A method for detecting adiponectin by polyaniline and polyacrylic modified silk screen carbon electrode

Abstract

This invention discloses a method and sensor for adiponectin detection using a polyaniline and polyacrylic acid-modified wire mesh carbon electrode, relating to the field of adiponectin detection technology. The method involves electrochemically activating the wire mesh carbon electrode, electropolymerizing it to form a polyaniline-polyacrylic acid composite modification layer, immobilizing adiponectin monoclonal antibody after activation with N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, blocking the antibody with bovine serum albumin, and reacting it with a serum sample. The adiponectin concentration is determined based on the peak potential shift and peak current change. This invention simultaneously achieves both conductive transport and antibody immobilization, reduces non-specific adsorption and serum interference, and improves detection sensitivity, repeatability, and quantitative accuracy.

Description

Technical Field

[0001] This invention relates to the field of adiponectin detection technology, and more specifically, to a method for adiponectin detection using a polyaniline and polyacrylic acid modified wire mesh carbon electrode. Background Technology

[0002] In existing technologies for constructing human serum adiponectin electrochemical immunosensors based on screen-printed carbon electrodes, the electrode interface typically needs to simultaneously perform both electron transport and recognition molecule immobilization functions. However, current common modification methods mainly rely on single conductive materials or single functional polymers, making it difficult to balance conductivity and bioimmobilization capabilities. When the modification layer prioritizes conductive transport, there are insufficient effective binding sites for antibody immobilization, limiting the loading capacity of the recognition layer. When the modification layer prioritizes the introduction of surface functional groups, the interfacial charge transfer efficiency decreases, thereby weakening the detection signal response. These contradictions are further amplified in serum sample scenarios because the serum matrix is ​​complex, making non-specific adsorption, background fluctuations, and decreased detection repeatability more likely to occur. Single polyaniline modification or single polyacrylic acid modification is less repeatable than the composite system formed by the simultaneous polymerization of the two, indicating that existing single-interface designs are difficult to maintain stable output under real serum detection conditions.

[0003] Based on this, it can be seen that the problem with the existing technology is that in the electrochemical detection of adiponectin in human serum, the screen carbon electrode modification interface lacks an interface construction mechanism that can simultaneously coordinate the conductive transmission capability and the antibody stable fixation capability, making it difficult for the sensor to achieve both high sensitivity and high repeatability detection in complex serum environments. Summary of the Invention

[0004] To overcome the aforementioned deficiencies of the prior art, this invention provides a method for adiponectin detection using polyaniline and polyacrylic acid-modified wire mesh carbon electrodes. This method addresses the problem that in existing electrochemical detection of human serum adiponectin based on wire mesh carbon electrodes, the electrode modification interface struggles to simultaneously achieve both conductivity and antibody stability, leading to insufficient detection sensitivity, repeatability, and anti-interference capabilities in complex serum environments.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] A method for detecting adiponectin using a polyaniline and polyacrylic acid modified carbon mesh electrode, comprising the following steps;

[0007] S1. Electrode activation treatment to obtain a wire mesh carbon electrode, and electrochemical activation treatment to increase the density of oxygen-containing functional groups on the surface of the wire mesh carbon electrode.

[0008] S2. Construction of composite modification layer: Aniline monomer and acrylic monomer are used to form an electropolymerization system. The polyaniline-polyacrylic acid composite modification layer is formed by electropolymerization on the surface of the activated wire mesh carbon electrode. In this layer, polyaniline forms a conductive transport network and polyacrylic acid provides carboxyl group fixation sites, so as to simultaneously form an electron transport channel and an antibody fixation interface on the same interface.

[0009] S3. Preparation of recognition electrode: The carboxyl groups on the surface of the polyaniline-polyacrylic acid composite modification layer are activated by N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and adiponectin monoclonal antibody is immobilized on the surface of the activated polyaniline-polyacrylic acid composite modification layer to obtain the recognition electrode.

[0010] S4. Non-specific site blocking: The recognition electrode is blocked with bovine serum albumin to reduce non-specific adsorption.

