Preparation method and application of electrochemical ratio sensor of new coronavirus N protein based on acupuncture needle microelectrode

Abstract

The invention discloses a preparation method and application of a ratio-type electrochemical sensor for detecting N protein, a stainless steel acupuncture needle is used as a substrate, after pretreatment, AuNPs modification, MCNT-PEDOT conductive layer construction, TH / IMP double-signal fixation, secondary AuNPs modification, 4-MPBA self-assembly, N protein incubation, pyrrole imprinting polymerization and template elution are sequentially carried out, and a surface molecular imprinting sensor is prepared. The sensor adopts AuNPs + MCNT-PEDOT to improve the conductivity, a ratio system is constructed by taking TH as an internal reference and IMP as a response signal to counteract interference, specific recognition of N protein is realized through molecular imprinting, and the sensor has the advantages of high sensitivity, high specificity, strong interference resistance, portability, low cost, preparation controllability and the like, and is suitable for N protein POCT detection.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of electrochemical detection technology and biosensing technology, and specifically relates to a preparation method of a ratio-type electrochemical sensor based on a stainless steel acupuncture needle, and application of the sensor in detection of a novel coronavirus nucleocapsid protein (N protein). BACKGROUND

[0002] The novel coronavirus nucleocapsid protein (N protein) has significant advantages as a diagnostic target. Compared with the highly mutated spike protein (S protein), the N protein is highly conserved in viral evolution, which can effectively avoid detection escape caused by variant strains. At the same time, the N protein has high abundance in the virus body (about 40% of the total protein), and can be enriched in body fluids in the early stage of infection, which is an ideal target for rapid detection. However, the current mainstream PCR detection is sensitive but time-consuming and requires professional equipment, and the traditional antigen test strip has insufficient sensitivity (the detection limit LOD is generally > 80 PFU / mL). Therefore, there is an urgent need to develop a high-sensitivity, portable N protein detection technology to achieve point-of-care testing (POCT) and precise epidemic prevention and control.

[0003] Microelectrode technology provides an ideal platform for in vivo detection with its miniaturization, low invasiveness and high spatiotemporal resolution. In recent years, the combination of molecularly imprinted polymers (MIPs) has significantly improved the selectivity and sensitivity of the sensor. For example, a high-conductivity interface is constructed on the surface of an acupuncture needle microelectrode (ANME) by electro-polymerizing a three-dimensional gold nanorod and a polydopamine layer, and then a MIP nanocavity is formed by co-polymerization of a template molecule and a monomer, which realizes specific capture of target molecules through size matching, hydrogen bonding and π-π interaction, and can maintain low error even in the presence of high-concentration interferents. In neuroscience research, a carbon fiber nanoelectrode modified with nanodiamonds combined with a MIP layer realizes in situ detection of trace substances at the single-cell level, highlighting the advantages of minimally invasive penetration and resistance to biological contamination. These advances show that the integration of MIPs and microelectrodes provides a new paradigm for solving the problem of recognizing trace analytes in complex matrices.

[0004] Based on the development of ratio-type electrochemical sensors, this strategy effectively offsets electrode fluctuations, environmental interference and matrix effects by coupling internal reference signals (such as metal phthalocyanine, MOF-loaded probes) with target response signals, significantly improving the stability and reliability of detection, with a relative standard deviation (RSD) of 1.8-7.55%, which is much better than that of single-signal mode (RSD: 7.50-19.75%). This strategy has shown strong advantages in ultra-sensitive detection (LOD reaching fg / mL or CFU / mL level) and actual sample verification such as serum, providing key technical support for miniaturized electrodes (such as acupuncture needle electrodes ANME) to achieve precise sensing (such as POCT for N protein) in complex in vivo environments. SUMMARY

[0005] The present application aims to provide a ratio type electrochemical sensor for detecting N protein and its preparation method and application. The SMIP for N protein is prepared on the acupuncture needle electrode through in-situ electropolymerization and self-assembly technology. Imipramine is embedded under the SMIP layer, and current can smoothly pass through the imprinted nanohole. In addition, there is exposed thionine on the surface to provide a reference signal. The developed ratio type electrochemical sensor has good sensitivity and the ability to selectively detect N protein biomarkers.

