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

Abstract

The invention relates to the technical field of electrochemical biosensors, and discloses an electrochemical biosensor for detecting COVID-19 nucleic acid in blood and saliva. The construction method comprises the following steps: electrodepositing gold nanoparticles on the surface of a carbon electrode, and then dispensing a poly (3, 4-ethylenedioxythiophene)-poly (carboxyl betaine) hydrogel liquid on the surface to obtain PEDOT-pCBMA hydrogel / AuNPs / SPCE (poly (3, 4-ethylenedioxythiophene)-poly (3, 4-ethylenedioxythiophene)-poly (3, 4-ethylenedioxythiophene)-poly (3, 4-ethylenedioxythiophene)-poly (carboxyl betaine)); then modifying the surface of an electrode with the captured DNA fixed with the ferrocene; and finally modifying the electrode with methylene blue to obtain the electrochemical biosensor. By adopting the electrochemical biosensor, the detection result adopts a ratio type electrochemical signal mode, so that the detection result is more intuitive and accurate; the ultra-low pollution detection in saliva or blood is realized by utilizing the high hydrophilicity and electric neutrality of the zwitterionic hydrogel; and the three-dimensional porous structure of the hydrogel effectively improves electron transfer and substance transport, and realizes high sensitivity.

Description

Technical Field

[0001] The present invention relates to the technical field of electrochemical biosensors, and in particular to an electrochemical biosensor for detecting COVID-19 nucleic acid in blood and saliva. Background Art

[0002] COVID-2019 is caused by the SARS-CoV-2 coronavirus. SARS-CoV-2 infections usually spread rapidly from person to person, with transmission routes including direct contact with infected individuals and through droplets and aerosol particles. Due to the immediacy and high infectivity of the SARS-CoV-2 coronavirus transmission, early screening and rapid detection are crucial for better managing, controlling infections, and treating diseases.

[0003] 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 during the acute phase of infection. Compared with antibody serological tests, nucleic acid testing can detect infected patients at an earlier stage and is currently the "gold standard" for novel coronavirus detection. However, the RT-PCR testing process is usually time-consuming (>3h) and complex. In addition, false-negative results often occur in nucleic acid testing, so many patients cannot be isolated and treated in a timely manner while waiting for RT-PCR test results or diagnosis, which leads to more widespread community transmission.

[0004] For COVID-2019 diagnostic testing, nasopharyngeal and oropharyngeal swabs are the official designated professional standards. However, sample collection causes discomfort to patients, and medical staff in contact with patients are also at risk of infection. Saliva is a clear and thin biological fluid with antibacterial, bacteriostatic, and rinsing effects and contains abundant disease markers, and has been used to diagnose various diseases. A large number of studies have also shown that saliva can transmit the SARS-CoV-2 virus through droplets, aerosols, coughing, and sneezing. In addition, saliva testing is a non-invasive, simple, and safe method.

[0005] Electrochemical biosensors have the characteristics of simple operation and high sensitivity and are widely used in quantitative detection of disease biomarkers. However, electrochemical biosensors are highly sensitive to the non-specific adsorption of interfering biomolecules on their sensing interfaces, which seriously hinders their clinical practical applications in complex biological fluids such as blood, serum, saliva, or sweat. Therefore, it is crucial to construct an anti-pollution electrochemical biosensor that can prevent the adsorption of non-specific substances on the sensing interface.

