Preparation method and application of portable sensor for rapid detection of panax ginseng root rot

Abstract

The application discloses a portable sensor for rapidly detecting panax ginseng root rot and a preparation method and application thereof, and belongs to the technical field of biosensors, and comprises double-luminescent ligand co-encapsulation materials, a capture antibody Ab1, ion-doped organic metal framework sulfides, a labeled antibody Ab2, dibutyl ethanolamine DBAE, a micro electrochemiluminescence ECL sensing and detecting platform and disposable disposable electrodes. The application aims to solve the technical bottleneck of the existing detection technology, that is, the dependence on large instruments, the complicated operation, the long detection period and the insufficient sensitivity. The double-luminescent ligand co-encapsulation materials realize signal superposition enhancement and background reduction, the composite material optimizes the conductivity and catalytic activity of the electrode to amplify the ECL signal, the DBAE is used as a co-reactant, the detection safety and the luminescence efficiency are considered, and a feasible technical scheme is provided for early warning and precise prevention and control of the panax ginseng industry diseases.

Description

Technical Field

[0001] This invention belongs to the field of biosensor technology, and specifically relates to a portable biosensor for treating root rot in Panax notoginseng, its preparation method, and its application. Background Technology

[0002] Panax notoginseng, a traditional and precious Chinese medicinal herb, has its dried roots and rhizomes containing active ingredients such as total saponins. These ingredients possess traditional medicinal properties such as promoting blood circulation and stopping bleeding, relieving pain, as well as modern pharmacological value such as cardiovascular protection, anti-inflammatory and analgesic effects, and immune regulation. It is widely used in the pharmaceutical and health care fields, and the industry has a significant scale. However, root rot of Panax notoginseng, caused by Fusarium sp., is a serious soil-borne disease that not only leads to plant death and a significant drop in yield, but also damages medicinal parts, causing quality deterioration, polluting the soil, and exacerbating continuous cropping obstacles. Furthermore, relying on chemical control can easily increase costs and cause ecological pollution. It has become a core bottleneck restricting the sustainable development of the Panax notoginseng industry. The development of rapid and accurate detection technology is crucial for early warning and comprehensive control of the disease.

[0003] Electrochemiluminescence (ECL) is an analytical technique that uses electrochemical methods to excite substances to produce light. Its core principle is to apply a specific voltage to the electrode surface, causing a redox reaction in the system to generate reactive intermediates (such as free radicals or excited-state molecules). These intermediates then transition to the excited state through energy transfer or electron transfer, releasing photons upon returning to the ground state. This technique combines the high selectivity of electrochemistry with the high sensitivity of luminescence analysis, offering advantages such as controllable reaction, low background signal, low detection limit, and wide linear range, making it a research hotspot in the field of biosensing. For target analytes like Fusarium sp., which have complex structures and are easily affected by sample matrices, ECL technology can meet the stringent requirements for trace detection. However, current ECL detection systems are often limited to laboratory settings due to their large size, resulting in practical bottlenecks such as long detection times and high costs.

[0004] Given that Panax notoginseng cultivation is mostly distributed in mountainous fields, existing high-sensitivity detection technologies have limitations such as reliance on large instruments, complex operation, and long detection cycles. Therefore, miniaturization and portability of ECL detection equipment are significantly necessary. Traditional laboratory testing requires sample collection and transport to other locations, taking several days or even weeks, which cannot meet the immediate need for early warning of root rot. Miniaturized ECL equipment, on the other hand, can be used directly at the planting site, allowing for timely control measures to reduce disease spread. Furthermore, miniaturized ECL equipment, with its high sensitivity, can accurately capture low concentrations of pathogens in the early stages of disease, while avoiding problems such as sample deterioration and changes in pathogen activity during off-site testing. Currently, portable ECL sensing detection technology for Panax notoginseng root rot has not been reported. Summary of the Invention

[0005] The core objective of this invention is to overcome the technical bottlenecks of existing Panax notoginseng root rot detection technologies, which rely on large instruments, are complex to operate, have long detection cycles, lack sufficient sensitivity, and cannot perform on-site real-time detection. This invention provides a portable sensor based on the ECL principle, its preparation method, and its application, enabling rapid, sensitive, and specific on-site detection of Fusarium sp. This fills the application gap of portable ECL technology in the field of Panax notoginseng root rot detection, and provides a practical technical solution for early warning and precise control of diseases in the Panax notoginseng industry. Based on this objective, this invention first prepares a dual-luminescent ligand co-encapsulation material, MSN-TCBPE / Ru(dcbpy)3. 2+ Two luminescent ligands were co-encapsulated using an MSN carrier to enhance signal intensity and reduce background interference through synergistic enhancement. Simultaneously, amination modification endowed the material with bioconjugation activity. Au@Co / Ni-MOF / S composite materials were prepared concurrently, and MOF sulfurization modification and gold ion doping optimized conductivity and catalytic performance, laying the foundation for enhanced electron transport and ECL reaction on the electrode surface. Dibutylethanolamine (DBAE) was used as a co-reactant in the detection system, replacing tripropylamine (TPrA) commonly used in traditional ECL detection, offering lower toxicity and higher luminescence efficiency. For on-site, real-time detection needs, a miniature ECL sensing platform was constructed by integrating a USB-disk-based electrochemical workstation, a photon counter, and a counting unit. Using disposable electrodes as a portable substrate, a portable ECL sensor for detecting root rot in Panax notoginseng was prepared through chemical bonding and layer-by-layer modification.

