Colorimetric detection method for daucoside based on enhanced octahedral silver oxide laccase activity and application of colorimetric detection method

Abstract

The invention discloses a colorimetric detection method for daucoside based on enhanced octahedral silver oxide laccase activity and application, and belongs to the technical field of agricultural product safety detection. According to the present invention, the nucleic acid aptamer capable of being specifically combined with the danioside is adopted as the original recognition element, and the colorimetric detection method for enhancing the activity of the octahedral silver oxide laccase by using the nucleic acid aptamer is constructed, and is used for rapid danioside detection; the method is high in sensitivity and strong in specificity, the lowest detection limit of the droomcorn glycoside is 0.0743 mu g / mL, the color change can be distinguished by naked eyes, the method has the advantages that the operation is simple and convenient, the reaction is rapid, large-scale instruments are not needed and the like, the recovery rate in a sorghum sample reaches 89.66%-100.87%, and the method is suitable for on-site screening of the droomcorn glycoside in practical samples such as sorghum.

Description

Technical Field

[0001] This invention belongs to the field of agricultural product safety testing technology, specifically relating to a colorimetric detection method and application of stigmosiderin based on enhancing the activity of octahedral silver oxide laccase. Background Technology

[0002] Dhurrin, a major cyanogenic glycoside in sorghum, is converted into hydrogen cyanide (HCN) through enzymatic or acidic hydrolysis during the brewing process of baijiu (Chinese liquor). HCN is then transformed into ethyl carbamate (EC), a Group 2A carcinogen, posing a serious threat to food safety. Therefore, efficient monitoring of dhurrin content in sorghum is crucial.

[0003] Currently, the detection of stigmosiderin mainly relies on techniques such as Raman spectroscopy imaging, high-performance liquid chromatography (HPLC), and ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS). For example, CN105092756A discloses a rapid method for detecting stigmosiderin in sorghum, a raw material for baijiu (Chinese liquor) brewing. This method involves identifying characteristic ions using HPLC-MS to detect stigmosiderin standards and establishing a multiple reaction monitoring (LC-MS / MS-MRM) method. The method is then used to qualitatively detect stigmosiderin in sorghum and to accurately quantify it using a quantitative standard curve. Zhou Hanling et al. (Zhou Hanling, An Mingzhe, Li Yanghua, et al. Determination of strychnoside content in sorghum by high performance liquid chromatography-tandem mass spectrometry [J]. Brewing Technology, 2020, (05): 78-82+86. DOI: 10.13746 / j.njkj.2019285.) established a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) method for the detection of strychnoside in sorghum. The sample was extracted with methanol-water (95:5, v / v), separated on a C18 column, detected by multiple reaction monitoring (MRM) mode of HPLC-MS / MS, and quantified by external standard method. Although these methods are accurate, they require expensive equipment, complex pretreatment and professional operation, making it difficult to achieve rapid on-site detection.

[0004] Colorimetry, as a classic optical analysis technique, can determine concentrations as low as 10. -4For trace components at mg / L, most colorimetric reactions are completed within seconds to minutes. Standard serial methods can process multiple samples simultaneously, requiring no instruments; semi-quantitative analysis is achieved using colorimetric cards or standard color scales, making it suitable for large-scale screening. Its unique advantages have led to its widespread application in environmental monitoring, food safety, and medical testing. Nucleic acid aptamers (single-stranded DNA / RNA that specifically binds to targets) serve as recognition elements, offering advantages such as high stability and ease of modification. They can be coupled with nanozymes to enhance detection specificity. Colorimetric detection using aptamers combined with nanozymes provides a fast, accurate, and cost-effective solution for monitoring sorghum glutenin, particularly suitable for pretreatment screening of raw materials in liquor brewing and real-time quality control on production lines, reducing the risk of EC formation from the source. Future improvements in aptamer sequence optimization (e.g., multivalent design) and nanozyme activity regulation (e.g., silver-based material morphology engineering) can further enhance the detection accuracy in complex food matrices.

[0005] In summary, there is an urgent need to develop a colorimetric biosensor based on the combination of aptamers and nanozymes to provide a new pathway for the efficient detection of sorghum glycosides. Summary of the Invention

[0006] The technical problem to be solved by this invention is that existing detection methods cannot achieve rapid detection of strychnine in sorghum.

