Colorimetric and fluorescent dual-mode nano-enzyme sensor, preparation method thereof and application of colorimetric and fluorescent dual-mode nano-enzyme sensor in hydrogen peroxide detection

Abstract

The invention discloses a colorimetric and fluorescent dual-mode nano-enzyme sensor. The colorimetric and fluorescent dual-mode nano-enzyme sensor comprises nano-enzyme, hydrogen peroxide and a colorimetric probe, wherein the nano-enzyme is prepared from ZrCl4, 2-aminobiphenyl dicarboxylic acid and Cu (NO3) 2 by a one-pot method; the colorimetric probe is TMB, can oxidize colorless TMB into blue oxTMB, generates an ultraviolet absorption peak at 652 nm, and has a strong fluorescence emission peak at 405 + / -5 nm. The zirconium-based metal organic framework UiO-67-NH2 with high stability is used as a carrier, Zr < 4 + > sites are replaced by part of Cu < 2 + >, the fluorescent nano-enzyme with peroxidase activity is formed, and the problems that free enzyme is difficult to recover, the stability is low and the like are solved.

Description

Technical Field

[0001] The present invention belongs to the field of biosensor technology, and in particular relates to a colorimetric and fluorescent dual-mode nanoenzyme sensor, a preparation method thereof, and an application in detecting hydrogen peroxide. Background Art

[0002] Hydrogen peroxide (H2O2) is a commonly used additive and is widely used in the food industry for disinfection, sterilization, and oxidative bleaching. Although food-grade H2O2 is considered safer than other common residues (e.g., pesticides), excessive H2O2 consumption may have some adverse consequences for human health. The biological toxicity of H2O2 is mainly manifested through three pathways: corrosive damage, oxygen release, and lipid peroxidation. Severe exposure to H2O2 can cause irritation and corrosion of the skin, eyes, and respiratory tract, and ingestion of H2O2 may lead to several serious pathological conditions, including abdominal pain, gas embolism, gastric distension, and even death. Therefore, it is urgent to establish a quantitative, rapid, intuitive, and low-cost detection method for hydrogen peroxide, which is crucial to ensure food safety and human health.

[0003] Natural enzymes are a type of environmentally friendly biocatalyst with the advantages of high specificity and high efficiency. However, natural enzymes are not very stable under adverse reaction conditions such as excessive acidity or alkalinity, high temperature, and organic solvents, and their short activity period and difficulty in recovery limit their large-scale industrial application. Compared with natural enzymes, nanozymes make up for the disadvantage of natural enzymes being easily inactivated and have many advantages: (1) high stability, able to maintain catalytic ability under extreme conditions; (2) easy to obtain. Compared with the preparation and purification process of natural enzymes, nanomaterials can be prepared and synthesized by chemical methods, which is simple to operate and low in cost; (3) tunable catalytic ability. In experiments, changing the ratio of raw materials, reaction time, and temperature can optimize the morphology and size of nanomaterials, and their catalytic activity can be improved through different surface modifications.

[0004] With the development of detection technology, various methods based on different principles have been applied to hydrogen peroxide detection, such as chromatography, electrochemical method, fluorescence method, colorimetry, etc. Among these methods, colorimetry has attracted the attention of many researchers due to its advantages such as accurate results and simple operation. Summary of the invention

[0005] In response to the above technical problems, the present invention provides a colorimetric fluorescence dual-mode nanozyme sensor and a preparation method thereof and an application in detecting hydrogen peroxide. The colorimetric fluorescence dual-mode nanozyme sensor can visually and quantitatively detect hydrogen peroxide in real time / on-site with high sensitivity. The detection limits of colorimetry and fluorescence are 0.0057 μM and 0.0020 μM, respectively. The detection process is convenient, fast and low-cost.

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0007] In the first aspect, a colorimetric fluorescence dual-mode nanozyme sensor is characterized in that the colorimetric fluorescence dual-mode nanozyme sensor comprises ZrCl4, Cu(NO3)2 and 2-aminobiphenyldicarboxylic acid to prepare a doped Cu 2+ A metal organic framework, hydrogen peroxide and a colorimetric probe; the colorimetric probe is TMB, the doped Cu 2+ The metal-organic framework is composed of Cu(NO3)2 and UiO-67-NH2, which can oxidize colorless TMB to blue and produce an ultraviolet absorption peak at 652nm, and has a strong fluorescence emission peak at 405±5nm. The metal-organic framework UiO-67-NH2 is prepared using ZrCl4 and 2-aminobiphenyldicarboxylic acid.