[0006] The specific technical solution adopted in this invention is as follows:

[0007] This invention provides a portable sensor for rapid detection of root rot in Panax notoginseng. The portable sensor comprises a dual-luminescent ligand co-encapsulation material MSN-TCBPE / Ru(dcbpy)3. 2+ The system comprises a capture antibody Ab1, a gold-loaded cobalt-doped nickel-ion organic framework sulfide Au@Co / Ni-MOF / S, a labeled antibody Ab2, co-reactants, a micro-electrochemiluminescence (ECL) sensing platform, and disposable electrodes; the method for diagnosing root rot in Panax notoginseng involves detecting the pathogen Fusarium sp.; the capture antibody Ab1 and labeled antibody Ab2 specifically recognize and capture Fusarium sp.; and the dual-luminescent ligand co-encapsulation material MSN-TCBPE / Ru(dcbpy)3. 2+ The dual-luminescent ligands are co-encapsulated in mesoporous silica MSN; the dual-luminescent ligands are tetracarboxylated biphenylethylene (H4TCBPE) and carboxylated terpyridine ruthenium (Ru(dcbpy)3). 2+The gold-loaded cobalt-doped nickel ion organic framework sulfide Au@Co / Ni-MOF / S is prepared by doping a Ni ion organic framework Ni-MOF with Co ions and then sulfiding it with gold; the co-reactant is dibutylethanolamine (DBAE).

[0008] Preferably, the dual-luminescent ligand co-encapsulation material MSN-TCBPE / Ru(dcbpy)3 2+ The preparation steps are as follows:

[0009] (1) Dissolve 0.5 ~ 1.5 g cetyltrimethylammonium bromide (CTAB) in a mixture of 75 ~ 225 mL ultrapure water and 50 ~ 150 mL anhydrous ethanol, then add 1 ~ 3 mL ammonia water and 0.5 ~ 1.5 mL tetraethyl silicate (TEOS) under stirring, and react at 30 ~ 40 °C for 8 ~ 12 hours. Centrifuge and dry to obtain MSN;

[0010] (2) Sonicate 100-300 mg of MSN obtained in step (1) in 50-150 mL of anhydrous ethanol, add 1-3 mL of 3-aminopropyltriethoxysilane APTES, and reflux at 60-80 °C for 4-8 hours under nitrogen protection. Then centrifuge and dry to obtain amination-modified mesoporous silica MSN-NH2.

[0011] (3) Take 13.5 ~ 40.5 mg H4TCBPE and 9.6 ~ 28.8 mg Ru(dcbpy)3 2+ Disperse the mixture in 4-12 mL of anhydrous ethanol, add 30-90 mg of MSN-NH2 obtained in step (2) to the mixture, and centrifuge and dry to obtain MSN-TCBPE / Ru(dcbpy)3. 2+ ,like Figure 1 As shown, this is the MSN-TCBPE / Ru(dcbpy)3 prepared under optimal conditions. 2+ The representation diagram.

[0012] Preferably, the preparation steps of the gold-loaded ion-doped metal-organic framework sulfide Au@Co / Ni-MOF / S are as follows:

[0013] (1) Mix 0.43 ~ 1.29 g nickel nitrate hexahydrate Ni(NO3)2·6H2O, 0.15 ~ 0.45 g trimesic acid H3BTC and 1.5 ~ 4.5 g polyvinylpyrrolidone PVP in 20 ~ 60 mL N,N-dimethylformamide DMF and 10 ~ 30 mL ultrapure water, stir and dissolve at room temperature, transfer the mixture to a polytetrafluoroethylene-lined autoclave, keep at 120 ~ 160 °C for 8 ~ 12 hours, centrifuge and dry to obtain green nickel ion organic framework Ni-MOF;

[0014] (2) Mix 50-150 mg of Ni-MOF obtained in step (1) and 75-225 mg of cobalt chloride hexahydrate CoCl2·6H2O in 30-90 mL of anhydrous ethanol. Heat the mixture in an oil bath at 60-90°C for 1-3 hours, then centrifuge and dry to obtain a blue-green mixture. Place 50-150 mg of the mixture and 25-75 mg of sulfur powder in a porcelain boat. Heat the furnace to 600-800°C and maintain the temperature under an argon flow for 2-4 hours. Centrifuge and dry to obtain cobalt-doped nickel ion organic framework sulfide Co / Ni-MOF / S.

[0015] (3) Disperse 20-60 mg of the Co / Ni-MOF / S obtained in step (2) in 50-150 mL of ultrapure water, add 2-6 mL of 1% chloroauric acid HAuCl4 solution and 5-15 mg of PVP, stir for 4-8 hours, add 2-6 mL of sodium citrate solution C6H9NaO7 and 1-3 mg of sodium borohydride NaBH4, and stir for 20-26 hours. Centrifuge and dry to obtain black Au@Co / Ni-MOF / S product, as shown below. Figure 2 The figure shown is a characterization diagram of Au@Co / Ni-MOF / S prepared under optimal conditions.

[0016] This invention also discloses a method for preparing a portable sensor for rapid detection of root rot in Panax notoginseng, comprising:

[0017] (1) Using a disposable, disposable electrode as the working electrode, drop 5 ~ 15 μL of Au@Co / Ni-MOF / S solution onto the electrode surface and dry at room temperature for 2 ~ 4 hours;

[0018] (2) Wash the electrode obtained in step (1) with phosphate buffer solution PBS, and continue to add 3~9 μL of Ab1 solution to the electrode surface and incubate at room temperature for 1~3 hours;

[0019] (3) The electrode obtained in step (2) is cleaned with PBS, and 8 ~ 12 µL of bovine serum albumin (BSA) solution is dropped onto the electrode surface. The electrode is dried at 4°C, then cleaned with PBS and stored at 4°C.

[0020] (4) Take 2-4 mg of the dual-luminescent ligand co-encapsulation material MSN-TCBPE / Ru(dcbpy)3. 2+ Add 900–2700 μL of PBS and 100–300 μL of 1-ethyl-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide EDC / NHS solution, shake at room temperature for 2–6 hours, centrifuge to remove the supernatant, add 950–2850 μL of PBS, then add 50–150 μL of Ab2 solution, shake at 4°C, centrifuge to remove the supernatant, and disperse with 1–3 mL of PBS to obtain MSN-TCBPE / Ru(dcbpy)3. 2+ -Ab2;

[0021] The aforementioned micro electrochemiluminescence (ECL) sensing and detection platform consists of a photon counter, a counting unit, and a USB-type electrochemical workstation.

