A tobacco mosaic virus (TMV) bi-modal biosensor, methods of making and uses thereof

Abstract

The application belongs to the technical field of biosensors, and provides a tobacco mosaic virus (TMV) dual-mode biosensor, a preparation method and application thereof. 2 The biosensor is composed of a dual-mode luminescent material Co / Ce-TCBPE@Ru(dcbpy)3 2 ⁺‑COOH, a lateral flow chromatography (LFIA) flexible electrode, a TMV antibody Ab1, a TMV antibody Ab1 and a self-assembled micro electrochemical luminescence (ECL) sensor, and the qualitative and quantitative dual-signal output of visual and ECL detection significantly improves the sensitivity and accuracy of TMV detection. The sensor detection range is 0.1 pg / mL to 100 ng / mL, the detection limit reaches 48.9 fg / mL, and the sensor has the advantages of simple operation, rapid response, high stability and dual-signal guarantee, and is suitable for rapid visual detection of plant diseases, and has a wide application prospect in agricultural detection.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biosensors, and particularly relates to a tobacco mosaic virus TMV dual-mode biosensor, a preparation method and application thereof. BACKGROUND

[0002] Tobacco mosaic virus (TMV) is a widely influential important plant virus that can cause serious diseases in various crops, leading to reduced yield and quality of agricultural crops, and causing huge economic losses to agricultural production. Traditional TMV detection methods such as enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) have high sensitivity and specificity, but have problems such as complex operation, long detection period, strong dependence on equipment, and are not suitable for on-site rapid detection.

[0003] As a rapid, portable and low-cost biological detection technology, lateral flow immunochromatographic assay (LFIA) has been widely used in plant disease detection in recent years. However, traditional LFIA mainly relies on single visual signal output, has low sensitivity, is easily interfered by background, and is difficult to meet the accurate detection requirements of low-concentration viruses. Therefore, how to improve the detection sensitivity and reliability on the basis of LFIA has become a research hotspot.

[0004] In recent years, electrochemiluminescence (ECL) technology has shown great development potential in the field of biosensing due to its high sensitivity, low background noise and easy operation. Combining ECL technology with LFIA platform is expected to overcome the limitations of traditional methods and realize efficient and accurate virus detection. However, there is still a lack of dual-mode (visual + electrochemiluminescence) detection sensors for TMV. SUMMARY

[0005] To solve the above technical problems, the application provides a preparation method of a tobacco mosaic virus TMV dual-mode biosensor and application thereof. The TMV dual-mode biosensor is a new type of biosensor based on dual-mode luminescent material and lateral flow chromatography flexible electrode integration, which can realize rapid, sensitive, visual and quantitative detection.

[0006] One of the purposes of the application is to prepare a tobacco mosaic virus TMV dual-mode biosensor, which is composed of dual-mode luminescent material Co / Ce-TCBPE@Ru(dcbpy)3²⁺-COOH, lateral flow chromatography LFIA flexible electrode, TMV antibody Ab1, TMV antibody Ab2 and self-assembled micro-ECL sensor.

[0007] The autonomous assembly micro ECL sensor, also known as the autonomous assembly micro electrochemiluminescence (ECL) sensor, is based on the detection mechanism of electrochemiluminescence, and the autonomous assembly photon detector includes a photon counter and a counting unit, and a micro electrochemical workstation provides a voltage, and a customized adaptive shell is provided to optimize the protection performance and portability of the equipment, so that the micro ECL sensor realizes the detection effect with high sensitivity and high stability similar to the large laboratory detection instrument, and the detection portability is improved, and the on-site detection is realized.

[0008] The second purpose of the present application is to disclose a preparation method of the above-mentioned tobacco mosaic virus (TMV) double-mode biosensor, characterized in that the method comprises the following steps:

[0009] (1) Preparation of detection line T line and quality detection line C line: taking TMV antibody Ab1 as the working electrode of LFIA flexible electrode, i.e. the coating of the detection line T line on the nitrocellulose NC membrane; taking the goat anti-mouse IgG antibody as the coating of the quality detection line C line; 2.5-7.5 μL of 0.5 mg·mL −1 of the TMV antibody Ab1 is dropped on the surface of the working electrode, and incubated at 4 °C for 2 hours; 1-3 mg·mL −1 of the goat anti-mouse IgG antibody is fixed on the C line of the NC membrane, so as to obtain the preparation of the NC membrane with the detection line T line and the quality detection line C line, i.e. the LFIA flexible electrode;

[0010] (2) Preparation of LFIA-ECL immunotest strip: the sample pad is pretreated with 10 mmol·L −1 of phosphate buffer solution (PBS) containing 2.5% Tween 20 and 0.1% curdlan X-100, and then heated at 37 °C for 6 hours;

[0011] The conjugate pad is pretreated with 10 mmol·L −1 of buffer solution PBS containing 2.5% bovine serum albumin (BSA) and 0.5% Tween 20, and then dried at 37 °C for 6 hours;

[0012] The solution containing 4-12 mg·mL −1 of Co / Ce-TCBPE@Ru(dcbpy)3 2+ -COOH-Ab2 is uniformly sprayed on the conjugate pad, and then dried at 37 °C for 6 hours;

[0013] The NC membrane, the conjugate pad, the sample pad and the water absorption pad prepared in (1) are sequentially pasted on the bottom plate to obtain the LFIA-ECL immunotest strip;

[0014] (3) Take 80-240 μL of TMV antigen containing 1.0% Tween 20 in 10 mM pH=7.4 PBS and 0.0001 ng·mL −1 , 0.001 ng·mL −1 , 0.1 ng·mL −1 , 1 ng·mL −1 , 10 ng·mL −1 , 100 ng·mL −1 Concentrations are dropped on the sample pad of LFIA-ECL immunochromatographic strip, and the color change of T line and C line of working electrode is observed for 5-10 min.

[0015] The third object of the application is to prepare a high-precision LFIA flexible electrode by using inkjet printing technology. By optimizing the formula of conductive ink (containing silver paste / carbon ink material) and printing parameters, a three-electrode system perfectly matched with the test strip is constructed on the NC film flexible substrate. The successful development of this technology provides important technical support for the construction of a portable and high-performance LFIA-ECL dual-mode detection platform.

