An electrochemical biosensor for detecting COVID-19 nucleic acid in blood and saliva

Abstract

This invention relates to the field of electrochemical biosensor technology, and discloses an electrochemical biosensor for detecting COVID-19 nucleic acid in blood and saliva. The construction method is as follows: gold nanoparticles are electrodeposited on the surface of a carbon electrode, and then a poly(3,4-ethylenedioxythiophene)-polycarboxylic acid betaine hydrogel is drop-coated onto the surface to obtain PEDOT-pCBMA hydrogel / AuNPs / SPCE; then, ferrocene-immobilized capture DNA is used to modify the electrode surface, and finally, methylene blue is applied to the electrode to obtain the electrochemical biosensor. This invention uses the above-mentioned electrochemical biosensor, and the detection results adopt a ratiometric electrochemical signal mode, which is more intuitive and accurate; it utilizes the high hydrophilicity and electroneutrality of zwitterionic hydrogels to achieve ultra-low contamination detection in saliva or blood; and the three-dimensional porous structure of the hydrogel effectively enhances electron transfer and mass transport, achieving high sensitivity.

Description

Technical Field

[0001] This invention relates to the field of electrochemical biosensor technology, and in particular to an electrochemical biosensor for detecting COVID-19 nucleic acid in blood and saliva. Background Technology

[0002] COVID-2019 is caused by the SARS-CoV-2 coronavirus. SARS-CoV-2 infection typically spreads rapidly from person to person, through direct contact with infected individuals as well as via droplets and aerosol particles. Due to the immediacy and high infectivity of the SARS-CoV-2 coronavirus, early screening and rapid testing are crucial for better management, control, and treatment of the infection.

[0003] The two main diagnostic methods for SARS-CoV-2 infection are molecular methods (detecting the RNA or antigen of the SARS-CoV-2 coronavirus) and serological tests (detecting antibodies against the SARS-CoV-2 coronavirus). Studies have shown that most patients develop an antibody response 7-14 days after infection, which limits the effectiveness of antibody-based diagnosis in the acute phase of infection. Compared to antibody serological testing, nucleic acid testing can detect infected patients at a much earlier stage and is currently the "gold standard" for novel coronavirus testing. However, the RT-PCR testing process is typically time-consuming (>3 hours) and complex. Furthermore, false negative results are frequent in nucleic acid testing, so many patients cannot receive timely isolation and treatment while awaiting RT-PCR test results or diagnosis, leading to wider community transmission.

[0004] For COVID-19 diagnostic testing, nasopharyngeal and oropharyngeal swabs are the officially designated professional standards. However, sample collection can cause discomfort for patients, and healthcare workers in contact with patients are also at risk of infection. Saliva, on the other hand, is a clear, thin biofluid with antibacterial, bacteriostatic, and flushing properties, and contains abundant disease biomarkers, and has been used to diagnose a variety of diseases. Numerous studies have also shown that saliva can transmit the SARS-CoV-2 virus through droplets, aerosols, coughing, and sneezing. Furthermore, saliva testing is a non-invasive, simple, and safe method.

[0005] Electrochemical biosensors, characterized by their simplicity and high sensitivity, are widely used in the quantitative detection of disease biomarkers. However, their high sensitivity to the non-specific adsorption of interfering biomolecules at their sensing interfaces severely hinders their practical clinical application in complex biofluids such as blood, serum, saliva, and sweat. Therefore, it is crucial to construct anti-fouling electrochemical biosensors capable of preventing the adsorption of non-specific substances at the sensing interface.