[0022] As a preferred option, the detection steps are as follows:

[0023] (1) Preparation of standard solutions: Prepare a set of Fusarium sp. standard solutions of different concentrations, including blank standard samples;

[0024] (2) Preparation of working electrode: 8-24 µL of Fusarium sp. standard solution of different concentrations was added dropwise to the modified electrode surface. After incubation for 30-60 minutes, 8-24 µL of MSN-TCBPE / Ru(dcbpy)3 was added dropwise. 2+ -Ab2, after incubation for 30 to 60 minutes, the working electrode is obtained;

[0025] (3) Plotting the working curve: Connect the disposable electrode to the USB flash drive electrochemical workstation; add DBAE solution as a co-reactant to the electrode to cover the three electrodes; apply cyclic voltage to the electrode using cyclic voltammetry, and detect the light signal intensity using a micro ECL sensing platform; the response light signal intensity containing different concentrations of Fusarium sp. standard solution is recorded as follows: I Response light signal intensity I Concentration of Fusarium sp. standard solution c The logarithmic functions are linearly related, plotted. I -lg c Working curve, such as Figure 3As shown, the limit of detection was 14.9 pg / mL;

[0026] (4) Detection of root rot in Panax notoginseng samples: Soak 5-10 g of Panax notoginseng root and leaf samples (slices or powders) in 10-30 mL of 50-75% ethanol aqueous solution for 30-60 minutes to obtain Fusarium sp. soaking solution; then replace the Fusarium sp. standard solution in step (1) with the Fusarium sp. soaking solution and perform detection according to the methods in steps (2) and (3), based on the intensity of the response light signal. I Concentration of Fusarium sp. standard solution c The logarithmic function and the working curve show a linear relationship, and the content of Fusarium sp. in Panax notoginseng samples is obtained, thus completing the diagnosis of root rot in Panax notoginseng.

[0027] Preferably, the disposable electrode is selected from one of the following conductive substrates: screen-printed electrode, conductive carbon cloth, and paper-based electrode; the Au@Co / Ni-MOF / S solution is prepared by dissolving Au@Co / Ni-MOF / S in ultrapure water, with a concentration of 0.8 mg / mL; the Ab1 solution is prepared by dissolving Ab1 in PBS, with a concentration of 10 μg / mL; the BSA solution is prepared by dissolving BSA in PBS, with a concentration of 100 µg / mL; the Ab2 solution is prepared by dissolving Ab2 in PBS, with a concentration of 10 μg / mL; the EDC / NHS solution is prepared by adding 76 mg EDC and 11.5 mg NHS to 1 mL of ultrapure water; the DBAE solution is prepared by dissolving DBAE in PBS, with a concentration of 10 mM; the PBS is a 150 mM phosphate buffer solution with a pH of 7.4; the different concentrations of Fusarium sp. standard solutions are 100 pg / mL, 500 pg / mL, and 1... The cyclic voltammetry detection parameters were set as follows: initial potential of 0 V, maximum potential of 1.2 V, and scan rate of 0.1 V / s.

[0028] The present invention has the following advantages and effects compared with the prior art:

[0029] (1) This invention creatively uses MSN as a carrier to co-encapsulate H4TCBPE and Ru(dcbpy)3 2+ Two luminescent ligands form MSN-TCBPE / Ru(dcbpy)3 2+This composite material design, through the synergistic effect of two luminescent ligands, achieves superposition enhancement of the luminescence signal, reduces the probability of nonradiative transitions, and significantly improves the luminescence quantum efficiency. Simultaneously, the uniform confinement effect of the MSN not only modulates the aggregation state of H4TCBPE to optimize its aggregation-induced emission effect but also effectively suppresses Ru(dcbpy)3. 2+ The dissolution of the ligand material avoids signal loss or system instability caused by dissolution and shortens the intermolecular distance, thereby effectively promoting resonance energy transfer and significantly enhancing the electrochemiluminescence signal intensity. Compared with single luminescent ligand materials, the ECL efficiency of this dual-luminescent system reaches 7.5% (the standard is 5%), providing a reliable signal output guarantee for trace detection of Fusarium sp.

[0030] (2) The Au@Co / Ni-MOF / S prepared in this invention serves as a substrate reinforcement material, integrating multiple core performance advantages. The unique hollow structure of Ni-MOF endows it with an ultra-large specific surface area, providing sufficient active sites for the immobilization of captured antibodies, while facilitating the rapid diffusion of reactants and products. After sulfurization modification, the internal electron transport path of the material is optimized, and the conductivity is significantly improved. The precise doping of Co ions further regulates the electronic structure of the material, greatly improving the catalytic activity. Its active surface area can reach 1.84 times that of the undoped sample. The loading of gold nanoparticles further accelerates the electron transfer efficiency at the electrode / electrolyte interface and enhances the catalytic performance through the surface plasmon resonance effect and excellent conductivity, significantly promoting the oxidation of co-reactant DBAE to generate more active intermediate DBAE. ・ Through multiple synergistic effects, the ECL luminescence signal is efficiently amplified. At the same time, the long-chain structure of DBAE has a stronger electron-donating ability and is easier to achieve amine oxidation process than the traditional co-reactant TPRA, achieving higher luminescence efficiency in the whole system and less toxicity, which is in line with the development concept of green agricultural detection. The multi-dimensional performance optimization of this substrate reinforcement material provides core structural support for the high sensitivity and fast response of the sensor, ensuring that the constructed sensor has excellent practical detection applicability and can accurately meet the actual needs of rapid detection of trace Fusarium sp.