[0008] As a preferred embodiment, Cu(NO3)2, ZrCl4 and 2-aminobiphenyl dicarboxylic acid are mixed and stirred to obtain NH2-UiO-67(Zr / Cu), and then the NH2-UiO-67(Zr / Cu) solution, the colorimetric probe TMB solution, and the hydrogen peroxide solution are physically mixed to obtain a colorimetric fluorescence dual-mode nanozyme sensor. After adding hydrogen peroxide, NH2-UiO-67(Zr / Cu) can catalyze the colorless TMB into blue oxTMB as a colorimetric detection signal, and the fluorescence of NH2-UiO-67(Zr / Cu) at 405±5nm is used as a fluorescence detection signal. A colorimetric fluorescence dual-mode nanozyme sensor is constructed based on the oxidative color development reaction. TMB and NH2-UiO-67(Zr / Cu) is affected by the analyte, and the colorimetric signal and the fluorescence intensity change. According to the change of the hydrogen peroxide concentration of the analyte, the colorless TMB undergoes an oxidation reaction to become colorless, and then the ultraviolet absorption intensity of oxTMB and the fluorescence intensity of the sensor at 405nm show a linear relationship with the concentration of the analyte, thereby realizing the quantitative detection of the concentration of hydrogen peroxide in the analyte. The detection limits of the colorimetric and fluorescent dual-mode nanozyme sensor for hydrogen peroxide are 0.0057μM and 0.0020μM, respectively, and hydrogen peroxide in the concentration range of 0-1000μM can be accurately detected; hydrogen peroxide can be visually and quantitatively detected in real time / on-site, making the detection process portable, fast and low-cost, meeting the detection requirements of food safety.

[0009] In a second aspect, the present invention also provides a method for preparing the above-mentioned colorimetric fluorescence dual-mode nanozyme sensor, which specifically comprises the following steps:

[0010] Step 1: Mix a DMF solution of ZrCl4, Cu(NO3)2 and 2-aminobiphenyldicarboxylic acid, and heat to react to obtain a solution A;

[0011] Step 2: Wash, filter and dry the solution A to obtain NH2-UiO-67(Zr / Cu), take 10 mg, add pure water to obtain solution B, and store at 4°C away from light;

[0012] Step 3: Weigh 24 mg of TMB and add anhydrous ethanol to obtain solution C;

[0013] Step 4: preparing a hydrogen peroxide solution, i.e., solution D;

[0014] Step 5: Physically mix solutions B, C, and D, and use buffer to make up the volume. React for 20-30 minutes to obtain the colorimetric and fluorescent dual-mode nanozyme sensor, which is stored at 4°C away from light.

[0015] As a preferred embodiment, the molar mass ratio of ZrCl4, Cu(NO3)2 and 2-aminobiphenyl dicarboxylic acid in solution A in step 1 and step 2 is 1:2:2, the volume of DMF is 10 mL, the DMF solution of ZrCl4 and Cu(NO3)2 is mixed by adding Cu(NO3)2 solution to ZrCl4 at one time and ultrasonically dispersing it uniformly, and the mixed solution of ZrCl4 and Cu(NO3)2 and the DMF solution of 2-aminobiphenyl dicarboxylic acid are mixed by adding the DMF solution of 2-aminobiphenyl dicarboxylic acid to the DMF solution for 3 seconds. A mixed solution of ZrCl4 and Cu(NO3)2 in DMF is added dropwise at a rate of one drop, the heating temperature is 80°C, the rotation speed is 200 rpm, the concentration of the DMF solution of ZrCl4 is 0.1 mol / L, the concentration of the DMF solution of Cu(NO3)2 is 0.2 mol / L, the concentration of the DMF solution of 2-aminoterephthalic acid is 0.2 mol / L, the volume of solution D in step 4 is 10 mL, the detergent used for washing is anhydrous ethanol, and the drying method is oven drying at a temperature range of 60-75°C.

[0016] As a preferred embodiment, the concentration of solution E in step 5 is 10 mM; the concentration of solution F in step 6 is 100 mM; the buffer solution in step 7 is acetic acid-sodium acetate buffer solution; the volume ratio of solutions D, E, and F is 1:1:1, the pH of the buffer solution is 4, the reaction system is dissolved to 3 mL, and the volumes of solutions D, E, and F in step 7 are 100 μL, 100 μL, and 100 μL, respectively.

[0017] In a third aspect, the present invention also provides the use of the above-mentioned colorimetric fluorescence dual-mode nanozyme sensor or the colorimetric fluorescence dual-mode nanozyme sensor obtained by the preparation method of the above-mentioned colorimetric fluorescence dual-mode nanozyme sensor in the detection of hydrogen peroxide.

