Visual semi-quantitative ascorbic acid test paper as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of test paper, and particularly relates to visual semi-quantitative test paper as well as a preparation method and application thereof. The visual semi-quantitative ascorbic acid detection test paper comprises a PVC substrate, a nitrocellulose membrane is arranged on the PVC substrate, one end of the nitrocellulose membrane is connected with a sample pad, the other end of the nitrocellulose membrane is connected with a water absorption pad, and a detection line is arranged on the surface of the nitrocellulose membrane. The detection line is prepared by scribing a manganese dioxide nanoneedle solution wrapped by bovine serum albumin. The bovine serum albumin and manganese ions on the surface of the manganese dioxide nanoneedle form coordinate bonds, so that the bovine serum albumin wraps the surface of the manganese dioxide nanoneedle. And the manganese dioxide nanoneedle is in a gamma-MnO2 needle shape. The test paper provided by the invention has no color vision dependence defect, can realize on-site rapid visual semi-quantitative detection, and has a good application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of test paper, and particularly relates to a visual semi-quantitative ascorbic acid test paper and a preparation method and application thereof. BACKGROUND

[0002] Efficient detection of ascorbic acid is crucial for food quality monitoring and clinical diagnosis, but the existing technology has significant limitations. Traditional laboratory methods such as titration or high-performance liquid chromatography require complex sample pretreatment and expensive instrument support, and electrochemical methods rely on electrode modification and precise temperature control, both of which cannot achieve on-site rapid detection. Although the colorimetric method of noble metal nanomaterials developed in recent years has visual potential, its application is still subject to three bottlenecks: (1) high material cost, traditional visual technology uses gold, silver and other nanoneedles to produce local surface plasmon resonance peak shift, thereby realizing multi-color visualization technology, but also leading to high cost of single detection; (2) complex preparation process, poor batch stability, difficult to mass production; (3) color vision dependence defect, the interpretation mode relying on multi-color signal conversion is difficult for color vision impaired people to identify, and environmental light interference easily reduces accuracy, limiting its application range.

[0003] Therefore, there is an urgent need to develop a detection method with low cost, simple preparation and no color vision dependence defect for detecting ascorbic acid. SUMMARY

[0004] In order to solve the problems and deficiencies of the prior art, the purpose of the present application is to provide a visual semi-quantitative ascorbic acid test paper and a preparation method and application thereof.

[0005] In order to facilitate the understanding of the present application, the materials used in the present application and their abbreviations are listed as follows: bovine serum albumin is abbreviated as BSA. Bovine serum albumin coated manganese dioxide nanoneedle is abbreviated as BSA@MnO2. Nitrocellulose membrane is abbreviated as NC membrane. Test line is abbreviated as T line.

[0006] In order to achieve the above-mentioned purpose, the first purpose of the present application is to provide a visual semi-quantitative ascorbic acid test paper, characterized in that it comprises a PVC substrate, the NC membrane is provided on the PVC substrate, one end of the NC membrane is connected with the sample pad, and the other end is connected with the water absorption pad, and the surface of the NC membrane is provided with the T line.

[0007] The T line is prepared by drawing a line with BSA@MnO2 solution. BSA forms a coordination bond with manganese ions on the surface of MnO2 nanoneedle, so that BSA is wrapped on the surface of MnO2 nanoneedle.

[0008] The carboxyl group of the BSA forms a coordination bond with the trivalent or tetravalent manganese ion on the surface of the MnO2 nanoneedle. The amino group of the BSA forms a coordination bond with the trivalent or tetravalent manganese ion on the surface of the MnO2 nanoneedle, so that the BSA is wrapped on the surface of the MnO2 nanoneedle;

[0009] The MnO2 nanoneedle is in a γ-MnO2 needle-like morphology.

[0010] Preferably, the crystal face of the MnO2 nanoneedle is (110) type, the crystal face spacing is 0.22 nm, the length is 5 nm to 22 nm, and the length is concentrated in 12 nm.

[0011] Preferably, the PVC substrate is self-adhesive and has three adhesive sections, which are 1.5 cm, 2.5 cm and 2 cm, respectively.

[0012] Preferably, the BSA@MnO2 concentrated solution is prepared by the following method: dissolving BSA in water to obtain a BSA solution. Add manganese sulfate monohydrate solution to the BSA solution and stir for 1 min to 3 min to obtain a mixed solution. Add sodium hydroxide solution to the mixed solution to obtain a reaction solution. Transfer the reaction solution to a dialysis bag with a molecular weight cut-off of 80KDa to 100KDa, dialyze in deionized water to obtain a dialysate. Concentrate the dialysate using an ultrafiltration tube with a molecular weight cut-off of 25KDa to 35KDa to obtain a BSA@MnO2 solution.

