Preparation and application of a dual-mode non-toxic instant detection biosensor based on black phosphorus quantum dots

Abstract

The present invention discloses the preparation and application of a dual-mode non-toxic instant detection biosensor based on black phosphorus quantum dots, and conceives and designs a dual-mode non-toxic instant rapid detection method that can use black phosphorus quantum dots as simulated peroxidase to detect hydrogen peroxide in the system. Compared with other quantum dots, black phosphorus quantum dots have simple synthesis steps, high yield, stable storage after synthesis, non-toxic and harmless, and the ultraviolet-visible light absorption curve constructed using the characteristic absorption peak of TMB at 652 nm has a good linear relationship. In addition, the rapid instant detection of test strips and mobile phones, the dual-mode detection system can be promoted and used as a model of safe and non-toxic rapid instant detection.

Description

Technical Field

[0001] The invention belongs to the technical field of nanomaterials and biosensors, and specifically relates to the preparation and application of a dual-mode non-toxic instant rapid detection biosensor based on black phosphorus quantum dots simulating peroxidase to detect hydrogen peroxide in a system. Background Art

[0002] Hydrogen peroxide (H2O2) is an inorganic volatile compound with strong oxidizing properties. It is usually used as a bleaching agent and disinfectant. In food and beverages, H2O2 residues may be found in products after pasteurization and packaging. In my country, H2O2 is mainly used in the production of dairy products and water-cured products, such as milk sterilization, squid, tendons, and tripe production disinfection. Considering that hydrogen peroxide can play other important process roles in addition to disinfection, it is widely used in the food field. It is worth noting that due to the long-term and large-scale use of H2O2 by people, many relevant literatures at home and abroad have reported the consequences of excessive accumulation of H2O2. For example, large-scale accumulation in organisms can lead to oxidative stress, gene mutations, and cell dysfunction in organisms, which will undoubtedly increase the risk of many progressive diseases and even cancer. At present, the international community generally believes that H2O2 concentrations exceeding a certain threshold are harmful to humans, especially in the case of long-term ingestion or inhalation, the toxicity will further manifest and deepen. Some regions, such as the Australian and New Zealand Food Standards Agency, have food safety regulations that the residual concentration of H2O2 in food should be strictly controlled below 5 mg / kg. In addition, H2O2 above 3% will seriously affect the survival of microorganisms and algae in the environment and disrupt the balance of the food chain. Therefore, ensuring that the H2O2 level is within a safe range is crucial to human health and environmental safety.

[0003] The colorimetric analysis of horseradish peroxidase (HRP) has special application significance due to its simple colorimetric reading, rapid detection pathway, and convenient experimental method. Among these methods, 3,3',5,5'-tetramethylbenzidine (TMB) is the most widely used HRP colorimetric substrate. More and more studies have shown that in addition to HRP, there are many substances that have a catalytic effect similar to HRP to catalyze TMB color development and these substances have better properties than biological enzymes and can be used in different environments, such as ferroferric oxide nanoparticles, DNA enzymes, quantum dots, etc. Among them, quantum dot catalysis of TMB is currently a hot topic of research. In addition to carbon quantum dots as the representative, new catalytic materials such as ferroferric oxide quantum dots and silicon quantum dots have also been reported recently. These studies use the excellent physical and chemical properties of quantum dots to continuously optimize the detection system, and the detection sensitivity is also getting higher and higher. However, this type of system still has some unavoidable disadvantages. For example, in this type of system, TMB can only be dissolved in dimethyl sulfoxide (DMSO), a "universal solvent" due to its low solubility. DMSO is a volatile liquid with vascular toxicity and liver toxicity. After volatilization, it is highly irritating to the skin and eyes. In addition, its strong permeability and burns to the skin require the drug to be operated in the laboratory and the waste liquid recovery is relatively strict.