[0031] (3) This invention creatively constructs DBAE-MSN-TCBPE / Ru(dcbpy)3 2+ A synergistic system of co-reactant and luminescent material was developed to address the shortcomings of traditional ECL detection co-reactant TPRA, which is characterized by high toxicity (LD50 96 mg / kg), safety hazards, and environmental risks. Extensive screening experiments were conducted to evaluate various amine co-reactants in combination with MSN-TCBPE / Ru(dcbpy)3. 2+The compatibility of luminescent materials was systematically investigated, with the screening scope covering TPRA, triethylamine, triethanolamine, diethylethanolamine, DBAE, and diisopropylamine. Experimental results showed that DBAE was compatible with MSN-TCBPE / Ru(dcbpy)3. 2+ It has the best compatibility and optimal ECL luminescence response, and its median lethal dose is as high as 1071.27 mg / kg. Its toxicity is far lower than that of traditional TPRA. In the field detection of Panax notoginseng root rot, it not only ensures the health and safety of operators, but also reduces the emission of environmental pollutants. It is more in line with the development concept of green agriculture and the trend of environmental protection of detection technology. Attached Figure Description

[0032] Figure 1 MSN-TCBPE / Ru(dcbpy)3 2+ Electron scanning electron microscopy (SEM) morphology images;

[0033] Figure 2 SEM morphology of Au@Co / Ni-MOF / S;

[0034] Figure 3 This is a standard curve for a portable sensor used to detect root rot in Panax notoginseng. Detailed Implementation

[0035] 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.

[0036] Example 1: MSN-TCBPE / Ru(dcbpy)3 dual-emitting ligand co-encapsulation material 2+ Preparation:

[0037] (1) Dissolve 0.5 g cetyltrimethylammonium bromide (CTAB) in a mixture of 75 mL ultrapure water and 50 mL anhydrous ethanol, then add 1 mL ammonia and 0.5 mL tetraethyl silicate (TEOS) under stirring, react at 30 °C for 8 hours, and centrifuge and dry to obtain MSN;

[0038] (2) 100 mg of MSN obtained in step (1) was sonicated in 50 mL of anhydrous ethanol, 1 mL of 3-aminopropyltriethoxysilane APTES was added, and the mixture was refluxed at 60 °C for 4 hours under nitrogen protection. The mixture was then centrifuged and dried to obtain amination-modified mesoporous silica MSN-NH2.

[0039] (3) Mix 13.5 mg H4TCBPE and 9.6 mg Ru(dcbpy)3 2+Dispersed in 4 mL of anhydrous ethanol, 30 mg of MSN-NH2 obtained in step (2) was added to the mixture, and the mixture was centrifuged and dried to obtain MSN-TCBPE / Ru(dcbpy)3. 2+ .

[0040] Example 2: Dual-luminescent ligand co-encapsulation material MSN-TCBPE / Ru(dcbpy)3 2+ Preparation:

[0041] (1) Dissolve 1 g of hexadecyltrimethylammonium bromide (CTAB) in a mixture of 150 mL of ultrapure water and 100 mL of anhydrous ethanol, then add 2 mL of ammonia and 1 mL of tetraethyl silicate (TEOS) under stirring, react at 35 °C for 10 hours, and centrifuge and dry to obtain MSN;

[0042] (2) 200 mg of MSN obtained in step (1) was sonicated in 100 mL of anhydrous ethanol, 2 mL of 3-aminopropyltriethoxysilane APTES was added, and the mixture was refluxed at 70 °C for 6 hours under nitrogen protection. The mixture was then centrifuged and dried to obtain amination-modified mesoporous silica MSN-NH2.

[0043] (3) Mix 27 mg H4TCBPE and 19.2 mg Ru(dcbpy)3 2+ Dispersed in 8 mL of anhydrous ethanol, 60 mg of MSN-NH2 obtained in step (2) was added to the mixture, and the mixture was centrifuged and dried to obtain MSN-TCBPE / Ru(dcbpy)3. 2+ .

[0044] Example 3: Dual-luminescent ligand co-encapsulation material MSN-TCBPE / Ru(dcbpy)3 2+ Preparation:

[0045] (1) Dissolve 1.5 g cetyltrimethylammonium bromide (CTAB) in a mixture of 225 mL ultrapure water and 150 mL anhydrous ethanol, then add 3 mL ammonia and 1.5 mL tetraethyl silicate (TEOS) under stirring, react at 40 °C for 12 hours, and centrifuge and dry to obtain MSN;

[0046] (2) 300 mg of MSN obtained in step (1) was sonicated in 150 mL of anhydrous ethanol, 3 mL of 3-aminopropyltriethoxysilane APTES was added, and the mixture was refluxed at 80 °C for 8 hours under nitrogen protection. The mixture was then centrifuged and dried to obtain amination-modified mesoporous silica MSN-NH2.

[0047] (3) Mix 40.5 mg H4TCBPE and 28.8 mg Ru(dcbpy)3 2+Dispersed in 12 mL of anhydrous ethanol, 90 mg of MSN-NH2 obtained in step (2) was added to the mixture, and the mixture was centrifuged and dried to obtain MSN-TCBPE / Ru(dcbpy)3. 2+ .

[0048] Example 4: Preparation of gold-loaded ion-doped metal-organic framework sulfide Au@Co / Ni-MOF / S:

[0049] (1) 0.43 g nickel nitrate hexahydrate Ni(NO3)2·6H2O, 0.15 g trimesic acid H3BTC and 1.5 g polyvinylpyrrolidone PVP were mixed in 20 mL N,N-dimethylformamide DMF and 10 mL ultrapure water and stirred to dissolve at room temperature. The mixture was then transferred to a polytetrafluoroethylene-lined autoclave and kept at 120 °C for 8 hours. After centrifugation and drying, green nickel ion organic framework Ni-MOF was obtained.

[0050] (2) Mix 50 mg of Ni-MOF obtained in step (1) and 75 mg of cobalt chloride hexahydrate CoCl2·6H2O in 30 mL of anhydrous ethanol. Heat the mixture in an oil bath at 60°C for 1 hour, then centrifuge and dry to obtain a blue-green mixture. Place 50 mg of the mixture and 25 mg of sulfur powder in a porcelain boat, heat the furnace to 600°C, and maintain the temperature for 2 hours under an argon flow. Centrifuge and dry to obtain cobalt-doped nickel ion organic framework sulfide Co / Ni-MOF / S.