[0006] In order to achieve the above-mentioned purpose, the present application is realized by the following technical scheme:

[0007] The present application is a preparation method of a ratio type electrochemical sensor for detecting N protein, which comprises the following steps:

[0008] Step 1) Pretreatment of acupuncture needle: polish the acupuncture needle, ultrasonically clean it in ethanol and deionized water for 4-6 minutes respectively, and dry it with nitrogen for standby;

[0009] Step 2) Modification of gold nanoparticles: take the pretreated acupuncture needle in step 1) as the working electrode, immerse the 9-11mm needle tip part in a 2.5mmol / L HAuCl4 solution containing 0.1-02M KCl, modify it by cyclic voltammetry scanning method, the scanning rate is 20-30mV / s, the potential range is-1.5V~0.5V, and scan for 4-6 cycles to obtain the electrode ANME / AuNPs;

[0010] Step 3) Modification of multi-walled carbon nanotubes: take the ANME / AuNPs obtained in step 2) as the working electrode, and in a water solution containing 0.01-0.03mol / L 3,4-ethylenedioxythiophene and 1-3mg / mL multi-walled carbon nanotubes, electropolymerize at a potential of 1.1-1.3V for 120-140 seconds, clean and dry to obtain the electrode ANME / AuNPs / MCNT;

[0011] Step 4) Modification of reference signal: immerse the ANME / AuNPs / MCNT electrode obtained in step 3) in a 4-6mM thionine solution (solvent is 0.1M PBS, pH=6.0), modify it by cyclic voltammetry scanning method, the scanning rate is 80-100mV / s, the potential range is-0.4V~0.4V, and scan for 50-70 times to obtain the electrode ANME / AuNPs / MCNT / TH;

[0012] Step 5) Modification of built-in signal: 1 / 2 tip portion of the ANME / AuNPs / MCNT / TH electrode obtained in step 4) is immersed in 10-30 mM imipramine solution (solvent is 0.1 M PBS, pH = 7.0), and is modified by cyclic voltammetry scanning method, scanning rate is 90-110 mV / s, potential range is -0.4 V-1.0 V, scanning is 5-7 times, to obtain 1 / 2 electrode ANME / AuNPs / MCNT / TH / IMP;

[0013] Step 6) Secondary modification of gold nanoparticles: ANME / AuNPs / MCNT / TH / IMP obtained in step 5) is used as working electrode, and is scanned for 1-2 cycles by cyclic voltammetry according to the parameters in step 2) to obtain electrode ANME / AuNPs / MCNT / TH / IMP / AuNPs;

[0014] Step 7) Modification of 4-mercapto phenylboronic acid: the electrode obtained in step 6) is immersed in 10 mM 4-mercapto phenylboronic acid ethanol solution, and is soaked at 0-4 ℃ for 40-60 minutes to obtain electrode ANME / AuNPs / MCNT / TH / IMP / AuNPs / 4-MPBA;

[0015] Step 8) Binding of template molecule: the electrode obtained in step 7) is immersed in 50 μg / mL N protein solution (solvent is 0.02 M PBS, pH = 7.4), and is incubated at 0-4 ℃ for 8-10 hours to obtain electrode ANME / AuNPs / MCNT / TH / IMP / AuNPs / 4-MPBA-N;

[0016] Step 9) Modification of imprinted polymer film: the electrode obtained in step 8) is used as working electrode, and is polymerized in 0.01-0.02 M PBS solution (pH = 7.4) containing 0.1-0.3 M pyrrole by cyclic voltammetry scanning method, scanning rate is 30-50 mV / s, potential range is -0.3 V-0.8 V, scanning is 11-13 times, to obtain electrode ANME / AuNPs / MCNT / TH / IMP / AuNPs / 4-MPBA-N / Ppy;

[0017] Step 10) Elution of template molecule: the electrode obtained in step 9) is immersed in eluent (0.2 M HCl mixed with 20% methanol, solvent is 0.1 M PBS, pH = 5.0), and is eluted for 80-100 minutes to obtain surface molecularly imprinted microsensor ANME / AuNPs / MCNT / TH / IMP / AuNPs@SMIP.

[0018] Preferably, in step 1), the acupuncture needle is made of stainless steel, with diameter of 0.2-0.3 mm and length of 30-50 mm.

[0019] Preferably, in step 2), the cyclic voltammetry scan adopts a three-electrode system, with a saturated calomel electrode as the reference electrode and a platinum wire electrode as the counter electrode.

[0020] Preferably, in step 3), the multi-walled carbon nanotubes have a diameter of 10-20 nm and a length of 5-15 μm, and are treated with nitric acid reflux before use to increase the surface carboxyl groups.

[0021] Preferably, in step 4), the thionine solution is filtered through a 0.22 μm filter before use to remove insoluble impurities.

[0022] Preferably, in step 5), the 1 / 2 needle tip portion refers to a region 5 mm from the needle tip end point, and the unmodified region is wrapped with insulating tape to avoid contact with the solution.

[0023] Preferably, in step 7), the 4-mercapto phenylboronic acid ethanol solution is prepared fresh before use, and the soaking reaction is carried out under nitrogen protection.

[0024] Preferably, in step 8), the concentration of the N protein solution is 50 μg / mL ± 5 μg / mL, and the incubation process is carried out in a constant temperature incubator to avoid light.

[0025] The present application also provides a ratio-type electrochemical sensor for detecting N protein prepared by the above method.

[0026] The present application also provides the use of the ratio-type electrochemical sensor for detecting N protein in detecting COVID-19 N protein, and the linear range of the use is 0.005 ng / mL-10000 ng / mL, and the detection limit is 0.001 ng / mL.