[0006] Currently, the application of electrochemical biosensors in complex biological environments (such as human serum, blood, urine, sweat, saliva) is still limited. The commonly used solution is to construct a sensing interface modified with anti-fouling materials to resist non-specific protein adsorption. These anti-fouling materials are usually highly hydrophilic and electro-neutral, and the dense coating formed by them also provides a physical barrier for non-specific protein adsorption. Among them, polyethylene glycol, zwitterionic polymers, and polypeptides are the most commonly used anti-fouling materials. Due to its high immunogenicity, polyethylene glycol is easily oxidized and degraded under physiological conditions, so its long-term stability is poor. Polypeptides have good hydrophilicity and are easy to design, but short straight-chain polypeptides are easily hydrolyzed and enzymatically degraded. Long-chain heteromorphic polypeptides have good anti-fouling performance and good stability, but they are complex to synthesize and have a high usage cost. Zwitterionic polymers are widely used in the construction of anti-fouling interfaces due to their large steric hindrance and neutral polymer backbone. However, the anti-fouling coatings reported currently, while providing good resistance to non-specific adsorption performance, will also form a passivation layer on the electrode surface due to the introduction of non-conductive anti-fouling materials with a high grafting density, which will make the sensing surface inert and lead to impaired detection sensitivity. This makes anti-interference, accuracy, and sensitivity in electrochemical sensing systems mutually contradictory and incompatible properties that cannot be achieved simultaneously. Summary of the Invention

[0007] The object of the present invention is to provide an electrochemical biosensor for the detection of COVID-19 nucleic acid in blood and saliva. The electrochemical biosensor of the present invention has a simple preparation process, low cost, universality, and is easy to be prepared on a large scale. When in use, a small amount of sample can be used to highly sensitively and accurately collect and analyze information, realizing trace detection of COVID-19 nucleic acid in blood or saliva, and having simple operation, low cost, wide detection range, low detection limit, high sensitivity, and high accuracy.

[0008] The detection result of the electrochemical biosensor prepared by the present invention adopts a ratio-type electrochemical signal mode, which is more intuitive and accurate; by utilizing the high hydrophilicity and electro-neutrality of the zwitterionic hydrogel, ultra-low pollution detection in saliva or blood is realized; and the three-dimensional porous structure of the hydrogel effectively improves electron transfer and mass transfer, realizing high sensitivity.

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

[0010] Step 1, mix chloroauric acid trihydrate HAuCl 4 ·3H 2 O, NaCl and water to obtain a mixed aqueous solution, immerse a screen-printed carbon electrode SPCE into the mixed aqueous solution, and prepare AuNPs / SPCE by cyclic voltammetry electrodeposition;

[0011] Step 2: Drop the poly(3,4-ethylenedioxythiophene)-poly(carboxybetaine methyl acrylate) (PEDOT-pCBMA) hydrogel solution onto the AuNPs / SPCE in the ungelled state, and spread it evenly on the entire working electrode surface. Then, refrigerate for reaction to obtain PEDOT-pCBMA hydrogel / AuNPs / SPCE;

[0012] Step 3: Mix N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), the capture DNA C1 immobilized with ferrocene, and water to obtain a mixed aqueous solution. React the mixed aqueous solution at room temperature for 30 minutes, and then drop the mixed aqueous solution onto the electrode for reaction for 1 - 5 h to prepare C1-Fc / PEDOT-pCBMA hydrogel / AuNPs / SPCE;

[0013] Step 4: Mix methylene blue (MB) and water to obtain a mixed aqueous solution. Drop the mixed aqueous solution onto the surface of the modified electrode in Step 3 and react for 2 - 30 min to obtain MB / C1-Fc / PEDOT-pCBMA hydrogel / AuNPs / SPCE, which is the electrochemical biosensor.

[0014] Furthermore, in the mixed aqueous solution of Step 1, the concentration of HAuCl 4 ·3H 2 O is 0.1 - 5.0 mM, and the concentration of NaCl is 0.1 - 1.0 M; in 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 cycles.

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

[0016] Mix CBMA monomer, 3,4-ethylenedioxythiophene (EDOT) monomer, N,N'-methylenebisacrylamide (MBAA), N,N,N',N'-tetramethylethylenediamine (TEMED), ammonium persulfate (APS), NaCl, and water to obtain a mixed aqueous solution. After ultrasonic treatment of the mixed aqueous solution, refrigerate and react at 1 - 4 °C for 6 - 12 hours to prepare the PEDOT-pCBMA hydrogel.