[0013] Preferably, the concentration of the BSA solution is 1.0 mg / mL to 1.9 mg / mL, and more preferably 1.5 mg / mL.

[0014] Preferably, the concentration of the manganese sulfate monohydrate solution is 95 mmol / L to 105 mmol / L, and more preferably 100 mmol / L.

[0015] Preferably, the concentration of the sodium hydroxide solution is 0.8 mol / L to 1.2 mol / L, and more preferably 1 mol / L.

[0016] Preferably, the volume ratio of the BSA solution, the manganese sulfate monohydrate solution and the sodium hydroxide solution is 50:1.5 to 2.5:0.5 to 1, and preferably 50:1.9:0.75.

[0017] Preferably, the reaction time of the reaction of adding sodium hydroxide solution is 50 min to 70 min, and more preferably 60 min.

[0018] Preferably, the dialysis time in deionized water is 1 day to 2 days, and more preferably 1 day.

[0019] A second object of the present application is to provide a preparation method of a visual semi-quantitative ascorbic acid detection test paper, comprising the following steps Preparation of NC membrane with T line: coating BSA@MnO2 solution to NC membrane, drying to obtain NC membrane with T line; Preparation of visual semi-quantitative ascorbic acid detection test paper: respectively assembling NC membrane with T line, sample pad and water absorption pad to the surface of PVC substrate, cutting the PVC substrate into strips to obtain visual semi-quantitative ascorbic acid detection test paper.

[0020] Preferably, the concentration of the BSA@MnO2 solution is 4 mg / mL-10 mg / mL, more preferably 10 mg / mL.

[0021] Preferably, the chromatography speed of the NC membrane is 95 s / 4 cm-140 s / 4 cm, more preferably 120 s / 4 cm.

[0022] Preferably, the drying temperature is 37°C-45°C, and the time is 6 h-9 h, more preferably 45°C, 7 h.

[0023] Preferably, the number of T lines on the nitrocellulose membrane with detection lines is 3-5, the interval is 3.8-4.2 mm, and each T line contains 1.28 µg-3.2 µg of BSA@MnO2.

[0024] Preferably, the number of T lines on the NC membrane with T line is 5, the interval is 4 mm, and each T line contains 3.2 µg of BSA@MnO2.

[0025] Preferably, the material of the sample pad is glass fiber, polyester fiber or filter paper, more preferably glass fiber.

[0026] A third object of the present application is to provide a visual semi-quantitative ascorbic acid detection test paper for detecting ascorbic acid.

[0027] Preferably, the application includes ascorbic acid detection in the fields of food, medicine or clinical detection.

[0028] A method for detecting ascorbic acid by using a visual semi-quantitative detection test paper, comprising the following steps: Dissolving the to-be-detected substance into a buffer solution with a pH value of 4-9 to obtain a detection solution, adding the detection solution to the sample pad, and reacting for 5 min-10 min, and calculating the concentration of the to-be-detected substance according to the number of T lines disappeared and the content of BSA@MnO2 on each detection line.

[0029] Preferably, the buffer solution is citric acid buffer, acetic acid buffer and phosphoric acid buffer, more preferably 10 mmol / L citric acid buffer.

[0030] Preferably, when the to-be-tested substance is dissolved, 0.4g kaolin is added into the buffer solution to adsorb the impurity color, and the filtrate is used as the detection liquid for detection.

[0031] Preferably, the to-be-tested liquid is added into a 96-well plate, and 100-200ul of the to-be-tested liquid is added into each well.

[0032] Preferably, the volume of the to-be-tested liquid is 100ul / well.

[0033] Compared with the prior art, the present application has the following beneficial effects: 1. A visual semi-quantitative ascorbic acid detection test paper, comprising a PVC substrate, wherein an NC membrane is arranged on the PVC substrate, one end of the NC membrane is connected with a sample pad, and the other end of the NC membrane is connected with a water absorption pad, and a T line is arranged on the surface of the NC membrane; the detection line is prepared by line marking of a BSA@MnO2 solution; the manganese ions on the surface of the BSA@MnO2 nanoneedle form a coordination bond with BSA, so that the BSA is wrapped on the surface of the MnO2 nanoneedle; the MnO2 nanoneedle is in a gamma-MnO2 needle-like morphology, and the MnO2 in the needle-like morphology can form a stable inclusion state with BSA; and other morphologies of the MnO2 needle-like morphology are not conducive to the inclusion of BSA, or the concentration of the MnO2 after inclusion is low, which is not conducive to the preparation of the detection test paper.