[0027] Advantages:

[0028] 1) In the process of modifying gold nanoparticles (AuNPs), the present application adopts cyclic voltammetry scan method, which can accurately control the nucleation and growth process of gold core, so that AuNPs are uniformly and densely deposited on the acupuncture needle electrode surface, forming a nanosphere structure with high specific surface area. This structure not only provides a stable and broad support for the subsequent fixation of multi-walled carbon nanotubes (MCNT) and conductive polymers, but also greatly increases the contact sites between the imprint layer and the N protein template after the formation of the molecular imprinting cavity, effectively promotes the transmission of electrons at the electrode interface, and significantly improves the response sensitivity of the sensor.

[0029] 2) The application innovatively introduces a composite material of multi-walled carbon nanotubes (MCNT) and conductive polymer PEDOT when constructing a sensing interface. MCNT itself has excellent conductivity and one-dimensional nanostructure, and through electrochemical copolymerization with PEDOT, a three-dimensional interconnected conductive network is formed, greatly accelerating the electron transfer rate of thionine (TH) and imipramine (IMP) probe molecules, and its huge specific surface area also provides sufficient sites for subsequent modification, thereby realizing significant amplification of electrochemical signals.

[0030] 3) The application ingeniously designs a double-signal system of thionine (TH) and imipramine (IMP) and constructs a ratio-type detection model. Among them, the TH signal serves as a stable internal reference, and its intensity does not change with the concentration of N protein; while the IMP signal serves as a response signal, and its intensity decreases regularly with the capture of N protein by the molecularly imprinted cavity. By calculating the signal ratio of the two for quantitative analysis, non-specific interference from sensor itself differences, environmental fluctuations and complex sample matrix can be effectively eliminated, fundamentally improving the accuracy, reproducibility and reliability of the detection.

[0031] 4) The application adopts surface molecular imprinting technology and uses 4-mercaptophenylboronic acid (4-MPBA) as a functional monomer to construct recognition sites in situ on the gold nanoparticle layer. The boronic acid group of 4-MPBA can specifically and reversibly bind to the glycosyl group of N protein, ensuring that the imprinted cavity is perfectly complementary to the target molecule in shape, size and chemical functional groups. The sensor can accurately identify N protein from complex biological samples, effectively avoiding interference from similar structured substances.

[0032] 5) The application uses stainless steel acupuncture needles as the base material of the sensor, which has outstanding advantages. Acupuncture needles themselves have the characteristics of miniaturization, high mechanical strength and low cost, making them very suitable for developing disposable or portable detection devices. The sensor prepared with the acupuncture needle as the base material is small in size, requires less sample, and is durable, laying a solid foundation for future implementation of point-of-care testing (POCT) and clinical instant diagnosis.

[0033] 6) The preparation process of the application integrates various electrochemical methods, which has high controllability and reproducibility. From the deposition of AuNPs, the mixing of PEDOT / MCNT, to the fixation of TH / IMP and the final polymerization of pyrrole imprinting layer, the whole process is precisely controlled by electrochemical techniques such as cyclic voltammetry. This method can form a uniform and firm modification layer on the electrode surface, ensuring that the performance of sensors prepared in different batches is consistent, which is conducive to standardized production and practical application. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 Scanning electron microscope images of the electrodes.

[0035] Figure 2 To progressively modify the electrode in 5.0 mM [Fe(CN)6] 3- / 4- Cyclic voltammetry in solution.

[0036] Figure 3 Cyclic voltammograms of the stepwise modified electrode in 0.1M PBS (pH=7.0) solution.

[0037] Figure 4 Differential pulse voltammograms of the stepwise modified electrode in 0.1M PBS (pH=7.0) solution.

[0038] Figure 5 Differential pulse voltammetry plots of the ratiometric electrochemical sensor used for detection of N protein solutions with different concentrations.

[0039] Figure 6 When the prepared ratiometric and built-in electrochemical sensors are used for the detection of N protein, respectively, I IMP / I TH and I IMP Linear relationship between N protein concentration and logarithmic value (1gC).

[0040] Figure 7 The figures show the response results of the prepared ratiometric and built-in electrochemical sensors after binding to N protein and nonspecific molecules, respectively.

[0041] Figure 8 Different modified substrates for embedded signals were used in 5.0 mM [Fe(CN)6] 3- / 4- Cyclic voltammetry comparison in solution and final signal expression in 0.1M PBS (pH=7.0) solution using cyclic voltammetry and differential pulse voltammetry.

