Cucumber mosaic virus photoelectrochemical biosensor and detection method thereof

Abstract

The application discloses a cucumber mosaic virus photoelectric chemical sensor and a detection method thereof, and belongs to the technical field of photoelectric chemical sensors.The existing PEC sensor has the problems of low photoelectric conversion efficiency and lack of efficient signal amplification strategy.The sensor provided by the application takes a screen-printed electrode as a substrate, a three-dimensional flower-shaped ZnIn2S4 nanomaterial is used as a light active layer to modify a working electrode, and a basic phosphatase-mediated enzymatic biological catalytic precipitation signal amplification system is matched;the detection method adopts a sandwich immunization mode to capture viruses, catalyzes the generation of BCP precipitation to shield the transmission of photo-generated carriers, and quantitatively detects through photocurrent attenuation, the linear range of the detection is 0.001-200 ng / mL, and the detection limit is 0.001 ng / mL, so that the cucumber mosaic virus can be rapidly screened in a field.

Description

Technical Field

[0001] This invention relates to a photoelectrochemical biosensor for cucumber mosaic virus and its detection method, belonging to the field of photoelectrochemical biosensing technology. Background Technology

[0002] Cucumber mosaic virus (CMV) is one of the most serious plant viruses in agricultural production, characterized by strong resistance, high infectivity, and strong concealment.

[0003] Currently, conventional CMV detection technologies mainly fall into three categories: the first is electron microscopy, which directly observes the morphology of viral particles for qualitative detection; the second is immunological detection methods, which rely on the specific binding reaction between antigens and antibodies for qualitative or quantitative detection; and the third is molecular biological detection methods, which identify viral characteristic sequences through nucleic acid amplification for highly specific detection. In recent years, photoelectrochemical (PEC) biosensors have been gradually applied to the CMV detection field due to their high sensitivity, fast response speed, and low cost. Among them, PEC sensors based on screen-printed electrodes (SPE) have become an important research direction for portable CMV detection technology due to the mass production, portability, and single-use nature of SPE. Currently, these PEC sensors typically use photonanomaterials to modify the working electrode of the SPE to construct the sensing interface, capturing target CMV based on a sandwich immunoreaction mode. Some schemes use alkaline phosphatase (ALP)-labeled secondary antibodies, utilizing ALP to catalyze the reaction of BCIP / NBT substrates to achieve signal conversion, ultimately achieving quantitative detection of the virus by detecting changes in photocurrent signals.

[0004] However, existing SPE-based PEC biosensors have limitations. First, the photoelectric conversion performance of the photonanomaterials they use is limited by the material properties themselves, making it difficult to achieve stable high photoelectric response under low energy consumption conditions. Second, these sensors have not yet developed efficient signal amplification strategies that are compatible with existing sensing systems, making it difficult to further improve detection sensitivity. As a result, existing technologies cannot simultaneously meet the requirements of CMV detection for high sensitivity, low cost, and portability for rapid field screening. Summary of the Invention

[0005] (a) Purpose of the invention

[0006] The purpose of this invention is to provide a photoelectrochemical biosensor and corresponding detection method for detecting cucumber mosaic virus, which has the advantages of high sensitivity, low cost and portability, so as to meet the needs of rapid field screening of cucumber mosaic virus.

[0007] (II) Technical Solution

[0008] In a first aspect, the present invention provides a cucumber mosaic virus photoelectrochemical biosensor, comprising:

[0009] The substrate electrode is a screen-printed electrode;

[0010] The working electrode surface of the screen-printed electrode is modified with a three-dimensional flower-shaped ZnIn2S4 nanomaterial as a photoactive layer.

[0011] The sensor is configured to match an alkaline phosphatase-mediated enzymatic biocatalytic precipitation signal amplification system: when cucumber mosaic virus is captured, the insoluble BCP precipitate generated by alkaline phosphatase catalyzing the BCIP / NBT substrate can cover the surface of the three-dimensional flower-shaped ZnIn2S4 nanomaterial, generating a corresponding photocurrent attenuation signal by shielding photogenerated carrier transmission, thereby realizing the quantitative detection of cucumber mosaic virus.

[0012] Preferably, the three-dimensional flower-shaped ZnIn2S4 nanomaterial is assembled from intersecting nanosheets.

[0013] Preferably, the alkaline phosphatase-labeled secondary antibody is an immune probe formed by conjugating alkaline phosphatase, gold nanoparticles, and cucumber mosaic virus secondary antibody.

[0014] Preferably, the sensor has a linear detection range of 0.001 ng / mL to 200 ng / mL for cucumber mosaic virus, and a detection limit of 0.001 ng / mL.