[0004] In addition, conventional TMB colorimetric system detection usually requires the use of an ultraviolet spectrophotometer, which is expensive, cumbersome to operate, and needs to be analyzed in a laboratory, which has become a significant drawback that restricts its on-site and rapid detection. As the most popular and powerful smart tool at present, smartphones have huge development and application potential, and it is expected that a rapid and instant detection application model based on smartphones can be designed. Summary of the invention

[0005] To solve the above problems, a dual-mode non-toxic instant rapid detection biosensor based on black phosphorus quantum dots was developed.

[0006] To achieve the above object, the present invention adopts the following technical solution:

[0007] A dual-mode non-toxic instant rapid detection biosensor based on black phosphorus quantum dots simulating peroxidase to detect hydrogen peroxide in the system, comprising a non-toxic colorimetric biosensor and a test paper-based visual biosensor. Both the non-toxic colorimetric biosensor and the test paper-based visual biosensor use ultra-small black phosphorus quantum dots BPQDs as simulated peroxidase to catalyze TMB to quickly detect hydrogen peroxide in the system;

[0008] The preparation of ultra-small black phosphorus quantum dots BPQDs includes the following steps: weigh black phosphorus crystals in a glove box, grind them in a mortar for a period of time, add a small amount of N-methyl-2-pyrrolidone NMP and grind them evenly, transfer them to a double-necked flask and add a saturated alkali-mixed NMP solution, react at 160 ° C for 10 h, keep stirring vigorously during the reaction, centrifuge for 20 min after the reaction is completed, collect the supernatant and dry it to obtain BPQDs solid. The grinding time for both steps is not less than 20 min.

[0009] Furthermore, the preparation of the non-toxic colorimetric biosensor comprises the following steps:

[0010] At room temperature, different concentrations of H2O2 were added to a mixed solution consisting of acetic acid-sodium acetate buffer, BPQDs and TMB. After standing for a period of time, UV-visible absorption spectra were measured. The change in absorbance was recorded at 652 nm and the absorption spectrum of the above solution was obtained. Then, the corresponding relationship between the concentration range and the absorbance was obtained for the detection of hydrogen peroxide in the system.

[0011] Furthermore, the standing time was 30 min.

[0012] Furthermore, the preparation of the test paper-based visual biosensor comprises the following steps:

[0013] (1) Take ordinary qualitative filter paper from the laboratory, cut it into small test paper squares, and evenly coat and fix it with sodium alginate solution before use. Then, evenly coat the mixed solution composed of acetic acid-sodium acetate buffer, BPQDs and TMB on the test paper, freeze-dry it and store it for later use;

[0014] (2) During the test, drop H2O2 of different concentrations onto the prepared test paper. After waiting for a while, use the Android phone colorimetric software "Color Grab" to perform color analysis.

[0015] Furthermore, the mixed solution consisting of acetic acid-sodium acetate buffer, BPQDs and TMB specifically refers to a mixed solution consisting of 790 mL, 0.2 mM, pH = 3 acetic acid-sodium acetate buffer, 100 μL, 1 mg / mL BPQDs solution and 100 μL, 10 mM TMB solution.

[0016] Furthermore, a saturated alkali-mixed NMP solution was prepared by stirring 50 mL of anhydrous NMP, 100 mg of sodium hydroxide, and 1 mg of potassium hydroxide in a glove box for 10 min.

[0017] Furthermore, in step (1), the concentration of sodium alginate solution is 0.5 mg / mL, and the freeze-drying time is 2 h.

[0018] Furthermore, in step (2), the amount of H2O2 added was 10 μL and the waiting time was 30 min.

[0019] Furthermore, in step (2), the color analysis is performed using the colorimetric software "Color Grab" APP to obtain the three color parameters of red R, green G, and blue B, and the ratio of B / (R + G + B) is used to evaluate the color difference between the paper-based sensors fed back by different concentrations of H2O2, thereby obtaining the corresponding relationship between the ratio of B / (R + G + B) and the H2O2 concentration range, which is used for the detection of hydrogen peroxide in the system.