[0018] In a fourth aspect, the present invention also provides a method for detecting hydrogen peroxide using a colorimetric fluorescence dual-mode nanozyme sensor, which specifically comprises the following steps:

[0019] Step S1: The colorimetric fluorescence dual-mode nanozyme sensor is mixed with a series of hydrogen peroxide solutions of different concentrations in proportion to prepare a reaction system. After reacting at 25-30°C for 20-30 minutes, a UV-visible spectrophotometer and a fluorescence spectrometer are used for spectral detection. Through the changes in ultraviolet absorption of the colorimetric fluorescence dual-mode nanozyme sensor at 652nm and the changes in fluorescence intensity at 405nm, a calibration curve is drawn with the logarithm of the hydrogen peroxide concentration as the horizontal coordinate and the ultraviolet peak value of the colorimetric fluorescence dual-mode nanozyme sensor at 652nm and the fluorescence intensity peak value at 405nm as the vertical coordinate;

[0020] Step S2: Mix the colorimetric fluorescence dual-mode nanozyme sensor with the hydrogen peroxide solution to be tested in proportion to form a reaction system. After reacting at 25-30°C for 20-30 minutes, use a UV-visible spectrophotometer and a fluorescence spectrometer to perform spectral detection. Through the changes in ultraviolet absorption of the colorimetric fluorescence dual-mode nanozyme sensor at 652nm and the changes in fluorescence intensity at 405nm, the hydrogen peroxide concentration in the sample is calculated according to the calibration curve obtained in step 1 to achieve detection of hydrogen peroxide.

[0021] As a preferred embodiment, the preparation method of the reaction system of step S1 and step S2 includes: adding 100 μL NH2-UiO-67 (Zr / Cu) solution, 100 μL TMB solution and 100 μL hydrogen peroxide solution, and using NaAc-HAc buffer with a pH value of 4.0 to make a 3 mL reaction system; the hydrogen peroxide solution uses ultrapure water as a solvent, pH 7-7.5, the TMB solution concentration is 10 mM, and the solvent is anhydrous ethanol; the concentration of the hydrogen peroxide solution is 100 mM.

[0022] As a preferred embodiment, a UV-visible spectrophotometer and a fluorescence spectrometer are used for spectral detection, and the absorption value A at the UV absorption peak at 652 nm is recorded. 652 , fluorescence intensity I at 405 nm 405 , using the UV absorption peak value and the fluorescence intensity I at 405nm 405 A calibration curve is obtained by a functional relationship between the colorimetric and fluorescent dual-mode nanozyme sensor and the concentration of hydrogen peroxide. The concentration of hydrogen peroxide to be measured is calculated using the calibration curve. The colorimetric and fluorescent dual-mode nanozyme sensor appears colorless and emits bright green fluorescence in the absence of hydrogen peroxide. As the concentration of hydrogen peroxide increases, the ultraviolet absorption intensity at 652nm increases, and the color gradually changes from colorless to blue; the fluorescence intensity at 405nm decreases, and the bright green fluorescence intensity gradually weakens until there is no fluorescence, thereby realizing colorimetric and fluorescent dual-mode detection of hydrogen peroxide content.

[0023] As a preferred embodiment, in the presence of hydrogen peroxide and under acidic conditions, NH2-UiO-67 (Zr / Cu) can oxidize colorless TMB into blue oxTMB; due to the ion activation in the buffer solution, NH2-UiO-67 (Zr / Cu) can emit bright green fluorescence at 405nm. When the amount of oxTMB produced increases, the fluorescence intensity of NH2-UiO-67 (Zr / Cu) decreases due to the inner filter effect; the ultraviolet absorption value at 652nm and the fluorescence intensity at 405nm of the colorimetric fluorescence dual-mode nanozyme sensor are linearly related to the concentration of the object to be detected, thereby realizing the detection of the concentration of hydrogen peroxide in the substance to be detected, and the conditions of the ultraviolet-visible light spectrum include: the observation range of the ultraviolet-visible spectrum is 550-750nm; the fluorescence excitation wavelength is 305-315nm, and the fluorescence emission spectrum observation range is 332-500nm.

[0024] The technical principle of the present invention is: in the presence of hydrogen peroxide, NH2-UiO-67 (Zr / Cu) can catalyze the colorless TMB into blue oxTMB as a colorimetric detection signal, and the fluorescence of NH2-UiO-67 (Zr / Cu) is used as a fluorescence detection signal. Based on the oxidation color development reaction and the fluorescence shutdown mechanism, the NH2-UiO-67 (Zr / Cu) colorimetric fluorescence dual-mode nanozyme sensor is constructed. TMB and NH2-UiO-67 (Zr / Cu) are affected by the object to be detected, and the colorimetric signal and fluorescence intensity change. According to the change in the concentration of hydrogen peroxide to be detected, the colorless TMB undergoes an oxidation reaction to turn blue, and the fluorescence intensity of NH2-UiO-67 (Zr / Cu) decreases. Then, the change in the ultraviolet absorption intensity of oxTMB and the fluorescence intensity of NH2-UiO-67 (Zr / Cu) present a linear relationship with the concentration of the object to be detected, thereby realizing the quantitative detection of the concentration of hydrogen peroxide in the substance to be detected.