[0011] S5. Sample reaction and signal acquisition: A human serum sample containing adiponectin is added to the surface of the blocked recognition electrode to allow adiponectin to specifically bind to the adiponectin monoclonal antibody. The peak potential shift and peak current change before and after binding are obtained by electrochemical detection.

[0012] S6. Concentration determination: Establish a quantitative relationship between adiponectin concentration and peak potential shift and peak current change, and determine the adiponectin concentration in human serum samples accordingly.

[0013] In a preferred embodiment, in step S2, the electropolymerization of aniline monomer and acrylic monomer is carried out in an acidic electrolyte, and the activated wire mesh carbon electrode is subjected to multiple scans using cyclic voltammetry, so that polyaniline and polyacrylic acid grow simultaneously on the surface of the wire mesh carbon electrode to form a polyaniline-polyacrylic acid composite modification layer.

[0014] In a preferred embodiment, the acidic electrolyte comprises hydrochloric acid, sodium chloride, aniline monomer, and polyacrylic acid, wherein the aniline monomer forms polyaniline chains after anodizing, and the carboxyl groups of the polyacrylic acid participate in regulating the growth orientation of the polyaniline chains, so that the polyaniline-polyacrylic acid composite modification layer forms a through-hole porous conductive network.

[0015] In a preferred embodiment, the polyaniline-polyacrylic acid composite modification layer formed in S2 has a three-dimensional porous structure with a pore size of 50-200 nm, so as to simultaneously provide electron transport channels and antibody immobilization interfaces, and improve the conductivity and antibody loading capacity of the composite modification layer.

[0016] In a preferred embodiment, in step S3, an activation solution containing N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide is used to activate the carboxyl groups on the surface of the polyaniline-polyacrylic acid composite modification layer, thereby converting the carboxyl groups into active ester groups that can undergo a coupling reaction with the amino groups in the adiponectin monoclonal antibody molecule.

[0017] In a preferred embodiment, in step S3, adiponectin monoclonal antibody at a concentration of 400-800 μg / mL is added to the surface of the activated polyaniline-polyacrylic acid composite modified layer and incubated to fix it, so as to obtain the recognition electrode.

[0018] In a preferred embodiment, in step S4, the bovine serum albumin blocking treatment is performed using a gradient blocking method. First, a lower concentration of bovine serum albumin is used to fill the pores and surface defects of the polyaniline-polyacrylic acid composite modification layer, and then a higher concentration of bovine serum albumin is used to cover the surface of the recognition electrode to reduce non-specific adsorption, background noise, and serum interference.

[0019] In a preferred embodiment, in step S5, the electrochemical detection is performed using square wave voltammetry or differential pulse voltammetry; wherein, square wave voltammetry is used to obtain the peak current change, and differential pulse voltammetry is used to obtain the peak potential shift and peak current change when detecting low concentrations of adiponectin.

[0020] In a preferred embodiment, in step S6, the peak potential shift and peak current change are used as dual-parameter signals to establish a quantitative relationship between adiponectin concentration and the dual-parameter signals, and the adiponectin concentration in the human serum sample is output according to the quantitative relationship; wherein, the peak potential shift reflects the interfacial potential change caused by antigen-antibody binding, and the peak current change reflects the interfacial charge transfer change caused by antigen-antibody binding.

[0021] A polyaniline and polyacrylic acid modified wire mesh carbon electrode adiponectin sensor includes a wire mesh carbon electrode substrate, a polyaniline-polyacrylic acid composite modification layer disposed on the surface of the wire mesh carbon electrode substrate, an adiponectin monoclonal antibody layer fixed on the surface of the polyaniline-polyacrylic acid composite modification layer, and a blocking layer covering the non-recognition area of ​​the adiponectin monoclonal antibody layer.

[0022] The polyaniline-polyacrylic acid composite modification layer is formed by electropolymerization of aniline monomers and acrylic monomers on the surface of a wire mesh carbon electrode substrate after electrochemical activation treatment. Polyaniline forms a conductive transport network in the polyaniline-polyacrylic acid composite modification layer, and polyacrylic acid provides carboxyl group fixation sites in the polyaniline-polyacrylic acid composite modification layer.