[0016] The preparation method of the LFIA flexible electrode is as follows:

[0017] (1) Cut the nitrocellulose NC film into a size of 30 mm in width and 90 mm in length, and prepare a wax channel with a width of 5 mm in the middle of the NC film by wax dyeing, so as to divide the NC film into wettable and non-wettable areas;

[0018] (2) Print the conductive area on the NC film, select BASE-CP10P conductive ink, adjust the air pressure gauge to 45 psi, and determine the positions of the two scales on the tray before formal inkjet printing. The printing platform is preheated at 40 DEG C before printing;

[0019] (3) The hole cover on the platform is cut and spliced to ensure the flatness and stability of the substrate during printing. When connecting the printer, select ink drop observation, point glue scraping, and 16B program;

[0020] (4) The limit voltage value of the waveform voltage is 40 V, and the conventional setting is 20 V. Adjust the parameters to determine the printed pattern and position on the NC film. Start printing the working electrode WE and the counter electrode CE. After printing, put the NC film into the drying box and heat it at 130 DEG C for 15 min for curing;

[0021] (5) When printing the reference electrode RE, select BASE-CD01 high-conductivity silver paste, and adjust the pattern and position again for printing. Then put the complete printed NC film into the drying box and heat it at 120 DEG C for 30 min to obtain the LFIA flexible electrode.

[0022] The fourth object of the application is to combine the rapid separation characteristics of lateral flow chromatography with the high sensitivity of electrochemiluminescence to construct a dual-mode detection system. LFIA realizes efficient capture of target objects, and ECL provides accurate quantification. The two complement each other to solve the problem of insufficient sensitivity or poor portability of a single technology, forming a one-stop detection scheme of "sample in-result out".

[0023] The application also includes a dual-mode luminescent material Co / Ce-TCBPE@Ru(dcbpy)3²⁺-COOH. In the material, Co / Ce-TCBPE@Ru(dcbpy)3²⁺-COOH realizes the synthesis of nanoflower metal-organic framework MOFs material with Co as the central ion, Ce as the doping atom, TCBPE with the property of aggregation-induced emission, and Ru(dcbpy)3²⁺-COOH with excellent ECL performance as a dual ligand. The material combines the characteristics of visible light fluorescence emission and electrochemiluminescence, and the luminescent performance can be further enhanced through resonance energy transfer between the dual ligands. The doping of Ce can improve the electrochemically active surface area of the material and enhance the reaction active center, further improving the luminescent performance. The tobacco mosaic virus antibody is functionally modified to prepare a dual-mode luminescent bioconjugate for target capture and signal amplification.

[0024] The preparation method of the dual-mode luminescent material Co / Ce-TCBPE@Ru(dcbpy)3²⁺-COOH is as follows:

[0025] Take 0.5-1.5 mg of cerium nitrate hexahydrate Ce(NO3)3·6H2O, 3-9 mg of cobalt nitrate hexahydrate Co(NO3)3·6H2O, 8-24 mg of tetracarboxy biphenyl ethylene H4TCBPE, and 5-15 mg of tris(4,4-dicarboxy biphenyl) ruthenium chloride C 36 H 19 ClN6O 12 Ru-4 as a reaction raw material is added to 6-18 mL of N,N-dimethylformamide DMF and 2-6 mL of ultrapure water, and then 10-30 μL of nitric acid HNO3 is added dropwise to dissolve the reaction raw material. After stirring uniformly, the reaction is carried out at 100 ℃ for 24 h. The orange product Co / Ce-TCBPE@Ru(dcbpy)3 2+ -COOH is obtained by centrifugal washing with DMF and ultrapure water and vacuum drying at 60 ℃ overnight.

[0026] Preferably, in (2), the preparation method of Co / Ce-TCBPE@Ru(dcbpy)3 2+ -COOH-Ab2 is as follows:

[0027] Take 2~6 mg Co / Ce-TCBPE@Ru(dcbpy)3 in claim 4 2+ -COOH is dispersed in 850~2250 μL PBS with pH=7.4, 50~150 μL 400 mmol·L −1 of 1-ethyl-(3-dimethylaminopropyl) carbonyl diimide hydrochloride EDC and 50~150 μL 100 mmol·L −1 of N-hydroxysuccinimide NHS, and shaken at 4 ℃ for 3 h;

[0028] Centrifugal removal of excess EDC and NHS, add 50~150 μL 10 μg·mL −1 Ab2 solution, shaken at 4 ℃ overnight, and the resulting biological conjugate Co / Ce-TCBPE@Ru(dcbpy)3 2+ -COOH-Ab2 is obtained, washed with PBS for 3~5 times, and finally Co / Ce-TCBPE@Ru(dcbpy)3 2+ -COOH-Ab2 is dissolved in 1~3 mL PBS with a concentration of 2 mg·L −1 , and stored at 4 ℃.

[0029] The fifth object of the present application is the application of the tobacco mosaic virus TMV dual-mode biosensor in detecting and / or diagnosing tobacco mosaic virus. It mainly relates to a novel sensing platform integrating qualitative screening and quantitative analysis, which is simple to operate, short in detection time, and can quickly complete sample processing and result interpretation in the field environment. The ECL-LFIA dual-mode detection sensor has a detection range of 0.1 pg·mL−1~100 ng·mL−1 for tobacco mosaic virus, a detection limit of 48.9 fg·mL−1, and a wide application prospect, especially for agricultural plant disease monitoring and early warning.

[0030] The steps of detecting and / or diagnosing tobacco mosaic virus TMV by using the tobacco mosaic virus TMV dual-mode biosensor are as follows:

[0031] (1) The NC membrane LFIA flexible electrode is removed for ECL three-electrode detection, and 20~60 μL 4~12 mmol·L −1 of tripropylamine with 0.10~0.15 mol·L −1 pH=7.4 PBS solution is added at WE;

[0032] (2) The TMV antigen is detected by time-intensity method, and the voltage is set to continuously cycle potential scanning from 0 V to 1.2~1.8 V;

[0033] (3) In the detection process, the photomultiplier tube is set to 400-800 V, and the scanning rate is 0.1-0.2 V·s −1 , record the light intensity, and draw a working curve;

[0034] (4) The TMV antigen sample solution to be detected is replaced by standard solution for detection, and the concentrations of the TMV antigen sample solution are 0.0001 ng·mL −1 , 0.001 ng·mL −1 , 0.1 ng·mL −1 , 1 ng·mL −1 , 10 ng·mL −1 , and 100 ng·mL −1 .

[0035] The detection process of the tobacco mosaic virus TMV double-mode biosensor for detecting and / or diagnosing the tobacco mosaic virus TMV involves a portable electrochemiluminescence detection device, which comprises a dark box body provided with a host warehouse and a detection warehouse, a photon counting unit fixedly arranged in the host warehouse, and an electrochemical pressure unit fixedly arranged in the detection warehouse.

[0036] The inside of the dark box body is provided with a horizontal partition plate to divide the internal space into the host warehouse at the top and the detection warehouse at the bottom, the horizontal partition plate is provided with a detection through hole communicating the host warehouse and the detection warehouse, and the photon counting unit performs electrochemiluminescence detection on the to-be-detected sensor connected with the electrochemical pressure unit through the detection through hole.