[0034] The visual semi-quantitative detection test paper of the present application is prepared by using BSA@MnO2 as raw material to prepare a T line, and then assembling the T line with a PVC substrate, an NC membrane, a sample pad and a water absorption pad; the T line prepared by BSA@MnO2 is dark brown; in an acidic environment, the MnO2 in the BSA@MnO2 is reduced to colorless Mn 2+ ions by ascorbic acid; therefore, the T line changes from dark brown to colorless before and after the reaction, the color change is very obvious, and the number of the disappeared T line is proportional to the concentration of ascorbic acid; therefore, the visual semi-quantitative detection test paper provided by the present application has no color vision dependence defect, does not need to rely on a multi-color level signal conversion judgment mode, is easy to identify by people with color vision deficiency, and is less affected by environmental light; and the concentration range of ascorbic acid can be determined according to the number of the disappeared T line, so that the detection test paper has the function of semi-quantitative ascorbic acid detection; compared with the traditional visual technology using gold and silver nanoneedles, the raw material cost of the detection test paper of the present application is low and easy to obtain; therefore, the detection test paper of the present application has the characteristics of low single detection cost and easy production; on the other hand, the detection test paper of the present application has only four components, so that the preparation process is simple, easy to mass production, and the batch quality is more stable during production.

[0035] 2. A visual semi-quantitative ascorbic acid detection test paper, the BSA@MnO2 solution is prepared by the following method: dissolving BSA in water to obtain a BSA solution. Adding a manganese sulfate monohydrate solution to the BSA solution to obtain a mixture. Adding a sodium hydroxide solution to the mixture to react to obtain a reaction solution. Transferring the reaction solution to a dialysis bag with a molecular weight cut-off of 80KDa-100KDa, dialyzing in deionized water, collecting the dialysate in the dialysis bag, and obtaining the BSA@MnO2 solution.

[0036] The BSA@MnO2 solution prepared by the method can specifically react with ascorbic acid, and the reaction is stable and sensitive. The color change from dark brown to colorless before and after the reaction of BSA@MnO2 and ascorbic acid is obvious, which lays the theoretical foundation for the visual semi-quantitative ascorbic acid detection test paper.

[0037] 3. A preparation method of a visual semi-quantitative ascorbic acid detection test paper, comprising the following steps: coating the BSA@MnO2 solution to the NC membrane, drying to obtain an NC membrane with a T line. Assembling the NC membrane with a T line, a sample pad and a water absorption pad to the surface of a PVC substrate respectively to obtain a visual semi-quantitative ascorbic acid detection test paper.

[0038] The method of the present application prepares the BSA@MnO2 solution and uniformly coats it on the surface of the NC membrane to form the T line, and then assembles it with the PVC substrate, the sample pad and the water absorption pad to divide it into the visual semi-quantitative ascorbic acid detection test paper. The method provided by the present application prepares the T line with uniform quality and stable reaction ratio with ascorbic acid. The T line reacts with the ascorbic acid solution to change from brown to colorless and disappear from the visual semi-quantitative ascorbic acid detection test paper, and the color change is very obvious. The number of the disappeared T line is proportional to the concentration of ascorbic acid. Therefore, the visual semi-quantitative ascorbic acid detection test paper of the present application can semi-quantitatively detect the concentration of ascorbic acid, and the color change is obvious and easy to judge.

[0039] 4. The visual semi-quantitative ascorbic acid detection test paper of the present application has the ability to selectively resist interference of common substances in samples such as tartaric acid, phosphoric acid, malic acid, citric acid, sucrose, vitamin E, sodium chloride and potassium chloride. Figure 6 As shown in the table, except for the T line on the test paper corresponding to ascorbic acid disappearing, the T lines on the other test papers are still clear, which shows that the visual semi-quantitative ascorbic acid detection test paper of the present application has good anti-interference ability and has more reliable accuracy in complex detection environment.

[0040] 5. The present invention provides a visual semi-quantitative ascorbic acid test strip for the detection of ascorbic acid, applicable to food, pharmaceutical, or clinical testing fields. The test strip of the present invention is simple to use, small in size, and easy to carry, making it ideal for on-site ascorbic acid detection. Furthermore, the test strip does not rely on a multi-level signal conversion interpretation mode, making it easily identifiable for people with color vision deficiencies, unaffected by ambient light, and highly accurate. The test strip of the present invention provides a completely new pathway for rapid on-site ascorbic acid detection. Attached Figure Description

[0041] Figure 1 This is a UV absorption spectrum scan of BSA@MnO2 of the present invention, with the inset in the upper right corner showing the appearance of BSA@MnO2 in a centrifuge tube.