[0015] Secondly, the present invention provides a photoelectrochemical detection method for cucumber mosaic virus, comprising the following steps:

[0016] S1. A screen-printed electrode with a surface modified with three-dimensional flower-shaped ZnIn2S4 nanomaterials is used as the working electrode.

[0017] S2. Using a sandwich immune recognition mode, cucumber mosaic virus capture antibody, test sample, alkaline phosphatase-labeled secondary antibody are sequentially incubated with the working electrode to specifically capture cucumber mosaic virus in the test sample.

[0018] S3. Add BCIP / NBT substrate solution, and generate insoluble BCP precipitate by alkaline phosphatase to cover the surface of the three-dimensional flower-like ZnIn2S4 nanomaterial;

[0019] S4. Detect the attenuation degree of the photocurrent signal of the working electrode and quantitatively obtain the concentration of cucumber mosaic virus in the sample to be tested.

[0020] Preferably, the three-dimensional flower-like ZnIn2S4 nanomaterial is assembled from intersecting nanosheets and is prepared by hydrothermal reaction using ZnCl2, InCl3 and thioacetamide as raw materials.

[0021] Preferably, in step S2, the alkaline phosphatase-labeled secondary antibody is an immune probe formed by conjugation of alkaline phosphatase, gold nanoparticles, and cucumber mosaic virus secondary antibody.

[0022] Preferably, in step S3, the reaction time of alkaline phosphatase catalyzing the BCIP / NBT substrate is 10 min to 20 min.

[0023] Preferably, in step S4, a visible light source with a wavelength of 405 nm is used to excite the working electrode to generate photocurrent.

[0024] Preferably, the linear range for the detection of cucumber mosaic virus by the method is from 0.001 ng / mL to 200 ng / mL, and the detection limit is 0.001 ng / mL.

[0025] (III) Beneficial Effects

[0026] This invention modifies the working electrode surface of a screen-printed electrode with a three-dimensional flower-shaped ZnIn2S4 nanomaterial as a photoactive layer. This three-dimensional flower-shaped structure is assembled from intersecting nanosheets and has the characteristics of being non-toxic, having a high specific surface area, high light-trapping ability, and low electron-hole recombination rate. It can generate a stable high photoelectric response under low energy consumption conditions, overcoming the problems of high toxicity, wide bandgap, and high electron-hole recombination rate of photoactive materials used in existing PEC sensors, which lead to low photoelectric conversion efficiency. Compared with similar PEC sensors using traditional photonanomaterials, its photoelectric conversion efficiency is significantly improved, providing a stable photoelectric basis for high-sensitivity detection.

[0027] This invention utilizes an insoluble BCP precipitate generated by alkaline phosphatase catalyzing BCIP / NBT as a signal modulation medium. When the target cucumber mosaic virus is captured, alkaline phosphatase catalyzes the substrate to generate an insoluble BCP precipitate that covers the surface of the photoactive layer, shielding the transmission path of photogenerated carriers and converting trace immune recognition events into significant photocurrent attenuation signals. This overcomes the problem of insufficient detection sensitivity caused by the lack of an adaptive signal amplification strategy in existing PEC sensors.

[0028] This invention utilizes a three-dimensional flower-shaped ZnIn2S4-modified screen-printed electrode and a BCP signal amplification system to construct an integrated detection sensor. The screen-printed electrode itself has the advantages of being mass-producible, low-cost, portable, and disposable. Combined with the aforementioned photoactive layer with high photoelectric conversion efficiency and high-magnification signal amplification strategy, sample detection can be completed directly without large equipment. This effectively solves the problems of existing cucumber mosaic virus detection methods, which rely on large equipment, are cumbersome to operate, and are time-consuming, and cannot simultaneously meet the needs of high sensitivity, low cost, and portability for rapid field screening. Compared with traditional electron microscopy, immunological, and molecular biological detection methods, the detection cycle is short, which can be directly adapted to the needs of rapid field screening. The detection limit is as low as 0.001 ng / mL, and the linear range covers 0.001 ng / mL to 200 ng / mL.

[0029] Furthermore, by employing an immune probe formed by loading alkaline phosphatase onto gold nanoparticles and using cucumber mosaic virus secondary antibody, this invention can increase the enzyme loading and immune recognition specificity, further reduce non-specific interference, and improve the accuracy of detection results. Attached Figure Description

[0030] Figure 1 This is a schematic diagram illustrating the principle of the photoelectrochemical biosensor based on CMV detection of the present invention.

[0031] Figure 2 The image shows the electron microscopy characterization of the three-dimensional flower-like ZnIn2S4 material prepared in this invention.

[0032] Figure 3 This is a diagram illustrating the charge transfer mechanism of the material of the present invention.

