Sensor for multichannel respiratory virus impedance detection and detection method thereof

Abstract

The invention provides a sensor for multichannel respiratory virus impedance detection and a detection method thereof. The synchronous detection of four respiratory viruses by the single-electrode chip is realized by electrodepositing nano-gold particles on the surface of a screen-printed carbon electrode and constructing a carboxyl self-assembled single layer, and fixing different virus specific antibodies on the surfaces of different working electrodes by adopting EDC / NHS (Ethylene Diamine Chloride / N-Hydroxysuccinimide). And recording an electrochemical impedance spectrum by using a red oxidation-reduction probe, and carrying out quantitative analysis based on total impedance change. The method has the advantages of high sensitivity, strong specificity, short detection period, low cost, suitability for on-site rapid screening and the like.

Description

Technical Field

[0001] This invention relates to the field of biosensors and on-site timely testing, specifically to a sensor and detection method for multi-channel respiratory virus impedance detection. Background Technology

[0002] Respiratory viral diseases pose a persistent threat to human health. Aerosols, as key vectors for the transmission of respiratory pathogens, can significantly increase individual exposure risk in enclosed or semi-enclosed environments such as hospital wards, school classrooms, and public transportation. Therefore, developing rapid, portable detection technologies for respiratory viruses in aerosols is crucial for early warning of individual exposure risks and for epidemic control.

[0003] However, pathogen detection in aerosols faces numerous challenges. First, the concentration of target viruses in aerosol samples is typically low; second, the aerosol matrix is ​​complex and may contain various non-specific interfering substances; and third, in high-risk environments, rapid screening for multiple pathogens simultaneously is often required. These characteristics make it exceptionally difficult to perform multi-target, simultaneous, and highly sensitive analyses on-site.

[0004] Currently, common respiratory virus detection methods mainly include traditional immunochromatography and nucleic acid analysis. Traditional immunochromatography, such as colloidal gold test strips, while offering advantages like portability, low cost, and ease of operation, has limited sensitivity and quantitative capabilities, making it difficult to accurately detect low-copy-number aerosol samples. Nucleic acid analysis methods, represented by quantitative polymerase chain reaction (PCR), while possessing high sensitivity and specificity and considered the "gold standard," require complex nucleic acid extraction steps, strict temperature control, and expensive equipment, resulting in long testing cycles, high costs, and limited applicability in resource-constrained on-site or point-of-care testing scenarios.

[0005] Therefore, there is an urgent need in this field for a new respiratory virus detection technology that can combine high sensitivity, high specificity, multi-channel simultaneous detection, speed, low cost, and ease of field use. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a sensor and detection method for multi-channel respiratory virus impedance detection. This sensor and method are designed for various common respiratory viruses, including H1N1 influenza A virus, SARS-CoV-2, respiratory syncytial virus (RSV), and influenza B virus, aiming to achieve parallel, highly sensitive, rapid detection suitable for portable and field applications.

[0007] To solve the above-mentioned technical problems, the technical solution proposed in this application is as follows: This invention provides a sensor for multi-channel respiratory virus impedance detection, comprising: Four independent working electrode zones, which share the same screen-printed carbon electrode array patch for the reference electrode and the counter electrode; A layer of gold nanoparticles is coated on the surface of each of the working electrodes; A carboxyl-containing self-assembled monolayer formed on the surface of the gold nanoparticle layer; Different respiratory pathogen-specific antibodies were immobilized on the surfaces of different working electrodes via EDC / NHS chemical coupling.

[0008] Furthermore, the nano-gold particle layer is formed by electrochemical deposition, with the electrodeposition solution containing 5.0 mmol / L chloroauric acid and 0.1 mol / L sulfuric acid, and the deposition conditions being a constant potential deposition of -0.20 V for 150 s.

[0009] Furthermore, the carboxyl-containing self-assembled monolayer is composed of 11-mercaptoundecanoic acid and 6-mercapto-1-hexanol.