[0037] The present application has the following advantages and effects compared with the prior art:

[0038] (1) The present application proposes a new TMV double-mode biosensor combining double-mode luminescent material and lateral flow chromatography flexible electrode, and a new material Co / Ce-TCBPE@Ru(dcbpy)3²⁺-COOH with double-mode luminescent performance is introduced into the biosensor;

[0039] The material takes tetracarboxy biphenyl ethylene (H4TCBPE) as a skeleton, and through the coordination of metal ions cobalt (Co²⁺) and the combination with tris(4,4-dicarboxy diphenylpyridine) ruthenium chloride [Ru(bpy)3]²⁺, the material is endowed with excellent visible light luminescence and ECL double signal characteristics, and the resonance energy transfer between the double ligands further improves the luminescence effect of the material; Co / Ce-TCBPE@Ru(dcbpy)3²⁺-COOH can not only produce bright visible light under ultraviolet excitation, but also improve the electrochemical activity surface area of the material through the doping of Ce, and can produce stable and strong electrochemiluminescence signals under electrochemical excitation conditions, greatly improving the detection sensitivity and the accuracy of signal reading;

[0040] (2) In order to realize flexible and low-cost electrode integration, the application introduces a nitrocellulose (NC) membrane surface printed electrode technology, and by customizing printing of conductive materials such as carbon ink and silver paste on the surface of the NC membrane, a working electrode (WE), a counter electrode (CE) and a reference electrode (RE) are constructed, and the organic integration of the lateral flow chromatography system and the three-electrode system is realized;

[0041] (3) The above preparation technology has the characteristics of simple process, flexible pattern design, good membrane flexibility, excellent liquid transport performance and the like, and can significantly improve the stability and detection efficiency of the sensor in complex environments such as high temperature, cold and high humidity;

[0042] (4) The LFIA-ECL dual-mode detection sensor prepared by the application has the advantages of low cost, high sensitivity, good specificity, rapid detection and easy preparation, and the related data in the examples show that the LFIA-ECL dual-mode detection sensor provided by the application has a qualitative detection speed of 5-7 minutes, and a quantitative detection speed of 2-3 minutes, realizing the one-stop rapid detection of "sample in-result out", and having the dual functions of qualitative screening and quantitative analysis, and effectively optimizing the current detection technology of TMV. BRIEF DESCRIPTION OF DRAWINGS

[0043] Figure 1 SEM morphology diagram of the dual-mode luminescent material Co / Ce-TCBPE@Ru(dcbpy)3²⁺-COOH;

[0044] Figure 2 Log calibration diagram of the dual-mode sensor for different concentrations of TMV;

[0045] Figure 3 Reproducibility detection data diagram of the dual-mode sensor for TMV;

[0046] Figure 4 Stability detection data diagram of the dual-mode sensor for TMV;

[0047] Figure 5 Specificity detection data diagram of the dual-mode sensor for TMV;

[0048] Figure 6 Preparation and application schematic diagram of the dual-mode sensor;

[0049] Figure 7 Structure schematic diagram of the portable electrochemical luminescence detection equipment in the embodiment of the application;

[0050] Figure 8 Internal structure schematic diagram of the portable electrochemical luminescence detection equipment in the embodiment of the application;

[0051] Figure 9This is a schematic diagram of the dark box in the portable electrochemiluminescence detection device according to an embodiment of the present invention;

[0052] Figure 10 This is a top view of the dark box in the portable electrochemiluminescence detection device of this invention.

[0053] Attached reference numerals: 1. Dark box housing; 11. Main unit compartment; 12. Detection compartment; 13. Reserved interface; 14. Placement position marker; 15. Second card limiting slot; 16. Top opening; 17. Front opening; 2. Horizontal partition; 21. Detection through hole; 22. First card limiting slot; 3. Counting main unit; 4. Induction probe; 5. Micro electrochemical workstation; 6. Electrode converter; 7. Top sliding door; 8. Front sliding door. Detailed Implementation

[0054] To enable those skilled in the art to better understand the present invention, the present invention will now be further described in conjunction with specific embodiments.

[0055] Example 1: Fabrication of the LFIA-ECL Dual-Mode Sensor

[0056] (1) Dual-mode luminescent material Co / Ce-TCBPE@Ru(dcbpy)3 2+ Preparation of -COOH

[0057] Take 0.5 mg of cerium nitrate hexahydrate Ce(NO3)3·6H2O, 3 mg of cobalt nitrate hexahydrate Co(NO3)3·6H2O, 8 mg of tetracarboxylated biphenyl ethylene H4TCBPE, and 5 mg of tris(4,4-dicarboxylated bipyridine)ruthenium chloride C. 36 H 19 ClN6O 12 Ru-4 was added to 6 mL of N,N-dimethylformamide (DMF) and 2 mL of ultrapure water, followed by the addition of 10 μL of nitric acid (HNO3) to dissolve the solution. The mixture was stirred until homogeneous, then transferred to a high-pressure reactor and reacted at 100 °C for 24 h. The mixture was then washed separately with DMF and ultrapure water by centrifugation, and dried overnight in a vacuum drying oven at 60 °C to obtain the orange product, the dual-mode luminescent material Co / Ce-TCBPE@Ru(dcbpy)3. 2+ -COOH.

[0058] like Figure 1 As shown, the dual-mode luminescent material Co / Ce-TCBPE@Ru(dcbpy)3 2+ Scanning electron microscope image of -COOH, showing the prepared Co / Ce-TCBPE@Ru(dcbpy)3. 2+ -COOH has a nano-flower-like structure with a large specific surface area and a high electrochemically active surface area.

[0059] (2) Preparation of lateral flow chromatography LFIA flexible electrode

[0060] a. Cut the nitrocellulose NC membrane of model JN140 into a size of 30 mm in width and 90 mm in length, prepare a wax channel with a width of 5 mm in the middle of the NC membrane by wax dyeing, and divide the NC membrane into wettable and non-wettable areas; it should be noted that the wax channel specifically refers to the sample flow channel located in the middle area, and the wax dyeing area on both sides of the wax channel is a non-wettable area.

[0061] b. Perform conductive area printing on the NC membrane, select BASE-CP10P conductive ink, adjust the air pressure gauge pointer to 45 psi, and determine the positions of the two scales on the tray before formal inkjet printing; the printing platform uses a carbon ink printing preheating of 40 ℃ before printing;

[0062] c. Use the cutting and splicing substrate to cover the holes on the platform to ensure the flatness and stability of the substrate during printing, and select ink drop observation, point glue scraping, and 16B program when connecting the printer;

[0063] d. The limit voltage value of the waveform voltage is 40 V, and the conventional setting is 20 V; adjust the parameters to determine the printed pattern and position on the NC membrane, start printing the working electrode WE and the counter electrode CE, and after printing, put the NC membrane into a drying oven at 130 ℃ for 15 min for curing;

[0064] e. When printing the reference electrode RE, select BASE-CD01 high-conductivity silver paste, re-adjust the pattern and position for printing, and then put the complete printed NC membrane into a drying oven at 120 ℃ for 30 min to obtain the LFIA flexible electrode.