[0033] Figure 4 This is a test diagram of the photocurrent response of the sensor of the present invention. Detailed Implementation

[0034] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention can be practiced in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0035] Example 1: Preparation and detection of cucumber mosaic virus photoelectrochemical biosensor

[0036] 1. Preparation of three-dimensional flower-like ZnIn2S4 nanomaterials

[0037] 0.3 mM ZnCl2, 0.6 mM InCl3, and 2.4 mM TAA were dissolved in 30 mL of ultrapure water and stirred for 30 minutes to obtain a precursor solution. The solution was transferred to a 100 mL high-pressure reactor and heated at 160 °C for 2 hours. After the reaction was completed, the product was washed with ultrapure water and dried to obtain three-dimensional flower-like ZnIn2S4 nanomaterials.

[0038] 2. Preparation of ZnIn2S4 / SPE electrode

[0039] The above-mentioned three-dimensional flower-like ZnIn2S4 nanomaterials were dispersed in ultrapure water to prepare a suspension with a concentration of 0.5 mg / mL. 0.005% (w / v) imine phthalate was added as a film-forming aid, and the mixture was ultrasonically dispersed for 10 min to obtain a homogeneous dispersion. 10 μL of the above dispersion was dropped onto the surface of an SPE working electrode, which had been ultrasonically cleaned sequentially with anhydrous ethanol and ultrapure water. After drying at room temperature, a ZnIn2S4 / SPE electrode was obtained.

[0040] 3. Preparation of immune probes

[0041] Au NPs (15-20 nm in diameter) were prepared according to literature methods. 1.0 mL of Au NPs solution was taken, and 100 μL of 5% Tween 20 was added. The solution was incubated at 37°C for 1 hour. The pH was adjusted to 8-9 with sodium carbonate solution. 20 μL of 100 μg / mL secondary antibody (pAb2) and 80 μL of 100 μg / mL alkaline phosphatase (ALP) were added, and the solution was incubated at 4°C with stirring for 12 hours. After centrifugation and washing, the obtained ALP-Au NPs-pAb2 was dispersed in 1.0 mL of buffer to obtain the immunoprobe.

[0042] 4. Sensor Construction and Detection Methods

[0043] The working area was the circular well (5 mm geometric diameter) of the ZnIn2S4 / SPE electrode. 25 μL of 0.1 mg / mL CMV capture antibody (Ab1) was immobilized in a 96-well plate and incubated overnight at 4°C. Non-specific sites were blocked with blocking buffer. 25 μL of different concentrations of CMV standards were added, and the plate was incubated at 37°C for 60 minutes. After washing, 25 μL of the above-mentioned immunoprobe (ALP-Au NPs-pAb2) at 0.1 mg / mL was added, and the plate was incubated at 37°C for 60 minutes. After washing, 25 μL of BCIP / NBT solution was added, and the BCP reaction was performed at 37°C for 15 minutes. 10 μL of the BCP product was added to the surface of the pre-prepared ZnIn2S4 / SPE electrode and incubated at 37°C for 15 minutes. After washing, photoelectrochemical measurements were performed.

[0044] 5. Photoelectrochemical detection

[0045] A three-electrode system (ZnIn2S4 / SPE as the working electrode, platinum wire as the counter electrode, and Ag / AgCl as the reference electrode) was used. The system was irradiated with a 405 nm LED light source in PBS buffer containing 0.1 M ascorbic acid, and the photocurrent signal was detected by applying a 0 V bias voltage. The CMV concentration was quantified by the degree of photocurrent attenuation.

[0046] 6. Experimental Results and Characterization

[0047] Material characterization: SEM and TEM showed that ZnIn2S4 has a three-dimensional flower-like hierarchical structure assembled from thin nanosheets. EDS confirmed the presence of Zn, In and S elements. The XRD pattern was a perfect match with the standard ZnIn2S4 card (JCPDS No. 65-2023) with no impurity peaks.

[0048] Performance testing: Photocurrent testing showed that after BCP deposition, the photocurrent significantly decreased from approximately 1.8 μA to approximately 0.4 μA, with a signal suppression rate of 77.8%. The sensor exhibited good stability during 10 optical switching cycles.

[0049] Sensing performance: Experimental results show that the CMV concentration exhibits a good linear relationship with the photocurrent signal change in the range of 0.001 ng / mL to 200 ng / mL (R0). 2 =0.9998), with a detection limit as low as 0.001 ng / mL. Selectivity tests showed that the signal inhibition rates against tobacco mosaic virus (TMV) and potato virus Y (PVY) at 100 ng / mL were both less than 5%, with no significant cross-reactivity; the spiked recoveries of actual tomato leaf samples were 92.3%–105.7%, with relative standard deviations (RSD) less than 3.2%.