[0006] Currently, the application of electrochemical biosensors in complex biological environments (such as human serum, blood, urine, sweat, and saliva) remains limited. A common solution is to construct sensing interfaces modified with antifouling materials to resist nonspecific protein adsorption. These antifouling materials are typically highly hydrophilic and electrically neutral, and the dense coating they form provides a physical barrier against nonspecific protein adsorption. Polyethylene glycol (PEG), zwitterionic polymers (ZIPs), and peptides are the most commonly used antifouling materials. PEG, due to its high immunogenicity, is easily oxidized and degraded under physiological conditions, resulting in poor long-term stability. Peptides have good hydrophilicity and are easy to design, but short linear peptides are easily hydrolyzed and enzymatically digested. Long-chain heteropeptides exhibit excellent antifouling properties and stability, but their synthesis is complex and their cost is high. Zwitterionic polymers, due to their large steric hindrance and neutral polymer backbone, are widely used in the construction of antifouling interfaces. However, while currently reported antifouling coatings offer excellent resistance to nonspecific adsorption, the introduction of highly grafted, non-conductive antifouling materials can lead to the formation of a passivation layer on the electrode surface. This inertization of the sensing surface and impaired detection sensitivity makes interference resistance, accuracy, and sensitivity in electrochemical sensing systems contradictory and mutually exclusive. Summary of the Invention

[0007] The purpose of this invention is to provide an electrochemical biosensor for detecting COVID-19 nucleic acid in blood and saliva. The electrochemical biosensor of this invention has a simple preparation process, low cost, universality, and is easy to scale up. When applied, it can collect and analyze information with high sensitivity and accuracy by taking a small amount of sample, realizing trace detection of COVID-19 nucleic acid in blood or saliva. It is also simple to operate, low in cost, has a wide detection range, low detection limit, high sensitivity, and high accuracy.

[0008] The electrochemical biosensor prepared in this invention uses a ratiometric electrochemical signal mode for detection results, which is more intuitive and accurate. It utilizes the high hydrophilicity and electroneutrality of zwitterionic hydrogels to achieve ultra-low contamination detection in saliva or blood. Furthermore, the three-dimensional porous structure of the hydrogels effectively enhances electron transfer and mass transport, achieving high sensitivity.

[0009] To achieve the above objectives, the present invention provides a method for constructing an electrochemical biosensor for detecting COVID-19 nucleic acid in blood and saliva, comprising the following steps:

[0010] Step 1: Mix chloroauric acid trihydrate HAuCl4·3H2O, NaCl and water to obtain a mixed aqueous solution. Immerse the screen-printed carbon electrode SPCE in the mixed aqueous solution and deposit AuNPs / SPCE by cyclic voltammetry.

[0011] Step 2: The poly(3,4-ethylenedioxythiophene)-polycarboxybetaine (PEDOT-pCBMA) hydrogel was drop-coated onto AuNPs / SPCE in a non-gelled state, allowing it to spread evenly over the entire working electrode surface. The reaction was then carried out under cold conditions to obtain PEDOT-pCBMA hydrogel / AuNPs / SPCE.

[0012] Step 3: N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride EDC, N-hydroxysuccinimide NHS, ferrocene-imposed capture DNA C1, and water are mixed to obtain a mixed aqueous solution. The mixed aqueous solution is reacted at room temperature for 30 minutes, and then dropped onto the electrode and reacted for 1-5 hours to obtain C1-Fc / PEDOT-pCBMA hydrogel / AuNPs / SPCE;

[0013] Step 4: Mix methylene blue (MB) and water to obtain a mixed aqueous solution. Add the mixed aqueous solution dropwise to the surface of the modified electrode from Step 3 and react for 2-30 minutes to obtain MB / Cl-Fc / PEDOT-pCBMA hydrogel / AuNPs / SPCE, which is the electrochemical biosensor.

[0014] Further, in step 1, the concentration of HAuCl4·3H2O in the mixed aqueous solution is 0.1-5.0 mM, and the concentration of NaCl is 0.1-1.0 M; in the cyclic voltammetry, the low potential for electrodeposition is -1.0 to -0.5 V, the high potential is 0.1 to 1.0 V, the scan rate is 10-100 mV / s, and the number of scan cycles is 2-10.

[0015] Furthermore, the preparation method of the poly(3,4-ethylenedioxythiophene)-polycarboxybenzene PEDOT-pCBMA hydrogel in step 2 is as follows:

[0016] CBMA monomer, 3,4-ethylenedioxythiophene monomer EDOT, N,N'-methylenebisacrylamide MBAA, tetramethylethylenediamine TEMED, ammonium persulfate APS, NaCl and water were mixed to obtain a mixed aqueous solution. After sonication, the mixed aqueous solution was refrigerated at 1-4℃ for 6-12 hours to obtain PEDOT-pCBMA hydrogel.