[0042] Figure 9 Different assembly methods of the N protein were studied at 5.0 mM [Fe(CN)6]. 3- / 4- A comparison of cyclic voltammetry in the solution. Detailed Implementation

[0043] The following will refer to the appendices in the embodiments of the present invention. Figures 1-9 The technical solutions in the embodiments of the present invention are clearly and completely described herein. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0044] Technical solution / principle:

[0045] The application discloses a preparation method of a ratio type electrochemical sensor for detecting N protein, the sensor and application thereof, and the core lies in that a stainless steel acupuncture needle is used as a substrate, a conductive network-anchored double signal forming ratio system-molecular imprinting technology path for specific recognition is constructed through layered modification, and the electrochemical sensor with high sensitivity and high specificity is prepared. Specifically, the preparation method of the miniaturized sensor with the acupuncture needle as the substrate is used, the miniaturization advantage of the acupuncture needle is used to reduce the sample dosage (only 100 microliters) and reduce the detection cost, and the POCT scene is adapted; the AuNPs+MCNT-PEDOT composite conductive layer design is used to improve the electron transmission efficiency and solve the problem of insufficient sensitivity of the microelectrode; the TH / IMP ratio signal system is constructed to offset the environmental and matrix interference and solve the problem of poor stability of single signal detection; the 4-MPBA mediated molecular imprinting technology is used to realize the specific recognition of the N protein and solve the problem that the interference substances in the complex sample affect the detection accuracy; and finally, the high sensitivity (detection limit is less than or equal to 0.001 ng / mL), high specificity, rapidness (detection time is less than or equal to 45 minutes) and portable detection of the novel coronavirus N protein are realized, and the needs of instant diagnosis and precise prevention and control are met.

[0046] The application can be directly applied to the instant diagnosis (POCT) scene, such as rapid screening of the novel coronavirus in primary medical institutions, field detection of epidemiology, emergency detection at entry ports and the like, and simultaneously provides a technical paradigm for high-sensitivity detection of protein markers of other pathogenic microorganisms.

[0047] In addition to the following embodiment 1, the specific embodiments of the application also include embodiments formed by using all the left end point values of the range values of the technical solutions, embodiments formed by using all the right end point values of the range values, and embodiments formed by using any end point value of the range values.

[0048] Embodiment 1: a preparation method of a ratio type electrochemical sensor for detecting N protein, the preparation method comprising the following steps:

[0049] Step 1) acupuncture needle pretreatment: a stainless steel acupuncture needle with a diameter of 0.25 mm and a length of 40 mm is selected, after polishing and polishing to remove the surface oxide layer, the acupuncture needle is sequentially ultrasonically cleaned in ethanol and deionized water for 5 minutes, and then dried by nitrogen for standby, so that the clean and activated electrode substrate is provided for subsequent modification;

[0050] 2) Gold nanoparticles modification: A three-electrode system was used (a needle as the working electrode, saturated calomel as the reference electrode, and a platinum wire as the counter electrode). The 10 mm tip of the needle was immersed in a 2.5 mmol / L HAuCl4 solution containing 0.1 M KCl. The electrode was modified by cyclic voltammetry scanning at a rate of 25 mV / s and a potential range of -1.5 V to 0.5 V for 5 cycles (5 times of potential cycling). The ANME / AuNPs electrode was obtained. A layer of gold nanoparticles with high specific surface area and excellent conductivity was formed on the tip surface by electrochemical deposition;

[0051] 3) Multi-walled carbon nanotube modification: The ANME / AuNPs electrode was placed in an aqueous solution containing 0.02 mol / L 3,4-ethylenedioxythiophene (PEDOT monomer) and 2 mg / mL multi-walled carbon nanotubes (MCNT, diameter 15 nm, length 10 μm). The electrode was electro-polymerized at a potential of 1.2 V for 130 seconds. After cleaning and drying, the ANME / AuNPs / MCNT electrode was obtained. A three-dimensional conductive network was constructed to further amplify the electrical signal;

[0052] 4) Reference signal modification: The ANME / AuNPs / MCNT electrode was immersed in a 5 mM thionine solution (solvent: 0.1 M PBS, pH = 6.0). The electrode was modified by cyclic voltammetry scanning at a rate of 90 mV / s and a potential range of -0.4 V to 0.4 V for 60 times. Thionine (TH) was fixed as the internal reference signal to obtain the ANME / AuNPs / MCNT / TH electrode. The thionine signal molecule, which was used as a stable internal reference, was fixed;

[0053] 5) Internal signal modification: The non-tip area of the electrode was wrapped with insulating tape, and only the 5 mm tip (i.e., 1 / 2 of the tip length) was exposed and immersed in a 20 mM imipramine solution (solvent: 0.1 M PBS, pH = 7.0). The electrode was modified by cyclic voltammetry scanning at a rate of 100 mV / s and a potential range of -0.4 V to 1.0 V for 6 times. Imipramine (IMP) was fixed as the response signal to obtain the 1 / 2 electrode ANME / AuNPs / MCNT / TH / IMP. The response signal molecule was fixed in a partial area, which laid the foundation for ratio detection;