[0065] (3) Co / Ce-TCBPE@Ru(dcbpy)3 2+ Preparation of -COOH-Ab2 biological conjugate

[0066] Disperse 2 mg of Co / Ce-TCBPE@Ru(dcbpy)3 2+ -COOH in 850 μL of pH=7.4 phosphate buffered saline solution PBS, 50 μL of 400 mmol·L −1 of 1-ethyl-(3-dimethylaminopropyl) carbonyl diimide hydrochloride EDC, and 50 μL of 100 mmol·L −1 of N-hydroxysuccinimide NHS, and shake at 4 ℃ for 3 h. Then centrifuge to remove excess activator, add 50 μL of 10 μg·mL −1Ab2 solution, overnight shaking at 4 ℃, and Co / Ce-TCBPE@Ru(dcbpy)3 2+ -COOH-Ab2 bioconjugate was washed with PBS for several times, and finally dispersed in 1 mL PBS and stored at 4 ℃.

[0067] (4) Preparation of LFIA-ECL sensor

[0068] a. TMV antibody Ab1 was used as the coating material of the detection line T line on the working electrode of the lateral flow chromatography LFIA flexible electrode and the coating material of the quality control line C line on the goat anti-mouse lgG antibody, respectively. 2.5 μL of 0.5 mg·mL −1 of Ab1 was dropped on the surface of the working electrode, and incubated at 4 °C for 2 hours. 1 mg·mL −1 of IgG was fixed on the C line of the paper electrode.

[0069] b. The sample pad was pretreated with 10 mmol·L −1 of PBS buffer containing 2.5% Tween 20 and 0.1% Triton X-100, pH=7.4, and then heated at 37 ℃ for 6 hours; the conjugate pad was pretreated with 10 mmol·L −1 of PBS buffer containing 2.5% bovine serum albumin BSA and 0.5% Tween 20, pH=7.4, and then dried at 37 ℃ for 6 hours. 4 mg·mL −1 of Co / Ce-TCBPE@Ru(dcbpy)3 2+ -COOH-Ab2 solution was evenly sprayed on the conjugate pad, and then dried at 37 ℃ for 6 hours. The LFIA-ECL immunostrip was prepared by sequentially pasting the NC membrane, the conjugate pad, the sample pad, and the water absorption pad on the bottom plate.

[0070] c. 80 μL of 10 mM PBS containing 1.0% Tween 20, pH=7.4, and different concentrations of TMV antigen were dropped on the sample pad of the LFIA-ECL immunostrip, and the color changes at the T line and the C line of the working electrode were observed after 5~10 min.

[0071] Example 2 Preparation of LFIA-ECL dual-mode sensor

[0072] (1) Preparation of dual-mode luminescent material Co / Ce-TCBPE@Ru(dcbpy)3 2+ -COOH

[0073] Take 1 mg of cerium nitrate hexahydrate Ce(NO3)3·6H2O, 6 mg of cobalt nitrate hexahydrate Co(NO3)3·6H2O, 16 mg of tetracarboxy biphenyl ethylene H4TCBPE and 10 mg of tris(4,4-dicarboxy dipyridine) ruthenium chloride C 36 H 19 ClN6O 12 Ru-4 is added to 12 mL of N, N-dimethylformamide DMF and 4 mL of ultrapure water, and then 20 μL of nitric acid HNO3 is added dropwise to dissolve the solution, and then the mixture is stirred uniformly, and then the mixture is transferred to a high-pressure reaction kettle, and reacted at 120 ℃ for 20 h, and then washed by centrifugation with DMF and ultrapure water, and then dried in a vacuum drying oven at 80 ℃ overnight to obtain an orange product Co / Ce-TCBPE@Ru(dcbpy)3 2+ -COOH.

[0074] (2) Preparation of LFIA flexible electrode

[0075] a. The nitrocellulose NC membrane with model number JN140 is cut into a size of 30 mm in width and 90 mm in length, a wax channel with a width of 5 mm is prepared in the middle of the NC membrane by wax dyeing, and the NC membrane is divided into wettable and non-wettable areas;

[0076] b. Conductive area printing is performed on the NC membrane, BASE-CP10P conductive ink is selected, the air compressor is adjusted to 1 gear, the pointer on the air pressure gauge is adjusted to 45 psi, the positions of the two scales on the tray are determined before formal inkjet printing, and the printing platform is preheated at 40 ℃ before printing with carbon ink printing;

[0077] c. The hole cover on the platform is completed by cutting and splicing the substrate to ensure the flatness and stability of the substrate during printing. When connecting the printer, select ink drop observation, point glue scraping, and 16B program;

[0078] d. The limit voltage value of the waveform voltage is 40 V, and the conventional setting is 20 V. Adjust the parameters to determine the printed pattern and position on the NC membrane. Start printing the working electrode WE and the counter electrode CE. After printing, put the NC membrane into the drying oven and heat it at 130 ℃ for 15 min for curing;

[0079] e. When printing the reference electrode RE, select BASE-CD01 high-conductivity silver paste, adjust the pattern and position again for printing. Then put the complete printed NC membrane into the drying oven and heat it at 120 ℃ for 30 min to obtain the LFIA flexible electrode.

[0080] (3) Preparation of Co / Ce-TCBPE@Ru(dcbpy)3 2+ -COOH-Ab2 biological conjugate

[0081] Co / Ce-TCBPE@Ru(dcbpy)3-COOH-Ab2 was prepared by the following steps: 3 mg Co / Ce-TCBPE@Ru(dcbpy)3-COOH was dispersed in 1700 μL PBS (pH=7.4), 100 μL 400 mmol·L-1 EDC and 100 μL 100 mmol·L-1 NHS, and shaken at 4 °C for 3 h. Then the excess activator was removed by centrifugation, 100 μL 10 μg·mL-1 Ab2 solution was added, and shaken at 4 °C overnight. The obtained Co / Ce-TCBPE@Ru(dcbpy)3-COOH-Ab2 was washed with PBS for several times, and finally dispersed in 2 mL PBS and stored at 4 °C. 2+ −1 −1 −1 2+