[0017] Furthermore, the concentrations of CBMA monomer in the mixed aqueous solution are 1-8 M, 3,4-ethylenedioxythiophene (EDOT) monomer is 0.1-1 M, N,N'-methylenebisacrylamide (MBAA) is 5-15 mM, tetramethylethylenediamine (TEMED) is 0.02-0.1 mM, ammonium persulfate (APS) is 0.01-0.1 M, and NaCl is 0.1-2 M.

[0018] Furthermore, in step 2, the drop volume of the PEDOT-pCBMA hydrogel solution is 0.5-5 μL.

[0019] Further, in step 3, the concentration of EDC in the mixed aqueous solution is 0.1-2M, the concentration of NHS is 0.1-2M, the concentration ratio of EDC to NHS is 4:1, the concentration of Cl is 0.1-5μM, and the volume of the mixed aqueous solution dropped onto the electrode is 10-100μL.

[0020] Furthermore, the concentration of MB in the mixed aqueous solution in step 4 is 10-50 μM.

[0021] The present invention also provides an electrochemical biosensor, which is constructed using the above-described method.

[0022] The present invention also provides a kit for detecting COVID-19 virus in blood and / or saliva, which is not intended for the diagnosis and treatment of the disease, and the kit contains the aforementioned electrochemical biosensor.

[0023] The present invention also provides the application of the above-described electrochemical biosensor in the preparation of a kit for detecting COVID-19 virus in blood and / or saliva.

[0024] Furthermore, the detection method of the electrochemical biosensor is as follows:

[0025] Step 1: Using a disposable screen-printed carbon electrode (SPCE), the prepared electrochemical biosensor is used as the working electrode, the carbon electrode as the counter electrode, and the silver / silver chloride electrode as the reference electrode to form a three-electrode system. N836 target DNA (T1) is dropped onto the working electrode for reaction. Then, the three-electrode system is immersed in a pH 7-8 buffer solution. The change in current signal is measured by an electrochemical workstation. Based on the relationship between the standard T1 concentration in the sample cell and the current signal, a standard working curve is plotted.

[0026] Step 2: Using the prepared electrochemical biosensor as the working electrode, the carbon electrode as the counter electrode, and the silver / silver chloride electrode as the reference electrode, a three-electrode system is formed. The same volume of the test solution as the T1 solution in Step 1 is added to the working electrode for reaction. The three-electrode system is then immersed in a pH 7-8 buffer solution. The change in current signal is measured using an electrochemical workstation. Based on the working curve, the concentration of T1 in the test solution is determined.

[0027] Furthermore, the pH 7-8 buffer solution is a 0.2M phosphate buffer solution.

[0028] This invention employs a ratiometric electrochemical signal mode for the detection of target N836 DNA; utilizing the high hydrophilicity and electroneutrality of zwitterionic hydrogels, it enables ultra-low contamination detection in complex saliva or blood; and utilizing the three-dimensional porous structure of conductive zwitterionic hydrogels, it effectively enhances electron transfer and mass transport performance, achieving highly sensitive electrochemical sensing detection.

[0029] In clinical applications of reagents, complex biological samples often contain numerous interfering proteins and cells, which indiscriminately and nonspecifically adhere to the electrochemical sensing interface, forming a high-impedance layer. This severely affects the accuracy and sensitivity of analytical detection, thus limiting the clinical use of electrochemical biosensors. Studies have shown that the nonspecific adsorption of interfering proteins is mainly based on charge attraction and hydrophobic interactions. The highly sensitive and anti-fouling electrochemical biosensor based on conductive zwitterionic hydrogels / gold nanoparticles prepared in this invention possesses a highly hydrophilic and electrically neutral conductive structure. The high hydrophilicity helps to bind more free water molecules at the sensing interface, forming a dense hydrated layer to counteract hydrophobic interactions and resist nonspecific protein adsorption. The electrically neutral biosensing interface effectively avoids electrostatic attraction of charged interfering substances at the interface. Therefore, the electrochemical biosensor of this invention exhibits excellent anti-fouling performance, achieving highly accurate and sensitive COVID-19 nucleic acid detection.