[0023] The adiponectin monoclonal antibody layer was formed by covalently coupling the carboxyl groups on the surface of the polyaniline-polyacrylic acid composite modification layer with N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

[0024] The blocking layer is formed using bovine serum albumin to block the exposure sites outside the adiponectin monoclonal antibody layer, thereby reducing non-specific adsorption.

[0025] A polyaniline-polyacrylic acid composite modification layer and an adiponectin monoclonal antibody layer together constitute the adiponectin recognition electrochemical interface. The specific binding of adiponectin to the adiponectin monoclonal antibody causes a shift in peak potential and a change in peak current at the electrode interface, thereby enabling the electrochemical detection of adiponectin in human serum samples.

[0026] The technical effects and advantages of this invention are as follows:

[0027] 1. This invention forms a polyaniline-polyacrylic acid composite modification layer by electropolymerization on the surface of an activated wire mesh carbon electrode. The polyaniline forms a conductive network and the polyacrylic acid provides carboxyl group fixation sites, thereby achieving electron transfer and antibody fixation on the same electrode interface. This improves the problem that it is difficult to balance conductivity and biomolecule loading capacity under single material modification methods, and enhances the signal response and detection sensitivity of adiponectin detection.

[0028] 2. This invention utilizes polyacrylic acid to regulate the growth process of polyaniline chains, enabling the polyaniline-polyacrylic acid composite modification layer to form a continuous porous conductive structure. This increases the antibody immobilization interface while ensuring charge transfer efficiency, thereby improving the stability and repeatability of the recognition interface.

[0029] 3. This invention activates the carboxyl groups on the surface of the polyaniline-polyacrylic acid composite modification layer with N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and covalently immobilizes the adiponectin monoclonal antibody at the electrode interface to form a stable recognition layer, thereby reducing antibody desorption and improving the reliability of the adiponectin recognition process and the consistency of the detection results.

[0030] 4. This invention reduces non-specific adsorption at the electrode interface by performing bovine serum albumin blocking treatment on the recognition electrode, especially by using a gradient blocking method that covers pores and surface exposed sites in stages, thereby reducing background noise and the proportion of serum interference signals, and thus improving the detection specificity and detection stability under complex serum sample conditions.

[0031] 5. This invention, by simultaneously acquiring peak potential shift and peak current change after adiponectin specifically binds to adiponectin monoclonal antibody, provides a joint characterization of the interface changes caused by antigen-antibody binding, thereby reducing the impact of single signal fluctuations on detection results, improving the accuracy of adiponectin concentration analysis and the anti-interference ability for low-concentration sample detection.

[0032] 6. This invention establishes a dual-parameter quantitative relationship involving both peak potential shift and peak current change, thereby achieving quantitative detection of adiponectin concentration in human serum samples. Compared with single-parameter analysis methods, it has a lower detection limit, higher recovery rate, and lower serum interference fluctuations, thus improving the sensitivity, quantitative accuracy, and repeatability of adiponectin detection. Attached Figure Description

[0033] Figure 1 This is a flowchart of the method of the present invention;

[0034] Figure 2 This is a schematic diagram of the electrochemical immunosensor system of the present invention. Detailed Implementation

[0035] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0036] Figure 2 middle

[0037] BaseSPCE: Describes the basic structure of screen-printed carbon electrodes.

[0038] Ceramic Plastic Substrate: A ceramic / plastic substrate that provides mechanical support and insulation.

[0039] Carbon Ink Working Electrode: Carbon ink working electrode, serving as an active interface for electrochemical reactions;

[0040] Silver Reference Electrode: Silver reference electrode, used to maintain potential stability;

[0041] ProposedPAn-PAACompositeLayer: Polyaniline-polyacrylic acid composite modification layer

[0042] ElectrodePretreatment: Electrode pretreatment unit, which activates the electrode surface through cyclic voltammetry;

[0043] Electropolymerization: Electropolymerization unit, constructing a three-dimensional porous structure of PAn-PAA on the electrode surface;

[0044] NHSEDCActivation: Carboxyl activation unit, which generates antibody immobilization sites through the NHS / EDC reaction;

[0045] Biorecognition Layer: Biometrics Layer

[0046] Antibody Immobilization: Antibody immobilization unit, covalently bound to adiponectin monoclonal antibody;

[0047] BSABlocking: A closed unit that uses bovine serum albumin to reduce nonspecific adsorption;

[0048] DetectionUnit: Signal Detection and Processing Module

[0049] ElectrochemicalWorkstation: An electrochemical workstation that performs square wave voltammetry / differential pulse voltammetry detection.