[0082] (4) Preparation of LFIA-ECL sensor

[0083] a. 5 μL 0.5 mg·mL-1 Ab1 was dropped on the surface of the working electrode of the LFIA flexible electrode, and incubated at 4 °C for 2 h, with TMV antibody Ab1 as the coating material of the detection line T line on the NC membrane, and goat anti-mouse lgG antibody as the coating material of the quality control line C line. 2 mg·mL-1 IgG was fixed on the C line of the paper electrode. −1 −1

[0084] b. The sample pad was pretreated with 10 mmol·L-1 PBS buffer (pH=7.4) containing 2.5% Tween 20 and 0.1% Triton X-100, and then heated at 37 °C for 6 h; the conjugate pad was pretreated with 10 mmol·L-1 PBS buffer (pH=7.4) containing 2.5% BSA and 0.5% Tween 20, and then dried at 37 °C for 6 h. A solution containing 8 mg·mL-1 Co / Ce-TCBPE@Ru(dcbpy)3-COOH-Ab2 was uniformly sprayed on the conjugate pad, and then dried at 37 °C for 6 h. The LFIA-ECL immunochromatographic strip was prepared by sequentially pasting the NC membrane, the conjugate pad, the sample pad, and the water absorption pad on the base plate. −1 −1 −1 2+

[0085] ​​​​​​​​​​​c. Take 160 μL of 10 mM PBS pH=7.4 containing 1.0% Tween 20 and different concentrations of TMV antigen and drop on the sample pad of LFIA-ECL immunochromatographic test strip, and wait for 5~10 min to observe the color change at the T line and C line of the working electrode.

[0086] Example 3 Preparation of LFIA-ECL dual-mode sensor

[0087] (1) Dual-mode luminescent material Co / Ce-TCBPE@Ru(dcbpy)3 2+ -COOH

[0088] Take 1.5 mg of cerium nitrate hexahydrate Ce(NO3)3·6H2O, 9 mg of cobalt nitrate hexahydrate Co(NO3)3·6H2O, 24 mg of tetracarboxy biphenyl ethylene H4TCBPE, and 15 mg of tris(4,4-dicarboxy bipyridine) ruthenium chloride C 36 H 19 ClN6O 12 Ru-4 is added to 18 mL of N,N-dimethylformamide DMF and 6 mL of ultrapure water, and then 30 μL of nitric acid HNO3 is added dropwise to dissolve the solution, and then the mixture is stirred uniformly, and then the mixture is transferred to a high-pressure reaction kettle, and the solution is reacted at 80 ℃ for 30 h, and then washed with DMF and ultrapure water by centrifugation, and then dried in a vacuum drying oven at 50 ℃ overnight to obtain an orange product Co / Ce-TCBPE@Ru(dcbpy)3 2+ -COOH.

[0089] (2) Preparation of LFIA flexible electrode for lateral flow chromatography

[0090] a. Cut the nitrocellulose NC membrane of model JN140 into a size of 30 mm wide and 90 mm long, and prepare a wax channel with a width of 5 mm in the middle of the NC membrane by wax dyeing, so as to divide the NC membrane into wettable and non-wettable areas;

[0091] b. Perform conductive area printing on the NC membrane, select BASE-CP10P conductive ink, adjust the air pressure gauge pointer to 45 psi, and determine the positions of the two scales on the tray before formal inkjet printing, and use a carbon ink printing platform to preheat at 40 ℃ before printing;

[0092] c. Use the cutting and splicing method to cover the holes on the platform to ensure the flatness and stability of the substrate during printing, and select ink drop observation, point glue scraping, and 16B program when connecting the printer;

[0093] d. The limit voltage value of the waveform voltage is 40 V, and the conventional setting is 20 V. The printing pattern and position on the NC film are determined by adjusting the parameters. The printing of the working electrode WE and the counter electrode CE of the conductive area is started. After the printing is completed, the NC film is placed in a drying oven at 130 ℃ for 15 min for curing;

[0094] e. The BASE-CD01 high-conductivity silver paste is selected for printing the reference electrode RE. The printing is performed again after adjusting the pattern and position. The complete printed NC film is placed in a drying oven at 120 ℃ for 30 min for heat treatment, and the LFIA flexible electrode is obtained.

[0095] (3) Co / Ce-TCBPE@Ru(dcbpy)3 2+ Preparation of the -COOH-Ab2 bioconjugate

[0096] 6 mg of Co / Ce-TCBPE@Ru(dcbpy)3 2+ -COOH is dispersed in 2250 μL of a phosphate buffered saline solution PBS with pH = 7.4, 150 μL of 400 mmol·L −1 of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride EDC, and 150 μL of 100 mmol·L −1 of N-hydroxysuccinimide NHS, and is shaken at 4 ℃ for 3 h. Then, the excess activator is removed by centrifugation. 150 μL of a 10 μg·mL −1 Ab2 solution is added, and is shaken at 4 ℃ overnight. The obtained Co / Ce-TCBPE@Ru(dcbpy)3 2+ -COOH-Ab2 bioconjugate is washed with PBS for multiple times. Finally, it is dispersed in 3 mL of PBS, and is stored at 4 ℃.

[0097] (4) Preparation of the LFIA-ECL sensor

[0098] a. TMV antibody Ab1 is used as the coating material of the detection line T on the NC film and the goat anti-mouse lgG antibody is used as the coating material of the quality control line C. 7.5 μL of 0.5 mg·mL −1 of Ab1 is dropped on the surface of the working electrode, and is incubated at 4 °C for 2 h. 3 mg·mL −1 of IgG is fixed on the C line of the paper electrode.

[0099] b. The sample pad is first treated with 10 mmol·L −1PBS buffer pH=7.4 pretreatment, then heated at 37 ℃ for 6 hours; the binding pad was first treated with 10 mmol·L −1 pH=7.4 buffer PBS pretreatment, then dried at 37 ℃ for 6 hours. The solution containing 12 mg·mL −1 of Co / Ce-TCBPE@Ru(dcbpy)3 2+ -COOH-Ab2 was evenly sprayed on the conjugate pad, and then dried at 37 ℃ for 6 hours. The LFIA-ECL immunization test strip was prepared by pasting the NC membrane, the binding pad, the sample pad, and the water absorption pad in sequence on the bottom plate.

[0100] c. 240 μL of 10 mM pH=7.4 PBS containing 1.0% Tween 20 and different concentrations of TMV antigen were dropped on the sample pad of the LFIA-ECL immunization test strip, and the color change at the T line and C line of the working electrode was observed after 5~10 min.

[0101] Example 4 Detection and / or diagnosis of tobacco mosaic virus antigen

[0102] A method for detecting tobacco mosaic virus by using a tobacco mosaic virus dual-mode biosensor, comprising the following steps:

[0103] (1) The NC membrane LFIA flexible electrode was removed for ECL three-electrode detection, and 20 μL of 0.10 mol·L −1 of 4 mmol·L −1 of tripropylamine in 0.10 mol·L −1 pH=7.4 PBS solution was added at the working electrode WE;

[0104] (2) The TMV antigen was detected by time-light intensity method, and the voltage was set to continuously cycle potential scanning from 0 V to 1.2 V;

[0105] (3) The photomultiplier tube was set to 400 V during the detection process, the scanning rate was 0.1 V·s −1 , the light intensity was recorded, and the working curve was drawn.