[0030] The advantages and positive effects of the electrochemical biosensor for detecting COVID-19 nucleic acid in blood and saliva described in this invention are as follows:

[0031] 1. The electrochemical biosensor of the present invention has a simple preparation process, low cost, strong versatility, and is easy to scale up.

[0032] 2. The electrochemical biosensor of the present invention is based on gold nanoparticles. The nano-sized spherical structure effectively increases the electroactive area and accelerates the electron transfer process, which is beneficial to enhancing the sensitivity of the electrochemical biosensor.

[0033] 3. The electrochemical biosensor of the present invention is based on a conductive zwitterionic hydrogel structure, which has a three-dimensional porous structure, high hydrophilicity and electroneutrality, and excellent anti-fouling performance and biocompatibility. Its three-dimensional network structure and gold nanoparticles accelerate electron transfer and ion transport, enabling anti-fouling and high-accuracy detection of COVID-19 nucleic acid in saliva or blood.

[0034] 4. The electrochemical biosensor of the present invention is used to detect COVID-19 SARS-CoV-2. It can sensitively and accurately collect and analyze information with a small amount of saliva or blood to achieve trace detection. Furthermore, it has a short detection time, simple operation, low cost, wide detection range, low detection limit, high sensitivity, and high accuracy, making it a rapid and efficient method for nucleic acid detection of COVID-19 SARS-CoV-2.

[0035] 5. The electrochemical biosensor of the present invention has a detection range of 4 μM-0.9 mM for the target analyte N836 DNA, with the linear equation being: I(μA)=0.0123C+0.571, a linear correlation coefficient of 0.996, and a limit of detection of 0.13 μM.

[0036] The electrochemical biosensor of this invention has a detection range of 1 fM to 1 nM for COVID-19 SARS-CoV-2 nucleic acid, and the linear equation is: I Fc / I MB (μA) = 0.0578LgC + 1.27, the linear correlation coefficient is 0.995, and the limit of detection is 0.26fM.

[0037] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0038] Figure 1 This is a scanning electron microscope image of the gold nanoparticles electrodeposited in Example 1 of the present invention;

[0039] Figure 2 A photograph of the PEDOT-pCBMA hydrogel synthesized in Example 1 of this invention;

[0040] Figure 3 This is a scanning electron microscope image of the PEDOT-pCBMA hydrogel / gold nanoparticles synthesized in Example 1 of this invention;

[0041] Figure 4 The Fourier transform infrared spectrum of the PEDOT-pCBMA hydrogel synthesized in Example 1 of this invention;

[0042] Figure 5 Thermogravimetric curve of the synthesized PEDOT-pCBMA hydrogel prepared in Example 1 of this invention;

[0043] Figure 6 The static water contact angle diagram of the PEDOT-pCBMA hydrogel / AuNPs modified electrode prepared in Example 1 of this invention;

[0044] Figure 7 This is a current response diagram of the electrochemical biosensor prepared in Example 2 of the present invention to saliva of different dilution concentrations;

[0045] Figure 8 This is a current response diagram of the electrochemical biosensor prepared in Example 2 of the present invention to fetal bovine serum at different dilution concentrations;

[0046] Figure 9 This is a graph showing the change in current signal for the detection of different concentrations of the target analyte N836 nucleic acid by the electrochemical biosensor prepared in Example 2 of this invention;

[0047] Figure 10 This is a standard curve diagram of the electrochemical biosensor prepared in Example 2 of the present invention for the detection of different concentrations of the target analyte N836 nucleic acid. Detailed Implementation

[0048] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0049] Unless otherwise defined, the technical or scientific terms used in this invention should be understood in their ordinary sense by those skilled in the art. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without inventive effort are within the scope of protection of this invention. Experimental methods not specifically described in the following embodiments are generally determined according to national standards. Experimental instruments, equipment, and reagents not specified in the following embodiments are all commercially available materials.

