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

Abstract

The invention provides a colorimetric / fluorescent dual-mode nano-enzyme sensor, a preparation method thereof and application of the colorimetric / fluorescent dual-mode nano-enzyme sensor in formaldehyde detection. The sensor comprises a metal organic framework nano-enzyme AIE-MOF coated PCN-222 loaded with an AIE-MOF material, hydrogen peroxide and a chromogenic substrate o-phenylenediamine OPD; the metal organic framework nano enzyme PCN-222 in the AIE-MOF-coated PCN-222 can oxidize colorless OPD into yellow 2, 3-diaminophenazine DAP, an ultraviolet absorption peak is generated at the position of 450 nm, a strong fluorescence emission peak is generated at the position of 560 nm, the AIE-MOF-coated PCN-222 has a strong fluorescence emission peak at the position of 446 nm, and the AIE-MOF-coated PCN-222 and a fluorescence signal of the DAP can form a ratio fluorescence sensor. According to the invention, the excellent catalytic ability of the nano-enzyme and the luminescence characteristic of AIE-MOF are integrated in one system, so that high-sensitivity formaldehyde detection is realized.

Description

Technical Field

[0001] This invention belongs to the field of biosensing technology, and particularly relates to a colorimetric / fluorescence dual-mode nanoenzyme sensor, its preparation method, and its application in formaldehyde detection. Background Technology

[0002] Formaldehyde (HCHO) is a highly reactive and toxic carbonyl compound that poses a serious and ongoing threat to global food safety and public health. According to the International Agency for Research on Cancer (IARC), formaldehyde is classified as a Group 1 carcinogen because it has a clear link to nasopharyngeal carcinoma, leukemia, and other malignant tumors. However, the illegal use of formaldehyde as a preservative in various foods, such as seafood, dairy products, fruits, and vegetables, remains a concern. These practices aim to extend shelf life, enhance appearance, or mask spoilage. In addition to the carcinogenic risk, formaldehyde can cause acute toxic reactions, including gastrointestinal discomfort, respiratory complications, neurological damage, and organ failure. Therefore, the sensitive, specific, rapid, and reliable detection of trace formaldehyde residues in complex environments is a significant challenge in food safety monitoring and the medical field. Innovative analytical methods, especially those suitable for on-site screening, are urgently needed to prevent contaminated products from entering the supply chain.

[0003] Natural enzymes, as environmentally friendly biocatalysts, have advantages such as high selectivity and high catalytic efficiency. However, under harsh conditions such as strong acids / bases, high temperatures, and organic solvents, natural enzymes are unstable, have short lifespans, and are difficult to recycle, which limits their industrial-scale application. In contrast, nanozymes (biomimetic enzymes made of nanomaterials) overcome the shortcomings of natural enzymes, which are prone to inactivation, and have the following advantages: (1) High stability: They can maintain catalytic activity even in extreme pH, high temperature, and organic solvent environments; (2) Easy to obtain and low cost: Compared with the complex preparation and purification process of natural enzymes, nanozymes can be conveniently prepared through chemical synthesis, with a simple process and easy scale-up; (3) Controllable catalytic performance: By adjusting synthesis parameters such as precursor ratio, reaction time, and temperature, their morphology and size can be precisely controlled, and their catalytic activity can be further improved through surface modification. The AIE effect (aggregation-induced emission) refers to the photophysical phenomenon where molecules emit almost no light in dilute solutions, but their luminescence is significantly enhanced when aggregated or in the solid state. Unlike traditional fluorescent materials, which suffer from reduced luminescence intensity due to aggregation-induced quenching (ACQ) in the condensed or solid state, AIE-MOFs exhibit the opposite behavior: they emit weak light in solution but strong fluorescence in the aggregated or solid state. This inherent AIE property makes them particularly suitable for integration into solid-state MOF structures for robust sensing applications.