[0025] Advantages of the present invention:

[0026] 1. The present invention uses a highly stable zirconium-based metal organic framework UiO-67-NH2 as a carrier to 2+ Doping on its surface solves the problems of difficulty in recovering free enzymes and low stability;

[0027] 2. The colorimetric fluorescence dual-mode nanozyme sensor constructed based on NH2-UiO-67 (Zr / Cu) of the present invention integrates the excellent catalytic ability of natural enzymes and the luminescent properties of UiO-67-NH2 into one system. It has a simple design, convenient operation, high selectivity and detection stability for hydrogen peroxide, and reduces detection time and cost;

[0028] 3. The hydrogen peroxide detection method established by the present invention has high detection sensitivity, and the detection limits are 0.0057 μM and 0.0020 μM, respectively, which meet the relevant requirements of national standards and have good anti-interference and stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are 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.

[0030] Figure 1 It is the SEM image of UiO-67-NH2 in Example 1 of the present invention and NH2-UiO-67 (Zr / Cu) in Example 2.

[0031] Figure 2 This is the EDS image of NH2-UiO-67 (Zr / Cu) in Example 2 of the present invention.

[0032] Figure 3 It is the XPS graph of UiO-67-NH2 in Example 1 of the present invention and NH2-UiO-67 (Zr / Cu) in Example 2.

[0033] Figure 4 It is a particle size distribution diagram of UiO-67-NH2 in Example 1 and NH2-UiO-67 (Zr / Cu) in Example 2 of the present invention.

[0034] Figure 5 This is a verification diagram of the catalytic ability of NH2-UiO-67 (Zr / Cu) in Verification Example 1 of the present invention.

[0035] Figure 6 This is a daylight image of the colorimetric-fluorescent dual-mode nanozyme sensor solution at different concentrations of hydrogen peroxide in Example 4 of the present invention.

[0036] Figure 7 It is a fluorescence image of the colorimetric and fluorescence dual-mode nanozyme sensor solution under different concentrations of hydrogen peroxide in Example 4 of the present invention.

[0037] Figure 8 It is the ultraviolet-visible spectrum and fluorescence emission spectrum of the colorimetric-fluorescent dual-mode nanozyme sensor solution under different concentrations of hydrogen peroxide in Example 4 of the present invention.

[0038] Fig. 9 This is the colorimetric detection curve of the colorimetric and fluorescent dual-mode nanozyme sensor solution under different concentrations of hydrogen peroxide in Example 4 of the present invention.

[0039] Fig.10 1 is the fluorescence detection curve of the colorimetric and fluorescence dual-mode nanozyme sensor solution under different concentrations of hydrogen peroxide in Example 4 of the present invention.

[0040] Fig.11 This is an analysis diagram of the selectivity and anti-interference properties of the colorimetric-fluorescence dual-mode nanozyme sensor in Verification Example 2 of the present invention. DETAILED DESCRIPTION

[0041] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0042] Unless otherwise specified, all raw materials used in the examples are commercially available conventional raw materials, and the technical means used are conventional means well known to those skilled in the art.

[0043] Example 1

[0044] This embodiment first provides a MOF material, and its synthesis method includes the following steps: 23 mg ZrCl4 and 51.5 mg 2-aminobiphenyl dicarboxylic acid are respectively dissolved in 10 mL DMF, and ultrasonic treatment is performed until they are completely dissolved.

[0045] The 2-aminobiphenyldicarboxylic acid solution was poured into a 50 mL eggplant bottle, placed in silicone oil at 120° C., and stirred at 200 rpm using a magnetic stirrer.

[0046] Add 400 μL of acetic acid, and then slowly drop the DMF solution of ZrCl4 into the eggplant bottle. After the addition is completed, stir for 30 minutes, turn off the magnetic stirrer, and keep heating at 120°C for 12 hours. After the heating is completed, extract the eggplant bottle from the silicone oil bath, wait for the solution to cool to room temperature, and transfer the solution equally to two 50mL centrifuge tubes, 10mL of solution in each tube, and then add 10mL of anhydrous ethanol respectively, and centrifuge at 12000rpm, 4°C, for 20min.

[0047] Remove the supernatant, add 10 mL of anhydrous ethanol, centrifuge, and repeat this step 3 times. Place the final precipitate in a 75°C oven until it is completely dry to obtain UiO-67-NH2 material.

[0048] UiO-67-NH2 was characterized. Figure 1 a is the SEM image of UiO-67-NH2. It can be seen from the figure that the synthesized UiO-67-NH2 is uniform in size. Figure 4It is the particle size distribution diagram of UiO-67-NH2.

[0049] Example 2

[0050] This embodiment first provides a MOF material, and its synthesis method includes the following steps: 23 mg ZrCl4, 37.6 mg Cu(NO3)2 and 51.5 mg 2-aminobiphenyldicarboxylic acid are respectively dissolved in 10 mL DMF, and ultrasonic treatment is performed until they are completely dissolved.