[0042] Figure 2 The images show the morphology and length distribution of BSA@MnO2 according to the present invention. (A) is a scanning electron microscope image of BSA@MnO2, and (B) is a length distribution diagram of BSA@MnO2.

[0043] Figure 3 This is a diagram illustrating the structure of the semi-quantitative test strip of this invention.

[0044] Figure 4 This is a schematic diagram illustrating the method of using the visual semi-quantitative test strip of the present invention.

[0045] Figure 5 The diagram shows the relationship between the detection concentration and the T-line of the semi-quantitative test strip of the present invention; (1) is the T-line diagram of the test strip corresponding to the ascorbic acid concentration of 0mg / 100mL~1.38mg / 100mL, (2) is the T-line diagram of the test strip corresponding to the ascorbic acid concentration of 1.38mg / 100mL~2.75mg / 100mL, (3) is the T-line diagram of the test strip corresponding to the ascorbic acid concentration of 2.75mg / 100mL~4.13mg / 100mL, (4) is the T-line diagram of the test strip corresponding to the ascorbic acid concentration of 4.13mg / 100mL~5.50mg / 100mL, and (5) is the T-line diagram of the test strip corresponding to the ascorbic acid concentration of 5.50mg / 100mL~6.88mg / 100mL.

[0046] Figure 6 This is an anti-interference experimental diagram of the semi-quantitative visual test strip of the present invention; wherein, (1) is 10 mmol / L ascorbic acid; (2) is 10 mmol / L tartaric acid; (3) is 10 mmol / L phosphoric acid; (4) is 100 mmol / L sucrose; (5) is 10 mmol / L sucrose; (6) is 10 mmol / L vitamin E. (7) 100 mmol / L sodium chloride; (8) 100 mmol / L malic acid; (9) 100 mmol / L citric acid; (10) 10 mmol / L sorbic acid; (11) 100 mmol / L potassium chloride. DETAILED DESCRIPTION

[0047] In order to make the skilled in the art better understand the technical solutions of the present application can be implemented, the following embodiments of the present application, with the preferred embodiments and the drawings with detailed description, the technical solutions of the embodiments of the present application are clearly and completely described, obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments of the present application, all other embodiments obtained by the skilled in the art without making creative labor, belong to the scope of protection of the present application.

[0048] It should be noted that all the professional terms used in the present application are only for the purpose of describing the specific embodiments, and are not intended to limit the scope of protection of the present application, unless otherwise specified, the various raw materials, reagents, instruments and equipment used in the following embodiments of the present application can be purchased or prepared by the existing method.

[0049] Example 1 A preparation method of visual semi-quantitative ascorbic acid detection test paper, comprising the following steps: Preparation of BSA@MnO2: 75 mg of BSA was dissolved in 50 mL of water to obtain a BSA solution. 1.9 mL of 100 mmol / L manganese sulfate monohydrate solution was added to the BSA solution, and stirred for 1 min to obtain a mixed solution. 0.75 mL of 1 mol / L sodium hydroxide solution was added to the mixed solution, and reacted for 60 min to obtain a reaction liquid. The reaction liquid was transferred to a dialysis bag with a molecular weight cutoff of 100 KDa, and dialyzed in deionized water for 1 day to obtain a dialysate. The dialysate was concentrated using an ultrafiltration tube with a molecular weight cutoff of 30 KDa to obtain a BSA@MnO2 concentrate.

[0050] Preparation of NC membrane with T line: the BSA@MnO2 concentrate was diluted with water to prepare a 10 mg / mL BSA@MnO2 solution. The BSA@MnO2 solution was coated on the NC membrane with a chromatographic speed of 120 s / 4 cm at an interval of 4 mm using a membrane marker to form 5 detection lines. The NC membrane was dried at a temperature of 45℃ for 7 h to obtain an NC membrane with T line, wherein each detection line contains 3.2 μg of BSA@MnO2.

[0051] Preparation of the visual semi-quantitative detection test paper: place the PVC substrate on the bottom layer. Among them, the PVC substrate is divided into 1.5 cm, 2.5 cm, and 2 cm adhesive sections. First, the NC membrane with T lines is pasted to the 2.5 cm adhesive section on the surface of the PVC substrate, and then the sample pad and the water absorption pad are installed on the 1.5 cm and 2 cm adhesive sections on the surface of the PVC, respectively, and the sample pad and the water absorption pad are connected with the NC membrane, to obtain the visual semi-quantitative detection test paper.

