Intelligent reproducible detection method for norovirus

Abstract

The invention relates to an intelligent reproducible detection method of norovirus, and relates to the technical field of aptamer electrochemical sensing, and the intelligent reproducible detection method of norovirus comprises the following steps: constructing a norovirus electrochemical sensor, dispensing a sample containing norovirus on the constructed norovirus electrochemical sensor, and detecting the norovirus in the sample. After incubation is conducted for 40-50 min, the sample is detected in a 10-15 mM phosphate buffer solution through pulse voltammetry DPV, the pulse amplitude ranges from 45 mV to 55 mV, the pulse width ranges from 16 ms to 17 ms, the pulse period ranges from 90 ms to 110 ms, and the potential range ranges from-0.5 V to 0.5 V. The electrochemical sensor for the norovirus is small in size and convenient to carry, detection can be carried out on site in a specific environment, the detection method is simple, and the detection result is accurate and reliable.

Description

Technical Field

[0001] The present invention relates to the field of aptamer electrochemical sensing technology, and in particular to an intelligent and regenerative detection method for norovirus. Background Art

[0002] Norovirus (NoV) is a positive-stranded RNA calicivirus that can frequently contaminate a variety of foods, especially livestock products such as beef and pork. Once in the food chain, norovirus often compromises human immunity and can cause illnesses including acute gastroenteritis, severe diarrhea, and even acute septic shock. Currently, methods for detecting norovirus have been developed, including enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), and reverse transcription loop-mediated isothermal amplification (RT-LAMP). ELISA primarily detects norovirus through an antigen-antibody reaction. This method is time-saving, cost-effective, and easy to use, but lacks specificity and sensitivity, and is expensive. PCR, which amplifies virus-specific RNA fragments for detection, offers high sensitivity and specificity, allowing accurate identification of viral genotypes, but requires high sample collection and processing requirements. RT-LAMP only requires a simple constant temperature device to complete the reaction. It is easy and quick to operate, has high detection sensitivity, and produces accurate results. However, it can only detect the presence of viral nucleic acids and cannot distinguish between infectious intact virus particles and non-infectious viral RNA fragments or degradation residues, which may lead to false positive results.

[0003] A Chinese invention patent application, publication number CN113155924A, discloses a method for detecting norovirus. The method utilizes AuNPs@ZnFe2O4@COF, Apt@AuNPs@ZnFe2O4@COF, and AuNPs@BP@Ti3C2MXene. The method comprises the following steps: 1) synthesizing ZnFe2O4@COF, synthesizing AuNPs@ZnFe2O4@COF, and synthesizing Apt@AuNPs@ZnFe2O4@COF; 2) exfoliating black phosphorus BP; synthesizing BP@Ti3C2MXene; synthesizing AuNPs@BP@Ti3C2MXene; and immobilizing a peptide (NoroBP); and 3) constructing a norovirus electrochemical sensor.

[0004] This invention creates an electrochemical sensor that can quickly and effectively detect norovirus concentrations in samples. However, when analyzing norovirus in specific environments, such as beef and pork samples from remote farms, the detection process is prone to problems such as complex background interference or limited shelf life. Therefore, current technology still has shortcomings in detecting and analyzing norovirus in specific environments. Summary of the Invention

[0005] To address the above technical issues, the present invention provides an intelligent and reproducible method for detecting norovirus. The electrochemical sensor for norovirus of the present invention is small and portable, can be used for on-site detection under specific conditions, has a simple detection method, and produces accurate and reliable detection results.

[0006] In a first aspect, the present invention provides an intelligent and reproducible detection method for norovirus, comprising the following steps: The detection method comprises the following steps: A norovirus electrochemical sensor was constructed, and a sample containing norovirus was drop-coated onto the constructed norovirus electrochemical sensor. After incubation for 40-50 minutes, the sample was detected by pulse voltammetry (DPV) in 10-15 mM phosphate buffer, with a pulse amplitude of 45-55 mV, a pulse width of 16-17 ms, a pulse period of 90-110 ms, and a potential range of -0.5-0.5 V.

[0007] Optionally, the norovirus electrochemical sensor includes a motherboard and a daughter board, the motherboard includes a PID controller, an error amplifier, a heating driver, and a main control board equipped with an MSP430 single-chip microcomputer, and the daughter board is divided into an electrochemical daughter board and a single-chip microcomputer daughter board, the electrochemical daughter board includes a hexagonally arranged three-electrode system arranged on the top layer, an integrated feedback heater arranged on the bottom layer, and a double-layer flexible printed circuit board base, the single-chip microcomputer daughter board includes an MCU memory and a single-control switch; the flexible printed circuit board substrate includes a 17μm copper layer and a 1μm gold-plated layer with a thickness of 0.25mm, the three-electrode system includes 10 norovirus working electrodes with a diameter of 1mm, an Ag / AgCl reference electrode and a counter electrode, the feedback heater includes 15 resistor arrays and a negative temperature coefficient thermistor, and the main control board is used to execute a programmable temperature control program.