[0010] Furthermore, the specific antibodies against different respiratory pathogens are specific antibodies against SARS-CoV-2, influenza A (H1N1), influenza B, and respiratory syncytial virus, respectively.

[0011] Furthermore, the sensor also includes a blocking reagent for blocking nonspecific sites, the blocking reagent comprising bovine serum albumin.

[0012] Furthermore, the sensor is connected to a readout module, which is configured to acquire EIS in the frequency range of 0.1 Hz to 100 kHz, fit it with a Randle equivalent circuit, and use the change in total impedance to perform quantitative analysis on different antigen concentrations.

[0013] On the other hand, this application also claims protection for a respiratory virus detection method using any of the sensors described above, comprising the following steps: a. Drop the sample to be tested onto the surface of the antibody-fixed working electrode, incubate for a period of time to allow the virus in the sample to specifically bind with the corresponding antibody, and then rinse to remove unbound components; b. Place the sensor processed in step a in a red redox probe solution, record the electrochemical impedance spectroscopy, and obtain the change in total impedance before and after the virus binds to the antibody; c. Based on the pre-established calibration curve, the four respiratory viruses are quantitatively determined according to the total impedance change value obtained in step b.

[0014] Furthermore, the red redox probe used for detection was 5.0 mmol / L. .

[0015] Furthermore, the incubation time is 30 minutes.

[0016] On the other hand, this application also claims protection for a test kit, comprising: The sensor, sample diluent, washing solution, red red oxidation-reduction probe solution, and blocking solution described in any of the above.

[0017] Compared with existing technologies, the sensor and detection method for multi-channel respiratory virus impedance detection of the present invention achieve the following beneficial technical effects: This application provides a multi-channel respiratory virus detection sensor and method. By integrating four independent detection units on a single chip, it achieves simultaneous and rapid detection of multiple respiratory viruses. This method features high sensitivity and strong specificity, effectively distinguishing different viruses while avoiding complex operations and expensive equipment, significantly reducing detection costs and time. It is particularly suitable for rapid on-site screening, and the platform is highly versatile, facilitating expansion to the detection of other pathogens. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 The schematic diagram illustrates a four-channel screen-printed electrode of a sensor according to an embodiment of the present disclosure, comprising four working electrodes sharing the same reference electrode and counter electrode; Figure 2 The illustration schematically shows the changes in electrochemical impedance signals caused by the binding of different concentrations of influenza A (H1N1) virus to H1N1 antibodies immobilized on the electrode surface according to embodiments of the present disclosure. Figure 3 The illustration schematically shows the changes in electrochemical impedance signals caused by the binding of different concentrations of SARS-CoV-2 virus to SARS-CoV-2 antibodies immobilized on the electrode surface according to embodiments of the present disclosure; Figure 4 The illustration schematically shows the changes in electrochemical impedance signal caused by the binding of different concentrations of respiratory syncytial virus (RSV) to respiratory syncytial virus antibodies immobilized on the electrode surface according to embodiments of the present disclosure. Figure 5The illustration schematically shows the changes in electrochemical impedance signal caused by the binding of different concentrations of influenza B virus antibodies immobilized on the electrode surface according to embodiments of the present disclosure. Figure 6 The diagram illustrates the changes in total impedance values ​​after H1N1 antibody reacts with different respiratory virus antigen proteins; Figure 7 The diagram illustrates the changes in total impedance values ​​after SARS-CoV-2 antibodies react with different respiratory viral antigen proteins; Figure 8 The diagram illustrates the changes in total impedance values ​​after RSV antibodies react with different respiratory virus antigen proteins; Figure 9 The diagram illustrates the changes in total impedance values ​​after influenza B antibodies react with different respiratory virus antigen proteins. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, 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, 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.