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

[0018] Further, the coating volume of the PEDOT-pCBMA hydrogel droplets in step 2 is 0.5 - 5 μL.

[0019] Further, in the mixed aqueous solution in step 3, the concentration of EDC is 0.1 - 2 M, the concentration of NHS is 0.1 - 2 M, the concentration ratio of EDC to NHS is 4:1, the concentration of C1 is 0.1 - 5 μM, and the coating volume of the mixed aqueous solution onto the electrode is 10 - 100 μL.

[0020] Further, 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 constructed by the above construction method.

[0022] The present invention also provides a kit for detecting COVID-19 virus in blood and / or saliva, which is not for the purpose of diagnosing and treating diseases, and the kit contains the above-mentioned electrochemical biosensor.

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

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

[0025] Step 1, use a disposable screen-printed carbon electrode (SPCE), take 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 to form a three-electrode system. Drop the N836 target DNA (T1) onto the working electrode for reaction, and then immerse the three-electrode system in a pH 7 - 8 buffer solution. Measure the change in the current signal through an electrochemical workstation, and draw a standard working curve according to the relationship between the standard T1 concentration in the sample pool and the current signal.

[0026] Step 2, take 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 to form a three-electrode system. Drop the same volume of the test solution as the T1 solution in step 1 onto the working electrode for reaction, immerse the three-electrode system in a pH 7 - 8 buffer solution, measure the change in the current signal through an electrochemical workstation, and test the concentration of T1 in the test solution according to the working curve.

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

[0028] The detection of the target N836 DNA in the present invention adopts a ratio-type electrochemical signal mode; by utilizing the high hydrophilicity and electrical neutrality of the zwitterionic hydrogel, ultra-low contamination detection in complex saliva or blood can be achieved; by utilizing the three-dimensional porous structure of the conductive zwitterionic hydrogel, the electron transfer and mass transfer performance can be effectively improved, realizing highly sensitive electrochemical sensing detection.

[0029] In the clinical application of reagents, due to the presence of a large number of interfering proteins and cells in complex biological samples, they non-specifically adhere to the electrochemical sensing interface without discrimination, thus forming a high-impedance layer, seriously affecting the accuracy of analytical detection and reducing the detection sensitivity, thereby limiting the clinical use of electrochemical biosensors. Some studies have shown that the non-specific adsorption of interfering proteins is mainly based on charge attraction and hydrophobic interaction. The highly sensitive anti-pollution electrochemical biosensor based on conductive zwitterionic hydrogel / gold nanoparticles prepared in the present invention has a conductive structure with high hydrophilicity and electrical neutrality. Among them, the high hydrophilicity is conducive to binding more free water molecules at the sensing interface to form a dense hydration layer, so as to counteract the hydrophobic interaction and resist the non-specific adsorption of proteins; the prepared electrically neutral biosensing interface can effectively avoid the electrostatic attraction of charged interfering substances on the interface. Therefore, the electrochemical biosensor of the present invention exhibits excellent anti-pollution performance and realizes highly accurate and highly sensitive COVID-19 nucleic acid detection.

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

[0031] 1. The preparation process of the electrochemical biosensor of the present invention is simple, low in cost, highly universal, and easy to prepare on a large scale.

[0032] 2. The electrochemical biosensor of the present invention is based on gold nanoparticles, and the spherical structure with nanoscale size 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, has a three-dimensional porous structure, high hydrophilicity and electrical neutrality, has excellent anti-pollution performance and biocompatibility, and its three-dimensional network structure and gold nanoparticles accelerate electron transfer and ion transport, and can realize anti-pollution and highly accurate detection of COVID-19 nucleic acid in saliva or blood.

[0034] 4. The electrochemical biosensor of the present invention is used for detecting the COVID-19 novel coronavirus. By taking a small amount of saliva or blood, it can sensitively and accurately collect and analyze information, achieving trace detection; and it has a short detection time, simple operation, low cost, wide detection range, low detection limit, high sensitivity, and high accuracy, and is a rapid and efficient method for detecting the nucleic acid of the COVID-19 novel coronavirus.