[0051] Add 5 mL of Cu(NO3)2 DMF solution into the ZrCl4 DMF solution at one time and perform ultrasonication until uniform dispersion

[0052] The 2-aminobiphenyldicarboxylic acid solution was poured into a 50 mL eggplant bottle, placed in silicone oil at 120° C., and stirred at 200 rpm using a magnetic stirrer.

[0053] Add 400 μL of acetic acid, and then slowly drop the DMF mixed solution of ZrCl4 and Cu(NO3)2 into the eggplant bottle.

[0054] After the addition was completed, the mixture was stirred for 30 min, the magnetic stirrer was turned off, and heating was maintained at 120 °C for 12 h.

[0055] After heating, extract the eggplant bottle from the silicone oil bath. After the solution cools to room temperature, transfer the solution equally into two 50 mL centrifuge tubes, 12.5 mL of solution in each tube. Then add 10 mL of anhydrous ethanol to each tube and centrifuge at 12000 rpm, 4°C, for 20 min.

[0056] Remove the supernatant, add 10 mL of anhydrous ethanol, centrifuge, and repeat this step 3 times.

[0057] The final precipitate was placed in an oven at 75°C until completely dried to obtain NH2-UiO-67 (Zr / Cu) material.

[0058] Characterize NH2-UiO-67(Zr / Cu). Figure 1 b is the SEM image of NH2-UiO-67(Zr / Cu). It can be seen from the figure that the synthesized NH2-UiO-67(Zr / Cu) is uniform in size and Figure 1 a has a similar structure to UiO-67-NH2. Figure 2 It can also be seen that the characteristic element Cu is evenly distributed on NH2-UiO-67(Zr / Cu), which also reflects the successful preparation of NH2-UiO-67(Zr / Cu). Figure 3It can also be found that NH2-UiO-67(Zr / Cu) contains the characteristic element Cu compared with UiO-67-NH2, which further confirms that NH2-UiO-67(Zr / Cu) was successfully synthesized. Figure 4 Particle size distribution of NH2-UiO-67(Zr / Cu).

[0059] Verification Example 1

[0060] In order to verify the catalytic activity of NH2-UiO-67(Zr / Cu), 10 mg of NH2-UiO-67(Zr / Cu) obtained in Example 2 was dissolved in 10 mL of ultrapure water and diluted 100 times.

[0061] Then, 100 μL of UiO-67-Cu solution, 100 μL of TMB solution and 100 μL of hydrogen peroxide solution were taken, and the volume was adjusted to 3 mL using NaAc-HAc buffer with a pH value of 4.0. After reacting for 15-30 minutes, the ultraviolet absorption intensity at 652 nm was recorded using a UV-visible spectrophotometer. At the same time, 100 μL of UiO-67 solution was taken, 100 μL of TMB solution and 100 μL of hydrogen peroxide solution were added, and the volume was adjusted to 3 mL using NaAc-HAc buffer with a pH value of 4.0. After reacting for 15-30 minutes, the ultraviolet absorption intensity at 652 nm was recorded using a UV-visible spectrophotometer, and the absorbance values ​​of UiO-67-NH2, TMB, hydrogen peroxide and a mixed solution of TMB and hydrogen peroxide at 652 nm were measured.

[0062] like Figure 2 It shows that NH2-UiO-67(Zr / Cu) still retains a high catalytic activity, and its catalytic activity comes from the doping of Cu 2+ The nanozymes formed later. Figure 5 Verification diagram of the catalytic ability of NH2-UiO-67(Zr / Cu).

[0063] Example 3

[0064] This embodiment further provides a method for preparing a colorimetric and fluorescent dual-mode nanozyme sensor, which specifically comprises the following steps:

[0065] 10 mg of NH2-UiO-67 (Zr / Cu) obtained in Example 2 was dissolved in 10 mL of ultrapure water and diluted 100 times. Then 100 μL of NH2-UiO-67 (Zr / Cu) solution, 100 μL of TMB solution and 100 μL of hydrogen peroxide solution were taken, and the volume was adjusted to 3 mL using NaAc-HAc buffer with a pH value of 4.0, and the reaction was carried out for 15-30 minutes to obtain a colorimetric fluorescence dual-mode nanozyme sensor.

[0066] The concentration of the TMB solution is 10 mM, the solvent is anhydrous ethanol; the concentration of the hydrogen peroxide solution is 100 mM.

[0067] Example 4

[0068] This embodiment provides a colorimetric and fluorescent dual-mode nanozyme detection method for detecting hydrogen peroxide, which specifically includes the following steps:

[0069] 100 μL of hydrogen peroxide solution of different concentrations to be tested was added to the colorimetric fluorescence dual-mode nanozyme sensor prepared in Example 3 to prepare a 3 mL reaction system, and the color change of the solution was observed under sunlight and ultraviolet light sources.

