A nanozyme-based biosensor, its preparation method and application in the detection of ascorbic acid

Abstract

The present invention discloses a nanozyme-based biosensor and its preparation method and application in the detection of ascorbic acid, belonging to the field of chemical analysis and detection. In the present invention, the eggshell membrane is soaked in silver nitrate to prepare the nanozyme-based biosensor Ag / ESM. Ag / ESM has excellent nanozyme-like activity. Based on this, the present invention also constructs a simple and feasible detection method for ascorbic acid, which can detect ascorbic acid with high sensitivity, and the lowest detection limit is 0.016 μM. Different from most powdered nanozymes dispersed in buffer solutions, the Ag / ESM has a flaky structure, and the addition and removal of the catalyst can be easily controlled to start and terminate the reaction. The Ag / ESM has the advantages of low cost, simple operation, rapid and accurate detection, and reusable multiple times in the detection of ascorbic acid, making it have broad application prospects in the fields of food safety and healthcare.

Description

Technical Field

[0001] The present invention relates to the field of chemical analysis and detection, and particularly to a nano-like oxidase biosensor, a preparation method thereof, and an application thereof in the detection of ascorbic acid. Background Art

[0002] Ascorbic acid (AA), namely vitamin C, is an essential water-soluble vitamin for the human body. As an important active substance for maintaining life activities and keeping healthy, although the content of ascorbic acid in the body is small, it is indispensable. Its deficiency will lead to scurvy, cardiovascular diseases, nervous system diseases, etc., seriously threatening human health. Since the human body cannot synthesize ascorbic acid by itself, it must be supplemented through food or drugs, such as vitamin tablets, fresh fruits and vegetables, especially dates, peppers, tomatoes, bitter gourds, oranges, lemons, etc. Therefore, it is crucial to construct a simple method for detecting the content of ascorbic acid in food or drugs.

[0003] There are various detection methods for AA, including electrochemistry, chemiluminescence, high performance liquid chromatography, fluorescence method, etc. However, the colorimetric method has attracted much attention due to its low cost, simple operation, and the need for no complex equipment. AA has strong reducibility and can directly reduce oxidized TMB, which provides the possibility for constructing a colorimetric detection method.

[0004] As a mimic enzyme, noble metal nanozymes exhibit excellent enzyme activities in extreme environments, including peroxidase, oxidase, catalase, and superoxide dismutase activities, due to their excellent stability, simple preparation, and designability. Among them, the oxidase-like mimics have the advantage of directly oxidizing TMB without the need for hydrogen peroxide, which simplifies the operation process. Currently, the reported noble metal nanozymes mainly include noble metals such as gold, platinum, iridium, ruthenium, palladium, and rhodium, which are in powder form, expensive, and difficult to recycle and reuse. Summary of the Invention

[0005] The purpose of the present invention is to provide a nano-like oxidase biosensor, a preparation method thereof, and an application thereof in the detection of ascorbic acid, so as to solve the problems existing in the above-mentioned prior art. By immersing the eggshell membrane in a silver nitrate solution, a nano-like oxidase biosensor is prepared. This biosensor can be used for rapid and accurate detection of ascorbic acid, overcoming the disadvantages of complex operation and expensive instruments in current traditional detection methods, and realizing low-cost and easy-operation detection of ascorbic acid in food.

[0006] To achieve the above purpose, the present invention provides the following solutions:

[0007] The present invention provides a nano-like oxidase biosensor, which is prepared by immersing an eggshell membrane in a silver nitrate solution.

[0008] The present invention also provides a method for preparing a nanozyme-based biosensor, which includes the step of soaking an eggshell membrane in a silver nitrate solution.

[0009] Preferably, the method for obtaining the eggshell membrane includes the following steps:

[0010] Peel the eggshell membrane from the eggshell, wash to remove the egg liquid on the surface, dry and cut it into pieces.

[0011] Preferably, the size range of the sheet-shaped eggshell membrane is 0.5×0.5 cm to 1.5×1.5 cm.

[0012] Preferably, each eggshell membrane is soaked in 2 mL of a 1-10 mM silver nitrate solution for 0.5-3 h, then rinsed with distilled water and ethanol, and air-dried to obtain the nanozyme-based biosensor.