[0008] Optionally, the MCU memory contains a programmable thermal curve program.

[0009] Optionally, the preparation method of the norovirus working electrode includes the following steps: (1) Rinse the screen-printed electrode with ethanol and deionized water, blow dry the electrode surface with nitrogen, and drop-coat 5-15 μL of 0.2-0.3 mg / mL signal amplification carrier suspension on the screen-printed electrode surface. Dry at room temperature to form a basement membrane on the electrode surface to obtain screen-printed electrode A. (2) Add 5-15 μL of 5-7 μM ssDNA to the surface of screen-printed electrode A and incubate for 50-70 min, then add 5-15 μL of 1-3 mM 6-mercapto-1-hexanol and passivate for 20-40 min to obtain screen-printed electrode B; (3) Add 5-15 μL of 5-7 μM aptamer (Apt) to screen-printed electrode B and hybridize for 20-40 min to form an Apt / ssDNA complex in screen-printed electrode B to obtain screen-printed electrode C; (4) Soak the screen-printed electrode C in a 5-15 mM methylene blue (MB) solution for 5-15 min to allow MB to adsorb to the Apt / ssDNA complex, thereby obtaining a norovirus working electrode.

[0010] Optionally, the signal amplification carrier is a MXene@MWCNT-Au-Fc nanocomposite material, and the synthesis steps of the MXene@MWCNT-Au-Fc nanocomposite material are as follows: Synthesis of MXene: 2-3 mg of Ti3AlC2 powder was slowly added to continuously stirred HF (40%, 10-15 mL). After standing for 48-50 hours, the concentrated product was centrifuged and diluted with deionized water to 10-15 mL and re-centrifuged until the pH value of the supernatant reached 6. The resulting precipitate was dried for 12-14 hours to obtain MXene powder. Synthesis of MXene@MWCNT: 1.5-2.5 mg of MXene was ultrasonically dispersed in 1.5-2.5 ml of deionized water. Simultaneously, 1.5-2.5 mg of MWCNTs was dispersed in 1.5-2.5 ml of deionized water. The MXene and MWCNT dispersions were mixed, ultrasonicated for 0.5-1.5 h, and then centrifuged at 3500-4500 rpm for 8-12 min to obtain solid MXene@MWCNT. Synthesis of MXene@MWCNT-Au: 1.4-2.4 mg of MXene@MWCNT and 0.7-1.2 ml of HAuCl4 were added to 2.8-4.8 ml of deionized water to form MXene@MWCNT-Au; Synthesis of MXene@MWCNT-Au-Fc: Disperse 0.5-1.5 mg of MXene@MWCNT-Au in 1-3 mL of ethanol, mix with 0.5-1.5 mL of 10 mM Fc C2H6O, stir for 25-35 minutes, and centrifuge at 2500-3500 rpm for 10 minutes to separate MXene@MWCNT-Au-Fc.

[0011] In the aforementioned technical scheme, two-dimensional layered MXenes possess remarkable properties, including large surface area, strong hydrophilicity, and high conductivity. MXenes are typically obtained by etching the "A" layer elements from the Mn+1AXn phase. The multilayer structure may agglomerate and restack, limiting electrochemical performance. Therefore, appropriate functional groups (-O, -OH, and -F) were introduced into the etched product, Mn+1XnTx. Multi-walled carbon nanotubes (MWCNTs) and gold nanoparticles (AuNPs) can be embedded and grown in situ on the MXene without the use of reducing agents or stabilizers, pursuing optimal electrochemical performance. The synergistic effect of these multiple components creates favorable conditions for the large-scale anchoring of the signal ferrocene (Fc) and semi-complementary strands (ssDNA). Methylene blue (MB) can be incorporated into double-stranded DNA (dsDNA) through electrostatic interactions with the phosphate backbone.