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

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

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

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

[0039] Figure 2 It is a photograph of the synthesized PEDOT-pCBMA hydrogel in Example 1 of the present invention;

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

[0041] Figure 4 It is a Fourier infrared spectrum of the synthesized PEDOT-pCBMA hydrogel in Example 1 of the present invention;

[0042] Figure 5 It is a thermogravimetric curve of the synthesized PEDOT-pCBMA hydrogel prepared in Example 1 of the present invention;

[0043] Figure 6 It is a static water contact angle image of the PEDOT-pCBMA hydrogel / AuNPs modified electrode prepared in Example 1 of the present invention;

[0044] Figure 7 It is a current response image of the electrochemical biosensor prepared in Example 2 of the present invention for saliva with different dilution concentrations;

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

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

[0047] Figure 10 This is the standard curve graph of the electrochemical biosensor prepared in Example 2 of the present invention for the nucleic acid detection of the target N836 at different concentrations. Detailed implementation manners

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

[0049] Unless otherwise defined, the technical terms or scientific terms used in the present invention should have the ordinary meanings understood by those of ordinary skill in the art to which the present invention belongs. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually determined according to national standards. The experimental instruments, equipment and reagents not indicated the sources in the following embodiments are all commercially available raw materials.

[0050] Unless otherwise defined or stated, all the professional and scientific terms used in the present invention have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to the described content can be applied to the method of the present invention.

[0051] In the embodiments of the present invention, the current signal is recorded by a Shanghai Chenhua CHI 660E electrochemical workstation. The current signal is tested on a traditional three-electrode electrochemical system. A disposable screen-printed carbon electrode (SPCE) is used. The carbon electrode is used as the working electrode, the carbon electrode is used as the counter electrode, and the silver / silver chloride electrode is used as the reference electrode to form a three-electrode system, which is purchased from Changsha Sanjun. C1 (N-836 capture: NH 2 -C6-ATT TCC TTG GGT TTG TTC TG-Fc) and T1 (N-836: CAGAAC AAA CCC AAG GAA AU) are both purchased from Shanghai Sangon.

[0052] Other chemical reagents and raw materials are all 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 nanoparticles (AuNPs) modified electrode:

[0056] Mix chloroauric acid trihydrate (HAuCl 4 ·3H 2 O), NaCl, and water. The concentration of chloroauric acid trihydrate (HAuCl 4 ·3H 2 O) is 1 mM, and the concentration of NaCl is 0.5 M. Mix and stir evenly to obtain the gold nanoparticle deposition solution. Take 500 μL of the above deposition solution and drop it on a disposable screen-printed carbon electrode (SPCE). Through cyclic voltammetry, the potential range is set to -0.9 - 0.5 V, the scan rate is 50 mV / s, and deposit for 5 cycles to obtain AuNPs / SPCE.

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

[0058] Mix CBMA monomer, 3,4-ethylenedioxythiophene monomer (EDOT), N,N'-methylenebisacrylamide (MBAA), tetramethylethylenediamine (TEMED), ammonium persulfate (APS), NaCl, and water to obtain a mixed aqueous solution. The concentration of CBMA monomer is 4 M, the concentration of EDOT monomer is 0.15 M, the concentration of N,N'-methylenebisacrylamide (MBAA) is 15 mM, the concentration of tetramethylethylenediamine (TEMED) is 0.04 mM, the concentration of ammonium persulfate (APS) is 0.04 M, and the concentration of NaCl is 1 M. After ultrasonicating the mixed aqueous solution for 1 min, refrigerate and react 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 solution synthesized in step (2) in the ungelled state and drop-coat it on the AuNPs / SPCE prepared in step (1), and spread it evenly on the entire working electrode. Place the electrode coated with PEDOT-pCBMA hydrogel in a 4°C refrigerator and refrigerate and react for 8 hours to obtain the PEDOT-pCBMA hydrogel / AuNPs modified electrode;

[0061] Result analysis

[0062] The scanning electron microscope image of the electro-deposited gold nanoparticles in Example 1 is as Figure 1As shown, it is a uniform spherical structure with an average diameter of about 50 nm and is densely attached to the electrode surface. The photograph of the PEDOT-pCBMA hydrogel synthesized in Example 1 is as Figure 2 shown, presenting a typical gel state.