[0013] The present invention also provides a method for detecting ascorbic acid using a nanozyme-based biosensor, including the following steps:

[0014] Prepare a nanozyme-based biosensor using the above-mentioned preparation method;

[0015] Add the nanozyme-based biosensor and a TMB solution to an acetate buffer solution for reaction, and then add standard ascorbic acid solutions with different concentrations to the reaction system. By analyzing the relationship between the absorbance and the ascorbic acid concentration, establish a standard working curve and obtain a linear regression equation;

[0016] Add a test sample containing ascorbic acid to the same reaction system as above, measure the absorbance, and substitute the obtained absorbance data into the linear regression equation to calculate the content of ascorbic acid in the test sample.

[0017] Preferably, the concentration of the acetate buffer solution is 0.5-5 mM, and the pH range is 3-7.

[0018] Preferably, each milliliter of the reaction system contains one nanozyme-based biosensor and 1-20 μL of a TMB solution; wherein, the concentration of the TMB solution is 50-200 μM.

[0019] The reaction conditions are: reaction at 15-45 °C for 1-15 min.

[0020] Preferably, the concentration of the ascorbic acid standard solution is 0-100 μM;

[0021] Measure the absorbance using a UV-visible spectrophotometer, and the wavelength range is 200-800 nm.

[0022] The present invention also provides the application of the nanozyme-like biosensor in the detection of ascorbic acid.

[0023] The present invention discloses the following technical effects:

[0024] (1) The Ag / ESM nanozyme prepared in the present invention catalyzes the oxidation of TMB to develop color without hydrogen peroxide, showing excellent oxidase activity. After adding ascorbic acid, TMB is reduced and the solution color fades from blue. There is a good linear relationship between the concentration of ascorbic acid and the absorbance, and the lowest detection limit is as low as 0.0016 μM, constructing a highly sensitive, rapid, and colorimetric method for the detection of ascorbic acid.

[0025] (2) Different from most powder nanozymes, the Ag / ESM nanozyme of the present invention has a flaky structure, which is very convenient to put Ag / ESM into or take out of the reaction system, control the occurrence and termination of the reaction, facilitate recycling, and be reused multiple times.

[0026] (3) The Ag / ESM nanozyme biosensor of the present invention can effectively resist common interfering substances in lemon juice, such as Na + , K + , Ca 2+ , Mg 2+ , glucose, fructose, vitamin B, threonine, histidine, lysine, glycine, tyrosine, arginine, etc. Compared with traditional biosensors, the accuracy of detecting amino acids is significantly improved.

[0027] (4) The present invention adopts a rapid detection method, which can effectively detect the lemon juice content in food. This method has advantages such as high spike recovery rate and low relative standard deviation, showing high accuracy and good sensitivity. This method has broad application prospects in the field of food detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0029] Figure 1 It is a schematic diagram of the preparation and action mechanism of the Ag / ESM nanozyme in Example 1;

[0030] Figure 2 It is the SEM and EDX diagrams of the Ag / ESM nanozyme in Example 1; in the figure, C-K, O-K, N-K, S-K, and Ag-K respectively represent the analysis of C, O, N, S, and Ag elements in the eggshell membrane;

[0031] Figure 3 is the oxidase activity of the Ag / ESM-like oxidase in Example 2;

[0032] Figure 4 are the optimal reaction conditions of the Ag / ESM-like oxidase in Example 3; a: Effect of AgNO3 concentration on the absorbance of the Ag / ESM-like oxidase; b: Effect of the soaking time of eggshell membrane in AgNO3 solution on the absorbance of the Ag / ESM-like oxidase; c: Effect of reaction temperature on the absorbance of the Ag / ESM-like oxidase when Ag / ESM reacts with TMB solution; d: Effect of the size of eggshell membrane on the absorbance of the Ag / ESM-like oxidase; e: Effect of reaction time on the absorbance of the Ag / ESM-like oxidase when Ag / ESM reacts with TMB solution; f: Effect of pH value on the absorbance of the Ag / ESM-like oxidase when Ag / ESM reacts with TMB solution;

[0033] Figure 5 is the catalytic reaction control of the Ag / ESM-like oxidase in Example 4;

[0034] Figure 6 is the relationship between absorbance and AA concentration in Example 5; a: Spectrogram of measuring absorbance by adding different concentrations of AA; b: Linear relationship diagram of measuring different concentrations of AA and absorbance;

[0035] Figure 7 is the influence of interferents on the detection of AA by Ag / ESM in Example 5. Detailed implementation manners

[0036] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0037] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0038] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although this invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the said documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0039] Without departing from the scope or spirit of this invention, various modifications and variations can be made to the specific embodiments of the specification of this invention, which are obvious to those skilled in the art. Other embodiments obtained from the specification of this invention are obvious to those skilled in the art. The specification and examples of this invention are merely exemplary.