[0012] Ti3AlC2 itself exhibits a solid, condensed structure, while MXene transforms into a multilayered structure due to the removal of aluminum after HF etching. The spacing between MXene layers is regular, and each layer has a smooth surface. MWCNTs exhibit a regular, twisted shape and spontaneously bond with the soft MXene after reaction. High-magnification SEM observations revealed that MWCNTs are tightly and densely attached to the multilayer surface of MXene, forming a composite material with a large specific surface area. After mixing MXene@MWCNT with HAuCl4, AuNPs grow in situ on the multilayer surface of MXene@MWCNTs, forming MXene@MWCNT-Au.

[0013] Optionally, the detection range of the detection method is 1-10 6 copies / mL, and the minimum detection limit was 0.67 copies / mL.

[0014] In a second aspect, the present invention provides an intelligent, renewable detection method for norovirus that uses the aforementioned method to detect beef and pork samples from remote farms or in specific environments such as complex background interference and limited shelf life.

[0015] In summary, the present invention includes the following beneficial technical effects: This application discloses a smart and renewable detection method for norovirus, wherein the MXene@MWCNTs-Au-Fc signal carrier is assembled on a screen-printed electrode, ssDNA is semi-complementary to Apt, accommodates the renewable Apt, and enables norovirus to conduct ΔI MB / I FcThe on-chip compatible programmable thermal profile facilitates thermal regeneration with a fully optimized and precisely controlled temperature profile, enhancing the regeneration of Apt-ssDNA, ensuring highly reliable multiple analyses, and preventing functional degradation caused by internal thermal stress. Therefore, this detection method enables the reuse of Apt-based biosensors. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a data representation diagram of Example 1; ( Figure 1 A is the SEM image of Ti3AlC2 in Example 1; Figure 1 B is the SEM image of MXene@MWCNTs in Example 1; Figure 1 C is the SEM image of MXene@MWCNTs-Au in Example 1; Figure 1 D is the XRD pattern of MXene@MWCNTs and MXene@MWCNTs-Au in Example 1; Figure 1 E is the XPS graph of MXene@MWCNTs-Au in Example 1; Figure 1 F is the elemental mapping of MXene@MWCNTs-Au in Example 1;) Figure 2 This is a data representation diagram of Example 2; ( Figure 2 A is a CV curve diagram of the preparation process of the norovirus working electrode in Example 2; Figure 2 B is the EIS graph of the preparation process of the norovirus working electrode in Example 2; Figure 2 C is the Zeta potential of MXene, MXene@MWCNTs, MXene@MWCNTs-Au, MXene@MWCNTs-Au-Fc, and ssDNA / MXene@MWCNTs-Au-Fc in Example 2; Figure 2 D is the CV curve of MXene@MWCNTs and MXene@MWCNTs-Au at 100 mV / s in Example 2; Figure 2 E is the electroactive area diagram of MXene@MWCNTs and MXene@MWCNTs-Au in Example 2. Figure 3 Schematic diagram of the detection method of Example 4; ( Figure 3 A is a diagram of the Apt-based norovirus electrochemical sensor; Figure 3 B is a graph showing compatibility of field regeneration via programmable thermal curves; Figure 3 C is a physical picture of the homemade SPE system; Figure 3 D is a diagram showing how to achieve on-site regeneration by coordinating the timing of the microcontroller. Figure 4 A quick comparison chart of Example 3 with and without thermal regeneration compared to Example 4; Figure 5 is the evaluation diagram of programmable regeneration; ( Figure 5 A is a comparison of the preset thermal distribution curve (filled area) and the chip's real-time tracking capability (line). The inset area is usually a magnified view (cycle: 120 seconds). Figure 5 B is the I before (hollow bar) and after (solid bar) recombination. MB picture) Figure 6 For the use of DPV in 1-10 6 Detection of norovirus ΔI within the range of copies / mL MB / I Fc with lgC NoV Linear correlation plot; Figure 7 Figure 2 is a diagram of Fc stability during regeneration. DETAILED DESCRIPTION

[0017] The present invention is further described in detail below with reference to the examples.

[0018] Materials used in the following examples: Norovirus (NoV) was purchased from Xiamen Wankelong Biotechnology Co., Ltd. (China). Chloroauric acid (HAuCl4), methylene blue (MB), ferrocene (Fc), hydrogen fluoride (HF), potassium ferricyanide (K3[Fe(CN)6]), and phosphate buffered saline (PBS, 10 mM, pH 7.4) were purchased from Beijing Chemical Reagent Company (China). Ti3AlC2 powder (200 mesh) was purchased from Jilin Yiyi Technology Co., Ltd. (China). Multi-walled carbon nanotube powder was purchased from Nanjing Pioneer Nanotechnology Co., Ltd. (China). Anhydrous ethanol (99.8%) and 6-mercapto-1-hexanol (MCH) were purchased from Sigma-Aldrich Co., Ltd. (China). Deionized water was used throughout the experiments.