[0052] Example 2 A preparation method of a visual semi-quantitative detection test paper, comprising the following steps: Preparation of BSA@MnO2: 50 mg of BSA was dissolved in 50 mL of water to obtain a BSA solution. 1.5 mL of 95 mmol / L manganese sulfate monohydrate solution was added to the BSA solution, stirred for 2 min to obtain a mixed solution. 0.5 mL of 0.8 mol / L sodium hydroxide solution was added to the mixed solution, and reacted for 50 min to obtain a reaction liquid. The reaction liquid was transferred to a dialysis bag with a molecular weight cut-off of 80 KDa, and dialyzed in deionized water for 2 days to obtain a dialysate. The dialysate was concentrated using an ultrafiltration tube with a molecular weight cut-off of 25 KDa, dried to obtain a BSA@MnO2 concentrate.

[0053] Preparation of NC membrane with T lines: the BSA@MnO2 concentrate was diluted with water to prepare a BSA@MnO2 solution of 4 mg / mL. The BSA@MnO2 solution was coated on the NC membrane with a chromatography speed of 95 s / 4 cm at an interval of 3.8 mm using a membrane marker to form 4 detection lines. The NC membrane was dried at a temperature of 37°C for 6h to obtain the NC membrane with T lines, wherein each detection line contains 1.28 µg of BSA@MnO2.

[0054] Preparation of the visual semi-quantitative detection test paper: place the PVC substrate on the bottom layer. Among them, the PVC substrate has 1.5 cm, 2.5 cm, and 2 cm adhesive sections. First, the NC membrane with T lines is pasted to the 2.5 cm adhesive section on the surface of the PVC substrate, and then the sample pad and the water absorption pad are installed on the 1.5 cm and 2 cm adhesive sections on the surface of the PVC, respectively, and the sample pad and the water absorption pad are connected with the NC membrane, to obtain the visual semi-quantitative detection test paper.

[0055] Example 3 A preparation method of a visual semi-quantitative detection test paper, comprising the following steps: Preparation of BSA@MnO2: 95 mg of BSA was dissolved in 50 mL of water to obtain a BSA solution. 2.5 mL of 105 mmol / L manganese sulfate monohydrate solution was added to the BSA solution, and stirred for 3 min to obtain a mixed solution. 1 mL of 1.2 mol / L sodium hydroxide solution was added to the mixed solution, and reacted for 70 min to obtain a reaction solution. The reaction solution was transferred to a dialysis bag with a molecular weight cut-off of 100 KD, and dialyzed in deionized water for 2 days to obtain a dialysate. The dialysate was concentrated using an ultrafiltration tube with a molecular weight cut-off of 30 KD, dried, and BSA@MnO2 was obtained.

[0056] Preparation of NC membrane with T line: BSA@MnO2 was dissolved in water to prepare a BSA@MnO2 solution with a concentration of 7 mg / mL. The BSA@MnO2 solution was coated on an NC membrane with a chromatographic speed of 140 s / 4 cm at a spacing of 4.2 mm using a membrane marker to form three detection lines. The NC membrane was dried at a temperature of 40°C for 9 h to obtain an NC membrane with T line, wherein each detection line contained 2.24 µg of BSA@MnO2.

[0057] Preparation of visual semi-quantitative detection test paper: a PVC substrate was placed on the bottom layer. The PVC substrate had three adhesive sections of 1.5 cm, 2.5 cm and 2 cm. The NC membrane with T line was first adhered to the 2.5 cm adhesive section on the surface of the PVC substrate, and then the sample pad and the water absorption pad were installed on the 1.5 cm and 2 cm adhesive sections on the surface of the PVC, respectively, and the sample pad and the water absorption pad were connected to the NC membrane to obtain the visual semi-quantitative detection test paper.

[0058] In order to further illustrate the advantages of BSA@MnO2 of the present application, the following experiments were also conducted: I. Instruments and materials 1. Experimental materials The materials used in the present application mainly include BSA, ascorbic acid, manganese sulfate monohydrate and sodium hydroxide.

[0059] Among them, the purity of BSA is 98%, the CAS number is 9048-46-8, and it is purchased from Shanghai Yuan Ye Biological Technology Co., Ltd. The purity of ascorbic acid is 99%, the CAS number is 50-81-7, and it is purchased from Shanghai Aladdin Biochem Technology Co., Ltd. The purity of manganese sulfate monohydrate is 99%, the CAS number is 10034-96-5, and it is purchased from Shanghai Aladdin Biochem Technology Co., Ltd. The purity of sodium hydroxide is 99%, the CAS number is 1310-73-2, and it is purchased from Tianjin Damao Chemical Reagent Partnership (Limited Partnership).