[0050] Unless otherwise defined or stated, all technical and scientific terms used in this invention have the same meaning as those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention.

[0051] In the embodiments of this invention, the current signal was recorded using a Shanghai Chenhua CHI 660E electrochemical workstation. The current signal was tested on a conventional three-electrode electrochemical system using a disposable screen-printed carbon electrode (SPCE). The carbon electrode served as the working electrode and the counter electrode, while a silver / silver chloride electrode served as the reference electrode, forming a three-electrode system purchased from Changsha Sanjun. C1 (N-836capture: NH2-C6-ATT TCC TTG GGT TTG TTC TG-Fc) and T1 (N-836: CAGAAC AAA CCC AAG GAA AU) were both purchased from Shanghai Sangon Biotech.

[0052] All other chemical reagents and raw materials are commercially available products, purchased from Sigma-Aldrich.

[0053] Example 1

[0054] Preparation of PEDOT-pCBMA hydrogel / AuNPs modified electrode.

[0055] Step (1) Preparation of gold nanoparticle (AuNPs) modified electrodes:

[0056] Chloroauric acid trihydrate (HAuCl4·3H2O), NaCl, and water were mixed, with the concentration of chloroauric acid trihydrate (HAuCl4·3H2O) being 1 mM and the concentration of NaCl being 0.5 M. The mixture was stirred until homogeneous to obtain a gold nanoparticle deposition solution. 500 μL of the above deposition solution was dropped onto a disposable screen-printed carbon electrode (SPCE). Cyclic voltammetry was performed with a potential range of -0.9–0.5 V and a scan rate of 50 mV / s for 5 cycles to obtain AuNPs / SPCE.

[0057] Step (2) Synthesis of PEDOT-pCBMA hydrogel:

[0058] A mixed aqueous solution was prepared by mixing CBMA monomer, 3,4-ethylenedioxythiophene monomer (EDOT), N,N'-methylenebisacrylamide (MBAA), tetramethylethylenediamine (TEMED), ammonium persulfate (APS), NaCl, and water. The concentrations of CBMA monomer, EDOT monomer, N,N'-methylenebisacrylamide (MBAA), tetramethylethylenediamine (TEMED), ammonium persulfate (APS), and NaCl were 4 M, 0.15 M, 15 mM, 0.04 mM, and 1 M respectively. After sonication for 1 min, the mixture was refrigerated for 8 hours to obtain PEDOT-pCBMA hydrogel.

[0059] Step (3) Preparation of PEDOT-pCBMA hydrogel / AuNPs modified electrode:

[0060] Take 3 μL of the PEDOT-pCBMA hydrogel liquid synthesized in step (2) and drop it onto the AuNPs / SPCE prepared in step (1) in the ungelled state. Spread it evenly on the entire working electrode. Place the electrode with PEDOT-pCBMA hydrogel drop onto a 4℃ refrigerator for 8 hours to obtain the PEDOT-pCBMA hydrogel / AuNPs modified electrode.

[0061] Results Analysis

[0062] Scanning electron microscope image of electrodeposited gold nanoparticles in Example 1 is shown below. Figure 1 As shown, the structure is a uniform spherical structure with an average diameter of approximately 50 nm, densely attached to the electrode surface. A photograph of the PEDOT-pCBMA hydrogel synthesized in Example 1 is shown below. Figure 2As shown, it exhibits a typical gel state.