[0054] 6) Secondary modification of gold nanoparticles: The obtained ANME / AuNPs / MCNT / TH / IMP is used as the working electrode (5 mm electrode), and cyclic voltammetry scanning is performed for one cycle (i.e. immersion in a 2.5 mmol / L HAuCl4 solution containing 0.1 M KCl, modification by cyclic voltammetry scanning, scanning rate 25 mV / s, potential range -1.5 V to 0.5 V) according to the parameters of step 2) to supplement AuNPs on the surface of the signal molecule layer and increase the subsequent functional monomer binding sites, obtaining the electrode ANME / AuNPs / MCNT / TH / IMP / AuNPs; the gold nanoparticles deposited this time are intended to provide more abundant binding sites for subsequent modification;

[0055] 7) Modification of 4-mercapto phenylboric acid: The electrode is immersed in a 10 mM 4-mercapto phenylboric acid (4-MPBA) ethanol solution and soaked at 0-4°C for 50 minutes to form a monolayer by Au-S bond self-assembly (gold-sulfur bond self-assembly to form a monolayer, which serves as a functional monomer, and its boric acid group can specifically bind to N protein), obtaining the electrode ANME / AuNPs / MCNT / TH / IMP / AuNPs / 4-MPBA;

[0056] 8) Binding of template molecules: The obtained electrode is immersed in a 50 μg / mL N protein solution (solvent is 0.02 M PBS, pH = 7.4) and incubated at 2°C for 9 hours to form a 4-MPBA-N protein complex, obtaining the electrode ANME / AuNPs / MCNT / TH / IMP / AuNPs / 4-MPBA-N;

[0057] 9) Modification of imprinted polymer film: The obtained electrode is used as the working electrode, and cyclic voltammetry scanning is performed in a 0.01 M PBS solution containing 0.1 M pyrrole (pH = 7.4) to polymerize, with a scanning rate of 40 mV / s and a potential range of -0.3 V to 0.8 V, for 12 scans, to form a polypyrrole (Ppy) film wrapped complex, embedding the template molecules therein, obtaining the electrode ANME / AuNPs / MCNT / TH / IMP / AuNPs / 4-MPBA-N / Ppy;

[0058] 10) Elution of template molecules: The obtained electrode is immersed in an eluent (0.2 M HCl mixed with 20% methanol, solvent is 0.1 M PBS, pH = 5.0) and eluted for 90 minutes to form a polypyrrole (Ppy) film wrapped complex, obtaining the surface molecularly imprinted microsensor ANME / AuNPs / MCNT / TH / IMP / AuNPs@SMIP. The sensor surface leaves a three-dimensional cavity that is exactly complementary to N protein in shape, size, and functional groups, achieving specific recognition of the target.

[0059] Specifically, the sensor uses a stainless steel acupuncture needle as a substrate, forming a layered structure from the inside out: an AuNPs bottom layer, an MCNT-PEDOT conductive layer, a TH / IMP dual signal layer, a secondary AuNPs layer, a 4-MPBA functional layer, and a Ppy imprint layer. The 5mm needle tip region simultaneously contains the response signal (IMP) and imprint cavities, while the remaining areas contain only the reference signal (TH). The imprint layer contains uniformly distributed three-dimensional cavities precisely matched to the size (approximately 50kDa) and functional groups of the N protein, enabling specific target capture.

[0060] like Figure 1 As shown, the surface morphology evolution of the electrode during the stepwise modification process was characterized using a scanning electron microscope (SEM) system: only longitudinal scratches formed by mechanical polishing were visible on the surface of the bare acupuncture needle electrode (ANME). Figure 1 -A, E); After electrochemical deposition, gold nanoparticles (AuNPs) are densely attached to the scratch edges and surface in anisotropic morphology, forming a rough ANME / AuNPs interface. Their particle size distribution is between 40-160 nm, and the interparticle porosity effectively increases the specific surface area. Figure 1 -B,F); subsequently, a composite layer of 3,4-ethylenedioxythiophene and multi-walled carbon nanotubes was introduced by electropolymerization. The filamentary structure formed by the interwoven carbon nanotubes uniformly covered the AuNPs surface, further constructing a composite matrix with high loading capacity and fast electron transport pathways. Figure 1 -C, G); Finally, after molecular imprinting polymerization and template elution, the electrode surface exhibits a continuous, dense, and porous imprinted polymer film. The uniformly distributed nanopores within the film provide a structural basis for the specific recognition of the N protein, while the porous structure also ensures the effectiveness of the electron transport channel. Figure 1 -D,H).