[0021] like Figure 1 As shown, a sensor for multi-channel respiratory virus impedance detection according to this application includes: Four independent working electrode zones, which share the same screen-printed carbon electrode array patch for the reference electrode and the counter electrode; A layer of gold nanoparticles is coated on the surface of each of the working electrodes; preferably, the gold nanoparticle layer is formed by electrochemical deposition, wherein the electrodeposition solution contains 5.0 mmol / L chloroauric acid and 0.1 mol / L sulfuric acid, and the deposition conditions are a constant potential deposition of -0.20 V for 150 s.

[0022] A carboxyl-containing self-assembled monolayer is formed on the surface of the gold nanoparticle layer; preferably, the carboxyl-containing self-assembled monolayer is composed of 11-mercaptoundecanoic acid and 6-mercapto-1-hexanol.

[0023] Different respiratory pathogen-specific antibodies were immobilized on the surfaces of different working electrodes via EDC / NHS chemical coupling. These antibodies were specific to SARS-CoV-2, influenza A (H1N1), influenza B, and respiratory syncytial virus.

[0024] The sensor also includes a blocking reagent for blocking nonspecific sites, the blocking reagent comprising bovine serum albumin.

[0025] The sensor is connected to a readout module, which is configured to acquire EIS in the frequency range of 0.1 Hz to 100 kHz and fit it with a Randle equivalent circuit, using the change in total impedance to perform quantitative analysis of different antigen concentrations.

[0026] This application also provides a respiratory virus detection method using the aforementioned sensor, comprising the following steps: a. Drop the sample to be tested onto the surface of the antibody-fixed working electrode, incubate for a period of time to allow the virus in the sample to specifically bind with the corresponding antibody, and then rinse to remove unbound components; b. Place the sensor processed in step a in a red redox probe solution, record the electrochemical impedance spectroscopy, and obtain the change in total impedance before and after the virus binds to the antibody; c. Based on the pre-established calibration curve, the four respiratory viruses are quantitatively determined according to the total impedance change value obtained in step b.

[0027] Preferably, the red redox probe used for detection is 5.0 mmol / L. .

[0028] This application also provides a detection kit, comprising: The aforementioned sensor, sample diluent, washing solution, red redox probe solution, and blocking solution.

[0029] The substrate electrode used in the sensor of this invention is a screen-printed carbon electrode (SPCE), employing a four-channel array design, including four independent working electrodes (WE), a shared Ag / AgCl reference electrode (RE), and a counter electrode (CE), as shown below. Figure 1 As shown, the entire SPCE has a width D of 24 mm, a length H of 35 mm, and a diameter R of WE of 3 mm. An insulating layer is provided between adjacent working electrodes. The four working electrodes are respectively immobilized with monoclonal antibodies against influenza A (H1N1) protein HA protein, SARS-CoV-2 S1 protein, RSV F protein, and influenza B HA protein.

[0030] Example 1 Electrode surface functionalization and antibody immobilization Nano-gold electrodeposition: After rinsing the SPCE electrode with water and allowing it to air dry, nano-gold was electrodeposited. The electrodeposition solution consisted of 5 mM HAuCl4 and 0.1 M H2SO4. Deposition was carried out at a constant potential of -0.2 V for 150 s. A gold layer was visible to the naked eye on the carbon electrode surface, and the working electrode gradually changed from black to gold. After deposition, the electrode was rinsed with ultrapure water and allowed to air dry.

[0031] Self-assembled monolayer construction: Prepare an anhydrous ethanol solution of 0.1 mM 11-mercaptoundecanoic acid (MUA) and 1.0 mM 6-mercapto-1-hexanol, immerse the above-mentioned gold nanoparticle-modified working electrode in the solution, and incubate overnight at 4 °C.

[0032] EDC / NHS activation and antibody fixation: Prepare a mixture of 0.4 M EDC and 25 mM NHS in MES buffer (pH 5.5), drop it onto the working electrode surface, and incubate at room temperature for 30 min to form active esters. Then, wash with 1×PBS buffer and immediately add 20 μg / mL of target antibody solution. Incubate at room temperature for 60 min. After modification, wash three times with PBST solution (composed of 1×PBS and 0.05% Tween 20) to completely remove unbound antibodies.