[0007] To achieve the above-mentioned objectives, the technical solution adopted in this application is as follows: Firstly, this invention provides a colorimetric detection method for stigmosiderin based on enhanced octahedral silver oxidase activity. This method has advantages such as being visually detectable, highly sensitive, easy to operate, and rapid in detection. It can be applied to the rapid detection of stigmosiderin in actual sorghum samples. The method specifically includes the following steps: S1. Preparation of standard detection solution: Mix octahedral silver oxide and nucleic acid aptamer DHU1-23 solution and incubate the reaction; then add succinate standard solution of different concentrations and mix well, and continue incubation; finally add HEPES buffer, 2,4-dichlorophenol solution and 4-aminoantipyrine solution and mix well to obtain standard detection solution; The sequence of the nucleic acid aptamer DHU1-23 solution is shown in SEQ ID NO:1: 5'-GCTCATCTCATATGAGTATGAGC-3'; S2. Preparation of blank detection solution: Using an equal volume of triple-distilled water to replace the glutathione standard solution in step S1, a blank detection solution was prepared under the same conditions according to step S1. S3. Construction of standard curve: Measure the absorbance of the standard test solution and the blank test solution, establish the quantitative relationship between absorbance and glutathione concentration, construct the standard curve, and obtain the regression equation; S4. Detection of stigmosiderin in the sample to be tested: Using an equal volume of the sample solution to be tested instead of the stigmosiderin standard solution in step S1, the sample test solution to be tested is prepared under the same conditions as in step S1; the absorbance of the sample test solution to be tested is measured, and the absorbance is substituted into the regression equation obtained in step S3 to obtain the concentration of stigmosiderin in the sample to be tested.

[0008] In step S1 above, the final concentration of octahedral silver oxide in the solution is detected to be 4~100 μg / mL.

[0009] In step S1 above, the final concentration of the aptamer DHU1-23 solution in the detection solution is 1.25~50 nM.

[0010] In step S1 above, the final concentration of the styracin standard solution in the detection solution is 0~500μg / mL and is not 0.

[0011] In step S1 above, the final concentration of HEPES buffer in the solution is 1~30mM and the pH is 4~10.

[0012] In step S1 above, the final concentration of the 2,4-dichlorophenol solution in the solution is detected to be 0.3~3 mg / mL.

[0013] In step S1 above, the final concentration of the 4-aminoantipyrine solution in the solution is detected to be 0.3~3 mg / mL.

[0014] In step S1 above, the incubation temperature is 28~30℃ and the time is 20~30 minutes.

[0015] In step S3 above, the absorbance is detected by placing the standard detection solution or blank detection solution in a 96-well microplate, scanning it with a microplate reader, and measuring the absorption peak at 510 nm.

[0016] In step S3 above, a standard curve for colorimetric detection of glutathione is established with the concentration of the glutathione standard solution as the abscissa and the absorbance difference ΔA as the ordinate. Wherein, the absorbance difference ΔA = absorbance of the standard test solution - absorbance of the blank test solution.

[0017] In step S3 above, the standard curve is fitted linearly to obtain the regression equation y = 0.25x + 0.31 (R²). 2 =0.995), the linear range was 0.4~2 μg / mL, and the limit of detection was 0.0743 μg / mL; In the regression equation, y represents the absorbance difference ΔA, and x represents the stigmosiderin standard solution, in μg / mL.

[0018] In step S4 above, the absorbance is detected by placing the test sample solution in a 96-well microplate, scanning it with a microplate reader, and measuring the absorption peak at 510 nm.

[0019] Secondly, the present invention provides the application of the above method in the detection of stigmosiderin content, the application including the quantitative detection of stigmosiderin in sorghum.

[0020] In the above applications, the sorghum to be tested is pretreated to obtain a sample solution for quantitative detection of stigmosiderin; The pretreatment process is as follows: mechanically crush the sorghum to be tested, place the sorghum powder in a centrifuge tube, add 95% methanol aqueous solution, and extract by ultrasonication at 4~18℃. Centrifuge to obtain the supernatant, add ultrapure water, mix well, and filter to obtain the sample solution to be tested.

[0021] Furthermore, 1.5g of sorghum powder was placed in a 50-100mL centrifuge tube, and 20-25mL of 95% methanol aqueous solution was added; the ultrasonic extraction time was 1-2h, and the mixture was centrifuged at 8000-10000r / min for 8-10min; 0.4-0.5mL of the supernatant was taken and 0.8-1mL of ultrapure water was added and mixed well; the mixture was filtered using a 0.2μm filter membrane.