[0020] The significant advantages of the present invention are:

[0021] (1) The synthesis method of the main nanomaterial (BPQDs) is simple and has a high yield. Compared with the traditional solvothermal method, the present invention first uses physical grinding to achieve a simple liquid phase exfoliation effect, thereby improving the reaction efficiency of the entire system. At the same time, the introduction of potassium ions in potassium hydroxide can increase the yield of black phosphorus quantum dots and improve the stability of the product. Therefore, the improved synthesized black phosphorus quantum dots have a higher yield, more excellent physical and chemical properties, and ultra-high biosafety.

[0022] (2) The dual-mode sensor has low toxicity, high sensitivity, rapid response, and strong anti-interference ability, and can be used for rapid on-site detection outside the laboratory.

[0023] (3) No large-scale equipment or operator training is required. By combining with a smartphone APP, the test data can be directly and instantly obtained, which is convenient and fast. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a schematic diagram of the detection principle of a dual-mode non-toxic instant detection biosensor based on black phosphorus quantum.

[0025] Figure 2 is a TEM image of the BPQDs nanoparticles prepared in Example 1;

[0026] Figure 3 is a diagram of cytotoxicity analysis of BPQDs in Example 1;

[0027] Figure 4 is a UV-visible light absorption spectrum of the non-toxic colorimetric biosensor in Example 2 after adding different concentrations of the target;

[0028] Figure 5 This is a real picture of the visual changes of the test paper-based visual biosensor in Example 2 after adding different concentrations of the target;

[0029] Figure 6It is the absorption intensity diagram of the UV-visible absorption spectrum at 652 nm after adding different interfering substances in Example 3. DETAILED DESCRIPTION

[0030] In order to make the contents of the present invention easier to understand, the technical solution of the present invention is further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.

[0031] Example 1 Preparation and cytotoxicity test of ultra-small BPQDs

[0032] (1) Weigh 50 mg of black phosphorus crystals in a glove box, grind them in a mortar for 20 min, add a small amount (10 mL) of N-methyl-2-pyrrolidone (NMP) and grind for another 20 min, transfer to a double-necked flask and add 40 mL of saturated alkali mixed NMP solution prepared by mixing 50 mL of NMP, 100 mg of sodium hydroxide and 1 mg of potassium hydroxide, react at 160 °C for 10 h, and keep stirring vigorously. After the reaction is completed, centrifuge at 7000 rpm for 20 min and collect the supernatant. After drying for 12 h, BPQDs solid is obtained.

[0033] (2) Take U87MG cells in the logarithmic growth phase and add 100 μL (about 5×10 3 The cells were seeded in a 96-well plate and cultured in a 37°C, 5% CO2 incubator for 24 h. Then, the old culture medium was replaced with 100 μL of fresh culture medium containing different concentrations of BPQDs (0, 1, 5, 10, 20, 50 and 100 μg / mL). After incubation in a 37°C, 5% CO2 incubator for 24 h, 10 μL of CCK-8 solution was added to each well. After incubation in the incubator for 1-2 h, the OD value of the sample at 450 nm was measured using a microplate reader, and the cell survival rate was calculated using the following formula (1-1).

[0034] Cell survival rate = [(As-Ab) / (Ac-Ab)] × 100% (1-1)

[0035] Wherein: As: absorbance of experimental wells (containing cells, culture medium, CCK-8 solution and drug solution); Ac: absorbance of control wells (containing cells, culture medium, CCK-8 solution, but not containing drugs); Ab: absorbance of blank wells (containing culture medium and CCK-8 solution, but not containing cells or drugs).

[0036] Figure 2 (A) shows that the present invention successfully synthesized ultra-small BPQDs with a particle size of about 2.8 ± 0.8 nm and good dispersion; Figure 2(B) clearly shows the lattice fringes of the (021) plane of BPQDs, once again confirming the successful synthesis of ultra-small BPQDs.