[0004] With the development of detection technology, various methods based on different principles have been applied to formaldehyde detection, such as chromatography, electrochemical methods, fluorescence methods, and colorimetry. Among these methods, colorimetry and fluorescence methods have attracted much attention from researchers due to their advantages such as accurate results and ease of operation. Summary of the Invention

[0005] To address the above technical problems, this invention provides a colorimetric / fluorescence dual-mode nanozyme sensor, its preparation method, and its application in formaldehyde detection. This colorimetric / fluorescence dual-mode nanozyme sensor can quantitatively detect formaldehyde in real time / on-site visually, with high sensitivity. The detection limits for the colorimetric method and the fluorescence method are 14.5 nM and 1.78 nM, respectively. The detection process is convenient, fast, and inexpensive.

[0006] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: In a first aspect, a colorimetric / fluorescence dual-mode nanozyme sensor is characterized by comprising an AIE metal-organic framework nanozyme (AIE-MOF@PCN-222) loaded with aggregation-induced emission metal-organic framework (AIE-MOF) material, hydrogen peroxide, and a chromogenic substrate o-phenylenediamine (OPD); wherein the AIE-MOF@PCN-222 is a zirconium-based metal-organic framework nanozyme PCN-222 material loaded with AIE-MOF, wherein the AIE-MOF is formed by the self-assembly of zirconium metal ions and the organic ligand tetrakis[4-(3,5-dicarboxyphenyl)]tetraphenylethylene, and the PCN-222 is formed by the self-assembly of zirconium metal ions and tetracarboxyphenylporphyrin iron; the PCN-222 nanozyme in the AIE-MOF@PCN-222 can oxidize the colorless chromogenic substrate o-phenylenediamine OPD to a yellow 2,3-diaminophenazine (DAP), generating an ultraviolet absorption peak at 450±25 nm and a peak at 560±5 nm. The AIE-MOF exhibits a strong fluorescence emission peak at 446 ± 10 nm, and can be combined with the fluorescence signal of 2,3-diaminophenazine DAP to form a ratiometric fluorescence sensor.

[0007] According to the present invention, the AIE-MOF@PCN-222 is composed of a MOF-on-MOF structure, with the core being a metal-organic framework nanozyme PCN-222 having peroxidase properties and AIE-MOF loaded on the surface, thus retaining both the peroxidase properties of PCN-222 and the fluorescence properties of AIE-MOF.

[0008] According to the present invention, the organic ligand tetra[4-(3,5-dicarboxyphenyl)]tetraphenylethylene with an AIE group is used with zirconium (Zr) metal ions (Zr 4+Zirconium-based AIE-MOF materials are formed by the self-assembly of zirconium ion clusters, exhibiting stronger fluorescence emission in the aggregated state compared to other MOF materials. This invention employs tetratetra[4-(3,5-dicarboxyphenyl)]tetraphenylethylene ligands and zirconium ion clusters to form zirconium-based AIE-MOF materials. These materials trigger strong fluorescence emission in the aggregated state, with the emission peak overlapping the excitation spectrum of the catalytic product DAP, achieving efficient energy transfer. Furthermore, the multi-carboxyl structure enhances coordination stability and PCN-222 surface loading capacity, maintaining structural integrity in the liquid-phase detection system, and possessing both high-sensitivity fluorescence response and excellent stability. According to the present invention, the organic ligand tetracarboxyphenylporphyrin iron with peroxidase activity is combined with zirconium (Zr) metal ions (Zr... 4+ Clusters self-assemble to form metal-organic framework nanozyme material PCN-222, which can produce peroxidase-like activity. This invention uses PCN-222 as the core and loads AIE-MOF, fully leveraging the high specific surface area and abundant catalytic sites provided by the open channels of PCN-222 to ensure efficient catalytic reactions within the core. This structure effectively avoids the potential quenching effect of metal ions on the fluorescence of AIE molecules, leading to signal attenuation. By maintaining the integrity of the PCN-222 channels, both catalytic activity and fluorescence signal are preserved.