[0051] (3) Disperse 20 mg of Co / Ni-MOF / S obtained in step (2) in 50 mL of ultrapure water, add 2 mL of 1% chloroauric acid HAuCl4 solution and 5 mg of PVP, stir for 4 hours, add 2 mL of sodium citrate solution C6H9NaO7 and 1 mg of sodium borohydride NaBH4, stir for 20 hours, centrifuge and dry to obtain black Au@Co / Ni-MOF / S product.

[0052] Example 5: Preparation of gold-loaded ion-doped metal-organic framework sulfide Au@Co / Ni-MOF / S:

[0053] (1) 0.86 g nickel nitrate hexahydrate Ni(NO3)2·6H2O, 0.3 g trimesic acid H3BTC and 3 g polyvinylpyrrolidone PVP were mixed in 40 mL N,N-dimethylformamide DMF and 20 mL ultrapure water and stirred to dissolve at room temperature. The mixture was then transferred to a polytetrafluoroethylene-lined autoclave and kept at 140 °C for 10 hours. After centrifugation and drying, green nickel ion organic framework Ni-MOF was obtained.

[0054] (2) Mix 100 mg of Ni-MOF obtained in step (1) and 150 mg of cobalt chloride hexahydrate CoCl2·6H2O in 60 mL of anhydrous ethanol. Heat the mixture in an oil bath at 80°C for 2 hours, then centrifuge and dry to obtain a blue-green mixture. Place 100 mg of the mixture and 50 mg of sulfur powder in a porcelain boat, heat the furnace to 700°C, and maintain the temperature for 3 hours under an argon flow. Centrifuge and dry to obtain cobalt-doped nickel ion organic framework sulfide Co / Ni-MOF / S.

[0055] (3) Disperse 40 mg of Co / Ni-MOF / S obtained in step (2) in 100 mL of ultrapure water, add 4 mL of 1% chloroauric acid HAuCl4 solution and 10 mg of PVP, stir for 6 hours, add 4 mL of sodium citrate solution C6H9NaO7 and 2 mg of sodium borohydride NaBH4, stir for 24 hours, centrifuge and dry to obtain black Au@Co / Ni-MOF / S product.

[0056] Example 6: Preparation of gold-loaded ion-doped metal-organic framework sulfide Au@Co / Ni-MOF / S:

[0057] (1) 1.29 g nickel nitrate hexahydrate Ni(NO3)2·6H2O, 0.45 g trimesic acid H3BTC and 4.5 g polyvinylpyrrolidone PVP were mixed in 60 mL N,N-dimethylformamide DMF and 30 mL ultrapure water and stirred to dissolve at room temperature. The mixture was then transferred to a polytetrafluoroethylene-lined autoclave and kept at 160 °C for 12 hours. After centrifugation and drying, green nickel ion organic framework Ni-MOF was obtained.

[0058] (2) Mix 150 mg of Ni-MOF obtained in step (1) and 225 mg of cobalt chloride hexahydrate CoCl2·6H2O in 90 mL of anhydrous ethanol. Heat the mixture in an oil bath at 90°C for 3 hours, then centrifuge and dry to obtain a blue-green mixture. Place 150 mg of the mixture and 75 mg of sulfur powder in a porcelain boat, heat the furnace to 800°C, and maintain the temperature for 4 hours under an argon flow. Centrifuge and dry to obtain cobalt-doped nickel ion organic framework sulfide Co / Ni-MOF / S.

[0059] (3) Disperse 60 mg of Co / Ni-MOF / S obtained in step (2) in 150 mL of ultrapure water, add 6 mL of 1% chloroauric acid HAuCl4 solution and 15 mg of PVP, stir for 8 hours, add 6 mL of sodium citrate solution C6H9NaO7 and 3 mg of sodium borohydride NaBH4, stir for 26 hours, centrifuge and dry to obtain black Au@Co / Ni-MOF / S product.

[0060] Example 7: Fabrication of a portable sensor electrode for detecting root rot in Panax notoginseng:

[0061] (1) Using a disposable, disposable electrode as the working electrode, 5 μL of Au@Co / Ni-MOF / S solution was dropped onto the electrode surface and dried at room temperature for 2 hours;

[0062] (2) The electrode obtained in step (1) was washed with phosphate buffer solution PBS, and 3 μL of Ab1 solution was added to the electrode surface and incubated at room temperature for 1 hour.

[0063] (3) The electrode obtained in step (2) is cleaned with PBS, and 8 µL of bovine serum albumin (BSA) solution is dropped onto the electrode surface. The electrode is dried at 4°C, then cleaned with PBS and stored at 4°C.

[0064] Example 8: Fabrication of a portable sensor electrode for detecting root rot in Panax notoginseng:

[0065] (1) Using a disposable, disposable electrode as the working electrode, 10 μL of Au@Co / Ni-MOF / S solution was dropped onto the electrode surface and dried at room temperature for 3 hours;

[0066] (2) The electrode obtained in step (1) was washed with phosphate buffer solution PBS, and 6 μL of Ab1 solution was added to the electrode surface and incubated at room temperature for 2 hours.

[0067] (3) The electrode obtained in step (2) is cleaned with PBS, and 10 µL of bovine serum albumin (BSA) solution is dropped onto the electrode surface. The electrode is dried at 4°C, then cleaned with PBS and stored at 4°C.

[0068] Example 9: Fabrication of a portable sensor electrode for detecting root rot in Panax notoginseng:

[0069] (1) Using a disposable, disposable electrode as the working electrode, 15 μL of Au@Co / Ni-MOF / S solution was dropped onto the electrode surface and dried at room temperature for 4 hours;

[0070] (2) The electrode obtained in step (1) was washed with phosphate buffer solution PBS, and 9 μL of Ab1 solution was added to the electrode surface and incubated at room temperature for 3 hours.

[0071] (3) The electrode obtained in step (2) was cleaned with PBS, and 12 µL of bovine serum albumin (BSA) solution was dropped onto the electrode surface. The electrode was dried at 4°C, then cleaned with PBS and stored at 4°C.