[0022] Thirdly, the present invention also provides a biosensor or kit for implementing the above method, the biosensor or kit comprising octahedral silver oxide, nucleic acid aptamer DHU1-23 solution, HEPES buffer, 2,4-dichlorophenol solution, 4-aminoantipyrine solution, and stigmosiderin standard solution. In the biosensor or kit, the final concentration of octahedral silver oxide is 4–100 μg / mL; the final concentration of the aptamer DHU1-23 solution is 1.25–50 nM; the final concentration of the styrax glycoside standard solution is 0–500 μg / mL and not 0; the final concentration of the HEPES buffer is 1–30 mM with a pH of 4–10; the final concentration of the 2,4-dichlorophenol solution is 0.3–3 mg / mL; and the final concentration of the 4-aminoantipyrine solution is 0.3–3 mg / mL. The sequence of the nucleic acid aptamer DHU1-23 solution is shown in SEQ ID NO:1: 15'-GCTCATCTCATATGAGTATGAGC-3'.

[0023] Compared with existing technologies, the advantages of this invention are as follows: the detection method provided by this invention does not rely on large-scale instruments and equipment, and qualitative and semi-quantitative analysis can be completed by colorimetric reaction, enzyme-linked immunosorbent assay (ELISA) or visual observation, which greatly reduces equipment costs and operational barriers; the entire detection process is simple and fast, and can realize the rapid detection of stigmosiderin. Furthermore, the method of this invention exhibits high detection sensitivity, with a detection limit of 0.0743 μg / mL for stigmosiderin, effectively meeting the needs of trace detection. The method also demonstrates excellent specificity; the nucleic acid aptamer DHU1-23 used accurately identifies stigmosiderin molecules, effectively avoiding cross-reactions with other interfering substances such as glycosides, amino acids, metal ions, and common organic acids in the complex sorghum matrix, thus ensuring the accuracy of the detection results. In practical sample testing, the method demonstrates reliability and practicality, achieving a spiked recovery rate of 89.66%–100.87% in sorghum samples, with a relative standard deviation (RSD) of less than 5%. It shows high agreement and reproducibility with HPLC-MS results, making it suitable for rapid detection of stigmosiderin in sorghum. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of the principle of a colorimetric detection method for stigmosiderin based on enhancing the activity of octahedral silver oxidase. Figure 2 Changes in color and absorbance values ​​caused by the addition of timosiderin and other components in the colorimetric detection method for timosiderin; Sample 1 consisted of 20 μL of 1.5 mg / ml octahedral silver oxide, 20 μL of 30 mg / ml 2,4-dichlorophenol, and 20 μL of 30 mg / ml 4-aminoantipyrine. Sample 2 consisted of 20 μL 1.5 mg / ml octahedral silver oxide + 10 μL 200 μg / ml stigmosiderin + 20 μL 30 mg / ml 2,4-dichlorophenol + 20 μL 30 mg / ml 4-aminoantipyrine; Sample 3 is Sample 1 plus 10 μL of 1 μM DHU1-23 aptamer; Sample 4 was Sample 3 plus 10 μL of 50 μg / mL stigmosiderin; Sample 5 was Sample 3 plus 10 μL of 100 μg / mL stigmosiderin; Sample 6 was Sample 3 plus 10 μL of 200 μg / mL stigmosiderin; Figure 3 The standard curve for the colorimetric detection method of stigmosiderin; Figure 4 This is a histogram showing the specificity of the colorimetric detection method for stigmosiderin. Detailed Implementation

[0025] To make the technical problems, solutions, and beneficial effects of this application clearer, the following detailed description is provided in conjunction with the embodiments. Unless otherwise defined, all technical terms used herein have the same meaning as understood by one of ordinary skill in the art.

[0026] The detection principle of the colorimetric detection method for stigmosiderin based on enhancing the activity of octahedral silver oxide laccase is as follows: Figure 1 As shown, in colorimetric detection, octahedral silver oxide catalyzes the formation of semiquinone radicals from 2,4-dichlorophenol under alkaline conditions. These semiquinone radicals react with 4-aminoantipyrine to generate a red quinone imine dye, giving the sensing solution a pink color with a characteristic peak at 510 nm. The addition of the nucleic acid aptamer DHU1-23 promotes electron transfer, deepening the solution color to red. The addition of arganin further enhances electron transfer, causing the solution to deepen to a deeper red.