[0033] Electrode blocking: The antibody-modified electrode was blocked with 0.5% BSA for 50 min at room temperature to remove non-specific adsorption. After blocking, the electrode was washed with 1×PBS buffer, dried, and stored in a sealed container at 4 °C protected from light for later use.

[0034] Quantitative analysis of impedance testing Different concentrations of target antigen protein solutions were added to four independent working electrodes at 10 μL each, and incubated at room temperature for 30 min to achieve quantitative analysis of SARS-CoV-2 S1 protein, H1N1 HA protein, RSV F protein, and influenza B HA protein. Unbound components were gently washed with PBS, and EIS test curves were recorded in a PBS mixture containing 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6, and 100 mM KCl. The test frequency was 0.1 Hz – 100 kHz, and the sinusoidal perturbation amplitude was 15 mV.

[0035] For data fitting, Bode plots were drawn, and the total impedance values ​​corresponding to a 1 Hz blank solution and antigen protein solutions of different concentrations were selected. ΔZ = Z was then calculated. 样品 - Z 空白Plot a calibration curve and calculate the detection limit based on the fact that the difference between the total impedance value of the sample solution and the total impedance value of the blank solution is more than three times the deviation of the total impedance value of the blank solution. Figure 2 Bode maps and calibration curves of H1N1 antibody-modified electrodes after incubation with different concentrations of HA protein are shown, such as... Figure 2 Show, Figure 2 The HA protein concentrations from bottom to top were 0, 0.01 pM, 0.1 pM, 1 pM, 10 pM, and 50 pM. As the HA protein concentration increased, the impedance value gradually increased, indicating that the impedance changed significantly before and after the antibody bound to the HA protein. The detection limit of the method was 1.15 fM. Figure 3 Bode maps and calibration curves of SARS-CoV-2 antibody-modified electrodes after incubation with different concentrations of S1 protein. Figure 3 The corresponding HA protein concentrations from bottom to top are 0, 0.01 pM, 0.1 pM, 1 pM, 10 pM, 50 pM, and 200 pM, with a method detection limit of 0.22 fM. Figure 4 Bode maps and calibration curves of RSV antibody-modified electrodes after incubation with different concentrations of F protein. Figure 4 The F protein concentrations from bottom to top were 0, 0.01 pM, 0.1 pM, 1 pM, 10 pM, and 50 pM, respectively, with a method detection limit of 1.36 fM. Figure 5 Bode spectra and calibration curves of influenza B antibody-modified electrodes after incubation with different concentrations of HA protein. Figure 5 The corresponding HA protein concentrations from bottom to top are 0, 0.01 pM, 0.1 pM, 1 pM, 10 pM, and 50 pM, with a method detection limit of 4.45 fM.

[0036] Anti-interference test Using the above testing conditions, the impedance values ​​of electrodes modified with H1N1 antibody, SARS-CoV-2 antibody, RSV antibody, and influenza B antibody were measured relative to 0.01 pM H1N1 HA membrane protein, SARS-CoV-2 S1 protein, RSV F protein, and influenza B HA protein, respectively. A bar chart was then plotted showing the total impedance values ​​corresponding to a 1 Hz frequency. Figure 6 As shown, for the electrode modified with H1N1 antibody, the difference between the total impedance value of H1N1 antigen binding and the total impedance value of the blank solution was 492.5 Ω, while the difference in total impedance value caused by the binding of other proteins was less than 100 Ω, indicating that the H1N1 antibody-modified electrode has high selectivity for the HA protein of H1N1; Figure 7As shown, for the electrode modified with SARS-CoV-2 antibody, the difference between the total impedance value of S1 protein binding and the total impedance value of the blank solution was 786.3 Ω, while the differences in total impedance values ​​caused by the binding of other proteins were all less than about 50 Ω, indicating that the SARS-CoV-2 antibody-modified electrode has high selectivity for S1 protein; Figure 8 As shown, for the electrode modified with RSV antibody, the difference between the total impedance value of RSV antigen binding and the total impedance value of the blank solution is 447.5 Ω, while the difference in total impedance value caused by the binding of other proteins at the same concentration is much lower than the impedance change induced by F protein binding, indicating that the RSV antibody modified electrode has high selectivity for F protein; Figure 9 As shown, for the electrode modified with influenza B antibody, the total impedance value of influenza B HA antigen binding is 388 Ω different from that of the blank solution, while the total impedance value difference caused by binding of other proteins is around 30 Ω, indicating that the influenza B antibody modified electrode has high immunogenicity to its HA protein.