[0009] According to the present invention, the present invention employs an organic ligand tetracarboxyphenylporphyrin iron and zirconium (Zr) metal ions (Zr 4+ The clusters self-assemble to form the metal-organic framework nanozyme PCN-222 material. Based on this, the organic ligand tetratetraphenylethylene [4-(3,5-dicarboxyphenyl)] and zirconium (Zr) metal ions (Zr) were synthesized in situ. 4+ The metal-organic framework (AIE-MOF) material formed by the self-assembly of clusters yields the final product, AIE metal-organic framework nanozyme (AIE-MOF@PCN-222). This nanozyme is then mixed with hydrogen peroxide and the chromogenic substrate o-phenylenediamine (OPD) to prepare a nanozyme biosensor. PCN-222 can oxidize colorless o-phenylenediamine (OPD) to yellow 2,3-diaminophenazine (DAP), producing an ultraviolet absorption peak at 450±5 nm and a strong fluorescence emission peak at 560±5 nm. The zirconium-based aggregation-induced emission metal-organic framework (AIE-MOF) in the AIE metal-organic framework nanozyme (AIE-MOF@PCN-222) exhibits a strong fluorescence emission peak at 446±10 nm, which can form a ratiometric fluorescence sensor with the fluorescence signal of 2,3-diaminophenazine (DAP).

[0010] In a preferred embodiment, the particle size of the AIE metal-organic framework nanozyme (AIE-MOF@PCN-222) material is 3860 nm. The colorimetric / fluorescence dual-mode nanozyme sensor of this invention has detection limits of 14.5 nM and 1.78 nM for formaldehyde, respectively, enabling real-time / on-site visual quantitative detection of formaldehyde. This makes the detection process portable, rapid, and cost-effective, meeting the needs of food safety and clinical medical testing.

[0011] Secondly, the present invention also provides a method for preparing the above-mentioned colorimetric / fluorescence dual-mode nanozyme sensor, specifically including the following steps: Step 1: Synthesis of metal-organic framework nanoenzyme PCN-222: Zirconium metal salt and tetracarboxyphenylporphyrin iron were dissolved in N,N-dimethylformamide (DMF) solvent, and then benzoic acid was added as a regulator to carry out a solvothermal reaction. The resulting solution was then washed, filtered and dried to obtain zirconium-based metal-organic framework nanoenzyme PCN-222 powder material. Step 2: Synthesis of AIE metal-organic framework nanozyme (AIE-MOF@PCN-222): The zirconium-based metal-organic framework nanozyme PCN-222 obtained in Step 1, zirconium metal salt, and tetrakis[4-(3,5-dicarboxyphenyl)]tetraphenylethylene were dissolved in N,N-dimethylformamide (DMF) solvent. Benzoic acid was then added as a regulator, and a solvothermal reaction was carried out. The resulting solution was then washed, filtered, and dried to obtain zirconium-based AIE metal-organic framework nanozyme (AIE-MOF@PCN-222) powder material. Step 3: Preparation of colorimetric / fluorescence dual-mode nanozyme sensor: The AIE metal-organic framework nanozyme (AIE-MOF@PCN-222) obtained in step 2 is dispersed in an aqueous medium and mixed with the chromogenic substrate OPD and hydrogen peroxide in a phosphate buffer solution to obtain the colorimetric / fluorescence dual-mode nanozyme sensor.

[0012] In a preferred embodiment, in step 1, the zirconium metal salt is selected from zirconium tetrachloride, and the mass ratio of zirconium metal salt to tetracarboxyphenylporphyrin iron is 1:(0.5-1); the solvothermal reaction temperature is 120°C, the reaction time is 24-48h, the washing agent used is anhydrous ethanol, and the drying method is oven drying at a temperature of 60-75°C.

[0013] In a preferred embodiment, in step 2, the zirconium metal salt is selected from zirconium tetrachloride, and the mass ratio of zirconium metal salt to tetra[4-(3,5-dicarboxyphenyl)]tetraphenylethylene is 1:(0.3-0.7); the solvothermal reaction temperature is 120°C, the reaction time is 24-48h, the washing agent used is anhydrous ethanol, and the drying method is oven drying at a temperature of 60-75°C.