[0063] The scanning electron microscope image of PEDOT-pCBMA hydrogel / AuNPs is as Figure 3 shown. The PEDOT-pCBMA hydrogel is a loose porous structure with stacked lamellae, which can effectively increase the electroactive area and enhance the electron transfer and ion transport capabilities. The Fourier transform infrared spectroscopy image of the PEDOT-pCBMA hydrogel is as Figure 4 shown, where the peaks at 1373 and 1583 cm -1 correspond to the C-C (inter-ring stretching mode) and C═C (asymmetric stretching mode) of the thiophene ring and the stretching vibration of the quinone structure respectively. The peaks at 1232 and 1141 cm -1 are related to the stretching vibration of the C-O-C bond in the methylenedioxy group. The peaks at 928 and 681 cm -1 correspond to the C-S peak in thiophene, which indicates the successful polymerization of PEDOT. The peaks at 1722 and 1475 cm -1 correspond to the stretching vibration of the C═O group and the C-N bending vibration in the monomer respectively. The band generated by the C-H stretching vibration in pCBMA is visible at 3012 cm -1 . The peak at 3365 cm -1 in all samples corresponds to the stretching vibration of the O-H group on the surface. It indicates the successful synthesis of pCBMA. The thermogravimetric curve of the PEDOT-pCBMA hydrogel is as Figure 5 shown. The residual carbon content at 800 °C is relatively low, being 7.16%; the water content is relatively high, being 64.11%. This is attributed to the fact that the PEDOT-pCBMA hydrogel has a large specific surface area, where more hydrophilic groups can bind more water molecules, showing high hydrophilicity. As Figure 6 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-pollution electrochemical biosensor based on a conductive zwitterionic hydrogel:

[0066] Step (1): N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), capture DNA (C1) immobilized with ferrocene, and water were mixed to obtain a mixed aqueous solution, where the concentration of EDC was 0.8 M and the concentration of NHS was 0.2 M. 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 h, thus successfully preparing C1-Fc / PEDOT-pCBMA hydrogel / AuNPs / SPCE.

[0067] Step (2): Methylene blue (MB) and water were mixed to obtain a mixed aqueous solution, where the concentration of MB was 20 μM. 50 μL of the mixed aqueous solution was dropped onto the C1-Fc / PEDOT-pCBMA hydrogel / AuNPs / SPCE obtained in step (1) and reacted for 10 min to prepare MB / C1-Fc / PEDOT-pCBMA hydrogel / AuNPs / SPCE, which is a highly sensitive anti-pollution electrochemical biosensor based on conductive zwitterionic hydrogel / gold nanoparticles.

[0068] Result analysis

[0069] Anti-pollution ability of the highly sensitive anti-pollution electrochemical biosensor based on conductive zwitterionic hydrogel.

[0070] The highly sensitive anti-pollution electrochemical biosensor based on conductive zwitterionic hydrogel in Example 2 was immersed in human saliva buffer solutions with different concentrations, and the current signals before and after soaking for half an hour in human saliva buffer solutions with different concentrations were measured. The human saliva buffer solutions with different concentrations were prepared by adding different volumes of human saliva to water and diluting the human saliva to different concentrations, which were 1%, 10%, 20%, 50%, and 100% (pure saliva, without water), respectively.

[0071] The results are as Figure 7 shown. The results indicate that the peak current signal of this biosensor changes very little after soaking in human saliva with different concentrations, and the peak potential is basically not shifted, indicating excellent anti-pollution performance.