[0050] Compared with other detection methods (Table 1), the SPE-based biosensor of this invention has higher sensitivity, providing an efficient, convenient, rapid and sensitive detection platform for future plant virus detection.

[0051] Table 1. Comparison of the present invention with published CMV detection methods.

[0052]

[0053] [1] Rafidah, AR, Faridah, S., Shahrul, AA Chrono-amperometry measurement for rapid cucumber mosaic virus detection in plants[J]. ProcediaChemistry, 2016, 20, 25.

[0054] [2] Srivastava, N., Kapoor, R., Kumar, R., Kumar, S., Saritha, R.,Kumar, S., Baranwal, VK Rapid diagnosis of cucumber mosaic virus in bananaplants using a fluorescence-based real-time isothermal reverse transcription-recombinase polymerase amplification assay[J]. Journal of VirologicalMethods, 2019, 270, 52.

[0055] [3] Sun, W., Jiao, K., Zhang, SS Electrochemical ELISA for the detection of cucumber mosaic virus using o-phenylene-diamine as substrate[J]. Talanta, 2001, 55, 1211.

[0056] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A photoelectrochemical biosensor for cucumber mosaic virus, characterized in that, include: The substrate electrode is a screen-printed electrode; The working electrode surface of the screen-printed electrode is modified with a three-dimensional flower-shaped ZnIn2S4 nanomaterial as a photoactive layer. The sensor is configured to match an alkaline phosphatase-mediated enzymatic biocatalytic precipitation signal amplification system: when cucumber mosaic virus is captured, the insoluble BCP precipitate generated by alkaline phosphatase catalyzing the BCIP / NBT substrate can cover the surface of the three-dimensional flower-shaped ZnIn2S4 nanomaterial, generating a corresponding photocurrent attenuation signal by shielding photogenerated carrier transmission, thereby realizing the quantitative detection of cucumber mosaic virus.

2. The cucumber mosaic virus photoelectrochemical biosensor according to claim 1, characterized in that, The three-dimensional flower-shaped ZnIn2S4 nanomaterial is assembled from intersecting nanosheets.

3. The cucumber mosaic virus photoelectrochemical biosensor according to claim 1 or 2, characterized in that, The alkaline phosphatase-labeled secondary antibody is an immune probe formed by conjugating alkaline phosphatase, gold nanoparticles, and cucumber mosaic virus secondary antibody.

4. The cucumber mosaic virus photoelectrochemical biosensor according to any one of claims 1 to 3, characterized in that, The sensor has a linear detection range of 0.001 ng / mL to 200 ng / mL for cucumber mosaic virus, with a detection limit of 0.001 ng / mL.

5. A photoelectrochemical detection method for cucumber mosaic virus, characterized in that, Includes the following steps: S1. A screen-printed electrode with a surface modified with three-dimensional flower-shaped ZnIn2S4 nanomaterials is used as the working electrode. S2. Using a sandwich immune recognition mode, cucumber mosaic virus capture antibody, test sample, alkaline phosphatase-labeled secondary antibody are sequentially incubated with the working electrode to specifically capture cucumber mosaic virus in the test sample. S3. Add BCIP / NBT substrate solution, and generate insoluble BCP precipitate by alkaline phosphatase to cover the surface of the three-dimensional flower-like ZnIn2S4 nanomaterial; S4. Detect the attenuation degree of the photocurrent signal of the working electrode and quantitatively obtain the concentration of cucumber mosaic virus in the sample to be tested.

6. The photoelectrochemical detection method for cucumber mosaic virus according to claim 5, characterized in that, The three-dimensional flower-like ZnIn2S4 nanomaterial is assembled from intersecting nanosheets and is prepared by hydrothermal reaction using ZnCl2, InCl3 and thioacetamide as raw materials.

7. The photoelectrochemical detection method for cucumber mosaic virus according to claim 5 or 6, characterized in that, In step S2, the alkaline phosphatase-labeled secondary antibody is an immune probe formed by conjugating alkaline phosphatase, gold nanoparticles, and cucumber mosaic virus secondary antibody.

8. The photoelectrochemical detection method for cucumber mosaic virus according to any one of claims 5 to 7, characterized in that, In step S3, the reaction time of alkaline phosphatase catalyzing the BCIP / NBT substrate is 10 min to 20 min.

9. The photoelectrochemical detection method for cucumber mosaic virus according to any one of claims 5 to 8, characterized in that, In step S4, a visible light source with a wavelength of 405nm is used to excite the working electrode to generate photocurrent.

10. The photoelectrochemical detection method for cucumber mosaic virus according to any one of claims 5 to 9, characterized in that, The method has a linear detection range of 0.001 ng / mL to 200 ng / mL for cucumber mosaic virus, and a detection limit of 0.001 ng / mL.

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