[0050] Signal Processing: The signal processing unit uses a two-parameter model (ΔEp+ΔIp) for quantitative analysis.

[0051] Refer to the instruction manual appendix Figure 1-2 A method for detecting adiponectin using polyaniline and polyacrylic acid modified carbon mesh electrodes.

[0052] S1, Electrode activation treatment

[0053] First, a carbon mesh electrode was obtained and immersed in 0.01M phosphate buffer solution. Electrochemical activation was performed using cyclic voltammetry. The activation parameters were set as follows: settling time 1.000 s, initial potential -800 mV, upper limit potential 1200 mV, lower limit potential -800 mV, potential gradient 10 mV, scan rate 100 mV / s, and 10 cycles. This process generates oxygen-containing functional groups on the electrode surface through a redox reaction. The reaction mechanism is as follows:

[0054]

[0055]

[0056] Wherein, C represents the carbon electrode surface; the density of generated hydroxyl and carboxyl functional groups is quantitatively analyzed in the C1s spectrum by X-ray photoelectron spectroscopy; the peak at 288.5 eV corresponds to carboxyl carbon, and the peak at 286.2 eV corresponds to hydroxyl carbon; the experiment shows that the coverage of oxygen-containing functional groups on the electrode surface is increased to 42.3±3.5% after activation, which lays the foundation for the uniform growth of the subsequent polymer composite layer.

[0057] S2, Construction of composite modification layer

[0058] After electrode activation, a polyaniline-polyacrylic acid composite modification layer was further constructed. The activated wire mesh carbon electrode was placed in an electrolyte containing 0.9M HCl, 0.1M NaCl, 0.1M aniline monomer, and 15 mg / mL PAA, and in-situ electropolymerization was carried out using cyclic voltammetry. The scanning parameters were: settling time 1.000 s, potential range -400 to 1200 mV, scan rate 100 mV / s, and 6 cycles. There were two key reaction stages during the polymerization process.

[0059] The first stage is the aniline oxidation stage. At around 200mV, aniline monomers are anoly oxidized to generate cationic free radicals:

[0060]

[0061] The free radical then forms an oligomer chain through head-to-tail coupling, the chain length of which is sterically controlled by the carboxyl group of PAA;

[0062] The second stage is the polymer network formation stage; the carboxyl groups of PAA form hydrogen bonds with the imine groups (-NH-) of the polyaniline chain. The binding energy of the PAn chain, calculated using density functional theory, is -23.4 kJ / mol. This interaction causes the PAn chains to exhibit an extended conformation, ultimately forming a three-dimensional porous structure with pore sizes of 50-200 nm. The electrical conductivity of the composite layer was measured using the four-probe method. S / cm, which is about two orders of magnitude higher than that of pure PAn membrane;

[0063] To verify the effect of the composite modification layer formed by the simultaneous polymerization of polyaniline and polyacrylic acid, serum of the same concentration was tested at the same antibody concentration. Six parallel tests were performed for each condition, and the results are shown in Table 1.

[0064]

[0065] Table 1 Comparison of parallel detection results for different electrode modification methods

[0066] Table 1 shows that the reproducibility is best when polyaniline and polyacrylic acid are polymerized simultaneously, with a CV of 2.37%, which is significantly better than polyaniline modification alone or polyacrylic acid modification alone.

[0067] S3. Preparation of recognition electrode

[0068] After the formation of the polyaniline-polyacrylic acid composite modification layer, the recognition electrode is further prepared; the carboxyl groups on the surface of the composite layer are activated by the NHS / EDC two-step method.