[0037] In summary, this disclosure presents a multi-channel electrochemical impedance spectroscopy (EIS) method based on a gold nanoparticle-functionalized screen-printed carbon electrode for rapid analysis of respiratory viruses. The method employs covalently coupled antibodies, and the impedance signal gradually increases with increasing antigen concentration, exhibiting significant changes in electrical signal. The method achieves detection limits of fM for antigens such as H1N1, SARS-CoV-2, RSV, and influenza B. It offers advantages such as ease of operation, multi-channel parallel detection, and versatility; simply replacing the corresponding antibody allows for the analysis of other pathogens.

[0038] 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 the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A sensor for multi-channel respiratory virus impedance detection, characterized in that, include: Four independent working electrode zones, which share the same screen-printed carbon electrode array patch for the reference electrode and the counter electrode; A layer of gold nanoparticles is coated on the surface of each of the working electrodes; A carboxyl-containing self-assembled monolayer formed on the surface of the gold nanoparticle layer; Different respiratory pathogen-specific antibodies were immobilized on the surfaces of different working electrodes via EDC / NHS chemical coupling.

2. The sensor according to claim 1, characterized in that, The gold nanoparticle layer was formed by electrochemical deposition. The electrodeposition solution contained 5.0 mmol / L chloroauric acid and 0.1 mol / L sulfuric acid, and the deposition conditions were a constant potential deposition of -0.20 V for 150 s.

3. The sensor according to claim 1 or 2, characterized in that, The carboxyl-containing self-assembled monolayer is composed of 11-mercaptoundecanoic acid and 6-mercapto-1-hexanol.

4. The sensor according to claim 1 or 2, characterized in that, The specific antibodies against different respiratory pathogens are specific antibodies against SARS-CoV-2, H1N1 influenza A, influenza B, and respiratory syncytial virus, respectively.

5. The sensor according to claim 1 or 2, characterized in that, The sensor also includes a blocking reagent for blocking nonspecific sites, the blocking reagent comprising bovine serum albumin.

6. The sensor according to claim 1 or 2, characterized in that, The sensor is connected to a readout module, which is configured to acquire EIS in the frequency range of 0.1 Hz to 100 kHz and fit it with a Randle equivalent circuit, using the change in total impedance to perform quantitative analysis of different antigen concentrations.

7. A method for detecting respiratory viruses using the sensor described in any one of claims 1-6, characterized in that, Includes the following steps: a. Drop the sample to be tested onto the surface of the antibody-fixed working electrode, incubate for a period of time to allow the virus in the sample to specifically bind with the corresponding antibody, and then rinse to remove unbound components; b. Place the sensor processed in step a in a red redox probe solution, record the electrochemical impedance spectroscopy, and obtain the change in total impedance before and after the virus binds to the antibody; c. Based on the pre-established calibration curve, the four respiratory viruses are quantitatively determined according to the total impedance change value obtained in step b.

8. The sensor according to claim 7, characterized in that, The red redox probe used for detection was 5.0 mmol / L. .

9. The detection method according to claim 7, characterized in that, The incubation time is 30 minutes.

10. A test kit, characterized in that, include: The sensor, sample diluent, washing solution, red redox probe solution, and blocking solution as described in any one of claims 1-6.

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