[0061] like Figure 2 As shown, the N protein sensor was modified using 5.0 mM [Fe(CN)6] during the stepwise modification process. 3- / 4-Cyclic voltammetry in 0.1M KCl solution. The bare acupuncture needle microelectrode (ANME) exhibits only a weak and irreversible oxidation peak, indicating weak surface electron transport and low electrochemical activity. After electrodeposition of gold nanoparticles (AuNPs), a pair of reversible redox peaks appear, mainly attributed to the high specific surface area and excellent electrocatalytic activity of AuNPs, which significantly promotes electron transfer between the probe and electrode. Subsequently, the PEDOT / MWCNT composite layer formed by electropolymerization further enhances the current response. The mechanism lies in the synergistic construction of a three-dimensional electron conduction network by the large specific surface area of ​​multi-walled carbon nanotubes (MWCNTs) and the high conductivity of PEDOT, effectively improving interfacial transport efficiency. After modification with thionine (TH) and imipramine (IMP) dual-signal molecules, the current response remains at a high level, indicating that these two molecules not only possess good electrochemical activity themselves but also synergize with the underlying conductive structure, providing a stable signal basis for the subsequent construction of a ratiometric sensing platform. The subsequent second electrodeposition of gold nanoparticles further provides an ideal interface for subsequent biomodification. After self-assembling and incubating the N protein template with 4-mercaptophenylboronic acid (4-MPBA), template immobilization was achieved using the specific borate ester bond between 4-MPBA and the protein glycosyl group. This step caused a slight change in current, reflecting the binding of biomolecules on the electrode surface. Further electropolymerization of pyrrole was used to form a molecularly imprinted polymer (MIP) film. This process involves the spatially oriented polymerization of monomers around the template, forming a structure with specific recognition capabilities. After eluting the template protein, the current response recovered to some extent, indicating the formation of the imprinted cavity, laying the foundation for subsequent rebinding of the target protein.

[0062] like Figure 3 and Figure 4 The figures shown are the cyclic voltammogram and differential pulse voltammogram of the sensor in 0.1M PBS (pH=7.0), respectively. Figure 3 In the assay, the ANME / AuNPs / MCNT / TH / IMP electrode clearly identified the redox peaks corresponding to thionine and imipramine, respectively. Subsequently, after pyrrole electropolymerization to coat the N-protein template, the peak currents of both significantly decreased due to the formation of a dense, non-conductive polypyrrole film, reflecting the physical effect of the template being embedded in the polymer matrix. After template elution, the peak current of IMP recovered significantly, while the TH signal remained stable, indicating that the formation of the imprint cavity reopened the electron transport pathway, and that TH, as an internal reference signal, ensured the reliability of the detection process. Compared to cyclic voltammetry, Figure 4 The differential pulse voltammogram in the image more clearly demonstrates this continuous change process, further verifying the ratiometric sensing mechanism with molecular imprinting as the core and dual-signal molecules as the output, highlighting the potential of this design to achieve highly specific detection in complex biological samples.

[0063] Application Example 1

[0064] The prepared ratio-type electrochemical sensor was incubated in the solution containing different concentrations of N protein (0.005 ng / mL-10000 ng / mL) for 30 minutes. As shown in Figure 5 , the peak current of the imprinted pramipexole gradually decreased with the increase of the concentration of N protein. This current decrease was due to the occupation of the imprinted nanocavity by the structure-complementary N protein molecules, thereby hindering the electron transfer of the imprinted pramipexole.

[0065] Example 2

[0066] As shown in Figure 6 , when the prepared ratio-type electrochemical sensor was used for N protein detection, the I IMP / I TH value had a strong linear relationship with the logarithmic value of the concentration of N protein, and the linear equation was I IMP / I TH =-0.08192lgC+0.57283 (R 2 =0.99600). The linear range was 0.005 ng / mL-10000 ng / mL, and the detection limit was 0.001 ng / ml. In comparison, when the imprinted electrochemical sensor was used for N protein detection, the I IMP value had a weak linear relationship with the logarithmic value of the concentration of N protein, and the linear equation was I IMP =-6.4717lgC+40.6697 (R 2 =0.99512). The linear range was 0.01 ng / mL-10000 ng / mL, and the detection limit was 0.007 ng / mL. The above results proved that the ratio-type electrochemical sensor could be successfully applied to the sensitive detection of N protein.

[0067] Example 3

[0068] The specificity of the micro electrochemical sensor was studied by detecting only 1 μg / mL N protein and mixing it with fibroblast growth factor (FGF), osteonectin (OPN), mesothelin (MSLN), Na + , Mg 2+ , Ca 2+ , Cl - , SO4 2- , uric acid (UA) and dopamine (DA), respectively. As shown in Figure 7 , the signals generated by the ratio-type and imprinted sensors after the addition of other molecules were similar to those in the presence of only N protein, indicating that the sensor had good specificity, and the specificity of the ratio-type sensor was slightly better than that of the imprinted sensor.