[0019] Electrochemical experiments were conducted using a CHI-660D analyzer (CHInstrument, USA). During the regeneration process, thermal images were obtained using an infrared thermal imager (Tis-20+, Fluke, USA), and the solution temperature was continuously monitored using a thermocouple system (Keysight, USA, U1185A & U1242B). The morphologies of Ti3AlC2, MXene, multi-walled carbon nanotubes, MXene@MWCNTs, and MXene@MWCNTs-Au were observed using a scanning electron microscope (JEOL, Japan, JSM-7800M SEM). The crystal structure and elemental distribution of MXene@MWCNTs and MXene@MWCNTs-Au were analyzed using an X-ray diffractometer (Bruker, Germany, D8ADVANCE XRD) and an X-ray photoelectron spectroscopy (Thermo Scientific, USA, K-Alpha XPS). The elemental composition of the MXene@MWCNTs-Au was investigated using an energy dispersive spectrometer (Octane SDD EDS, AMETEK, USA). Zeta potential measurements were performed using a Zetasize rnano ZSP (Malvern Panalytica, UK). Norovirus control readings were obtained using RT-qPCR (EZNA® Viral RNA Kit, Omega Bio-tek, USA).

[0020] Example 1: Preparation and characterization of MXene@MWCNT-Au-Fc composites The preparation steps of MXene@MWCNT-Au-Fc composite materials are as follows: S1. Add 2.5 mg Ti3AlC2 powder to 10 mL 40% HF and etch for 48 h. Centrifuge and wash until pH is 6. After drying for 12 h, multilayer MXene is obtained. The SEM image of MXene is shown in Figure 2. Figure 1 As shown in A.

[0021] S2. 2 mg MXene and 2 mg MWCNTs were ultrasonically dispersed in 2 mL deionized water, mixed, and ultrasonically treated for 1 h. The MXene@MWCNT composite material was obtained by centrifugation at 4000 rpm for 10 min. The SEM image of the MXene@MWCNT composite material is shown in FIG. Figure 1 As shown in B.

[0022] S3, 2mgMXene@MWCNT and 1mL10mMHAuCl4 were reacted in 4mL deionized water to form MXene@MWCNT-Au. The SEM image of MXene@MWCNT-Au is shown in Figure 1 C, the XRD pattern is as follows Figure 1 As shown in D.

[0023] S4. Disperse 1 mg of MXene@MWCNT-Au in 2 mL of ethanol, mix with 1 mL of 10 mM FcC2H6O, stir for 30 min, and centrifuge at 3000 rpm for 10 min to separate MXene@MWCNT-Au-Fc. The XPS graph is shown in Figure 4. Figure 1 As shown in E.

[0024] Through SEM, we can see that Figure 1 B shows that the carbon nanotubes in MXene@MWCNT are tightly attached to the MXene layers. Figure 1 C shows that AuNPs are in situ grown on the MXene surface. Figure 1 The XRD of D showed Au characteristic peaks at 38.36° and 44.58°.

[0025] Through XPS, we can see that Figure 1 E shows that the Au4f peak is located at 82.99 eV.

[0026] It can be seen from EDS that Figure 1 Figure 5 shows elemental mapping, which shows that Ti, C, and O are evenly distributed. Among them, C is particularly dispersed, indicating that the bonding between MXene and MWCNTs is reliable and efficient. Au is distributed in a regular dot-like manner.

[0027] Example 2: Preparation of Norovirus Working Electrode The preparation method of the norovirus working electrode comprises the following steps: S1. Preparation of MXene@MWCNT-Au-Fc composite material: S11, 2.5 mg Ti3AlC2 powder was added to 10 mL 40% HF and etched for 48 h, centrifuged and washed to pH 6, and dried for 12 h to obtain multilayer MXene; S12, ultrasonically disperse 2 mg of MXene and 2 mg of MWCNTs in 2 mL of deionized water, mix, and ultrasonically treat for 1 h, and centrifuge at 4000 rpm for 10 min to obtain a MXene@MWCNT composite material; S13, 2 mg of MXene@MWCNT was reacted with 1 mL of 10 mM HAuCl4 in 4 mL of deionized water to form MXene@MWCNT-Au; S14. Disperse 1 mg of MXene@MWCNT-Au in 2 mL of ethanol, mix with 1 mL of 10 mM Fc C2H6O, stir for 30 min, and centrifuge at 3000 rpm for 10 min to separate MXene@MWCNT-Au-Fc.