[0027] Colorimetric detection method for arganin: Changes in color and absorbance values ​​caused by the addition of arganin and other components, such as... Figure 2 As shown. By Figure 2 It was observed that Sample 1 (containing only octahedral silver oxide and the chromogenic substrate) and Sample 2 (containing octahedral silver oxide, arganin, and the chromogenic substrate) both showed a light pink color and low absorbance values, indicating that the addition of arganin did not significantly enhance the chromogenic reaction in the absence of an aptamer. Sample 3, with the addition of the aptamer DHU1-23 to Sample 1, showed a significantly deeper red solution color and a significantly increased absorbance value, demonstrating that the introduction of the aptamer effectively enhanced the enzyme-like catalytic activity of octahedral silver oxide and promoted the electron transfer process. As the concentration of arganin increased (from Sample 4 to Sample 6), the solution color gradually changed from red to dark red, and the absorbance value at 510 nm also gradually increased accordingly, indicating that the specific binding between arganin and the aptamer can further accelerate electron transfer, thereby achieving sensitive and visualized semi-quantitative detection of arganin.

[0028] The following specific embodiments will be provided to explain the solution of the present invention. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0029] Example 1: Sensitivity Test of the Colorimetric Detection Method for Diosmin Based on Enhanced Octahedral Silver Oxidation Laccase Activity (1) Take 17 centrifuge tubes with a specification of 1.5 mL, add 20 μL of octahedral silver oxide with a concentration of 1.5 mg / mL and 10 μL of aptamer DHU1-23 solution with a concentration of 1 μM to each tube, mix thoroughly and incubate at 30℃ for 20 minutes; then add 10 μL of styrax glycoside standard solution of different concentrations to each tube, so that the final concentrations of styrax glycoside are 20, 30, 40, 50, 60, 70, 80, 90, 100, 110 and 125 respectively. 150, 175, 200, 300, 400, and 500 μg / mL were added to each tube and incubated at 30°C for 20 minutes. Finally, 420 μL of 10 mM HEPES buffer (pH 8), 20 μL of 30 mg / mL 2,4-dichlorophenol, and 20 μL of 30 mg / mL 4-aminoantipyrine were added to each tube and mixed thoroughly to obtain a standard detection solution with a total volume of 500 μL and different stigmosiderin concentrations.

[0030] (2) Take a 1.5 mL centrifuge tube, add 20 μL of 1.5 mg / mL octahedral silver oxide and 10 μL of 1 μM aptamer DHU1-23 solution to the tube, mix thoroughly and incubate at 30 °C for 20 minutes; then add 10 μL of triple-distilled water and continue incubating at 30 °C for 20 minutes; finally add 420 μL of 10 mM HEPES buffer (pH 8), 20 μL of 30 mg / mL 2,4-dichlorophenol and 20 μL of 30 mg / mL 4-aminoantipyrine to the tube, mix thoroughly to obtain a blank detection solution with a total volume of 500 μL.

[0031] (3) Take 200 μL of the standard detection solution with different concentrations of succinin prepared in step (1) and place it in a 96-well microplate. Use a microplate reader to scan and measure the absorption peak at 510 nm. The full wavelength scanning range is 300~800 nm to obtain its absorption spectrum. Take 200 μL of the blank detection solution prepared in step (2) and place it in a 96-well microplate. Use a microplate reader to scan and measure the absorption peak at 510 nm. The full wavelength scanning range is 300~800 nm to obtain its absorption spectrum.

[0032] (4) Calculate ΔA using the formula (absorbance difference ΔA = absorbance of standard test solution - absorbance of blank test solution), and plot the standard curve with the concentration of styrax glycoside standard solution as the x-axis and the absorbance difference ΔA as the y-axis, as shown below. Figure 3 As shown; Based on the standard curve of changes in glutathione concentration and absorbance value, a regression equation was established to determine the relationship between glutathione concentration and absorbance value: y = 0.25x + 0.31 (R²). 2=0.995); where: y is the absorbance difference ΔA in the regression equation, and x is the stigmosiderin standard solution (μg / mL); the linear range of stigmosiderin was found to be 0.4~2μg / mL, and the limit of detection was 0.0743μg / mL.