[0106] (4) The TMV antigen sample solution to be detected was 0.0001 ng·mL −1 in concentration instead of the standard solution for detection.

[0107] As shown in Figure 2 , in the LFIA-ECL dual-mode sensing detection working curve, it can be seen that the TMV antigen concentration has a good linear relationship with the electrochemiluminescence intensity in the range of 0.1 pg / mL~100 ng / mL, R 20.991, the relationship equation is I = 6465 + 534lgc, the detection limit is calculated by linear equation, the calculation method is: first test five empty numerical values, calculate the average value of ECL response IB = 3867, the standard deviation is SB = 98.8. K is a numerical factor selected according to the desired confidence level, generally set to 3, so the minimum detection signal IL can be calculated by equation: IL = IB + KSB = 4163, the lowest detection limit is 48.9 fg / mL by the relationship equation, the detection performance is excellent.

[0108] Example 5 Detection and / or diagnosis of tobacco mosaic virus antigen

[0109] A method for detecting and / or diagnosing tobacco mosaic virus using a tobacco mosaic virus bimodal biosensor, comprising the following steps:

[0110] (1) The NC membrane LFIA flexible electrode is taken off for ECL three-electrode detection, 40 μL of 8 mmol·L −1 0.12 mol·L −1 pH = 7.4 PBS solution is added at WE;

[0111] (2) The TMV antigen is detected by time-light intensity method, and the voltage is set to continuously cycle potential scanning from 0 V to 1.4 V;

[0112] (3) The photomultiplier tube is set to 600 V during the detection, the scanning rate is 0.15 V·s −1 , the light intensity is recorded, and the working curve is drawn.

[0113] (4) The TMV antigen sample solution to be detected is 0.001 ng·mL −1 Concentration instead of standard solution for detection and / or diagnosis.

[0114] Example 6 Detection and / or diagnosis of tobacco mosaic virus antigen

[0115] A method for detecting tobacco mosaic virus using a tobacco mosaic virus bimodal biosensor, comprising the following steps:

[0116] (1) The NC membrane LFIA flexible electrode is taken off for ECL three-electrode detection, 60 μL of 12 mmol·L −1 0.15 mol·L −1 pH = 7.4 PBS solution is added at WE;

[0117] (2) The TMV antigen is detected by time-light intensity method, and the voltage is set to continuously cycle potential scanning from 0 V to 1.8 V;

[0118] (3) In the detection process, the photomultiplier tube is set to 800 V, and the scanning rate is 0.2 V·s −1 , and the working curve is drawn.

[0119] (4) The TMV antigen sample solution to be detected is replaced by the standard solution at a concentration of 0.1 ng·mL −1 .

[0120] Example 7: Detection and / or diagnosis of tobacco mosaic virus antigen

[0121] A method for detecting tobacco mosaic virus using a tobacco mosaic virus bimodal biosensor, which is different from example 4:

[0122] (4) The TMV antigen sample solution to be detected is replaced by the standard solution at a concentration of 1 ng·mL −1 .

[0123] Example 8: Detection and / or diagnosis of tobacco mosaic virus antigen

[0124] A method for detecting tobacco mosaic virus using a tobacco mosaic virus bimodal biosensor, which is different from example 4:

[0125] (4) The TMV antigen sample solution to be detected is replaced by the standard solution at a concentration of 10 ng·mL −1 .

[0126] Example 9: Detection and / or diagnosis of tobacco mosaic virus antigen

[0127] A method for detecting tobacco mosaic virus using a tobacco mosaic virus bimodal biosensor, which is different from example 4:

[0128] (4) The TMV antigen sample solution to be detected is replaced by the standard solution at a concentration of 100 ng·mL −1 .

[0129] Example 10: Detection and / or diagnosis of actual samples in tobacco leaves

[0130] Take healthy tobacco leaves, add water, crush and grind to extract juice, and divide the juice into four groups, respectively, add 0, 1, 3, and 5 ng·mL −1 of TMV antigen. After testing each group of samples 3 times, calculate the average detection concentration, and calculate the corresponding recovery rate and RSD according to the obtained detection concentration. The recovery range is 97.4-103%, and the RSD range is 2.2-2.4%, with high detection accuracy.

[0131] Group Amount added (ng mL −1 ) Average amount detected (ng mL −1 ) RSD (n=3, %) Recovery (%) 1 0 0 2 1 1.03 2.4 103 3 3 2.94 2.7 98 4 5 4.87 2.2 97.4

[0132] Performance of LFIA-ECL dual-mode sensor detection

[0133] As Figure 3 The LFIA-ECL dual-mode sensor detection reproducibility; by the same TMV antigen concentration 10 ng·mL −1 The ECL detection of 7 prepared LFIA flexible electrodes is carried out, and the columnar relationship diagram of sample number and luminescence intensity is made, the signal intensity difference is within a reasonable range, and the relative standard deviation RSD is 4.16%;

[0134] Figure 4 The LFIA-ECL dual-mode sensor detection stability, the figure shows that under the scanning of the continuous voltage, the ECL signal of the sensor is very stable, and the relative standard deviation RED is only 1.32%; It is proved that the detection stability is excellent;

[0135] Figure 5 The LFIA-ECL dual-mode sensor detection specificity; the leaf roll disease, the bacterial wilt disease and the special disease are taken as interference, as Figure 5 As shown in the figure, the ECL response of the sensor is only enhanced when the sample containing TMV antigen is detected, which shows that the sensor has high selectivity in detecting TMV.

[0136] Example 13

[0137] The detection and / or diagnosis of tobacco mosaic virus in examples 4-12 of the application all use the portable electrochemical luminescence detection equipment provided in this example, which includes a dark box body 1 provided with a host warehouse 11 and a detection warehouse 12, a photon counting unit fixedly arranged in the host warehouse 11, and an electrochemical pressure unit fixedly arranged in the detection warehouse 12. Referring to Figure 8 As shown in the figure, the dark box body 1 specifically divides the internal space into the host warehouse 11 located at the top and the detection warehouse 12 located at the bottom through the horizontal partition plate 2 arranged inside.