[0037] like Figure 3 As shown, even at the highest concentration of 100 μg / mL, BPQDs had no obvious cytotoxicity, demonstrating that BPQDs have very low cytotoxicity and good biocompatibility in biological systems.

[0038] Example 2 Preparation and application of a dual-mode non-toxic instant detection biosensor based on black phosphorus quantum dots:

[0039] (1) The black phosphorus quantum dots prepared in Example 1 were used as simulated peroxidase to catalyze TMB to quickly detect hydrogen peroxide in the system. At room temperature, different concentrations of H2O2 were added to a mixed solution of acetic acid-sodium acetate buffer (790 mL, 0.2 mM, pH = 3), BPQDs (100 μL, 1 mg / mL) and TMB (100 μL, 10 mM). After standing for 30 min, UV-visible absorption spectra were measured, and the absorbance change was recorded at 652 nm to obtain the absorption spectrum of the above solution.

[0040] (2) Take ordinary laboratory qualitative filter paper and cut it into small test paper squares of 0.5 cm*0.5 cm. Before use, evenly coat and fix it with 0.5 mg / mL sodium alginate solution. Then, evenly coat the test paper with a mixed solution of acetic acid-sodium acetate buffer (790 mL, 0.2 mM, pH = 3), BPQDs (100 μL, 1 mg / mL) and TMB (100 μL, 10 mM). Freeze-dry and store for later use.

[0041] (3) Apply 10 μL of H2O2 of different concentrations onto the prepared test paper squares, wait for 30 min, and then use the Android phone colorimetric software "Color Grab" for color analysis.

[0042] Figure 4 The relationship between the change in UV absorption intensity and the target concentration in the system is shown. The figure shows that the UV absorption intensity increases with the increase in the target concentration (concentrations 1-6 are 10 μM, 20 μM, 50 μM, 100 μM, 150 μM, and 200 μM, respectively), and the absorbance recorded at 652 nm is linearly related to the target concentration in the range of 10~200 μM (with R 2 =0.9916). Meanwhile, within the linear detection range, the detection limit was calculated to be 0.64 μM according to the blank plus 3σ (3 times the standard deviation) method.

[0043] Figure 5The color change of the paper-based visual sensor shown in the test strip under fluorescent light is visible to the naked eye. The H2O2 concentrations from bottom to top are 10 μM, 20 μM, 50 μM, 100 μM, 200 μM and 500 μM. The color difference between the paper-based sensors with different concentrations of H2O2 is evaluated by calculating the B / (R + G + B) ratio. When the H2O2 concentration ranges from 10 to 200 μM, an R 2 = 0.9921, indicating that the dual-mode system can be used for quantitative detection of H2O2.

[0044] Example 3 Selectivity study of a dual-mode non-toxic instant detection biosensor based on black phosphorus quantum dots:

[0045] (1) Prepare a mixed solution of acetic acid-sodium acetate buffer (790 mL, 0.2 mM, pH = 3), BPQDs (100 μL, 1 mg / mL), and TMB (100 μL, 10 mM).

[0046] (2) Take H2O2 and common interferences (NA + , K + 、Zn 2+ , Cu 2+ Mg 2+ , Ca 2+ , lysine, glucose, antibiotics, etc.) are prepared into a 1 mM concentration solution.

[0047] (3) The solutions described in (2) were added to (1) respectively, and different control groups were set up. After half an hour of reaction, the UV-visible light absorption intensity of the system was measured at 652 nm to examine the anti-interference ability of the system.

[0048] like Figure 6 As shown in the figure, after adding H2O2, the UV absorbance of the sensor increased significantly. However, after adding other interferents, no obvious change in UV absorbance intensity was found, which shows that the detection system has high selectivity for detecting H2O2. Therefore, this system can be used as an effective detection system for the detection of H2O2 in different environments.