[0070] Figure 6 This is a daylight image of the colorimetric fluorescence dual-mode nanozyme sensor solution under different hydrogen peroxide concentrations. It can be seen from the figure that as the concentration of sodium hydrogen peroxide increases, the color of the solution gradually changes from colorless to blue.

[0071] The UV-visible absorption spectrum and the fluorescence emission spectrum were measured using a UV-visible spectrophotometer and a fluorescence spectrophotometer. The observation range of the UV-visible spectrum was 550-750nm; the fluorescence excitation wavelength was 305-315nm, and the observation range of the fluorescence emission spectrum was 332-500nm.

[0072] Figure 7 It is a fluorescence image of the colorimetric and fluorescence dual-mode nanozyme sensor solution under different concentrations of hydrogen peroxide in Example 4 of the present invention.

[0073] Figure 8 is the UV-visible spectrum and fluorescence emission spectrum of the colorimetric fluorescence dual-mode nanozyme sensor solution under different concentrations of hydrogen peroxide, where Figure 8 a is the UV-visible spectrum, Figure 8 b is the fluorescence emission spectrum. The functional relationship between the different concentrations of hydrogen peroxide values ​​of the solution and the UV-visible absorption peak and the fluorescence emission peak values ​​obtains the concentration of hydrogen peroxide values ​​corresponding to the UV-visible absorption peak and the fluorescence emission peak values ​​in the solution.

[0074] A 652 : The absorption value of the sensor solution corresponding to the UV-visible absorption spectrum at 652nm;

[0075] A 652 The UV-visible absorption peak near 652nm was monitored by multiple parallel experiments and the average value was calculated.

[0076] The functional relationship between the hydrogen peroxide values ​​of different concentrations and the UV-visible absorption peak values ​​of the solution is used to obtain the hydrogen peroxide concentration value corresponding to the UV-visible absorption peak value in the solution.

[0077] Fig. 9 This is the colorimetric detection curve of the colorimetric fluorescence dual-mode nanozyme sensor solution under different concentrations of hydrogen peroxide, where the x-axis is the hydrogen peroxide concentration value and the y-axis is the UV-visible absorption peak.

[0078] like Figure 8 As shown, the functional relationship between the UV-visible absorption peak value and the hydrogen peroxide concentration is: y = 0.0013x + 0.0166 (R 2 =0.99), where y represents the UV-visible absorption peak at 652 nm and x represents the hydrogen peroxide concentration.

[0079] The fluorescence emission peak is: 405 .

[0080] I 405 : The fluorescence intensity at 405nm in the fluorescence emission spectrum of the sensor solution; the emission peak near 405±5nm was monitored by multiple parallel experiments and the average value was calculated to obtain

[0081] The functional relationship between different hydrogen peroxide concentration values ​​of the solution and the fluorescence emission peak value is used to obtain the hydrogen peroxide concentration value corresponding to the fluorescence emission peak value in the solution.

[0082] Fig.10 This is the fluorescence detection curve of the colorimetric fluorescence dual-mode nanozyme sensor solution under different concentrations of hydrogen peroxide. The x-axis is the hydrogen peroxide concentration and the y-axis is the fluorescence intensity value.

[0083] like Fig.10 As shown, the functional relationship between the fluorescence emission peak and the sodium nitrite concentration is: y = -2.4558x + 1778.0220 (R 2 =0.99), where y represents the peak fluorescence intensity at 405 nm and x represents the logarithmic value of the hydrogen peroxide concentration.

[0084] Verification Example 2

[0085] According to Example 3, a colorimetric and fluorescent dual-mode nanozyme sensor was prepared, and Na 2+ Mg 2+ , K + , Ca 2+ , His, Suc, Glu, Arg, etc. were used as interfering ions to explore the selectivity and anti-interference ability of the colorimetric fluorescence dual-mode nanozyme sensor in detecting hydrogen peroxide. The concentration of the interfering ions was 100 times that of the hydrogen peroxide solution. After reacting at 25-30°C for 20-30 minutes, spectral detection was carried out using a UV-visible spectrophotometer and a fluorescence spectrometer. The colorimetric fluorescence dual-mode nanozyme sensor was detected by the changes in UV absorption at 652nm and the changes in fluorescence intensity at 405nm.

[0086] Fig.11 a is the selectivity and anti-interference analysis of the colorimetric mode, Fig.11 b is the selectivity and anti-interference analysis of the fluorescence mode. It can be seen from the figure that the ultraviolet absorption value and fluorescence intensity of the colorimetric fluorescence dual-mode nanozyme sensor change significantly only in the presence of hydrogen peroxide, indicating that the sensor has good selectivity and anti-interference for hydrogen peroxide detection.