[0040] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0041] Example 1 Preparation of Ag / ESM Nanozyme Biosensor

[0042] (1) Preparation of eggshell membrane (ESM): Peel the eggshell membrane from the eggshell, wash the egg liquid, and cut it into pieces of different sizes after drying.

[0043] (2) Preparation of Ag / ESM nanozyme biosensor: Dissolve 0.0849 g of silver nitrate (AgNO3) in 50 mL of distilled water at room temperature. Take 2 mL of the solution into a centrifuge tube, and immerse the treated eggshell membrane in the solution for 1 h (repeat this step to immerse the material, 2 mL for one piece). Then rinse it with distilled water and ethanol multiple times and dry it naturally. The schematic diagram of the preparation and mechanism of action is shown in Figure 1 .

[0044] The prepared Ag / ESM was observed by transmission electron microscopy, and its elements were analyzed by energy dispersive X-ray spectrometer. The SEM image and EDX image are shown in Figure 2 , and it can be seen from the figure that Ag is uniformly loaded on ESM.

[0045] Example 2 Oxidase Performance of Ag / ESM

[0046] 20 μL of 100 μM TMB or the mixed solution of ESM and TMB was added and incubated in 0.98 mL of 5 mM acetate buffer for 10 minutes, and no absorption peak was observed at 652 nm. However, after adding one piece of Ag / ESM and 20 μL of 100 μM TMB solution successively in the same acetate buffer, an absorption peak appeared at 652 nm after the reaction system was incubated at room temperature for 10 minutes, and colorless TMB was converted into blue TMB. ox (See Figure 3 ).

[0047] Example 3 Influence of Preparation and Reaction Conditions on the Oxidase Performance of Ag / ESM

[0048] The oxidase performance was investigated by changing the eggshell membrane size (0.5×0.5, 0.75×0.75, 1.0×1.0, 1.25×1.25, and 1.5×1.5 cm), AgNO3 concentration (1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 mM), soaking time (0.5, 1.0, 1.5, 2.0, 2.5, and 3 h), reaction temperature (15, 20, 25, 30, 35, 40, and 45 °C), pH (3, 4, 5, 6, and 7), and incubation time (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15 min).

[0049] The results are shown in Figure 4 , and the results show that the eggshell membrane size, AgNO3 concentration, soaking time, reaction temperature, pH, and reaction time all have great influences on the oxidase performance of Ag / ESM. The optimal size of the eggshell membrane is 1.5×1.5 cm, the optimal concentration of AgNO3 is 10 mM, the optimal soaking time of the eggshell membrane is 2.0 h; the optimal reaction temperature of the reaction system is 20 °C, the optimal pH value is 4, and the reaction time is 11 min.

[0050] Example 4 Only one pair of tweezers is needed to control the start and stop of the reaction

[0051] Different from most reported powdered nanozymes, the synthesized Ag / ESM nanozyme in this example is in the form of flakes. This unique structure enables the rapid and convenient addition and removal of nanozyme materials in the reaction system simply by using tweezers. Figure 5 This property was demonstrated. The nanozyme material was put into or taken out of the solution by tweezers, and the absorbance changes at each time point were recorded. When the Ag / ESM nanozyme was first added to the solution, the catalytic reaction started rapidly, resulting in a rapid increase in absorbance. When the nanozyme was removed by tweezers, the catalytic process was immediately paused, the oxidation of TMB stopped, and the absorbance only increased slightly. It shows that the reaction system can be quickly terminated after removing the Ag / ESM nanozyme, thus achieving precise control of the reaction process.

[0052] Example 5 Detection of Ascorbic Acid by Ag / ESM Nanozyme-like Biosensor

[0053] By reacting Ag / ESM with TMB under specific conditions and adding different concentrations of AA from 0 - 70 μM to the system, a linear relationship between the AA concentration and absorbance was established. The results showed that within the concentration range of 3 - 45 μM, there was a good linear relationship between the absorbance and the AA concentration (see Figure 6 ), and the linear equation was y = -0.0171x + 1.2467 (R 2 = 0.99). The lowest detection limit of this method was 0.016 μM.