[0049] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A dual-mode non-toxic instant detection biosensor based on black phosphorus quantum dots, characterized in that: include: Non-toxic colorimetric biosensors, test paper-based visual biosensors; The non-toxic colorimetric biosensor and the test paper-based visual biosensor both use ultra-small black phosphorus quantum dots BPQDs as simulated peroxidase to catalyze TMB to quickly detect hydrogen peroxide in the system; The preparation of ultra-small black phosphorus quantum dots BPQDs includes the following steps: weigh 50 mg of black phosphorus crystals in a glove box, grind them in a mortar for 20 min, add 10 mL of N-methyl-2-pyrrolidone NMP and grind them for another 20 min, transfer them to a double-necked flask, add 40 mL of a saturated alkali-mixed NMP solution prepared by mixing 50 mL of NMP, 100 mg of sodium hydroxide and 1 mg of potassium hydroxide, react at 160 ° C for 10 h, keep stirring vigorously during the reaction, centrifuge at 7000 rpm for 20 min after the reaction is completed, collect the supernatant and dry it for 12 h to obtain BPQDs solid with a particle size of 2.8±0.8 nm; The preparation of the non-toxic colorimetric biosensor comprises the following steps: At room temperature, different concentrations of H2O2 were added to a mixed solution consisting of acetic acid-sodium acetate buffer, BPQDs and TMB. After standing for 30 min, UV-visible absorption spectra were measured. The absorbance change was recorded at 652 nm and the absorption spectrum of the above solution was obtained. The corresponding relationship between the concentration range and the absorbance was obtained for the detection of hydrogen peroxide in the system. The preparation of the test paper-based visual biosensor comprises the following steps: (1) Take ordinary qualitative filter paper from the laboratory, cut it into small test paper squares, and evenly coat and fix it with sodium alginate solution before use. Then, evenly coat the mixed solution composed of acetic acid-sodium acetate buffer, BPQDs and TMB on the test paper, freeze-dry it and store it for later use; (2) During the test, drop H2O2 of different concentrations onto the prepared test paper square, wait for 30 minutes, and then use the Android phone colorimetric software "Color Grab" for color analysis.

2. The dual-mode non-toxic instant detection biosensor based on black phosphorus quantum dots according to claim 1, characterized in that: The mixed solution consisting of acetic acid-sodium acetate buffer, BPQDs and TMB specifically refers to a mixed solution consisting of 790 mL, 0.2 mM, pH = 3 acetic acid-sodium acetate buffer, 100 μL, 1 mg / mL BPQDs solution and 100 μL, 10 mM TMB solution.

3. The dual-mode non-toxic instant detection biosensor based on black phosphorus quantum dots according to claim 1, characterized in that: The grinding time of both steps was no less than 20 min.

4. The dual-mode non-toxic instant detection biosensor based on black phosphorus quantum dots according to claim 1, characterized in that: The saturated alkali-mixed NMP solution was prepared by stirring 50 mL of anhydrous NMP, 100 mg of sodium hydroxide, and 1 mg of potassium hydroxide in a glove box for 10 min.

5. The dual-mode non-toxic instant detection biosensor based on black phosphorus quantum dots according to claim 1, characterized in that: In step (1), the concentration of sodium alginate solution is 0.5 mg / mL and the freeze-drying time is 2 h.

6. The dual-mode non-toxic instant detection biosensor based on black phosphorus quantum dots according to claim 1, characterized in that: The amount of H2O2 added in step (2) is 10 μL.

7. The dual-mode non-toxic instant detection biosensor based on black phosphorus quantum dots according to claim 1, characterized in that: In step (2), the color analysis is performed using the colorimetric software "Color Grab" APP to obtain the three color parameters of red R, green G, and blue B, and the ratio of B / (R + G + B) is used to evaluate the color difference between the paper-based sensors of different concentrations of H2O2 feedback, and then the corresponding relationship between the ratio of B / (R + G + B) and the H2O2 concentration range is obtained for the detection of hydrogen peroxide in the system.

Images