[0087] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

Claims

1. A colorimetric fluorescence dual-mode nanozyme sensor, characterized in that: The colorimetric fluorescence dual-mode nanozyme sensor comprises ZrCl4, Cu(NO3)2 and 2-aminobiphenyldicarboxylic acid to prepare a Cu doped 2+ A metal organic framework, hydrogen peroxide and a colorimetric probe; the colorimetric probe is TMB, the doped Cu 2+ The metal-organic framework is composed of Cu(NO3)2 and UiO-67-NH2, which can oxidize colorless TMB to blue and produce an ultraviolet absorption peak at 652nm, and has a strong fluorescence emission peak at 405±5nm. The metal-organic framework UiO-67-NH2 is prepared using ZrCl4 and 2-aminobiphenyldicarboxylic acid.

2. A colorimetric fluorescence dual-mode nanozyme sensor according to claim 1, characterized in that: Cu(NO3)2, ZrCl4 and 2-aminobiphenyl dicarboxylic acid were mixed and stirred to obtain NH2-UiO-67(Zr / Cu), and then the NH2-UiO-67(Zr / Cu) solution, the colorimetric probe TMB solution and the hydrogen peroxide solution were physically mixed to obtain a colorimetric fluorescence dual-mode nanozyme sensor. After adding hydrogen peroxide, NH2-UiO-67(Zr / Cu) can catalyze the colorless TMB into blue oxTMB as a colorimetric detection signal, and the fluorescence of NH2-UiO-67(Zr / Cu) at 405±5nm is used as a fluorescence detection signal. A colorimetric fluorescence dual-mode nanozyme sensor is constructed based on the oxidative color development reaction. The device is used to detect the colorimetric signal and fluorescence intensity of TMB and NH2-UiO-67 (Zr / Cu) under the influence of the analyte. The colorimetric signal and fluorescence intensity change. According to the change of the concentration of hydrogen peroxide in the analyte, the colorless TMB undergoes an oxidation reaction to become colorless. Then, the ultraviolet absorption intensity of oxTMB and the fluorescence intensity of the sensor at 405 nm show a linear relationship with the concentration of the analyte, thereby realizing the quantitative detection of the concentration of hydrogen peroxide in the analyte. The detection limits of the colorimetric and fluorescent dual-mode nanozyme sensor for hydrogen peroxide are 0.0057 μM and 0.0020 μM, respectively, and the hydrogen peroxide concentration range of 0-1000 μM can be accurately detected. Real-time / on-site visual quantitative detection of hydrogen peroxide can be achieved, making the detection process portable, rapid and low-cost, meeting the testing needs of food safety.

3. The method for preparing a colorimetric and fluorescent dual-mode nanozyme sensor according to claim 1, characterized in that: The steps include: Step 1: Mix a DMF solution of ZrCl4, Cu(NO3)2 and 2-aminobiphenyldicarboxylic acid, and heat to react to obtain a solution A; Step 2: Wash, filter and dry the solution A to obtain NH2-UiO-67(Zr / Cu), take 10 mg, add pure water to obtain solution B, and store at 4°C away from light; Step 3: Weigh 24 mg of TMB and add anhydrous ethanol to obtain solution C; Step 4: preparing a hydrogen peroxide solution, i.e., solution D; Step 5: Physically mix solutions B, C, and D, and use buffer to make up the volume. React for 20-30 minutes to obtain the colorimetric and fluorescent dual-mode nanozyme sensor, which is stored at 4°C away from light.

4. The method for preparing a colorimetric-fluorescence dual-mode nanozyme sensor according to claim 3, characterized in that: The molar mass ratio of ZrCl4, Cu(NO3)2 and 2-aminobiphenyldicarboxylic acid in solution A in step 1 and step 2 is 1:2:2, the volume of DMF is 10 mL, the DMF solution of ZrCl4 and Cu(NO3)2 is mixed by adding Cu(NO3)2 solution to ZrCl4 at one time and ultrasonically dispersing it uniformly, and the mixed solution of ZrCl4 and Cu(NO3)2 and the DMF solution of 2-aminobiphenyldicarboxylic acid are mixed by adding one drop of ZrCl4 to the DMF solution of 2-aminobiphenyldicarboxylic acid at a speed of one drop per 3 seconds. A DMF mixed solution of ZrCl4 and Cu(NO3)2 is added dropwise at a heating temperature of 80°C and a rotation speed of 200 rpm. The concentration of the DMF solution of ZrCl4 is 0.1 mol / L, the concentration of the DMF solution of Cu(NO3)2 is 0.2 mol / L, and the concentration of the DMF solution of 2-aminoterephthalic acid is 0.2 mol / L. The volume of solution D in step 4 is 10 mL. The detergent used for washing is anhydrous ethanol. The drying method adopts oven drying at a temperature range of 60-75°C.

5. The method for preparing a colorimetric and fluorescent dual-mode nanozyme sensor according to claim 3, characterized in that: The concentration of solution C in step 3 is 10 mM; the concentration of solution D in step 4 is 100 mM; the buffer solution in step 5 is acetic acid-sodium acetate buffer solution; the volume ratio of solutions B, C, and D is 1:1:1, the pH of the buffer solution is 4, the reaction system is dissolved to 3 mL, and the volumes of solutions B, C, and D in step 5 are 100 μL, 100 μL, and 100 μL, respectively.