[0060] 2. Experimental instruments The instruments used in the present application mainly include a membrane marker and a UV spectrophotometer.

[0061] The film drawing instrument is XYZ three-dimensional film drawing gold spraying instrument produced by Shanghai Jinbiao Biotechnology Co., Ltd., model HM3030. The ultraviolet spectrophotometer is a micro-spectrophotometer produced by Thermo Fisher Scientific, model Nanodrop2000.

[0062] II. Experimental scheme 1. UV absorption spectrum scanning of BSA@MnO2 Take the BSA@MnO2 concentrated solution prepared in Example 1 and dilute it with water to 1 mg / mL solution, add 1 cm cuvette, use ultraviolet spectrophotometer to scan in the wavelength range of 200 nm~800 nm, and record the absorbance value.

[0063] 2. Morphology and length distribution of BSA@MnO2 Take the BSA@MnO2 concentrated solution of Example 1 and dilute it with water to 0.5 mg / mL solution, use transmission electron microscope to obtain BSA@MnO2 morphology image, use imageJ image processing software to measure BSA@MnO2 nanoneedle length, then use OriginPro2024 data analysis software to process and analyze to obtain the length distribution result of BSA@MnO2.

[0064] 3. Preparation of visual semi-quantitative ascorbic acid detection test paper Take the BSA@MnO2 concentrated solution prepared in Example 1 and dilute it with ultrapure water to 1 mg / mL to obtain BSA@MnO2 solution. Use the film drawing instrument to coat BSA@MnO2 solution to NC membrane, the liquid output is 0.8 μL / cm, the T line interval is 4 mm, and the drying is carried out at 45℃ for 7h. After drying, the NC membrane is pasted to the PVC substrate, and then the glass fiber sample pad and the water absorption pad are pasted. Use the strip cutting machine to cut the chromatography test paper strip, the width is 4 mm, and the visual semi-quantitative ascorbic acid detection test paper is obtained.

[0065] 4. Preparation of ascorbic acid standard solution Dilute 10 mmol / L ascorbic acid standard solution to prepare ascorbic acid standard solution with concentrations of 0 μg / mL, 13.8 μg / mL, 27.5 μg / mL, 41.3 μg / mL, 55.0 μg / mL and 68.8 μg / mL, respectively, add 200 μL per well into 96-well microplate, then insert the test paper strip into the microhole in the manner of Figure 4 , wait for 10 minutes, take out the test paper strip, and read the results.

[0066] 5. Blind sample experiment 5.1 Test paper strip method Three beverage samples were selected and diluted to 50 mL with 10 mmol / L citric acid buffer at pH 4.0. If the sample color is deep, add 0.4 g of kaolin per gram of sample to adsorb impurities and filter. Then take 100 μL of the filtrate and insert the test strip according to Figure 4 , wait for 10 min and read the results.

[0067] 5.1 2,6-dichloroindophenol titration method The specific operation steps refer to the national standard "GB 5009.86-2016 Determination of Ascorbic Acid in Food".

[0068] 6. Anti-interference experiment Prepare 10 mmol / L ascorbic acid solution, 10 mmol / L tartaric acid solution, 10 mmol / L phosphoric acid solution, 100 mmol / L sucrose solution, 10 mmol / L sucrose solution, 10 mmol / L vitamin E solution, 100 mmol / L sodium chloride solution, 100 mmol / L malic acid solution, 100 mmol / L citric acid solution, 10 mmol / L sorbic acid solution, and 100 mmol / L potassium chloride solution, respectively. Then take 100 μL of the filtrate and insert the test strip according to Figure 4 , wait for 10 min and observe the color of the T line.

[0069] Three, experimental results 1. UV absorption spectrum scanning of BSA@MnO2 The UV-Vis absorption spectrum of BSA@MnO2 composite material is shown in Figure 1 . In the wavelength range of 200-400 nm, the material shows significant absorption and presents a maximum absorption peak at 229 nm. This unique optical property endows BSA@MnO2 with a deep brown appearance, making it easy to observe with the naked eye, and therefore very suitable for the preparation of T lines.

[0070] 2. Morphology and length distribution of BSA@MnO2 The morphology and length distribution of BSA@MnO2 are shown in Figure 2 . Figure 2 (A) in the figure is a scanning electron micrograph of BSA@MnO2, which shows that the shape of BSA@MnO2 is fine needle-like. Figure 2 (B) in the figure is a length distribution graph of BSA@MnO2, which shows that the length distribution is concentrated around 12 nm, with a range of 5 nm to 22 nm. The nanoscale structure of this material endows it with a high specific surface area, significantly increasing the contact sites with ascorbic acid, so that it can be efficiently reduced and decolorized by ascorbic acid.