[0069] Comparative Example 1

[0070] First, at 5.0 mM [Fe(CN)6] 3- / 4- The cyclic voltammetric (CV) responses of each modification step were compared in solution. For example... Figure 8 As shown in AC, the CV signal showed a weak change after introducing the built-in signaling molecule imipramine (IMP) onto the ANME substrate, indicating that IMP modification was unsuccessful; while the CV signal showed a weak change after introducing AuNPs (…). Figure 8 -B) and further compounding of MCNTs ( Figure 8 -C), IMP immobilization caused a significant change in the CV signal, confirming that these two nanomaterials can effectively promote IMP immobilization. Finally, signal readout was performed on the three functionalized interfaces in 0.1M PBS (pH=7.0). ANME / AuNPs / MCNT / IMP all exhibited clear, characteristic responses attributable to IMP in both CV and differential pulse voltammetry (DPV) curves. Figure 8 -D, E) demonstrate the successful construction of the composite substrate.

[0071] Comparative Example 2

[0072] To investigate the efficient assembly mechanism of the N protein, three immobilization strategies were tested on the optimal substrate ANME / AuNPs / MCNT / IMP, and the assembly was carried out at 5.0 mM [Fe(CN)6]. 3- / 4- Monitoring in solution is performed using cyclic voltammetry (CV). For example... Figure 9 As shown in Figure -A, direct adsorption of the N protein onto the interface (ANME / AuNPs / MCNT / IMP / N) did not induce significant changes in the CV signal. Similarly, as... Figure 9 As shown in Figure -B, the CV response of introducing AuNPs first and then adsorbing N protein (ANME / AuNPs / MCNT / IMP / AuNPs / N) was not significantly different from that before modification. These results indicate that neither of these physical adsorption methods achieved efficient and large-scale immobilization of N protein. Conversely, when 4-MPBA was used as the molecular cross-linking agent (ANME / AuNPs / MCNT / IMP / AuNPs / 4-MPBA~N), the CV signal changed significantly. Figure 9 -C), which clearly demonstrates that the specific covalent interaction between 4-MPBA and N protein successfully achieved efficient and targeted fixation of N protein.

[0073] In a preferred embodiment, in step 1), the acupuncture needle is made of stainless steel, with a diameter of 0.2-0.3 mm and a length of 30-50 mm.

[0074] In a preferred embodiment, in step 2), the cyclic voltammetric scan uses a three-electrode system, with a saturated calomel electrode as the reference electrode and a platinum wire electrode as the counter electrode.

[0075] In a preferred embodiment, the multi-walled carbon nanotubes in step 3) have a diameter of 10-20 nm and a length of 5-15 μm, and are treated with nitric acid reflux before use to increase the surface carboxyl groups.

[0076] In a preferred embodiment, the thionine solution in step 4) is filtered through a 0.22 μm filter before use to remove insoluble impurities.

[0077] In a preferred embodiment, the 1 / 2 needle tip portion in step 5) refers to a region 5 mm from the needle tip end point, and the unmodified region is wrapped with insulating tape to avoid contact with the solution.

[0078] In a preferred embodiment, the 4-mercapto phenyl boronic acid ethanol solution in step 7) is prepared fresh and used immediately, and the soaking reaction is carried out under nitrogen protection.

[0079] In a preferred embodiment, the N protein solution in step 8) has a concentration of 50 μg / mL ± 5 μg / mL, and the incubation process is carried out in a constant temperature incubator to avoid light.

[0080] The application also provides a ratio-type electrochemical sensor for detecting N protein prepared by the above method.

[0081] The application also provides application of the ratio-type electrochemical sensor for detecting N protein in detecting coronavirus N protein, characterized in that the application has a detection linear range of 0.005 ng / mL-10000 ng / mL and a detection limit of 0.001 ng / mL.

[0082] Finally, it should be noted that the application is not limited to the above embodiments, and many variations are possible. All variations that can be directly derived or thought of by those of ordinary skill in the art from the disclosure of the application should be considered within the scope of the application.