[0069] During the EDC activation stage, 8 μL of a mixed solution containing 60 mM NHS and 100 mM MEDC was drop-coated onto the electrode surface and reacted at 37 °C for 20 min. EDC first forms an O-acylisourea intermediate with the carboxyl group:

[0070]

[0071] The intermediate is converted into a more stable succinimide ester (R-COO-NHS) in the presence of NHS.

[0072] During the antibody conjugation stage, 8 μL of adiponectin monoclonal antibody at a concentration of 400-800 μg / mL was added. The adiponectin monoclonal antibody was dissolved in 0.01 M phosphate buffer and incubated at 37°C for 40 min. The antibody underwent a nucleophilic substitution reaction with the active ester via a primary amino group (-NH2).

[0073]

[0074] The antibody fixation density was determined to be 3.2 ± 0.4 ng / mm² by fluorescence labeling, which is about 60% higher than that of the traditional glutaraldehyde cross-linking method.

[0075] S4, Nonspecific site blocking

[0076] After the adiponectin monoclonal antibody was fixed, nonspecific site blocking was further performed; 1.5% bovine serum albumin solution was used to block nonspecific sites, which has a dual mechanism of action.

[0077] The first is the physical covering mechanism; BSA is adsorbed onto the unmodified area of ​​the electrode surface through hydrophobic interactions, and its adsorption free energy is: ΔG=-15.7kJ / mol. Atomic force microscopy shows that the surface roughness decreases from 12.3±1.2nm to 8.7±0.9nm.

[0078] The second mechanism is charge shielding; at pH 7.4, BSA carries a net negative charge with a Zeta potential of -18.6 mV, which can repel negatively charged interfering proteins in serum; experiments have shown that this treatment reduces nonspecific adsorption to <2%;

[0079] Gradient BSA sealing was employed to reduce non-specific adsorption by stepwise filling of pores and surface defects; the results for different sealing processes are shown in Table 2.

[0080]

[0081] Table 2 Comparison of interface and interference indices for different sealing processes

[0082] As shown in Table 2, gradient BSA blocking reduces the nonspecific adsorption rate to 2.28%, the background noise to 0.361 μA, and the proportion of serum interference signal to 4.47% by stepwise filling of pores and surface defects.

[0083] S5. Sample Response and Signal Acquisition

[0084] After completing the non-specific site blocking, further sample reaction and signal acquisition are performed; serum samples are dropped onto the surface of the recognition electrode, allowing adiponectin antigen to bind to the immobilized antibody; antigen-antibody binding leads to interfacial charge transfer resistance (...). The quantitative relationship of the changes is as follows:

[0085]

[0086] in: The initial charge transfer resistance is approximately 1.2 kΩ. This represents the maximum resistance change (approximately 850Ω). This refers to the concentration of adiponectin antigen. The dissociation constant ( );

[0087] Within the standard concentration detection range, square wave voltammetry was used for detection; the parameters were set as follows: settling time 2.000 s, potential range -400 to 600 mV, step size 10 mV, frequency 15 Hz, pulse amplitude 80 mV; the oxidation peak current appeared near 300 mV. It is negatively correlated with antigen concentration, and its linear equation is:

[0088]

[0089] The linear detection range of this method is 0.1-100 ng / mL.

[0090] S6. Concentration determination

[0091] After obtaining the peak potential shift and peak current change, the concentration of adiponectin in the serum sample was further determined; for ultra-low concentration detection, i.e., <1 ng / mL, a two-parameter model of differential pulse voltammetry was used.

[0092]

[0093] This model extends the detection limit to 0.05 ng / mL by combining analysis of peak potential shift (ΔEp) and current change (ΔIp).

[0094] The general form of a two-parameter signal analytical model:

[0095]

[0096] Through potential-current co-calibration, the detection limit was extended from 0.1 ng / mL to 0.05 ng / mL, and the anti-interference capability was improved by 3 times;

[0097] To compare the performance of the single-parameter model and the two-parameter model, supplementary data is shown in the table below:

[0098]

[0099] Table 3 Comparison of detection performance between single-parameter and two-parameter models

[0100] Table 3 shows that the two-parameter model outperforms the single-parameter model in terms of detection limit, batch-specific RSD, recovery rate, and serum interference RSD.