[0071] 3. Preparation of Visualized Semi-Quantitative Ascorbic Acid Detection Test Strips The structure of the visualized semi-quantitative ascorbic acid test strip is as follows: Figure 3 As shown in the image, the visual semi-quantitative ascorbic acid test strip has a sample pad at the bottom, an NC membrane in the middle, and an absorbent pad at the top. Five T-lines are distributed on the NC membrane, with a spacing of 4 mm between them. The distance between the bottom T-line and the top of the sample pad is 4.5 mm, and the distance between the top T-line and the bottom of the absorbent pad is 4.5 mm.

[0072] 4. Preparation of ascorbic acid standard solution The relationship between the concentration of ascorbic acid standard solution and the disappearance of the T line on the test strip is as follows: Figure 5 As shown in the figure. The results indicate that when the ascorbic acid concentration reaches 1.38 mg / 100 mL, one T-line disappears. When the ascorbic acid concentration reaches 2.75 mg / 100 mL, two T-lines disappear. When the ascorbic acid concentration reaches 4.13 mg / 100 mL, three T-lines disappear. When the ascorbic acid concentration reaches 5.50 mg / 100 mL, four T-lines disappear. When the ascorbic acid concentration reaches 6.88 mg / 100 mL, five T-lines disappear. BSA@MnO2 undergoes an etching reaction and fading under the action of ascorbic acid, causing the T-lines to disappear sequentially. The number of T-lines disappearing is directly proportional to the concentration of ascorbic acid.

[0073] 5. Blind sample testing The blind sample test results are shown in Table 1. The results indicate that the ascorbic acid concentration in the samples measured by titration was within the range detected by the test strip. Comparison of the test strip of this invention with the third method (2,6-dichlorophenolindophenol titration) in the national standard GB 5009.86—2016 "Determination of Ascorbic Acid in Food", the test strip's measurement range conforms to the national standard value. This demonstrates the accuracy and reliability of this method.

[0074] Table 1 Results of blind sample testing In Table 1, "+" a "This indicates the number of lines that remain after the test strip has reacted for 8 minutes; the relative standard deviation (RSD)" indicates the number of lines that have not disappeared. b =Standard deviation ÷ Mean × 100%.

[0075] 6. Anti-interference experiment The visual semi-quantitative ascorbic acid test strip of this invention exhibits strong resistance to interference from tartaric acid, phosphoric acid, malic acid, citric acid, sucrose, vitamin E, sodium chloride, and potassium chloride. Figure 6As shown in the figure. The results show that, in addition to the T line on the test paper corresponding to ascorbic acid disappears, the T line on the test paper strip of other groups is still clear, which shows that the visual semi-quantitative ascorbic acid detection test paper of the application has good anti-interference ability.

[0076] IV. Conclusion The application realizes visual detection of ascorbic acid by using the color reaction of BSA@MnO2 and ascorbic acid. The visual semi-quantitative ascorbic acid detection test paper of example 2 and example 3 of the application has the same effect as example 1. The application uses BSA@MnO2 to prepare a T line, which will fade when reacting with ascorbic acid during the detection of ascorbic acid, and the amount of T line fading is proportional to the concentration of ascorbic acid. Therefore, the visual semi-quantitative detection of ascorbic acid is realized. The visual semi-quantitative ascorbic acid detection test paper provided by the application can quickly detect the concentration of ascorbic acid, and has the characteristics of convenient use, low cost, easy operation and convenient carrying. It is suitable for on-site detection environment of ascorbic acid.

[0077] On the other hand, the visual semi-quantitative ascorbic acid detection test paper of the application has the ability to select anti-interference for common tartaric acid, phosphoric acid, malic acid, citric acid, sucrose, vitamin E, sodium chloride and potassium chloride in the sample, such as Figure 6 As shown in the figure. The results show that, in addition to the T line on the test paper corresponding to ascorbic acid disappears, the T line on the test paper strip of other groups is still clear, which shows that the visual semi-quantitative ascorbic acid detection test paper of the application has good anti-interference ability, and has more reliable accuracy in complex detection environment.

[0078] It should be noted that when numerical ranges are involved in the present application, both endpoints of each numerical range and any number between the two endpoints are intended to be selected. Since the same steps and examples are used, in order to prevent repetition, the preferred embodiments of the application are described. Although the preferred embodiments of the application have been described, those skilled in the art can make further changes and modifications to these embodiments once they know the inventive concept of the application, and these changes and modifications all fall within the scope of the application.