Claims

1. A method of preparing a ratiometric electrochemical sensor for detecting N protein, characterized by The preparation method comprises the following steps: Step 1) acupuncture needle pretreatment: polishing and polishing the acupuncture needle, and sequentially ultrasonic cleaning in ethanol and deionized water for 4-6 minutes, and drying with nitrogen for standby; Step 2) modification of gold nanoparticles: taking the acupuncture needle pretreated in step 1) as a working electrode, immersing the 9-11mm needle tip part into a 2.5mmol / L HAuCl4 solution containing 0.1-02M KCl, and modifying by cyclic voltammetry scanning at a scanning rate of 20-30mV / s and a potential range of-1.5V~0.5V for 4-6 cycles to obtain the electrode ANME / AuNPs; Step 3) modification of multi-walled carbon nanotubes: taking the ANME / AuNPs obtained in step 2) as a working electrode, and electro-polymerizing in a water solution containing 0.01-0.03mol / L 3,4-ethylenedioxythiophene and 1-3mg / mL multi-walled carbon nanotubes at a potential of 1.1-1.3V for 120-140 seconds, and cleaning and drying to obtain the electrode ANME / AuNPs / MCNT; Step 4) modification of reference signal: immersing the ANME / AuNPs / MCNT electrode obtained in step 3) into a 4-6mM thionine solution (solvent: 0.1M PBS, pH=6.0), and modifying by cyclic voltammetry scanning at a scanning rate of 80-100mV / s and a potential range of-0.4V~0.4V for 50-70 times to obtain the electrode ANME / AuNPs / MCNT / TH; Step 5) modification of built-in signal: immersing 1 / 2 needle tip part of the ANME / AuNPs / MCNT / TH electrode obtained in step 4) into a 10-30mM imipramine solution (solvent: 0.1M PBS, pH=7.0), and modifying by cyclic voltammetry scanning at a scanning rate of 90-110mV / s and a potential range of-0.4V~1.0V for 5-7 times to obtain the 1 / 2 electrode ANME / AuNPs / MCNT / TH / IMP; Step 6) secondary modification of gold nanoparticles: taking the ANME / AuNPs / MCNT / TH / IMP obtained in step 5) as a working electrode, and cyclic voltammetry scanning for 1-2 cycles according to the parameters of step 2) to obtain the electrode ANME / AuNPs / MCNT / TH / IMP / AuNPs; Step 7) modification of 4-mercapto phenylboric acid: immersing the electrode obtained in step 6) into a 10mM 4-mercapto phenylboric acid ethanol solution, and soaking at 0℃-4℃ for 40-60 minutes to obtain the electrode ANME / AuNPs / MCNT / TH / IMP / AuNPs / 4-MPBA; Step 8) binding of template molecules: immersing the electrode obtained in step 7) into a 50μg / mL N protein solution (solvent: 0.02M PBS, pH=7.4), and incubating at 0℃-4℃ for 8-10 hours to obtain the electrode ANME / AuNPs / MCNT / TH / IMP / AuNPs / 4-MPBA~N; ​ ​ Step 9) Modification of the imprinted polymer film: using the electrode obtained in step 8) as the working electrode, polymerization was carried out by cyclic voltammetry scanning in a 0.01-0.02 M PBS solution (pH = 7.4) containing 0.1-0.3 M pyrrole, at a scanning rate of 30-50 mV / s, in a potential range of -0.3 V to 0.8 V, for 11-13 scans, to obtain the electrode ANME / AuNPs / MCNT / TH / IMP / AuNPs / 4-MPBA~N / Ppy; Step 10) Elution of the template molecules: the electrode obtained in step 9) was immersed in an eluent (0.2 M HCl mixed with 20% methanol, solvent: 0.1 M PBS, pH = 5.0) for 80-100 min, to obtain the surface molecularly imprinted microsensor ANME / AuNPs / MCNT / TH / IMP / AuNPs@SMIP.

2. The preparation method of the ratio-type electrochemical sensor for detecting N protein according to claim 1, characterized in that, In step 1), the acupuncture needle is made of stainless steel, with a diameter of 0.2-0.3 mm and a length of 30-50 mm.

3. The preparation method of the ratio-type electrochemical sensor for detecting N protein according to claim 1 or 2, characterized in that, In step 2), the cyclic voltammetry scanning was performed using a three-electrode system, with a saturated calomel electrode as the reference electrode and a platinum wire electrode as the counter electrode.

4. The preparation method of the ratio-type electrochemical sensor for detecting N protein according to claim 3, characterized in that, In step 3), the multi-walled carbon nanotubes had a diameter of 10-20 nm and a length of 5-15 μm, and were treated with nitric acid reflux before use to increase the surface carboxyl groups.

5. The method for preparing a ratiometric electrochemical sensor for detecting N protein according to claim 1, characterized in that, In step 4), the thionine solution was filtered through a 0.22 μm filter before use to remove insoluble impurities.

6. The method of claim 1 or 2 or 4 or 5, wherein the ratio-type electrochemical sensor for detecting N protein is prepared by the steps of: In step 5), the 1 / 2 needle tip portion refers to a region 5 mm from the needle tip end point, and the non-modified region was wrapped with insulating tape to avoid contact with the solution.

7. The method for preparing a ratiometric electrochemical sensor for detecting N protein according to claim 1, characterized in that, In step 7), the 4-mercapto phenyl boronic acid ethanol solution was prepared fresh before use, and the immersion reaction was carried out under nitrogen protection.

8. The method for preparing a ratiometric electrochemical sensor for detecting N protein according to claim 1, characterized in that, In step 8), the concentration of the N protein solution was 50 μg / mL ± 5 μg / mL, and the incubation process was carried out in a constant-temperature incubator to avoid light.

9. A ratiometric electrochemical sensor for detecting N protein, prepared by the method of any one of claims 1-8.

10. Use of the ratiometric electrochemical sensor for detecting N protein according to claim 9 for detecting SARS-CoV-2 N protein. The application has a detection linear range of 0.005 ng / mL-10000 ng / mL and a detection limit of 0.001 ng / mL.

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