[0072] Example 10 Portable sensor for detecting root rot in Panax notoginseng: luminescent material MSN-TCBPE / Ru(dcbpy)3 2+ Preparation of Ab2 conjugates:

[0073] Take 2 mg MSN-TCBPE / Ru(dcbpy)3 2+ Add 900 μL of PBS and 100 μL of 1-ethyl-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide EDC / NHS solution, shake at room temperature for 2 hours, centrifuge to remove the supernatant, add 950 μL of PBS, then add 50 μL of Ab2 solution, shake at 4°C, centrifuge to remove the supernatant, and disperse with 1 mL of PBS to obtain MSN-TCBPE / Ru(dcbpy)3. 2+ -Ab2.

[0074] Example 11 Portable sensor for detecting root rot in Panax notoginseng: luminescent material MSN-TCBPE / Ru(dcbpy)3 2+ Preparation of Ab2 conjugates:

[0075] Take 3 mg of MSN-TCBPE / Ru(dcbpy)3 2+ Add 1800 μL of PBS and 200 μL of 1-ethyl-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide EDC / NHS solution, shake at room temperature for 2-6 hours, centrifuge to remove the supernatant, add 1900 μL of PBS, then add 100 μL of Ab2 solution, shake at 4°C, centrifuge to remove the supernatant, and disperse with 2 mL of PBS to obtain MSN-TCBPE / Ru(dcbpy)3. 2+ -Ab2.

[0076] Example 12 Portable sensor for detecting root rot in Panax notoginseng: luminescent material MSN-TCBPE / Ru(dcbpy)3 2+ Preparation of Ab2 conjugates:

[0077] Take 4 mg MSN-TCBPE / Ru(dcbpy)3 2+ Add 2700 μL of PBS and 300 μL of 1-ethyl-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide EDC / NHS solution, shake at room temperature for 6 hours, centrifuge to remove the supernatant, add 2850 μL of PBS, then add 150 μL of Ab2 solution, shake at 4°C, centrifuge to remove the supernatant, and disperse with 3 mL of PBS to obtain MSN-TCBPE / Ru(dcbpy)3. 2+ -Ab2.

[0078] Example 13: The portable biosensor for detecting root rot in Panax notoginseng prepared in Examples 7 and 10 was applied to the detection of Fusarium sp., and the steps are as follows:

[0079] (1) Preparation of standard solutions: Prepare a set of Fusarium sp. standard solutions of different concentrations, including blank standard samples;

[0080] (2) Preparation of working electrode: 8 µL of Fusarium sp. standard solution of different concentrations was added dropwise to the modified electrode surface. After incubation for 30 minutes, 8 µL of MSN-TCBPE / Ru(dcbpy)3 was added dropwise. 2+ -Ab2, after 30 minutes of incubation, the working electrode is obtained;

[0081] (3) Plotting the working curve: Connect the disposable electrode to the USB flash drive electrochemical workstation; add DBAE solution as a co-reactant to the electrode to cover the three electrodes; apply cyclic voltage to the electrode using cyclic voltammetry, and detect the light signal intensity using a micro ECL sensing platform; the response light signal intensity containing different concentrations of Fusarium sp. standard solution is recorded as follows: I ; I Concentration of Fusarium sp. standard solution c The logarithmic functions are linearly related, plotted. I -lg c The working curve has a limit of detection of 14.9 pg / mL.

[0082] (4) Detection of root rot in Panax notoginseng samples: Soak 5 g of Panax notoginseng root and leaf samples (slices or powders) in 10 mL of 50% ethanol aqueous solution for 30 minutes to obtain Fusarium sp. soaking solution; then replace the Fusarium sp. standard solution in step (1) with the Fusarium sp. soaking solution and perform detection according to the methods in steps (2) and (3), based on the intensity of the response light signal. I Concentration of Fusarium sp. standard solution c The logarithmic function and the working curve show a linear relationship, and the content of Fusarium sp. in Panax notoginseng samples is obtained, thus completing the diagnosis of root rot in Panax notoginseng.

[0083] Example 14: The portable biosensor for detecting root rot in Panax notoginseng, prepared in Examples 8 and 11, was applied to the detection of Fusarium sp., and the steps are as follows:

[0084] (1) Preparation of standard solutions: Prepare a set of Fusarium sp. standard solutions of different concentrations, including blank standard samples;

[0085] (2) Preparation of working electrode: 16 µL of Fusarium sp. standard solution of different concentrations was added dropwise to the modified electrode surface. After incubation for 45 minutes, 16 µL of MSN-TCBPE / Ru(dcbpy)3 was added dropwise. 2+ -Ab2, after 45 minutes of incubation, the working electrode is obtained;

[0086] (3) Plotting the working curve: Connect the disposable electrode to the USB flash drive electrochemical workstation; add DBAE solution as a co-reactant to the electrode to cover the three electrodes; apply cyclic voltage to the electrode using cyclic voltammetry, and detect the light signal intensity using a micro ECL sensing platform; the response light signal intensity containing different concentrations of Fusarium sp. standard solution is recorded as follows: I ; I Concentration of Fusarium sp. standard solution c The logarithmic functions are linearly related, plotted. I -lg c The working curve has a limit of detection of 14.9 pg / mL.

[0087] (4) Detection of root rot in Panax notoginseng samples: 8 g of Panax notoginseng root and leaf samples, sliced ​​or crushed, were soaked in 20 mL of 60% ethanol aqueous solution for 45 minutes to obtain Fusarium sp. soaking solution; then, the Fusarium sp. soaking solution was used to replace the Fusarium sp. standard solution in step (1), and the detection was performed according to the methods in steps (2) and (3). The results were based on the intensity of the response light signal. I Concentration of Fusarium sp. standard solution c The logarithmic function and the working curve show a linear relationship, and the content of Fusarium sp. in Panax notoginseng samples is obtained, thus completing the diagnosis of root rot in Panax notoginseng.