[0079] Obviously, those skilled in the art can make various modifications and variations to the application without departing from the spirit and scope of the application. If these modifications and variations of the application fall within the scope of the equivalent technology of the application, the application also intends to include these modifications and variations.

Claims

1. A visual semi-quantitative ascorbic acid test strip, characterized in that, The PVC substrate comprises a nitrocellulose membrane, a sample pad and a water absorption pad, one end of the nitrocellulose membrane is connected with the sample pad, the other end of the nitrocellulose membrane is connected with the water absorption pad, and a detection line is arranged on the surface of the nitrocellulose membrane. The detection line is prepared by drawing a line with the bovine serum albumin-wrapped manganese dioxide nanoneedle solution; the bovine serum albumin is wrapped on the surface of the manganese dioxide nanoneedle by forming a coordination bond between the bovine serum albumin and manganese ions on the surface of the manganese dioxide nanoneedle. The manganese dioxide nanoneedle has a needle-like morphology of γ-MnO2.

2. The visual semi-quantitative ascorbic acid test strip according to claim 1, wherein, The crystal face of the manganese dioxide nanoneedle is (110) type, the crystal face spacing is 0.22 nm, the length distribution range is 5 nm to 22 nm, and the length is mainly distributed at 12 nm.

3. The visual semi-quantitative ascorbic acid detection test paper according to claim 1, wherein the bovine serum albumin-wrapped manganese dioxide nanoneedle solution is prepared by the following method: The bovine serum albumin is dissolved in water to obtain a bovine serum albumin solution; a manganese sulfate monohydrate solution is added to the bovine serum albumin solution to obtain a mixed solution; a sodium hydroxide solution is added to the mixed solution to obtain a reaction solution; the reaction solution is transferred to a dialysis bag with a molecular weight cut-off of 80KDa to 100KDa, and dialysis is performed in deionized water; the dialysate in the dialysis bag is collected to obtain the bovine serum albumin-wrapped manganese dioxide nanoneedle solution. The concentration of the bovine serum albumin solution is 1.0 mg / mL to 1.9 mg / mL; 4. The visual semi-quantitative ascorbic acid test strip according to claim 3, wherein, The concentration of the manganese sulfate monohydrate solution is 95 mmol / L to 105 mmol / L; The concentration of the sodium hydroxide solution is 0.8 mol / L to 1.2 mol / L; The volume ratio of the bovine serum albumin solution, the manganese sulfate monohydrate solution and the sodium hydroxide solution is 50:1.5 to 2.5:0.5 to 1; The reaction time of adding the sodium hydroxide solution is 50 min to 70 min. The preparation of the nitrocellulose membrane with a detection line comprises the following steps:

5. The method for preparing visual semi-quantitative ascorbic acid test paper according to claim 1, characterized in that, The bovine serum albumin-wrapped manganese dioxide nanoneedle solution is coated on the nitrocellulose membrane, and the coated nitrocellulose membrane is dried to obtain the nitrocellulose membrane with a detection line. The preparation of the visual semi-quantitative ascorbic acid detection test paper comprises the following steps: The number of the detection lines is 3 to 5, the distance between adjacent detection lines is 3.8 mm to 4.2 mm, and each detection line contains 1.28 μg to 3.2 μg of the bovine serum albumin-wrapped manganese dioxide nanoneedle material.

6. The method of claim 5, wherein the visual semi-quantitative ascorbic acid test strip is prepared by the steps of: The material of the sample pad is glass fiber, polyester fiber or filter paper.

7. The visual semi-quantitative ascorbic acid test strip according to claim 5, wherein, 8. The visual semi-quantitative ascorbic acid detection test paper according to claim 1 is used for detecting ascorbic acid. The preparation of the nitrocellulose membrane with a detection line comprises the following steps:

9. Use of a visual semi-quantitative ascorbic acid test strip according to claim 8 for the detection of ascorbic acid, characterized in that, The sample to be detected is dissolved in a buffer solution with a pH value of 4 to 9 to obtain a detection solution, the detection solution is added to the sample pad to react, and the concentration of ascorbic acid in the sample to be detected is calculated according to the number of disappeared detection lines and the content of the bovine serum albumin-wrapped manganese dioxide nanoneedle on each detection line. The buffer solution is a citric acid buffer solution, an acetic acid buffer solution or a phosphoric acid buffer solution. ​ 10. Use of a visual semi-quantitative ascorbic acid test strip according to claim 9 for the detection of ascorbic acid, characterized in that, When the sample is dissolved, add kaolin into the buffer solution according to the proportion of 0.4g kaolin per gram of sample to adsorb impurities, filter, and take the filtrate as the detection solution for detection.

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