[0028] S2. Rinse the screen-printed electrode with ethanol and deionized water, blow dry the electrode surface with nitrogen, and drop-coat 10 μL of the 0.25 mg / mL MXene@MWCNT-Au-Fc suspension prepared in Example 1 on the screen-printed electrode surface. Dry at room temperature to form a basement membrane on the electrode surface to obtain screen-printed electrode A.

[0029] S3. Add 10 μL of 6 μM ssDNA to the surface of screen-printed electrode A and incubate for 60 min. Then add 10 μL of 2 mM 6-mercapto-1-hexanol and passivate for 30 min to obtain screen-printed electrode B.

[0030] S4. Add 10 μL of 6 μM aptamer (Apt) to the screen-printed electrode B and hybridize for 30 minutes to form an Apt / ssDNA complex in the screen-printed electrode B to obtain the screen-printed electrode C.

[0031] S5. Soak the screen-printed electrode C in a 10 mM methylene blue (MB) solution for 10 min to allow MB to adsorb to the Apt / ssDNA complex, thereby obtaining a norovirus working electrode.

[0032] The electrochemical performance of the norovirus working electrode was verified by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The CV potential window was -0.2-0.6 V, and the EIS scanning frequency range was 10 -2 -10 5 Hz. The CV curve of the Norovirus working electrode is as follows Figure 2 As shown in A, the redox peak of MXene@MWCNT-Au-Fc modified significantly increases, indicating that the electrochemical active area is expanded. Figure 2 As shown in B, the high-frequency semicircle diameter reflects the interfacial charge transfer resistance (R et ), which effectively responds to the changes in the electrode / solution interface. R of MXene@CNTs-Au-Fc / GCE et shows the lowest value, which means that the charge transfer process is fast. et The values increased gradually with the addition of ssDNA, Apt, and MB. All the results of the EIS experiments were consistent with those of the CV.

[0033] Zeta potential Figure 2As shown in Figure C, the zeta potentials of MXene and MXene@MWCNTs are -24.5 and -17.7 mV, respectively. The zeta potential of MXene@MWCNTs-Au decreases (-19.6 mV) due to the introduction of negatively charged AuNPs. Similarly, the phosphate backbone of ssDNA is negatively charged, resulting in a lower zeta potential of ssDNA / MXene@MWCNTs-Au-Fc (-16.8 mV) than that of MXene@MWCNTs-Au-Fc (-14.1 mV).

[0034] The CV curve at 100mV / s is as follows Figure 2 As shown in Figure 4, MXene@MWCNTs-Au / SPE brings higher redox peaks than MXene@MWCNTs / SPE or bare sensor, indicating that AuNPs can accelerate electron transfer and increase the active area, indicating that MXene@CNTs-Au has significant electrochemical performance.

[0035] Figure 2 E shows that the electrochemical active area of MXene@MWCNT-Au / SPE reaches 0.099 cm², which is significantly higher than that of the bare electrode (0.047 cm²), showing better electron transfer, proving that MXene@MWCNT-Au is an effective signal amplification carrier.

[0036] Example 3: Construction of smart electrochemical sensor S1. Preparation of MXene@MWCNT-Au-Fc composite material: S11, 2.5 mg Ti3AlC2 powder was added to 10 mL 40% HF and etched for 48 h, centrifuged and washed to pH 6, and dried for 12 h to obtain multilayer MXene; S12, ultrasonically disperse 2 mg of MXene and 2 mg of MWCNTs in 2 mL of deionized water, mix, and ultrasonically treat for 1 h, and centrifuge at 4000 rpm for 10 min to obtain a MXene@MWCNT composite material; S13, 2 mg of MXene@MWCNT was reacted with 1 mL of 10 mM HAuCl4 in 4 mL of deionized water to form MXene@MWCNT-Au; S14. Disperse 1 mg of MXene@MWCNT-Au in 2 mL of ethanol, mix with 1 mL of 10 mM Fc C2H6O, stir for 30 min, and centrifuge at 3000 rpm for 10 min to separate MXene@MWCNT-Au-Fc.