[0033] Example 2: Specificity test of the colorimetric detection method for stigmosiderin based on enhancing the activity of octahedral silver laccase. (1) Single-specific detection: Take a 1.5 mL centrifuge tube and operate according to step (1) of Example 1, but add 10 μL of different concentrations of styrax glycoside standard solution, and replace it with 10 μL of one of the following solutions with a concentration of 100 μg / mL: styrax glycoside, amygdalin, Zn²⁺, K⁺, tryptophan, leucine, cysteine, glucose, dextran, ascorbic acid solution; to obtain test solutions of different single-target systems.

[0034] (2) Mixed specificity detection: Take a 1.5 mL centrifuge tube and operate according to step (1) of Example 1, but add 10 μL of different concentrations of arganin standard solution to replace it with 10 μL of arganin solution with a concentration of 100 μg / mL. At the same time as adding arganin solution, add 10 μL of one of the following solutions with a concentration of 100 μg / mL: distilled water, amygdalin, Zn²⁺, K⁺, tryptophan, leucine, cysteine, glucose, dextran, ascorbic acid solution; to obtain test solutions of different mixed target systems.

[0035] (3) Prepare blank detection solutions according to steps (2) to (4) of Example 1 and measure absorbance, calculate ΔA and draw histogram as shown below. Figure 4 As shown, the results indicate that the above-mentioned target has little interference with the reaction system, and the detection method of the present invention has good specificity.

[0036] Example 3: Application of the present invention in the quantitative detection of strychnine in sorghum 10g of sorghum grains were ground into powder. 1g of powder was mixed with solid mossin (mossin spiking mass set at 0, 4, 8, 12, and 16 μg) standard in 100mL centrifuge tubes. Then, 20mL of 95% cold methanol (95:5) was added to each tube. The mixture was sonicated at 18℃ for 60min, centrifuged at 8000r / min for 10min, and 800μL of supernatant was collected. 600μL of the supernatant was added to 2400μL of ultrapure water, filtered through a 0.22μm filter membrane, and directly injected into HPLC-MS. The remaining 200μL of supernatant was filtered and used for colorimetric detection to obtain the mossin concentration. The spiked recovery rate was calculated, and the results are shown in Table 1.

[0037] HPLC-MS: Liquid chromatography conditions: C 18Column (100 mm × 2.0 mm, 3 μm). Mobile phase: Phase A: ultrapure water; Phase B: methanol. Flow rate: 0.2 mL / min. Column temperature: 30 °C. Mass spectrometry conditions: Ion source: electrospray ionization source (ESI⁺ mode). Monitoring mode: multiple reaction monitoring (MRM). Auxiliary parameters: capillary voltage: 5500 V; ion source temperature: 500 °C; curtain gas: 30 psi; collision gas: 8 psi.

[0038] Table 1 Test Results

[0039] As shown in Table 1, the detection results of the method of the present invention are basically consistent with those of the instrumental method (HPLC-MS): the recovery rate obtained by colorimetric method is 89.66%~100.87%, and the average relative deviation (RSD) is 2.07%~3.21%; the recovery rate verified by HPLC-MS is 95.91%~101.50%, and the average relative deviation is 2.15%~3.62%, proving that the method is reliable.

[0040] In summary, the method of the present invention has the advantages of high sensitivity, good detection specificity, visual identification and simple operation. It can realize the rapid detection of stigmosiderin without large instruments, so it can be widely used for the rapid detection of stigmosiderin in sorghum.

Claims

1. A colorimetric detection method for stigmosiderin based on enhancing the activity of octahedral silver oxidase, characterized in that, Includes the following steps; S1. Preparation of standard detection solution: Mix octahedral silver oxide and nucleic acid aptamer DHU1-23 solution and incubate the reaction; then add succinate standard solution of different concentrations and mix well, and continue incubation; finally add HEPES buffer, 2,4-dichlorophenol solution and 4-aminoantipyrine solution and mix well to obtain standard detection solution; The sequence of the nucleic acid aptamer DHU1-23 solution is shown in SEQ ID NO:1: 5'-GCTCATCTCATATGAGTATGAGC-3'; S2. Preparation of blank detection solution: Using an equal volume of triple-distilled water to replace the glutathione standard solution in step S1, a blank detection solution was prepared under the same conditions according to step S1. S3. Construction of standard curve: Measure the absorbance of the standard test solution and the blank test solution, establish the quantitative relationship between absorbance and glutathione concentration, construct the standard curve, and obtain the regression equation; S4. Detection of stigmosiderin in the sample to be tested: Using an equal volume of the sample solution to be tested instead of the stigmosiderin standard solution in step S1, the sample test solution to be tested is prepared under the same conditions as in step S1; the absorbance of the sample test solution to be tested is measured, and the absorbance is substituted into the regression equation obtained in step S3 to obtain the concentration of stigmosiderin in the sample to be tested.