[0063] Scanning electron microscope image of PEDOT-pCBMA hydrogel / AuNPs as shown below Figure 3 As shown, the PEDOT-pCBMA hydrogel has a loose, porous structure with stacked layers, which effectively increases the electroactive surface area and enhances electron transfer and ion transport capabilities. The Fourier transform infrared spectrum of the PEDOT-pCBMA hydrogel is shown below. Figure 4 As shown, 1373 and 1583 cm -1 The peaks at 1232 and 1141 cm⁻¹ correspond to the C=C (interring stretching mode) and C=C (asymmetric stretching mode) vibrations of the thiophene ring, respectively, as well as the stretching vibrations of the quinone structure. -1 The peaks at 928 and 681 cm⁻¹ are related to the stretching vibration of the COC bond in the methylene dioxy group. -1 The peaks at 1722 and 1475 cm⁻¹ correspond to the CS peak in thiophene, indicating successful polymerization of PEDOT. -1 The peaks at these locations correspond to the stretching vibration of the C=O group and the bending vibration of the CN group in the monomer, respectively. The band generated by the CH stretching vibration in pCBMA is at 3012 cm⁻¹. -1 Visible at 3365cm in all samples. -1 The peak at this point corresponds to the stretching vibration of the OH groups on the surface, indicating the successful synthesis of pCBMA. The thermogravimetric curve of the PEDOT-pCBMA hydrogel is shown below. Figure 5 As shown, the residual carbon content at 800℃ is relatively low, at 7.16%, while the water content is relatively high, at 64.11%. This is attributed to the large specific surface area of ​​the PEDOT-pCBMA hydrogel, where more hydrophilic groups can bind more water molecules, resulting in high hydrophilicity. Figure 6 As shown, the static water contact angle of the PEDOT-pCBMA hydrogel / AuNPs modified electrode is 20.5 degrees, indicating that the modified interface has high hydrophilicity.

[0064] Example 2

[0065] Construction of a highly sensitive, anti-fouling electrochemical biosensor based on conductive zwitterionic hydrogels:

[0066] Step (1) N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), ferrocene-imposed capture DNA (C1), and water were mixed to obtain a mixed aqueous solution, wherein the concentration of EDC was 0.8M and the concentration of NHS was 0.2M. The mixed aqueous solution was reacted at room temperature for 30 minutes, and then 50 μL of the mixed aqueous solution was dropped onto the electrode prepared in step (3) of Example 1 and reacted for 3 hours, thus successfully preparing C1-Fc / PEDOT-pCBMA hydrogel / AuNPs / SPCE.

[0067] In step (2), methylene blue (MB) and water are mixed to obtain a mixed aqueous solution with a concentration of 20 μM of MB. 50 μL of the mixed aqueous solution is added dropwise to the Cl-Fc / PEDOT-pCBMA hydrogel / AuNPs / SPCE obtained in step (1) and reacted for 10 min to obtain MB / Cl-Fc / PEDOT-pCBMA hydrogel / AuNPs / SPCE, which is a highly sensitive and anti-fouling electrochemical biosensor based on conductive zwitterionic hydrogel / gold nanoparticles.

[0068] Results Analysis

[0069] The antifouling capability of a highly sensitive, antifouling electrochemical biosensor based on conductive zwitterionic hydrogels.

[0070] The highly sensitive, anti-fouling electrochemical biosensor based on conductive zwitterionic hydrogel from Example 2 was immersed in human saliva buffer solutions of different concentrations. The current signals before and after immersion in the human saliva buffer solutions of different concentrations for half an hour were tested. The different concentrations of human saliva buffer solutions were obtained by adding different volumes of human saliva to water and diluting the human saliva to different concentrations, namely 1%, 10%, 20%, 50%, and 100% (pure saliva, anhydrous).

[0071] The results are as follows Figure 7 As shown, the results indicate that the peak current signal of the biosensor changes very little after being immersed in human saliva of different concentrations, and the peak potential remains basically unchanged, indicating excellent anti-fouling performance.

[0072] The highly sensitive, anti-fouling electrochemical biosensor based on conductive zwitterionic hydrogel from Example 2 was immersed in fetal bovine serum (FBS) buffer solutions of different concentrations. The current signals before and after immersion in the FBS buffer solutions of different concentrations for half an hour were tested. The different concentrations of FBS buffer solutions were obtained by adding different volumes of FBS to water and diluting the FBS to different concentrations, namely 1%, 10%, 20%, 50%, and 100% (pure FBS, anhydrous).

[0073] Fetal bovine serum contains abundant proteins and cells, and can mimic 40% of the environment of human serum. Results are as follows... Figure 8 As shown, the results indicate that the highly sensitive anti-fouling electrochemical biosensor based on conductive zwitterionic hydrogels exhibits minimal signal changes after different concentrations of fetal bovine serum, demonstrating excellent anti-biofouling performance even in highly complex biological environments. This effectively enhances analytical detection accuracy and avoids false positive results.