[0037] S2. Preparation of Norovirus Working Electrode: S21. Rinse the screen-printed electrode with ethanol and deionized water, blow dry the electrode surface with nitrogen, and drop-coat 10 μL of a 0.25 mg / mL MXene@MWCNT-Au-Fc suspension on the screen-printed electrode surface. Dry at room temperature to form a basement membrane on the electrode surface, thereby obtaining a screen-printed electrode A. S22, adding 10 μL of 6 μM ssDNA to the surface of screen-printed electrode A and incubating for 60 min, and then adding 10 μL of 2 mM 6-mercapto-1-hexanol and passivating for 30 min to obtain screen-printed electrode B; S23, adding 10 μL of 6 μM aptamer (Apt) to screen-printed electrode B and hybridizing for 30 min to form an Apt / ssDNA complex in screen-printed electrode B to obtain screen-printed electrode C; S24. Soak the screen-printed electrode C in a 10 mM methylene blue (MB) solution for 10 min to allow MB to adsorb to the Apt / ssDNA complex, thereby obtaining a norovirus working electrode.

[0038] S3. Assembly of the sensor: The norovirus electrochemical sensor includes a motherboard and a daughterboard. The motherboard includes an MCU memory, a PID amplifier, a main control board equipped with an MSP430 microcontroller, and a drive heater. The daughterboard includes a hexagonally arranged three-electrode system arranged on the top layer, an integrated feedback heater arranged on the bottom layer, and a double-layer flexible printed circuit board substrate; the flexible printed circuit board substrate contains a 17μm copper layer and a 1μm gold-plated layer with a thickness of 0.25mm. The three-electrode system includes 10 norovirus working electrodes with a diameter of 1mm, an Ag / AgCl reference electrode and a counter electrode. The feedback heater includes 15 resistor arrays and a negative temperature coefficient thermistor. The main control board is used to execute a programmable temperature control program.

[0039] S4, 10 μL of concentrations of 0, 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 A norovirus sample with a concentration of 10 copies / mL was drop-coated on the constructed norovirus electrochemical sensor. After incubation for 45 minutes, the sample was detected by pulse voltammetry (DPV) in 10 mM phosphate buffer with a pulse amplitude of 50 mV, a pulse width of 16.7 ms, a pulse period of 100 ms, and a potential range of -0.5-0.5 V.

[0040] S5, Temperature control module: MSP430 single chip microcomputer executes PID algorithm to dynamically adjust the heating power, and the temperature error is <3℃.

[0041] Example 4: The specific detection steps of a smart and renewable detection method for norovirus are as follows: S1. Preparation of MXene@MWCNT-Au-Fc composite material: S11, 2.5 mg Ti3AlC2 powder was added to 10 mL 40% HF and etched for 48 h, centrifuged and washed to pH 6, and dried for 12 h to obtain multilayer MXene; S12, ultrasonically disperse 2 mg of MXene and 2 mg of MWCNTs in 2 mL of deionized water, mix, and ultrasonically treat for 1 h, and centrifuge at 4000 rpm for 10 min to obtain a MXene@MWCNT composite material; S13, 2 mg of MXene@MWCNT was reacted with 1 mL of 10 mM HAuCl4 in 4 mL of deionized water to form MXene@MWCNT-Au; S14. Disperse 1 mg of MXene@MWCNT-Au in 2 mL of ethanol, mix with 1 mL of 10 mM Fc C2H6O, stir for 30 min, and centrifuge at 3000 rpm for 10 min to separate MXene@MWCNT-Au-Fc.

[0042] S2. Preparation of Norovirus Working Electrode: S21. Rinse the screen-printed electrode with ethanol and deionized water, blow dry the electrode surface with nitrogen, and drop-coat 10 μL of a 0.25 mg / mL MXene@MWCNT-Au-Fc suspension on the screen-printed electrode surface. Dry at room temperature to form a basement membrane on the electrode surface, thereby obtaining a screen-printed electrode A. S22, adding 10 μL of 6 μM ssDNA to the surface of screen-printed electrode A and incubating for 60 min, and then adding 10 μL of 2 mM 6-mercapto-1-hexanol and passivating for 30 min to obtain screen-printed electrode B; S23, adding 10 μL of 6 μM aptamer (Apt) to screen-printed electrode B and hybridizing for 30 min to form an Apt / ssDNA complex in screen-printed electrode B to obtain screen-printed electrode C; S24. Soak the screen-printed electrode C in a 10 mM methylene blue (MB) solution for 10 min to allow MB to adsorb to the Apt / ssDNA complex, thereby obtaining a norovirus working electrode.