[0072] The highly sensitive anti-pollution electrochemical biosensor based on conductive zwitterionic hydrogel in Example 2 was immersed in fetal bovine serum buffer solutions with different concentrations, and the current signals before and after soaking for half an hour in fetal bovine serum buffer solutions with different concentrations were measured. The fetal bovine serum buffer solutions with different concentrations were prepared by adding different volumes of fetal bovine serum to water and diluting the fetal bovine serum to different concentrations, which were 1%, 10%, 20%, 50%, and 100% (pure fetal bovine serum, without water), respectively.

[0073] Fetal bovine serum contains abundant proteins and cells and can simulate a 40% human serum environment. The results are asFigure 8 As shown, the results indicate that the signal change of the highly sensitive anti-fouling electrochemical biosensor based on conductive zwitterionic hydrogel is small after exposure to different concentrations of fetal bovine serum, suggesting that it can maintain excellent anti-biofouling performance even in extremely complex biological environments, effectively enhancing the accuracy of analytical detection and avoiding false positive results.

[0074] Example 3

[0075] Application of the highly sensitive anti-fouling electrochemical biosensor based on conductive zwitterionic hydrogel for the detection of COVID-19 nucleic acid.

[0076] The method for detecting COVID-19 nucleic acid is as follows:

[0077] Step (1): Use the highly sensitive anti-fouling electrochemical biosensor based on the conductive zwitterionic hydrogel prepared in Example 2 as the working electrode, a carbon electrode as the counter electrode, and a silver / silver chloride electrode as the reference electrode to form a three-electrode system. Incubate the working electrode with 50 μL of COVID-19 nucleic acid N836 target DNA (T1). Then, immerse the three-electrode system in 50 μL of phosphate buffer solution with pH 7.4, and measure the change in current signal by differential pulse voltammetry using an electrochemical workstation. According to the relationship between the standard T1 concentration in the sample pool and the current signal, plot the standard working curve.

[0078] Step (2): Use the highly sensitive anti-fouling electrochemical biosensor based on the conductive zwitterionic hydrogel prepared in Example 2 as the working electrode, a carbon electrode as the counter electrode, and a silver / silver chloride electrode as the reference electrode to form a three-electrode system. Incubate the working electrode with 50 μL of the test solution, and measure the change in current signal by differential pulse voltammetry using an electrochemical workstation. According to the working curve, test the concentration of COVID-19 nucleic acid N836 target DNA (T1) in the test solution.

[0079] Result analysis

[0080] The change in current signal of the electrochemical biosensor for the detection of target N836 nucleic acid at different concentrations (10 -15 M - 10 -8 M) is as Figure 9 shown, and the standard working curve is as Figure 10 shown. The detection results indicate that the detection range of the MB / C1-Fc / PEDOT-pCBMA hydrogel / AuNPs modified electrode for COVID-19 nucleic acid is from 1 fM to 1 nM, and the linear equation is: I Fc / I MB(μA) = 0.0578LgC + 1.27, with a linear correlation coefficient of 0.995 and a minimum detection limit of 0.26 fM. The pCBMA-pSBMA hydrogel modified electrode has high sensitivity for the detection of COVID-19 nucleic acid, which is attributed to the high porosity and high specific surface area provided by the three-dimensional network structure of the hydrogel, shortening the ion and mass transfer paths, and the conductive zwitterionic backbone providing high conductivity.