[0088] Example 15: The portable biosensor for detecting root rot in Panax notoginseng, prepared in Examples 9 and 12, was applied to the detection of Fusarium sp., and the steps are as follows:

[0089] (1) Preparation of standard solutions: Prepare a set of Fusarium sp. standard solutions of different concentrations, including blank standard samples;

[0090] (2) Preparation of working electrode: 24 µL of Fusarium sp. standard solution of different concentrations was added dropwise to the modified electrode surface. After incubation for 60 minutes, 24 µL of MSN-TCBPE / Ru(dcbpy)3 was added dropwise. 2+ -Ab2, after 60 minutes of incubation, the working electrode is obtained;

[0091] (3) Plotting the working curve: Connect the disposable electrode to the USB flash drive electrochemical workstation; add DBAE solution as a co-reactant to the electrode to cover the three electrodes; apply cyclic voltage to the electrode using cyclic voltammetry, and detect the light signal intensity using a micro ECL sensing platform; the response light signal intensity containing different concentrations of Fusarium sp. standard solution is recorded as follows: I ; I Concentration of Fusarium sp. standard solution c The logarithmic functions are linearly related, plotted. I -lg c The working curve has a limit of detection of 14.9 pg / mL.

[0092] (4) Detection of root rot in Panax notoginseng samples: Soak 10 g of Panax notoginseng root and leaf samples (slices or powders) in 30 mL of 75% ethanol aqueous solution for 60 minutes to obtain Fusarium sp. soaking solution; then replace the Fusarium sp. standard solution in step (1) with the Fusarium sp. soaking solution and perform detection according to the methods in steps (2) and (3), based on the intensity of the response light signal. I Concentration of Fusarium sp. standard solution c The logarithmic function and the working curve show a linear relationship, and the content of Fusarium sp. in Panax notoginseng samples is obtained, thus completing the diagnosis of root rot in Panax notoginseng.

[0093] Table 1. Concentration of root rot pathogens in different Panax notoginseng samples and verification results.

[0094] 1 0 0 2 0.50 0.51 2.2 102 3 1.30 1.28 2.6 98 4 3.00 3.02 1.8 101

[0095] Table 2 Comparison of detection range and detection limit of different detection methods for root rot pathogens in Panax notoginseng

[0096] PCR technology 1 - 480 102 Enzyme-linked immunosorbent assay kit 1.5 - 10 680 This platform 0.1 - 1000 14.9

[0097] The above are merely preferred embodiments of the present invention and do not limit the scope of the patent. All equivalent changes and modifications made within the scope of the present invention should still fall within the scope of the present invention.

Claims

1. A method for preparing a portable sensor for rapid detection of root rot in Panax notoginseng, characterized in that, The portable sensor includes a dual-luminescent ligand co-encapsulation material MSN-TCBPE / Ru(dcbpy)3. 2+ The system comprises a capture antibody Ab1, a gold-loaded cobalt-doped nickel ion organic framework sulfide Au@Co / Ni-MOF / S, a labeled antibody Ab2, co-reactants, a micro-electrochemiluminescence (ECL) sensing platform, and disposable electrodes; the method for diagnosing root rot in Panax notoginseng involves detecting the pathogen Fusarium sp.; the capture antibody Ab1 and labeled antibody Ab2 specifically recognize and capture Fusarium sp.; and the dual-luminescent ligand co-encapsulation material MSN-TCBPE / Ru(dcbpy)3. 2+ The dual-luminescent ligands are co-encapsulated in mesoporous silica MSN; the dual-luminescent ligands are tetracarboxylated biphenylethylene (H4TCBPE) and carboxylated terpyridine ruthenium (Ru(dcbpy)3). 2+ The gold-loaded cobalt-doped nickel ion organic framework sulfide Au@Co / Ni-MOF / S is prepared by doping a Ni ion organic framework Ni-MOF with Co ions and then sulfiding it with gold; the co-reactant is dibutylethanolamine (DBAE).

2. The method for preparing a portable sensor for rapid detection of root rot in Panax notoginseng as described in claim 1, characterized in that, The dual-luminescent ligand co-encapsulation material MSN-TCBPE / Ru(dcbpy)3 2+ The preparation steps are as follows: (1) Dissolve 0.5 ~ 1.5 g cetyltrimethylammonium bromide (CTAB) in a mixture of 75 ~ 225 mL ultrapure water and 50 ~ 150 mL anhydrous ethanol, then add 1 ~ 3 mL ammonia water and 0.5 ~ 1.5 mL tetraethyl silicate (TEOS) under stirring, and react at 30 ~ 40 °C for 8 ~ 12 hours. Centrifuge and dry to obtain MSN; (2) Sonicate 100-300 mg of MSN obtained in step (1) in 50-150 mL of anhydrous ethanol, add 1-3 mL of 3-aminopropyltriethoxysilane APTES, and reflux at 60-80 °C for 4-8 hours under nitrogen protection. Then centrifuge and dry to obtain amination-modified mesoporous silica MSN-NH2. (3) Take 13.5 ~ 40.5 mg H4TCBPE and 9.6 ~ 28.8 mg Ru(dcbpy)3 2+ Disperse the mixture in 4-12 mL of anhydrous ethanol, add 30-90 mg of MSN-NH2 obtained in step (2) to the mixture, and centrifuge and dry to obtain MSN-TCBPE / Ru(dcbpy)3. 2+ .