[0138] As Figure 7 As shown in the figure, the top wall of the dark box body 1 is provided with a top opening 16 communicated with the host warehouse 11 and a top sliding door 7 movably arranged at the position of the top opening 16, and the front side wall of the dark box body 1 is provided with a front side opening 17 communicated with the detection warehouse 12 and a front sliding door 8 movably arranged at the position of the front side opening 17, so as to place the biological sensor (NC membrane LFIA flexible electrode) to be detected in the detection warehouse 12 for electrochemical luminescence detection through the opening and closing of the front sliding door 8.

[0139] Further, referring to Figure 8As shown, the photon counting unit comprises a counting host 3 and an inductive probe 4 electrically connected thereto, and it should be noted that the inductive probe 4 is electrically connected to the counting host 3 through a connecting cable (not shown in the figure). Among them, the counting host 3 can specifically adopt the C8855-01 photon counter provided by HAMAMATSU PHOTONICS K.K. (Hamamatsu), and the inductive probe 4 can specifically adopt a photon counting probe matched with the above-mentioned photon counter. Since it is a mature existing technology in the art, its specific structure and principle will not be repeated here.

[0140] In combination Figure 8 And Figure 10 As shown, the counting host 3 is fixedly arranged in the host warehouse 11, and the upper end surface of the horizontal partition plate 2 is provided with a first clamping groove 22 matched with the counting host 3. The bottom of the counting host 3 is fixedly clamped in the first clamping groove 22, so as to avoid the counting host 3 from shaking or shifting, and improve the stability of the fixed counting host 3.

[0141] Further, referring to Figure 9 As shown, the side wall of the dark box body 1 is provided with a reserved interface 13 communicated with the host warehouse 11, and the counting host 3 is arranged close to the reserved interface 13, so as to connect the photon counting unit located in the host warehouse 11 and the external data receiving and processing equipment (such as PC equipment) through the wiring cable (such as USB connecting line). Specifically, the counting host 3 in the photon counting unit is arranged in the host warehouse 11 close to the reserved interface 13, and the interface of the counting host 3 is located at the reserved interface 13. For details, please refer to Figure 7 As shown.

[0142] As shown in Figure 8 And Figure 10 The inductive probe 4 is fixedly arranged in the host warehouse 11, and the horizontal partition plate 2 is provided with a detection hole 21 communicated with the host warehouse 11 and the detection warehouse 12. The inductive probe 4 is fixedly embedded in the detection hole 21, and the detection end of the inductive probe 4 is arranged towards the detection warehouse 12, so as to detect and count the optical signal generated by the sensor to be detected located directly below.

[0143] Further, referring to Figure 7As shown, the electrochemical pressure applying unit includes a micro electrochemical workstation 5 fixedly arranged in the detection chamber 12, and an electrode converter 6 connected with the micro electrochemical workstation 5. The electrode converter 6 is connected with the electrode of the sensor to be detected, so as to connect the micro electrochemical workstation 5 and the sensor to be detected through the electrode converter 6, so as to apply a pulse voltage to the sensor to be detected through the micro electrochemical workstation 5, so as to promote the sensor to be detected to generate an electrochemical reaction to generate a detectable light signal. It should be noted that the micro electrochemical workstation 5 can specifically adopt a Sensit Smart U flash drive electrochemical workstation (analyzer) of PalmSens BV Company in the Netherlands, or a Sensit BT mini electrochemical workstation (analyzer), and the electrode converter 6 can specifically adopt a three-electrode converter, a paper electrode, or a three-electrode electrode clamp which is matched with the micro electrochemical workstation 5. Since it is a mature prior art in the art, the specific structure and principle will not be described here. In addition, the micro electrochemical workstation 5 can adopt a portable electrochemical workstation with built-in lithium battery and Bluetooth function, so that the micro electrochemical workstation 5 can be remotely controlled to apply a pulse voltage without external power supply and computer (or mobile phone), so as to ensure the airtightness and light shielding of the detection chamber 12.

[0144] In combination Figure 8 and Figure 9 As shown, the micro electrochemical workstation 5 is fixedly arranged in the detection chamber 12, and the upper end surface of the inner bottom wall of the dark box body 1 is provided with a second clamping groove 15 matched with the micro electrochemical workstation 5. The micro electrochemical workstation 5 is fixedly clamped in the second clamping groove 15, so as to avoid shaking or displacement of the micro electrochemical workstation 5.

[0145] Further, the upper end surface of the inner bottom wall of the dark box body 1 is further provided with a placement position mark 14 located directly below the detection hole 21. The placement position mark 14 is directly opposite to the detection end of the inductive probe 4, so as to ensure that the sensor to be detected is directly opposite to the detection end of the inductive probe 4 during detection, and improve the accuracy of the detection result data. At the same time, the electrode converter 6 is located below the detection hole 21 and is arranged towards the placement position mark 14, so as to connect the electrode converter 6 and the sensor to be detected and ensure that the sensor to be detected is placed in the placement position mark 14.

[0146] In specific use process: the counting host 3 is connected to the external data processing receiving equipment through the connecting cable, the front sliding door 8 is opened, the to-be-detected biosensor (NC membrane LFIA flexible electrode) is placed at the placement position mark 14 in the detection bin 12, and the working electrode WE, the counter electrode CE and the reference electrode RE are connected to the three electrodes of the electrode converter 6 respectively, so that the to-be-detected biosensor is located directly below the detection through hole 21; the front sliding door 8 is closed to make the inside of the detection bin 12 be closed and lightproof, the micro electrochemical workstation 5 is started through the mobile phone or the computer, so that the to-be-detected biosensor is applied with pulse voltage, and the light signal (released photon) generated by the to-be-detected biosensor is detected by the sensing probe 4 and the counting host 3; the external data processing receiving equipment processes and outputs the electrochemical luminescence detection result.

[0147] In conclusion, the novel tobacco mosaic virus TMV double-mode biosensor combining the double-mode luminescent material and the lateral flow chromatography flexible electrode has simple preparation method process, and the prepared LFIA-ECL double-mode detection sensor has low cost, high sensitivity, good specificity, rapid detection and easy preparation.

[0148] The above is only the preferred embodiment of the present application, and does not limit the patent scope of the present application, and any equivalent change and modification made within the scope of the present application shall still be within the scope of the present application.

Claims

1. A dual-mode biosensor for tobacco mosaic virus (TMV), characterized in that, The tobacco mosaic virus (TMV) dual-mode biosensor LFIA-ECL comprises the dual-mode luminescent material Co / Ce-TCBPE@Ru(dcbpy)3. 2+ -COOH, lateral flow chromatography LFIA flexible electrode, TMV antibody Ab1, TMV antibody Ab2 and self-assembled micro ECL sensor; The lateral flow chromatography LFIA flexible electrode was prepared using nitrocellulose NC membrane as raw material. The LFIA flexible electrode is loaded with a detection line (T-line) and a quality control line (C-line). TMV antibody Ab1 is used as the coating material at the detection line (T-line); goat anti-mouse IgG antibody is used as the coating material at the quality control line (C-line); and the binding pad is sprayed with the bioconjugate Co / Ce-TCBPE@Ru(dcbpy3). 2+ -COOH-Ab2 solution.