[0081] Currently reported anti-fouling coatings, while providing good resistance to non-specific adsorption, will also form a passivation layer on the electrode surface due to the introduction of non-conductive anti-fouling materials with a high grafting density, which will make the sensing surface inert and result in impaired detection sensitivity. This makes anti-interference, accuracy, and sensitivity in electrochemical sensing systems mutually contradictory and incompatible attributes that cannot be achieved simultaneously. To solve this problem, gold nanoparticles are added to the electrochemical biosensor in the present invention. Gold nanoparticles can effectively increase the electroactive area of the electrode and enhance the detection sensitivity. The modified PEDOT-pCBMA hydrogel with its excellent hydrophilicity and electro-neutral design can resist the non-specific adsorption of interfering proteins and improve the detection accuracy. Through activation with EDC / NHS activators, the amino group of the captured DNA (C1-Fc) modified with ferrocene is covalently bonded to the carboxyl group of the PEDOT-pCBMA hydrogel. Subsequently, methylene blue is modified and electrostatically bound to the phosphate backbone of the single-stranded DNA. The constructed ratio-type electrochemistry can achieve precise and sensitive detection of target DNA in complex biological fluids, providing a theoretical basis for promoting the clinical application of electrochemical biosensors.

[0082] Therefore, the present invention adopts the above-mentioned electrochemical biosensor for the detection of COVID-19 nucleic acid in blood and saliva. The detection result adopts a ratio-type electrochemical signal mode, which is more intuitive and accurate; by utilizing the high hydrophilicity and electro-neutrality of the zwitterionic hydrogel, ultra-low pollution detection in saliva or blood is achieved; and the three-dimensional porous structure of the hydrogel effectively improves electron transfer and mass transfer, 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 are not intended to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that they can still modify or equivalently replace the technical solutions of the present invention, and these modifications or equivalent replacements cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for constructing an electrochemical biosensor for COVID-19 nucleic acid detection in blood and saliva, characterized in that: The following steps are involved: Step 1, mixing chloroauric acid trihydrate HAuCl4·3H2O, NaCl and water to obtain a mixed aqueous solution, immersing a screen-printed carbon electrode SPCE in the mixed aqueous solution, and preparing AuNPs / SPCE by electrodeposition through cyclic voltammetry; Step 2, drop-coating the poly (3,4-ethylenedioxythiophene)-poly (carboxybetaine) PEDOT-pCBMA hydrogel solution onto AuNPs / SPCE in an ungelled state, so that it is evenly spread on the entire working electrode surface, and refrigerating the reaction to obtain PEDOT-pCBMA hydrogel / AuNPs / SPCE; Step 3, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride EDC, N-hydroxysuccinimide NHS, ferrocene-immobilized 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 the mixed aqueous solution is drop-coated on the electrode for reaction 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, and drop the mixed aqueous solution onto the modified electrode surface in step 3, react for 2-30 minutes to obtain MB / C1-Fc / PEDOT-pCBMA hydrogel / AuNPs / SPCE, which is an electrochemical biosensor.

2. The construction method according to claim 1, characterized in that: 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 of electrodeposition is -1.0 to -0.5 V, the high potential is 0.1 to 1.0 V, the scanning rate is 10-100 mV / s, and the number of scanning circles is 2 to 10 circles.

3. The construction method according to claim 1, characterized in that: The preparation method of poly (3,4-ethylenedioxythiophene)-polycarboxybetaine 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 are mixed to obtain a mixed aqueous solution. After ultrasonication of the mixed aqueous solution, it is refrigerated at 1-4°C for reaction for 6-12 hours to obtain a PEDOT-pCBMA hydrogel.

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

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

6. The construction method according to claim 1, characterized in that: Step 3: The concentration of EDC in the mixed aqueous solution is 0.1-2M, the concentration of NHS is 0.1-2M, the ratio of EDC to NHS is 4:1, the concentration of C1 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: Step 4: The concentration of MB in the mixed aqueous solution is 10-50 μM.

8. An electrochemical biosensor, characterized in that: The method is constructed by any one of claims 1 to 7.

9. A kit for detecting COVID-19 virus in blood and / or saliva, characterized in that: The kit comprises the electrochemical biosensor according to claim 8, not for the purpose of diagnosing or treating a disease.

10. Use of the electrochemical biosensor as claimed in claim 8 in preparing a kit for detecting COVID-19 virus in blood and / or saliva.

Images