2. The colorimetric detection method for stigmosiderin based on enhanced octahedral silver laccase activity according to claim 1, characterized in that: In step S1, the detection solution must satisfy at least one of the following: The final concentration of octahedral silver oxide is 4~100 μg / mL; The final concentration of the aptamer DHU1-23 solution was 1.25~50 nM; The final concentration of the styracin standard solution is 0~500μg / mL and is not 0; The final concentration of HEPES buffer is 1~30mM, and the pH is 4~10; The final concentration of the 2,4-dichlorophenol solution is 0.3~3 mg / mL; The final concentration of 4-aminoantipyrine solution is 0.3~3 mg / mL.

3. The colorimetric detection method for stigmosiderin based on enhanced octahedral silver laccase activity according to claim 1, characterized in that: In step S1, the incubation reaction is carried out at a temperature of 28-30°C for 20-30 minutes.

4. The colorimetric detection method for stigmosiderin based on enhanced octahedral silver laccase activity according to claim 1, characterized in that: In step S3, the absorbance is detected by placing the standard detection solution or blank detection solution in a 96-well microplate, scanning it with a microplate reader, and measuring the absorption peak at 510 nm.

5. The colorimetric detection method for stigmosiderin based on enhanced octahedral silver laccase activity according to claim 1, characterized in that: In step S3, a standard curve for colorimetric detection of glutathione is established with the concentration of the glutathione standard solution as the abscissa and the absorbance difference ΔA as the ordinate. Wherein, the absorbance difference ΔA = absorbance of the standard test solution - absorbance of the blank test solution.

6. The colorimetric detection method for stigmosiderin based on enhanced octahedral silver laccase activity according to claim 5, characterized in that: In step S3, the standard curve is fitted linearly to obtain the regression equation y = 0.25x + 0.31 (R²). 2 =0.995), the linear range was 0.4~2 μg / mL, and the limit of detection was 0.0743 μg / mL; In the regression equation, y represents the absorbance difference ΔA, and x represents the stigmosiderin standard solution, in μg / mL.

7. The colorimetric detection method for stigmosiderin based on enhanced octahedral silver laccase activity according to claim 1, characterized in that: In step S4, the absorbance is detected by placing the test sample solution in a 96-well microplate, scanning it with a microplate reader, and measuring the absorption peak at 510 nm.

8. The application of the method according to any one of claims 1 to 7 in the detection of stigmosiderin content, characterized in that: The application includes the quantitative detection of sorghum glycosides.

9. The application according to claim 8, characterized in that: The sorghum to be tested was pretreated to obtain the sample solution, which was used for the quantitative detection of stigmosiderin. The pretreatment process is as follows: mechanically crush the sorghum to be tested, place the sorghum powder in a centrifuge tube, add 95% methanol aqueous solution, and extract by ultrasonication at 4~18℃. Centrifuge to obtain the supernatant, add ultrapure water, mix well, and filter to obtain the sample solution to be tested.

10. A biosensor or kit that implements the method of any one of claims 1 to 7, characterized in that: The biosensor or kit includes octahedral silver oxide, nucleic acid aptamer DHU1-23 solution, HEPES buffer, 2,4-dichlorophenol solution, 4-aminoantipyrine solution, and stigmosiderin standard solution. In the biosensor or kit, the final concentration of octahedral silver oxide is 4–100 μg / mL; the final concentration of the aptamer DHU1-23 solution is 1.25–50 nM; the final concentration of the styrax glycoside standard solution is 0–500 μg / mL and not 0; the final concentration of the HEPES buffer is 1–30 mM with a pH of 4–10; the final concentration of the 2,4-dichlorophenol solution is 0.3–3 mg / mL; and the final concentration of the 4-aminoantipyrine solution is 0.3–3 mg / mL. The sequence of the nucleic acid aptamer DHU1-23 solution is shown in SEQ ID NO:1: 15'-GCTCATCTCATATGAGTATGAGC-3'.

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