Nanometer oxidase biosensor, preparation method thereof and application of nanometer oxidase biosensor in ascorbic acid detection

Abstract

The invention discloses a nano oxidase biosensor as well as a preparation method and application thereof in ascorbic acid detection, and belongs to the field of chemical analysis and detection. According to the invention, an eggshell membrane is soaked in silver nitrate to prepare the nano oxidase biosensor Ag / ESM. The Ag / ESM has excellent oxidase-like activity, so that the invention also constructs a simple and feasible ascorbic acid detection method, the detection method can detect ascorbic acid with high sensitivity, and the lowest detection limit is 0.016 [mu] M. The Ag / ESM is different from most powdery nano-enzymes dispersed in a buffer solution, is of a sheet structure, and can easily control the adding and taking out of a catalyst to realize the start and stop of the reaction. The Ag / ESM has the advantages of being low in cost, simple and convenient to operate, rapid, accurate and capable of being repeatedly used in the aspect of ascorbic acid detection, and has wide application prospects in the fields of food safety and medical care.

Description

Technical Field

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

[0002] Ascorbic acid (AA), also known as vitamin C, is an essential water-soluble vitamin for the human body. As an important active substance for maintaining life activities and health, ascorbic acid is indispensable in the body despite its low content. Its deficiency can lead to scurvy, cardiovascular disease, nervous system diseases, etc., which seriously threatens human health. Since the human body cannot synthesize ascorbic acid on its own, it must be supplemented through food or medicine, such as vitamin tablets, fresh fruits and vegetables, especially dates, peppers, tomatoes, bitter melon, citrus, lemons, etc. Therefore, it is crucial to construct a simple method to detect the content of ascorbic acid in food or medicine.

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

[0004] As enzyme mimics, precious 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, oxidase mimics have the advantage of directly oxidizing TMB without hydrogen peroxide compared to peroxidase mimics, which simplifies the operation process. At present, the reported precious metal nanozymes mainly include precious metals such as gold, platinum, iridium, ruthenium, palladium, and rhodium, which are in powder form, expensive and difficult to recycle. Summary of the invention

[0005] The purpose of the present invention is to provide a nano-oxidase biosensor and a preparation method thereof and application in ascorbic acid detection, so as to solve the problems existing in the above-mentioned prior art. Silver is loaded on eggshell membrane by an immersion method to prepare a nano-oxidase biosensor. The biosensor can be used for rapid and accurate detection of ascorbic acid, overcoming the disadvantages of the current traditional detection methods such as complex operation and expensive instruments, and realizing low-cost and easy-to-operate detection of ascorbic acid in food.

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

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

[0008] The present invention also provides a method for preparing a nano-oxidase biosensor, which comprises the step of soaking eggshell membrane in a silver nitrate solution.

[0009] Preferably, the method for obtaining eggshell membranes comprises the following steps:

[0010] The eggshell membrane is peeled off from the eggshell, washed to remove the egg liquid on the surface, dried and cut into slices.

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

[0012] Preferably, each piece of eggshell membrane is 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 obtain the nano-oxidase biosensor.

[0013] The present invention also provides a method for detecting ascorbic acid using a nano-oxidase biosensor, comprising the following steps:

[0014] A nano-oxidase biosensor is prepared by using the preparation method;

[0015] 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;

[0016] A sample to be tested containing ascorbic acid is added to the same reaction system as above, the absorbance is measured, and the obtained absorbance data is substituted into the linear regression equation to calculate the content of ascorbic acid in the sample to be tested.

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

[0018] Preferably, 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.

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

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

[0021] The absorbance was measured using a UV-visible spectrophotometer in the wavelength range of 200 to 800 nm.

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

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

[0024] (1) The Ag / ESM oxidase prepared by the present invention catalyzes the oxidation of TMB in the absence of hydrogen peroxide and exhibits excellent oxidase activity. After the addition of ascorbic acid, TMB is reduced and the color of the solution fades from blue. There is a good linear relationship between the ascorbic acid concentration and the absorbance, and the minimum detection limit is as low as 0.0016 μM. A highly sensitive, rapid, and colorimetric ascorbic acid detection method is constructed.

[0025] (2) Unlike most powdered nanozymes, the Ag / ESM-based oxidase of the present invention has a sheet-like structure, which makes it easy to put Ag / ESM into or take it out of the reaction system, control the occurrence and termination of the reaction, and facilitate recycling and repeated use.

[0026] (3) The Ag / ESM oxidase 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 amino acid detection is significantly improved.

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

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

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

[0030] Figure 2 The SEM and EDX images of the Ag / ESM oxidase in Example 1; CK, OK, NK, SK and Ag-K in the figure represent the analysis of C, O, N, S and Ag elements in the eggshell membrane, respectively;

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

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

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

[0034] Figure 6 The relationship between absorbance and AA concentration in Example 5; a: Spectrum of absorbance measured by adding different concentrations of AA; b: Linear relationship between absorbance and adding different concentrations of AA;

[0035] Figure 7 This is the effect of the interferents in Example 5 on the detection of AA by Ag / ESM. DETAILED DESCRIPTION

[0036] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0037] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0038] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0039] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0040] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0041] Example 1 Preparation of Ag / ESM Nano-Oxidase Biosensor

[0042] (1) Preparation of eggshell membrane (ESM): peel the ESM from the chicken egg shell, wash the egg liquid, dry it, and cut it into sheets of different sizes;

[0043] (2) Preparation of Ag / ESM nano-oxidase biosensor: Dissolve 0.0849 g of silver nitrate (AgNO3) in 50 mL of distilled water at room temperature. Take 2 mL of the solution in a centrifuge tube and soak the treated eggshell membrane in the solution for 1 h (repeat this step to soak the material, 2 mL soaking one piece). Then rinse with distilled water and ethanol several times and dry naturally. See the schematic diagram of the preparation and mechanism of action for details. 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 and EDX images are shown in Figure 2 , it can be seen from the figure that Ag is evenly loaded on the ESM.