3. The method for preparing a portable sensor for rapid detection of root rot in Panax notoginseng as described in claim 1, characterized in that, The preparation steps of the gold-loaded ion-doped metal-organic framework sulfide Au@Co / Ni-MOF / S are as follows: (1) Mix 0.43 ~ 1.29 g nickel nitrate hexahydrate Ni(NO3)2·6H2O, 0.15 ~ 0.45 g trimesic acid H3BTC and 1.5 ~ 4.5 g polyvinylpyrrolidone PVP in 20 ~ 60 mL N,N-dimethylformamide DMF and 10 ~ 30 mL ultrapure water, stir and dissolve at room temperature, transfer the mixture to a polytetrafluoroethylene-lined autoclave, keep at 120 ~ 160 °C for 8 ~ 12 hours, centrifuge and dry to obtain green nickel ion organic framework Ni-MOF; (2) Mix 50-150 mg of Ni-MOF obtained in step (1) and 75-225 mg of cobalt chloride hexahydrate CoCl2·6H2O in 30-90 mL of anhydrous ethanol. Heat the mixture in an oil bath at 60-90°C for 1-3 hours, then centrifuge and dry to obtain a blue-green mixture. Place 50-150 mg of the mixture and 25-75 mg of sulfur powder in a porcelain boat. Heat the furnace to 600-800°C and maintain the temperature under an argon flow for 2-4 hours. Centrifuge and dry to obtain cobalt-doped nickel ion organic framework sulfide Co / Ni-MOF / S. (3) Disperse 20-60 mg of Co / Ni-MOF / S obtained in step (2) in 50-150 mL of ultrapure water, add 2-6 mL of 1% chloroauric acid HAuCl4 solution and 5-15 mg of PVP, stir for 4-8 hours, add 2-6 mL of sodium citrate solution C6H9NaO7 and 1-3 mg of sodium borohydride NaBH4, stir for 20-26 hours, centrifuge and dry to obtain black Au@Co / Ni-MOF / S product.

4. The method for preparing a portable sensor for rapid detection of root rot in Panax notoginseng as described in claim 1, characterized in that, The preparation steps of the portable sensor for Panax notoginseng root rot disease are as follows: (1) Using a disposable, disposable electrode as the working electrode, drop 5 ~ 15 μL of Au@Co / Ni-MOF / S solution onto the electrode surface and dry at room temperature for 2 ~ 4 hours; (2) Wash the electrode obtained in step (1) with phosphate buffer solution PBS, add 3 ~ 9 μL of Ab1 solution to the electrode surface and incubate at room temperature for 1 ~ 3 hours; (3) The electrode obtained in step (2) is cleaned with PBS, and 8 ~ 12 µL of bovine serum albumin (BSA) solution is dropped onto the electrode surface. The electrode is dried at 4°C, then cleaned with PBS and stored at 4°C. (4) Take 2-4 mg of the dual-luminescent ligand co-encapsulation material MSN-TCBPE / Ru(dcbpy)3. 2+ Add 900–2700 μL of PBS and 100–300 μL of 1-ethyl-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide EDC / NHS solution, shake at room temperature for 2–6 hours, centrifuge to remove the supernatant, add 950–2850 μL of PBS, then add 50–150 μL of Ab2 solution, shake at 4°C, centrifuge to remove the supernatant, and disperse with 1–3 mL of PBS to obtain MSN-TCBPE / Ru(dcbpy)3. 2+ -Ab2; The aforementioned micro electrochemiluminescence (ECL) sensing and detection platform consists of a photon counter, a counting unit, and a USB-type electrochemical workstation.

5. The method for preparing a portable sensor for rapid detection of root rot in Panax notoginseng as described in claim 4, wherein the prepared portable sensor for root rot in Panax notoginseng is applied to the detection of Fusarium sp., characterized in that, The testing steps are as follows: (1) Preparation of standard solutions: Prepare a set of Fusarium sp. standard solutions of different concentrations, including blank standard samples; (2) Preparation of working electrode: 8-24 µL of Fusarium sp. standard solution of different concentrations was added dropwise to the modified electrode surface. After incubation for 30-60 minutes, 8-24 µL of MSN-TCBPE / Ru(dcbpy)3 was added dropwise. 2+ -Ab2, after incubation for 30 to 60 minutes, the working electrode is obtained; (3) Plotting the working curve: Connect the disposable electrode to the USB flash drive electrochemical workstation; add DBAE solution as a co-reactant to the electrode to cover the three electrodes; apply a cyclic voltage to the electrode using cyclic voltammetry, and detect the light signal intensity using the micro electrochemiluminescence ECL sensing platform; the response light signal intensity containing different concentrations of Fusarium sp. standard solution is recorded as . I ; I Concentration of Fusarium sp. standard solution c The logarithmic functions are linearly related, plotted. I -lg c The working curve has a limit of detection of 14.9 pg / mL. (4) Detection of root rot in Panax notoginseng samples: Soak 5-10 g of Panax notoginseng root and leaf samples (slices or powders) in 10-30 mL of 50-75% ethanol aqueous solution for 30-60 minutes to obtain Fusarium sp. soaking solution; then replace the Fusarium sp. standard solution in step (1) with the Fusarium sp. soaking solution and perform detection according to the methods in steps (2) and (3), based on the intensity of the response light signal. I Concentration of Fusarium sp. standard solution c The logarithmic function and the working curve show a linear relationship, and the content of Fusarium sp. in Panax notoginseng samples is obtained, thus completing the diagnosis of root rot in Panax notoginseng.

6. A method for preparing a portable sensor for rapid detection of root rot in Panax notoginseng as described in claim 5, characterized in that, The disposable, disposable electrode is selected from one of the following conductive substrates: screen-printed electrode, conductive carbon cloth, and paper-based electrode; the Au@Co / Ni-MOF / S solution is prepared by dissolving Au@Co / Ni-MOF / S in ultrapure water, with a concentration of 0.8 mg / mL; the Ab1 solution is prepared by dissolving Ab1 in PBS, with a concentration of 10 μg / mL; the BSA solution is prepared by dissolving BSA in PBS, with a concentration of 100 µg / mL; the Ab2 solution is prepared by dissolving Ab2 in PBS, with a concentration of 10 μg / mL; the EDC / NHS solution is prepared by adding 76 mg EDC and 11.5 mg NHS to 1 mL of ultrapure water; the DBAE solution is prepared by dissolving DBAE in PBS, with a concentration of 10 mM; the PBS is a 150 mM phosphate buffer solution with a pH of 7.4; the different concentrations of Fusarium sp. standard solutions are 100 pg / mL, 500 pg / mL, and 1... The cyclic voltammetry detection parameters were set as follows: initial potential of 0 V, maximum potential of 1.2 V, and scan rate of 0.1 V / s.

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