[0101] Clinical testing process

[0102] In a clinical testing process, serum samples from patients with metabolic syndrome were used as the testing subjects. Traditional enzyme-linked immunosorbent assays (ELISA) require 50 μL of sample, take 4-6 hours to detect, have a detection limit of 1 ng / mL, and exhibit 15-20% non-specific binding. Using the PAn-PAA modified electrode of this invention, only 8 μL of serum sample is required, analysis is completed within 80 minutes, and the detection limit reaches 0.05 ng / mL. Actual test data shows that when the adiponectin concentration in the sample is 10 ng / mL, the electrode response current is 12.3 μA, compared to 8.7 μA for the traditional electrode, representing an improvement of approximately 41%, with a non-specific binding rate of <5%. This testing process demonstrates that this invention has good overall applicability in terms of small sample volumes, rapid detection, and high sensitivity, making it particularly suitable for point-of-care diagnostic scenarios in community hospitals.

[0103] System Implementation Process

[0104] In addition to the method and process described in steps S1 to S6 above, this invention can also be implemented in a system form. Corresponding to Figure 2 The system architecture is as follows: the electrode modification module is responsible for electrode activation and the construction of the polyaniline-polyacrylic acid composite modification layer; the signal conversion module is responsible for adiponectin monoclonal antibody immobilization, BSA blocking, and three-electrode system connection; the data analysis module is responsible for executing SWV and DPV detection modes and calling a two-parameter model for concentration conversion; each module can be integrated into the same detection device or set separately in the electrode preparation unit, detection unit, and software analysis unit; the system adopts a modular design, which is easy to adapt to electrochemical analyzers of different specifications, and can be used with a micro-injection device to achieve high-throughput automated detection.

[0105] Extended Implementation Process 1: Portable Detection Device Integration Solution

[0106] In one extended implementation, the PAn-PAA modified electrode can be integrated with a miniaturized electrochemical analysis module into a portable detection device. The main body of the device measures 8×5×2cm³. It includes a replaceable electrode slot, a microfluidic sample introduction system, a low-power detection circuit, and a Bluetooth data transmission module. The replaceable electrode slot uses a spring-loaded contact design, supporting rapid insertion and removal of standard three electrodes to accommodate different batches of modified electrodes. The microfluidic sample introduction system integrates a 5μL quantitative injection channel and a waste liquid storage chamber, achieving automatic sample aspiration through capillary action. The low-power detection circuit is based on an STM32 microcontroller, supporting SWV and DPV dual-mode detection with an operating current of <10mA. The Bluetooth data transmission module transmits the detection results to a smartphone application in real time, enabling data visualization and analysis. This implementation is suitable for home health monitoring scenarios.

[0107] Extended Implementation Process Two: Multi-Indicator Joint Detection Electrode Array

[0108] In another extended implementation, single-index adiponectin detection can be expanded to multi-index simultaneous analysis. Specifically, four independent carbon electrodes are printed on a 6×6cm² ceramic substrate, each modified with a PAn-PAA composite layer and immobilized with different antibodies, including antibodies corresponding to adiponectin, leptin, resistin, and C-reactive protein. The channel switching circuit uses an analog switch chip to achieve four-channel alternating detection, sharing the same reference electrode. Hydrophobic isolation walls made of polydimethylsiloxane are set between each electrode to prevent sample diffusion and crosstalk. This implementation can be used for joint screening of biomarkers related to metabolic syndrome, and multiple index data can be obtained in a single test.

[0109] Extended Implementation Process 3: Dry Storage Electrode Fabrication Technology

[0110] To address the issue of poor electrode stability in traditional wet storage, this invention also provides a dry storage process. After antibody fixation, a phosphate buffer solution containing 5% trehalose and 1% mannitol is coated onto the electrode surface. Subsequently, the electrode is pre-frozen at -40°C for 4 hours and then dried under a vacuum of 0.1 mbar for 24 hours to form a porous protective layer. Before detection, 20 μL of phosphate buffer solution is added, and the electrode activity is restored within 5 minutes. This process allows the electrode to be stored at 4°C in a dry environment for 6 months with a sensitivity decay of <15%, making it suitable for storage in remote medical facilities.