[0045] Example 2 Oxidase Performance of Ag / ESM

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

[0047] Example 3 Effect of preparation and reaction conditions on the performance of Ag / ESM oxidase

[0048] The oxidase performance of eggshell membrane was investigated by changing the size of eggshell membrane (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), immersion 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] Results Figure 4 The results showed that the eggshell membrane size, AgNO3 concentration, immersion time, reaction temperature, pH and reaction time all had a significant effect on the oxidase performance of Ag / ESM. The optimal eggshell membrane size was 1.5×1.5cm, the optimal AgNO3 concentration was 10mM, and the optimal immersion time of the eggshell membrane was 2.0h; the optimal reaction temperature of the reaction system was 20℃, the optimal pH value was 4, and the reaction time was 11min.

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

[0051] Unlike most reported powdered nanozymes, the Ag / ESM nanozymes synthesized in this example are in sheet form. This unique structure enables the nanozyme material to be quickly and conveniently added and removed from the reaction system by simply using tweezers. Figure 5 This property was demonstrated. The nanozyme material was placed in or taken out of the solution using tweezers, and the absorbance change at each time point was recorded. When the Ag / ESM nanozyme was first added to the solution, the catalytic reaction started quickly, resulting in a rapid increase in absorbance. When the nanozyme was removed with tweezers, the catalytic process was immediately paused, TMB oxidation stopped, and the absorbance only increased slightly. This shows that the reaction system can be quickly terminated after the Ag / ESM nanozyme is removed, thereby achieving precise control of the reaction process.

[0052] Example 5: Ag / ESM Nano-Oxidase Biosensor for Detecting Ascorbic Acid

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

[0054] This method can detect the content of AA in fresh lemon juice diluted 20 times (5 mL of lemon juice is placed in a centrifuge tube, diluted to 10 mL with distilled water, centrifuged at 10000 r / min for 5 minutes, 1 mL of supernatant is taken, and then diluted to 10 mL with distilled water), and its content is 4.49 μM. On this basis, spike recovery is performed, and the spike recovery rate ranges from 96.80% to 104.27%, and the relative standard deviation (RSD) is less than 3.1%.

[0055] In order to test the selectivity of Ag / ESM for AA detection, 50 μM AA was replaced by 13 common food ions or amino acids, with the concentrations of each ion or amino acid being 100 times that of AA (5 mM) while keeping other steps unchanged. The results are shown in Figure 7 After adding these ions or amino acids, the absorbance of the system hardly changed. Only after adding AA did the absorbance of the system change significantly.

[0056] It can be seen from the experimental results of the above embodiments that the present invention uses a simple immersion method to load silver on eggshell membrane, and successfully constructs a flaky and efficient Ag / ESM oxidase mimetic. Under the condition of no hydrogen peroxide, the catalytic oxidation of TMB is colorized, showing excellent oxidase simulation activity. After adding AA, TMB is reduced and the color of the solution fades from blue. Using the excellent oxidase-like activity of Ag / ESM, a simple and easy AA detection method is constructed. The detection range of this method is 3-45μM, and the minimum detection limit (LOD) is 0.016μM, showing excellent sensitivity. This detection method can effectively detect the ascorbic acid content in lemon juice. The spiked recovery of lemon juice is good, with a recovery range of 96.80% to 104.27% and a relative standard deviation (RSD) of less than 3.1%. And unlike most powdered nanozymes dispersed in buffer, Ag / ESM oxidases have a flaky structure, which can easily control the addition and removal of the catalyst to achieve the start and termination of the reaction. The oxidase-like nanobiosensor prepared by the present invention has the advantages of low cost, simple operation, rapidity, accuracy and multiple reuse in detecting ascorbic acid, so that it has broad application prospects in the fields of food safety and medical care.

[0057] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A nano-oxidase biosensor, characterized in that: It is prepared by soaking eggshell membranes in a silver nitrate solution.

2. A method for preparing a nano-oxidase biosensor, characterized in that: The preparation method comprises the step of soaking the eggshell membrane in a silver nitrate solution.

3. The preparation method according to claim 2, characterized in that: The method for obtaining eggshell membranes comprises the following steps: The eggshell membrane is peeled off from the eggshell, washed to remove the egg liquid on the surface, dried and cut into slices.

4. The preparation method according to claim 3, characterized in that: The size of the sheet-like eggshell membrane ranges from 0.5×0.5 cm to 1.5×1.5 cm.

5. The preparation method according to claim 4, characterized in that: Each piece of 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.

6. A method for detecting ascorbic acid using a nano-oxidase biosensor, characterized in that: The following steps are involved: A nano-oxidase biosensor is prepared by the preparation method according to any one of claims 2 to 5; 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; A sample to be tested containing ascorbic acid is added to the same reaction system as above, the absorbance is measured, and the obtained absorbance data is substituted into the linear regression equation to calculate the content of ascorbic acid in the sample to be tested.

7. The method according to claim 6, characterized in that The concentration of the acetate buffer is 0.5-5 mM, and the pH range is 3-7.

8. The method according to claim 6, characterized in that 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.

9. The method according to claim 6, characterized in that The concentration of the ascorbic acid standard solution is 0-100 μM; The absorbance was measured using a UV-visible spectrophotometer in the wavelength range of 200 to 800 nm.

10. Use of the nano-oxidase biosensor as claimed in claim 1 in ascorbic acid detection.

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