[0074] Example 3

[0075] Application of highly sensitive and anti-fouling electrochemical biosensors based on conductive zwitterionic hydrogels for COVID-19 nucleic acid detection.

[0076] The methods for COVID-19 nucleic acid testing are as follows:

[0077] Step (1) Using the highly sensitive and anti-fouling electrochemical biosensor of the conductive zwitterionic hydrogel prepared in Example 2 as the working electrode, the carbon electrode as the counter electrode, and the silver / silver chloride electrode as the reference electrode, a three-electrode system was formed. 50 μL of COVID-19 SARS-CoV-2 target DNA N836 was incubated in the working electrode (T1). Then, 50 μL of phosphate buffer solution (pH 7.4) was used to soak the three-electrode system. The change in current signal was measured using differential pulse voltammetry on an electrochemical workstation. A standard working curve was plotted based on the relationship between the standard T1 concentration in the sample cell and the current signal.

[0078] Step (2) uses the highly sensitive and anti-fouling electrochemical biosensor of the conductive zwitterionic hydrogel prepared in Example 2 as the working electrode, the carbon electrode as the counter electrode, and the silver / silver chloride electrode as the reference electrode to form a three-electrode system. 50 μL of the test solution is incubated with the working electrode, and the change in current signal is measured using differential pulse voltammetry on an electrochemical workstation. Based on the working curve, the concentration of COVID-19 SARS-CoV-2 target DNA (T1) in the test solution is tested.

[0079] Results Analysis

[0080] Electrochemical biosensors for different concentrations (10 -15 M-10 -8 M) Changes in the current signal for nucleic acid detection of target analyte N836, as shown in... Figure 9 As shown, the standard working curve is as follows: Figure 10 As shown, the detection results indicate that the MB / C1-Fc / PEDOT-pCBMAhydrogel / AuNPs modified electrode has a detection range of 1 fM to 1 nM for COVID-19 SARS-CoV-2 nucleic acid, and the linear equation is: I Fc / I MB (μA) = 0.0578LgC + 1.27, with a linear correlation coefficient of 0.995 and a limit of detection of 0.26 fM. The pCBMA-pSBMA hydrogel-modified electrode exhibits high sensitivity for the detection of COVID-19 SARS-CoV-2 nucleic acid. This is attributed to the high porosity and high specific surface area provided by the three-dimensional network structure of the hydrogel, which shortens the transport path of ions and substances, and the high conductivity provided by the conductive zwitterionic framework.

[0081] Currently reported antifouling coatings, while providing excellent resistance to non-specific adsorption, also introduce high-graft-density non-conductive antifouling materials, resulting in a passivation layer on the electrode surface. This makes the sensing surface inert and impairs detection sensitivity. This makes anti-interference, accuracy, and sensitivity in electrochemical sensing systems contradictory and mutually exclusive. To address this issue, this invention adds gold nanoparticles to the electrochemical biosensor. Gold nanoparticles effectively increase the electrode's electroactive area and enhance detection sensitivity. The modified PEDOT-pCBMA hydrogel, with its excellent hydrophilicity and electroneutrality, resists the non-specific adsorption of interfering proteins, improving detection accuracy. Through activation with EDC / NHS activator, the amino groups of ferrocene-modified DNA-capturing groups (C1-Fc) are covalently bonded to the carboxyl groups of the PEDOT-pCBMA hydrogel. Subsequently, methylene blue is modified to electrostatically bind to the phosphate backbone of the DNA single strand. This ratiometric electrochemical method enables precise and sensitive detection of target DNA in complex biofluids, providing a theoretical basis for advancing the clinical application of electrochemical biosensors.

[0082] Therefore, the present invention employs the above-mentioned electrochemical biosensor for detecting COVID-19 nucleic acid in blood and saliva. The detection results adopt a ratiometric electrochemical signal mode, which is more intuitive and accurate. It utilizes the high hydrophilicity and electroneutrality of zwitterionic hydrogels to achieve ultra-low contamination detection in saliva or blood. Furthermore, the three-dimensional porous structure of the hydrogels effectively enhances electron transfer and mass transport, achieving high sensitivity.

