanol and deionized water to remove thiol moieties not bound to the electrode array;
rinsing the electrode array by sonicating it in absolute ethanol;
rinsing the electrode array with 70% ethanol and ultrapure water; and
The electrode manufacturing method further comprising the step of drying the electrode array with N _{2 gas.}
delete delete 9. The method of claim 8,
After step (c), washing the electrode array with phosphate-buffered saline (PBS) to remove the human IgG-Fc fragment not bound to the TA-functionalized SAM.
delete diluting the human serum sample to prepare a diluent;
applying the diluent to the bio-electrode;
culturing the bio-electrode in a humidity chamber; and
Comprising the step of determining the concentration of IgM-RF (rheumatoid factors) in the human serum sample based on the impedance signal generated by EIS (Electrochemical impedance spectroscopy) analysis of the cultured bio-electrode,
The bioelectrode,
Board;
an electrode array formed on the substrate, in which a plurality of fingers of a first electrode and a plurality of fingers of a second electrode are alternately disposed; and
a sensing region formed on the electrode array;
Each of the plurality of fingers of the first electrode and the plurality of fingers of the second electrode has an interval and a width in micrometers and is patterned in a regular wave shape without straight edges,
A self-assembled monolayer (SAM) functionalized with TA (Thotic Acid) is fixed to the plurality of fingers of the first electrode and the plurality of fingers of the second electrode patterned in the wave shape,
A human IgG-Fc fragment is covalently bound to the TA-functionalized SAM,
A method for detecting a rheumatism factor in which BSA (Bovine Serum Albumin) is fixed to the SAM to which the human IgG-Fc fragment is covalently bound.
delete

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