[0111] Extended Implementation Process 4: Flexible Wearable Sensors

[0112] In another extended implementation, the rigid ceramic substrate can be replaced with a flexible polyimide material; carbon electrode patterns are formed on a 50μm thick polyimide film using inkjet printing technology, with a bending radius of up to 3mm; the thickness of the electrode surface modification layer is controlled within 200nm, and a microneedle array with a length of 300μm is integrated to penetrate the stratum corneum to obtain tissue fluid; the system obtains energy through a near-field communication coil, eliminating the need for a built-in battery; this implementation process enables continuous dynamic monitoring and is suitable for tracking adiponectin levels in postoperative patients.

[0113] Extended Implementation Process Five: Automated Batch Inspection System

[0114] To address the high-throughput requirements of large medical institutions, this invention can also construct an automated batch testing system. Specifically, the SPCE array is arranged in an 8×12 configuration and matched to the standard 96-well plate size, with each well containing an independent three-electrode system. The system integrates an XYZ three-axis robotic arm sample loading platform with a positioning accuracy of ±0.1mm, supporting 96 samples / times of parallel sample loading. It is also equipped with a multi-channel electrochemical analyzer with 24 channels of simultaneous detection, increasing the detection throughput to 480 samples per hour. The system directly uploads test results through a laboratory information system interface, realizing an automated process from sample entry to report generation.

[0115] All of the above implementation processes revolve around the polyaniline-polyacrylic acid composite modification layer, adiponectin monoclonal antibody immobilization, bovine serum albumin blocking, and a two-parameter signal analysis model. Regardless of whether a desktop detection platform, portable detection device, multi-index detection array, dry storage, flexible wearable form, or automated batch detection system is used, the core detection mechanism remains consistent. That is, first, a highly reactive surface is formed through electrode activation treatment, then a polyaniline-polyacrylic acid composite modification layer is constructed through electropolymerization, then a recognition layer is formed by carboxyl activation and covalent coupling with the antibody, then non-specific adsorption is reduced by bovine serum albumin blocking, and finally, the peak potential shift and peak current change are obtained through SWV or DPV to determine the adiponectin concentration.

[0116] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for detecting adiponectin using a polyaniline and polyacrylic acid modified wire mesh carbon electrode, characterized in that, Includes the following steps; S1. Electrode activation treatment to obtain a wire mesh carbon electrode, and electrochemical activation treatment to increase the density of oxygen-containing functional groups on the surface of the wire mesh carbon electrode. S2. Construction of composite modification layer: Aniline monomer and acrylic monomer are used to form an electropolymerization system. The polyaniline-polyacrylic acid composite modification layer is formed by electropolymerization on the surface of the activated wire mesh carbon electrode. In this layer, polyaniline forms a conductive transport network and polyacrylic acid provides carboxyl group fixation sites, so as to simultaneously form an electron transport channel and an antibody fixation interface on the same interface. S3. Preparation of recognition electrode: The carboxyl groups on the surface of the polyaniline-polyacrylic acid composite modification layer are activated by N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and adiponectin monoclonal antibody is immobilized on the surface of the activated polyaniline-polyacrylic acid composite modification layer to obtain the recognition electrode. S4. Non-specific site blocking: The recognition electrode is blocked with bovine serum albumin to reduce non-specific adsorption. S5. Sample reaction and signal acquisition: A human serum sample containing adiponectin is added to the surface of the blocked recognition electrode to allow adiponectin to specifically bind to the adiponectin monoclonal antibody. The peak potential shift and peak current change before and after binding are obtained by electrochemical detection. S6. Concentration determination: Establish a quantitative relationship between adiponectin concentration and peak potential shift and peak current change, and determine the adiponectin concentration in human serum samples accordingly.

2. The method according to claim 1, characterized in that: In step S2, the electropolymerization of aniline monomer and acrylic monomer is carried out in an acidic electrolyte. Cyclic voltammetry is used to perform multiple scans on the activated wire mesh carbon electrode, so that polyaniline and polyacrylic acid grow simultaneously on the surface of the wire mesh carbon electrode to form a polyaniline-polyacrylic acid composite modification layer.