[0054] The content of AA in freshly squeezed lemon juice diluted 20 times (5 mL of lemon juice was placed in a centrifuge tube, diluted to 10 mL with distilled water, centrifuged at 10000 r / min for 5 min, 1 mL of the supernatant was taken and diluted to 10 mL with distilled water) could be detected by this method, and its content was 4.49 μM. On this basis, standard addition and recovery were carried out, and the range of standard addition recovery was 96.80% - 104.27%, and the relative standard deviation (RSD) was less than 3.1%.

[0055] To test the selectivity of Ag / ESM for AA detection, with other steps remaining unchanged, 50 μM of AA was replaced by 13 common food ions or amino acids, and their concentrations were 100 times the AA concentration (5 mM). The results are shown in Figure 7 . After adding these ions or amino acids, the absorbance of the system hardly changed, and only when AA was added did the absorbance of the system change significantly.

[0056] As can be seen from the experimental results of the above embodiments, the present invention uses a simple immersion method to load silver on the eggshell membrane, successfully constructing a flaky and highly efficient Ag / ESM oxidase mimetic. It catalyzes the oxidation of TMB to develop color without hydrogen peroxide, showing excellent oxidase mimetic activity. After adding AA, TMB is reduced and the solution color fades from blue. Utilizing the excellent peroxidase-like activity of Ag / ESM, a simple and feasible AA detection method is constructed. The detection range of this method is 3 - 45 μM, and the lowest detection limit (LOD) is 0.016 μM, showing excellent sensitivity. This detection method can effectively detect the ascorbic acid content in lemon juice. The spiked recovery rate determination results of lemon juice are good, with the recovery rate range being 96.80% - 104.27%, and the relative standard deviation (RSD) being less than 3.1%. And different from most powdered nanozymes dispersed in buffer solutions, the Ag / ESM peroxidase-like enzyme has a flaky structure, enabling easy control of the addition and removal of the catalyst to start and terminate the reaction. The peroxidase-like nano-biosensor prepared by the present invention has the advantages of low cost, simple operation, rapid and accurate detection, and reusability in the detection of ascorbic acid, making it have broad application prospects in the fields of food safety and healthcare.

[0057] The above-described embodiments are only used to describe the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for detecting ascorbic acid using a nano-oxidase biosensor, characterized in that: The following steps are involved: Preparation of nano-oxidase biosensors; The nano-oxidase biosensor and TMB solution are added to an acetate buffer for reaction, and then ascorbic acid standard solutions of different concentrations are added to the reaction system, and a standard working curve is established and a linear regression equation is obtained by analyzing the relationship between absorbance and ascorbic acid concentration; Adding a sample to be tested containing ascorbic acid into the same reaction system as above, measuring the absorbance, and substituting the obtained absorbance data into the linear regression equation to calculate the content of ascorbic acid in the sample to be tested; The method for preparing the nano-oxidase biosensor comprises the steps of soaking the eggshell membrane in a silver nitrate solution; The method for obtaining eggshell membranes comprises the following steps: Peel off the eggshell membrane from the eggshell, wash and remove the egg liquid on the surface, dry and cut into slices; The size of the sheet of eggshell membrane ranges from 0.5×0.5 cm to 1.5×1.5 cm; Each eggshell membrane was immersed in 2 mL of 1-10 mM silver nitrate solution for 0.5-3 h, then rinsed with distilled water and ethanol, and dried naturally to prepare the nano-oxidase biosensor; The concentration of the acetate buffer is 0.5-5 mM, and the pH range is 3-7; Each milliliter of the reaction system contains one nano-oxidase biosensor and 1-20 μL of TMB solution; wherein the concentration of the TMB solution is 50-200 μM; The reaction conditions are: 15-45°C for 1-15 min.

2. The method according to claim 1, characterized in that The concentration of the ascorbic acid standard solution is 0-100 μM; the absorbance is measured by using an ultraviolet-visible spectrophotometer, and the wavelength range is 200-800 nm.

3. Application of nano-oxidase biosensor in ascorbic acid detection, characterized in that: The nano-oxidase biosensor is prepared by soaking eggshell membrane in silver nitrate solution.

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