2. The method for preparing the tobacco mosaic virus (TMV) dual-mode biosensor as described in claim 1, characterized in that, The steps include the following: (1) Preparation of LFIA-ECL immunoassay strips: The sample pad was first soaked in 10 μL of a solution containing 2.5% Tween 20 and 0.1% Triton X-100 at a concentration of 10 mmol·L⁻¹. -1 Pre-treatment by soaking in phosphate-buffered saline (PBS) and then drying; The conjugate pad was first pretreated by soaking it in phosphate-buffered saline (PBS) containing 2.5% bovine serum albumin and 0.5% Tween 20, and then dried. 10 μL of a solution containing 4–12 mg / mL was then added. -1 Co / Ce-TCBPE@Ru(dcbpy)3 2+ The -COOH-Ab2 solution was sprayed evenly onto the conjugate pad and then dried. The sample pad, conjugate pad, NC membrane, and absorbent pad are pasted sequentially on a polyvinyl chloride base plate to obtain the LFIA-ECL immunoassay strip. (2) Preparation of the detection line T-line and the quality inspection line C-line: 2.5–7.5 μL of 0.5 mg / mL -1 TMV antibody Ab1 was dropped onto the surface of the T-line detection line of the working electrode of the LFIA flexible electrode in side-flow chromatography and incubated; 10 μL of 1~3 mg·mL⁻¹ was added. -1 The sheep anti-mouse IgG antibody was streaked and fixed on the quality control line C to obtain the detection line T and the quality control line C. (3) Take 80~240 μL of phosphate buffer PBS containing 1.0% Tween 20 and TMV antigen respectively and drop them onto the sample pad of the LFIA-ECL immunoassay strip. Wait 5~10 min and observe the color change at the T line and C line of the working electrode.

3. The preparation method as described in claim 2, characterized in that, In (2), the preparation process of the side-flow chromatography LFIA flexible electrode is as follows: S1. After cutting the nitrocellulose NC membrane, wax channels are prepared in the middle of the nitrocellulose NC membrane by wax dyeing, dividing the nitrocellulose NC membrane into wettable and non-wettable areas; S2. The wax channel is completely covered by cutting and splicing the substrate, and conductive ink is used to print the conductive area on the nitrocellulose NC film; Before printing, adjust the waveform voltage to 15-30V. After determining the printing pattern and position, start printing the working electrode WE and counter electrode CE in the conductive area. After printing, heat and cure the nitrocellulose NC film. S3. Readjust the pattern and position, print the reference electrode RE using highly conductive silver paste, and then heat-treat the fully printed nitrocellulose NC film to obtain the LFIA flexible electrode.

4. The preparation method as described in claim 2, characterized in that, In (1), the Co / Ce-TCBPE@Ru(dcbpy)3 2+ The preparation method of the -COOH-Ab2 solution is as follows: Take 2-6 mg of the dual-mode luminescent material Co / Ce-TCBPE@Ru(dcbpy)3 2+ -COOH was dispersed in 850–2250 μL of phosphate-buffered saline (PBS) or 50–150 μL of 400 mmol·L⁻¹ PBS. -1 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride EDC and 50~150 μL 100 mmol·L -1 In N-hydroxysuccinimide NHS, shake at 2-8 °C for 1-5 h; Centrifuge to remove excess 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), then add 50–150 μL of 10 μg·mL⁻¹ -1 TMV antibody Ab2 solution was shaken overnight at 2-8℃ to obtain the bioconjugated Co / Ce-TCBPE@Ru(dcbpy)3. 2+ -COOH-Ab2, wash 3-5 times with phosphate-buffered saline (PBS), and finally add Co / Ce-TCBPE@Ru(dcbpy)3. 2+ -COOH-Ab2 is dissolved in 1-3 mL of PBS, at a concentration of 4-12 mg·L⁻¹. -1 Obtain Co / Ce-TCBPE@Ru(dcbpy)3 2+ -COOH-Ab2 solution.

5. The preparation method as described in claim 4, characterized in that, The dual-mode luminescent material Co / Ce-TCBPE@Ru(dcbpy)3 2+ The preparation method for -COOH is as follows: Take 0.5-1.5 mg of cerium nitrate hexahydrate (Ce(NO3)3·6H2O), 3-9 mg of cobalt nitrate hexahydrate (Co(NO3)3·6H2O), 8-24 mg of tetracarboxylated biphenyl ethylene (H4TCBPE), and 5-15 mg of tris(4,4-dicarboxylated bipyridine)ruthenium chloride (C). 36 H 19 ClN6O 12 Ru-4 was added as a reactant to 6–18 mL of N,N-dimethylformamide (DMF) and 2–6 mL of ultrapure water. Then, 10–30 μL of nitric acid (HNO3) was added dropwise to dissolve the reactant. The mixture was stirred thoroughly and reacted at 100 °C for 24 h. The mixture was then washed by centrifugation with N,N-dimethylformamide (DMF) and ultrapure water, respectively, and vacuum-dried overnight at 50–60 °C to obtain the dual-mode luminescent material Co / Ce-TCBPE@Ru(dcbpy)3. 2+ -COOH.

6. The application of the tobacco mosaic virus TMV dual-mode biosensor as described in claim 1 and / or the tobacco mosaic virus TMV dual-mode biosensor prepared by any one of the preparation methods in claims 2-5 in the detection and / or diagnosis of tobacco mosaic virus.

7. A method for detecting and / or diagnosing tobacco mosaic virus (TMV) using the dual-mode biosensor of tobacco mosaic virus (TMV) as described in claim 1, characterized in that, The steps include the following: a. Remove the LFIA flexible electrode for ECL three-electrode detection. Add 20-60 μL of solution containing 4-12 mmol·L⁻¹ to the working electrode WE. -1 0.10~0.15 mol·L⁻¹ of tripropylamine -1 Phosphate-buffered saline (PBS) solution with pH 7.4; b. The TMV antigen was detected using the time-intensity method, with a continuous cyclic potential scan of voltage from 0 V to 1.2~1.8 V. c. During the detection process, the photomultiplier tube is set to 400~800 V, and the scanning rate is 0.1~0.2 V·s. -1 Record light intensity and plot working curves; d. Replace the standard solution with the TMV antigen sample solution to be tested.

8. The method for detecting tobacco mosaic virus (TMV) using a dual-mode biosensor as described in claim 7, characterized in that, In step d, the concentration of the TMV antigen sample solution to be tested is 0.0001-100 ng·mL. -1 .

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