[0083] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for constructing an electrochemical biosensor for detecting COVID-19 nucleic acid in blood and saliva, characterized in that, Includes the following steps: Step 1: Mix chloroauric acid trihydrate HAuCl4·3H2O, NaCl and water to obtain a mixed aqueous solution. Immerse the screen-printed carbon electrode SPCE in the mixed aqueous solution and deposit AuNPs / SPCE by cyclic voltammetry. Step 2: The poly(3,4-ethylenedioxythiophene)-polycarboxybetaine (PEDOT-pCBMA) hydrogel was drop-coated onto AuNPs / SPCE in a non-gelled state, allowing it to spread evenly over the entire working electrode surface. The reaction was then carried out under cold conditions to obtain PEDOT-pCBMA hydrogel / AuNPs / SPCE. Step 3: N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), ferrocene-imposed capture DNA C1, and water are mixed to obtain a mixed aqueous solution. The mixed aqueous solution is reacted at room temperature for 30 minutes, and then dropped onto the electrode and reacted for 1-5 hours to obtain C1-Fc / PEDOT-pCBMA. hydrogel / AuNPs / SPCE; Step 4: Mix methylene blue (MB) and water to obtain a mixed aqueous solution. Add the mixed aqueous solution dropwise to the surface of the modified electrode from Step 3 and react for 2-30 minutes to obtain MB / Cl-Fc / PEDOT-pCBMA hydrogel / AuNPs / SPCE, which is the electrochemical biosensor.

2. The construction method according to claim 1, characterized in that, In step 1, the concentration of HAuCl4·3H2O in the mixed aqueous solution is 0.1-5.0 mM, and the concentration of NaCl is 0.1-1.0 M; in the cyclic voltammetry method, the low potential for electrodeposition is -1.0 to -0.5 V, the high potential is 0.1 to 1.0 V, the scan rate is 10-100 mV / s, and the number of scan cycles is 2-10.

3. The construction method according to claim 1, characterized in that, The preparation method of poly(3,4-ethylenedioxythiophene)-polycarboxybenzene (PEDOT-pCBMA) hydrogel in step 2 is as follows: CBMA monomer, 3,4-ethylenedioxythiophene monomer EDOT, N,N'-methylenebisacrylamide MBAA, tetramethylethylenediamine TEMED, ammonium persulfate APS, NaCl and water were mixed to obtain a mixed aqueous solution. After sonication, the mixed aqueous solution was refrigerated at 1-4℃ for 6-12 hours to obtain PEDOT-pCBMA hydrogel.

4. The construction method according to claim 3, characterized in that: The concentrations of CBMA monomer in the mixed aqueous solution are 1-8 M, 3,4-ethylenedioxythiophene (EDOT) monomer is 0.1-1 M, N,N'-methylenebisacrylamide (MBAA) is 5-15 mM, tetramethylethylenediamine (TEMED) is 0.02-0.1 mM, ammonium persulfate (APS) is 0.01-0.1 M, and NaCl is 0.1-2 M.

5. The construction method according to claim 1, characterized in that: In step 2, the drop volume of PEDOT-pCBMA hydrogel solution is 0.5-5 μL.

6. The construction method according to claim 1, characterized in that: In step 3, the concentration of EDC in the mixed aqueous solution is 0.1-2M, the concentration of NHS is 0.1-2M, the concentration ratio of EDC to NHS is 4:1, the concentration of Cl is 0.1-5μM, and the volume of the mixed aqueous solution dropped onto the electrode is 10-100μL.

7. The construction method according to claim 1, characterized in that: In step 4, the concentration of MB in the mixed aqueous solution is 10-50 μM.

8. An electrochemical biosensor, characterized in that: It is constructed using the construction method described in any one of claims 1-7.

9. A kit for detecting COVID-19 virus in blood and / or saliva, characterized in that, Not intended for the diagnosis and treatment of diseases, the kit contains the electrochemical biosensor as described in claim 8.

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