6. The method for preparing a colorimetric-fluorescence dual-mode nanozyme sensor according to claim 3, characterized in that: Application of the colorimetric fluorescence dual-mode nanozyme sensor obtained by the preparation method of the colorimetric fluorescence dual-mode nanozyme sensor in hydrogen peroxide detection.

7. The method for detecting hydrogen peroxide using a colorimetric fluorescence dual-mode nanozyme sensor according to claim 1, characterized in that: The specific steps include: Step S1: The colorimetric fluorescence dual-mode nanozyme sensor is mixed with a series of hydrogen peroxide solutions of different concentrations in proportion to prepare a reaction system. After reacting at 25-30°C for 20-30 minutes, a UV-visible spectrophotometer and a fluorescence spectrometer are used for spectral detection. Through the changes in ultraviolet absorption of the colorimetric fluorescence dual-mode nanozyme sensor at 652nm and the changes in fluorescence intensity at 405nm, a calibration curve is drawn with the logarithm of the hydrogen peroxide concentration as the horizontal coordinate and the ultraviolet peak value of the colorimetric fluorescence dual-mode nanozyme sensor at 652nm and the fluorescence intensity peak value at 405nm as the vertical coordinate; Step S2: Mix the colorimetric fluorescence dual-mode nanozyme sensor with the hydrogen peroxide solution to be tested in proportion to form a reaction system. After reacting at 25-30°C for 20-30 minutes, use a UV-visible spectrophotometer and a fluorescence spectrometer to perform spectral detection. Through the changes in ultraviolet absorption of the colorimetric fluorescence dual-mode nanozyme sensor at 652nm and the changes in fluorescence intensity at 405nm, the hydrogen peroxide concentration in the sample is calculated according to the calibration curve obtained in step 1 to achieve detection of hydrogen peroxide.

8. The method for detecting hydrogen peroxide using a colorimetric fluorescence dual-mode nanozyme sensor according to claim 7, characterized in that: The preparation method of the reaction system of step S1 and step S2 includes: adding 100 μL NH2-UiO-67 (Zr / Cu) solution, 100 μL TMB solution and 100 μL hydrogen peroxide solution, and using NaAc-HAc buffer with a pH value of 4.0 to make a 3 mL reaction system; the hydrogen peroxide solution uses ultrapure water as a solvent, pH 7-7.5, the TMB solution concentration is 10 mM, and the solvent is anhydrous ethanol; the hydrogen peroxide solution concentration is 100 mM.

9. The method for detecting hydrogen peroxide using a colorimetric fluorescence dual-mode nanozyme sensor according to claim 7, characterized in that: The spectrum was detected by UV-visible spectrophotometer and fluorescence spectrometer, and the absorption value A at the UV absorption peak of 652 nm was recorded. 652 , fluorescence intensity I at 405 nm 405 , using the UV absorption peak value and the fluorescence intensity I at 405nm 405 A calibration curve is obtained by a functional relationship between the colorimetric and fluorescent dual-mode nanozyme sensor and the concentration of hydrogen peroxide. The concentration of hydrogen peroxide to be measured is calculated using the calibration curve. The colorimetric and fluorescent dual-mode nanozyme sensor appears colorless and emits bright green fluorescence in the absence of hydrogen peroxide. As the concentration of hydrogen peroxide increases, the ultraviolet absorption intensity at 652nm increases, and the color gradually changes from colorless to blue; the fluorescence intensity at 405nm decreases, and the bright green fluorescence intensity gradually weakens until there is no fluorescence, thereby realizing colorimetric and fluorescent dual-mode detection of hydrogen peroxide content.

10. The method for detecting hydrogen peroxide using a colorimetric fluorescence dual-mode nanozyme sensor according to claim 7, characterized in that: In the presence of hydrogen peroxide and under acidic conditions, NH2-UiO-67 (Zr / Cu) can oxidize colorless TMB into blue oxTMB. Due to the ion activation in the buffer solution, NH2-UiO-67 (Zr / Cu) can emit bright green fluorescence at 405 nm. When the amount of oxTMB produced increases, the fluorescence intensity of NH2-UiO-67 (Zr / Cu) decreases due to the inner filter effect. The ultraviolet absorption value at 652 nm and the fluorescence intensity at 405 nm of the colorimetric fluorescence dual-mode nanozyme sensor are linearly related to the concentration of the detected object, thereby realizing the detection of the concentration of hydrogen peroxide in the test substance. The conditions of the ultraviolet-visible light spectrum include: the observation range of the ultraviolet-visible spectrum is 550-750 nm; the fluorescence excitation wavelength is 305-315 nm, and the fluorescence emission spectrum observation range is 332-500 nm.

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