[0043] S3. Assembly of norovirus electrochemical sensor: The norovirus electrochemical sensor includes a motherboard and a daughterboard. The motherboard includes a PID controller, an error amplifier, a heating driver, and a main control board equipped with an MSP430 single-chip microcomputer. The daughterboards are divided into electrochemical daughterboards and single-chip microcomputer daughterboards. The electrochemical daughterboard includes a hexagonally arranged three-electrode system arranged on the top layer, an integrated feedback heater arranged on the bottom layer, and a double-layer flexible printed circuit board base. The single-chip microcomputer daughterboard includes an MCU memory and a single-control switch; the flexible printed circuit board substrate contains a 17μm copper layer and a 1μm gold-plated layer with a thickness of 0.25mm. The three-electrode system includes 10 norovirus working electrodes with a diameter of 1mm, an Ag / AgCl reference electrode and a counter electrode. The feedback heater includes 15 resistor arrays and a negative temperature coefficient thermistor. The main control board is used to execute a programmable temperature control program.

[0044] S4, 10 μL of concentrations of 0, 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 A norovirus sample with a concentration of 10 copies / mL was drop-coated on the constructed norovirus electrochemical sensor. After incubation for 45 minutes, the sample was detected by pulse voltammetry (DPV) in 10 mM phosphate buffer with a pulse amplitude of 50 mV, a pulse width of 16.7 ms, a pulse period of 100 ms, and a potential range of -0.5-0.5 V.

[0045] S5, Temperature control module: MSP430 single chip microcomputer executes PID algorithm to dynamically adjust the heating power, and the temperature error is <3℃.

[0046] S6, thermal profile design: using spline curve to increase temperature ( Figure 5 A-III), maintain at 80℃ for 30s and then slowly cool (120s) to avoid thermal stress damage.

[0047] Figure 3 This is a schematic diagram of the detection method in Example 4. MXene / MWCNTs-Au is used as a signal amplification carrier and combined with the internal reference signal ferrocene (Fc) to be modified on the surface of a homemade screen-printed electrode. ssDNA, Apt, and MB are sequentially modified onto the electrode surface through Au-S bonding, base complementarity, and electrostatic adsorption. NoV competitively binds Apt to ssDNA, and MB and the aptamer are excreted with NoV, inhibiting I MB The signal I Fc The signal remains constant, thus triggering the ratiometric measurement signal (ΔI MB / I Fc )like Figure 3A, achieved specific detection of norovirus. This method is based on the competitive binding strategy of auxiliary DNA chains to establish a sensing system, so the detection platform can be regenerated after the analysis is completed, such as Figure 3 B By integrating programmable thermal curves into the sensing system, such as Figure 3 C,With the hardware support of screen-printed electrodes, Apt can be updated regularly, e.g. Figure 3 D thus achieving smooth timing and optimal performance.

[0048] Figure 4 A quick comparison of Example 3 with and without thermal regeneration is shown for Example 4. Figure 5 A) The advantages of thermal regeneration with and without thermal contribution were semi-quantitatively evaluated. As shown in curve I, in the analysis of 10 6 Even after the high concentration of norovirus (100 copies / mL), a small MB peak was still observed. This indicates the presence of Apt-ssDNA residues that serve as MB containers, although most Apt-ssDNA structures are competitively dissociated due to Apt-NoV binding. Unreacted residues tend to accumulate on the sensing surface and bind to the newly formed Apt-ssDNA structure, resulting in I MB Thermal regeneration effectively removed Apt-ssDNA residues by accelerating and promoting base dissociation, thus providing clean conditions (curve III) for sensor regeneration (curve IV).

[0049] like Figure 6 As shown, using DPV in 1-10 6 Norovirus was detected within the range of copies / mL, ΔI MB / I Fc with lgC NoV The linear correlation was found (R²=0.996) and the detection limit was 0.67 copies / mL, indicating that the detection sensitivity of this application was high and trace amounts of norovirus in samples could be detected.

[0050] Regeneration effect analysis: Figure 5 After 7 cycles, the thermal curve III of I MB The signal RSDs is <2.07%, which is the lowest relative standard deviation compared with other thermal curves, such as Figure 7 As shown, thermal curve III of I Fc The stability is the best. The two sets of data can prove that curve III is better than conventional heating and has better regeneration effect.

[0051] The above are all preferred embodiments of the present invention, and are not intended to limit the scope of protection of the present invention. Therefore, any equivalent changes made based on the structure, shape, and principle of the present invention should be included in the scope of protection of the present invention.

Claims

1. An intelligent and reproducible detection method for norovirus, characterized in that: The detection method comprises the following steps: A norovirus electrochemical sensor was constructed, and a sample containing norovirus was drop-coated onto the constructed norovirus electrochemical sensor. After incubation for 40-50 minutes, the sample was detected by pulse voltammetry (DPV) in 10-15 mM phosphate buffer, with a pulse amplitude of 45-55 mV, a pulse width of 16-17 ms, a pulse period of 90-110 ms, and a potential range of -0.5-0.5 V.