3. The method according to claim 2, characterized in that: The acidic electrolyte includes hydrochloric acid, sodium chloride, aniline monomer, and polyacrylic acid. The aniline monomer forms polyaniline chains after anodizing, and the carboxyl groups of the polyacrylic acid participate in regulating the growth orientation of the polyaniline chains, so that the polyaniline-polyacrylic acid composite modification layer forms a through-hole conductive network.

4. The method according to claim 3, characterized in that: The polyaniline-polyacrylic acid composite modification layer formed in S2 has a three-dimensional porous structure with a pore size of 50-200 nm, which simultaneously provides electron transport channels and antibody immobilization interfaces, and improves the conductivity and antibody loading capacity of the composite modification layer.

5. The method according to claim 1, characterized in that: In step S3, an activation solution containing N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide is used to activate the carboxyl groups on the surface of the polyaniline-polyacrylic acid composite modification layer, thereby converting the carboxyl groups into active ester groups that can undergo coupling reactions with the amino groups in the adiponectin monoclonal antibody molecule.

6. The method according to claim 5, characterized in that: In step S3, adiponectin monoclonal antibody at a concentration of 400-800 μg / mL is added to the surface of the activated polyaniline-polyacrylic acid composite modified layer and incubated to fix it, so as to obtain the recognition electrode.

7. The method according to claim 1, characterized in that: In step S4, the bovine serum albumin blocking treatment is carried out in a gradient blocking manner. First, a lower concentration of bovine serum albumin is used to fill the pores and surface defects of the polyaniline-polyacrylic acid composite modification layer, and then a higher concentration of bovine serum albumin is used to cover the surface of the recognition electrode to reduce non-specific adsorption, background noise and serum interference.

8. The method according to claim 1, characterized in that: In step S5, electrochemical detection is performed using square wave voltammetry or differential pulse voltammetry; square wave voltammetry is used to obtain peak current changes, while differential pulse voltammetry is used to obtain peak potential shifts and peak current changes during low-concentration adiponectin detection.

9. The method according to claim 1, characterized in that: In step S6, the peak potential shift and peak current change are used as dual-parameter signals to establish a quantitative relationship between adiponectin concentration and the dual-parameter signals, and the adiponectin concentration in the human serum sample is output according to the quantitative relationship; wherein, the peak potential shift reflects the interfacial potential change caused by antigen-antibody binding, and the peak current change reflects the interfacial charge transfer change caused by antigen-antibody binding.

10. A polyaniline and polyacrylic acid modified wire mesh carbon electrode adiponectin sensor, characterized in that, It includes a screen carbon electrode substrate, a polyaniline-polyacrylic acid composite modification layer disposed on the surface of the screen carbon electrode substrate, an adiponectin monoclonal antibody layer fixed on the surface of the polyaniline-polyacrylic acid composite modification layer, and a blocking layer covering the non-recognition area of ​​the adiponectin monoclonal antibody layer. The polyaniline-polyacrylic acid composite modification layer is formed by electropolymerization of aniline monomers and acrylic monomers on the surface of a wire mesh carbon electrode substrate after electrochemical activation treatment. Polyaniline forms a conductive transport network in the polyaniline-polyacrylic acid composite modification layer, and polyacrylic acid provides carboxyl group fixation sites in the polyaniline-polyacrylic acid composite modification layer. The adiponectin monoclonal antibody layer was formed by covalently coupling the carboxyl groups on the surface of the polyaniline-polyacrylic acid composite modification layer with N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The blocking layer is formed using bovine serum albumin to block the exposure sites outside the adiponectin monoclonal antibody layer, thereby reducing non-specific adsorption. The polyaniline-polyacrylic acid composite modification layer and the adiponectin monoclonal antibody layer together constitute the adiponectin recognition electrochemical interface. After adiponectin specifically binds to the adiponectin monoclonal antibody, it causes a shift in the peak potential and a change in the peak current at the electrode interface, so as to realize the electrochemical detection of adiponectin in human serum samples.

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