2. The intelligent and reproducible detection method for norovirus according to claim 1, characterized in that: The norovirus electrochemical sensor includes a motherboard and a daughterboard. The motherboard includes a PID controller, an error amplifier, a heating driver, and a main control board equipped with an MSP430 single-chip microcomputer. The daughterboard is divided into an electrochemical daughterboard and a single-chip microcomputer daughterboard. The electrochemical daughterboard includes a hexagonally arranged three-electrode system arranged on the top layer, an integrated feedback heater arranged on the bottom layer, and a double-layer flexible printed circuit board base. The single-chip microcomputer daughterboard includes an MCU memory and a single-control switch; the flexible printed circuit board substrate includes a 17μm copper layer and a 1μm gold-plated layer with a thickness of 0.25mm. The three-electrode system includes 8-12 norovirus working electrodes with a diameter of 1mm, an Ag / AgCl reference electrode and a counter electrode. The feedback heater includes a 14-16 resistor array and a negative temperature coefficient thermistor. The main control board is used to execute a programmable temperature control program.

3. The intelligent and reproducible detection method for norovirus according to claim 2, characterized in that: The MCU memory contains a programmable thermal curve program.

4. The intelligent and reproducible detection method for norovirus according to claim 3, characterized in that: The preparation method of the norovirus working electrode comprises the following steps: (1) Rinse the screen-printed electrode with ethanol and deionized water, blow dry the electrode surface with nitrogen, and drop-coat 5-15 μL of 0.2-0.3 mg / mL signal amplification carrier suspension on the screen-printed electrode surface. Dry at room temperature to form a basement membrane on the electrode surface to obtain screen-printed electrode A. (2) Add 5-15 μL of 5-7 μM ssDNA to the surface of screen-printed electrode A and incubate for 50-70 min, then add 5-15 μL of 1-3 mM 6-mercapto-1-hexanol and passivate for 20-40 min to obtain screen-printed electrode B; (3) Add 5-15 μL of 5-7 μM aptamer (Apt) to screen-printed electrode B and hybridize for 20-40 min to form an Apt / ssDNA complex in screen-printed electrode B to obtain screen-printed electrode C; (4) Soak the screen-printed electrode C in a 5-15 mM methylene blue (MB) solution for 5-15 min to allow MB to adsorb to the Apt / ssDNA complex, thereby obtaining a norovirus working electrode.

5. The intelligent and reproducible detection method for norovirus according to claim 4, characterized in that: The signal amplification carrier is a MXene@MWCNT-Au-Fc nanocomposite material, and the synthesis steps of the MXene@MWCNT-Au-Fc nanocomposite material are as follows: Synthesis of MXene: 2-3 mg of Ti3AlC2 powder was slowly added to continuously stirred HF (40%, 10-15 mL). After standing for 48-50 h, the concentrated product was centrifuged and diluted with deionized water to 10-15 mL and re-centrifuged until the pH value of the supernatant reached 6. The resulting precipitate was dried for 12-14 h to obtain MXene powder. Synthesis of MXene@MWCNT: 1.5-2.5 mg of MXene was ultrasonically dispersed in 1.5-2.5 ml of deionized water. Simultaneously, 1.5-2.5 mg of MWCNTs was dispersed in 1.5-2.5 ml of deionized water. The MXene and MWCNT dispersions were mixed, ultrasonicated for 0.5-1.5 h, and then centrifuged at 3500-4500 rpm for 8-12 min to obtain solid MXene@MWCNT. Synthesis of MXene@MWCNT-Au: 1.4-2.4 mg of MXene@MWCNT and 0.7-1.2 ml of HAuCl4 were added to 2.8-4.8 ml of deionized water to form MXene@MWCNT-Au; Synthesis of MXene@MWCNT-Au-Fc: Disperse 0.5-1.5 mg of MXene@MWCNT-Au in 1-3 mL of ethanol, mix with 0.5-1.5 mL of 10 mM Fc C2H6O, stir for 25-35 minutes, and centrifuge at 2500-3500 rpm for 10 minutes to separate MXene@MWCNT-Au-Fc.

6. The intelligent and reproducible detection method for norovirus according to claim 5, characterized in that: The detection range of the detection method is 1-10 6 copies / mL, and the minimum detection limit was 0.67 copies / mL.

7. An intelligent, reproducible detection method for norovirus according to any one of claims 1 to 6 for use in beef and pork samples from remote farms or in specific environments such as complex background interference and limited shelf life.

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