[0014] In a preferred embodiment, in step 3, the OPD concentration in the colorimetric / fluorescence dual-mode nanozyme sensor is 1.4±0.1mM, the hydrogen peroxide concentration is 0.4±0.1mM, and the mass concentration of AIE-MOF@PCN-222 is 0.04-0.08mg / mL.

[0015] Thirdly, the present invention also provides the application of the above-mentioned colorimetric / fluorescence dual-mode nanozyme sensor or the colorimetric / fluorescence dual-mode nanozyme sensor prepared by the above-mentioned method in formaldehyde detection.

[0016] Fourthly, the present invention also provides a method for detecting formaldehyde using a colorimetric / fluorescence dual-mode nanozyme sensor, specifically comprising the following steps: Step S1: The colorimetric fluorescence dual-mode nanozyme sensor is mixed with a series of formaldehyde solutions of different concentrations in a certain proportion to prepare a reaction system. After reacting at 25-30 ℃ for 20-30 min, the spectral detection is performed using a UV-Vis spectrophotometer and a fluorescence spectrometer. The UV absorption change at 450 nm and the fluorescence intensity change at 446 nm and 560 nm of the colorimetric / fluorescence dual-mode nanozyme sensor are used as the abscissa, and the ratio of the UV peak value at 450 nm and the fluorescence intensity peak value at 446 nm and 560 nm of the colorimetric / fluorescence dual-mode nanozyme sensor is used as the ordinate to plot the calibration curves. Step S2: Mix the colorimetric / fluorescence dual-mode nanozyme sensor with the formaldehyde solution to be tested in a certain proportion to prepare a reaction system. After reacting at 25-30 ℃ for 20-30 min, perform spectral detection using a UV-Vis spectrophotometer and a fluorescence spectrometer. Calculate the formaldehyde concentration in the sample based on the changes in UV absorption at 450 nm and fluorescence intensity at 446 nm and 560 nm of the colorimetric / fluorescence dual-mode nanozyme sensor, according to the calibration curve obtained in step S1, thus achieving the detection of formaldehyde.

[0017] As a preferred embodiment, the preparation method of the reaction system in steps S1 and S2 includes: adding 100 μL of LAIE-MOF@PCN-222 solution, 100 μL of OPD solution, 100 μL of hydrogen peroxide solution and 100 μL of formaldehyde solution, and adjusting the volume to 2.5 mL using NaAc-HAc buffer to prepare a 2.5 mL reaction system; the formaldehyde solution uses ultrapure water as the solvent, the OPD solution concentration is 35 mM, and the solvent is anhydrous ethanol; the hydrogen peroxide solution concentration is 10 mM.

[0018] In a preferred embodiment, a UV-Vis spectrophotometer and a fluorescence spectrometer were used for spectral detection, and the absorbance value A at the UV absorption peak of 450 nm was recorded.450 Fluorescence intensity F at 446 nm 446 Fluorescence intensity F at 560 nm 560 Calculate F 446 / F 560 The ratio of fluorescence intensity is obtained by using the ratio of the UV absorption peak value A. 450 The ratio of fluorescence intensity to numerical value F 446 / F 560 A calibration curve was obtained by analyzing the functional relationship between formaldehyde concentration and the formaldehyde concentration. The concentration of the formaldehyde to be tested was calculated using the calibration curve. The colorimetric / fluorescence dual-mode nanoenzyme sensor exhibits a yellow color and emits yellow fluorescence in the absence of formaldehyde. As the formaldehyde concentration increases, the ultraviolet absorption intensity at 450 nm decreases, and the color gradually changes from yellow to colorless. The fluorescence intensity at 446 nm increases, and the fluorescence intensity at 560 nm decreases, with the fluorescence color changing from yellow to light green. This enables the colorimetric / fluorescence dual-mode detection of formaldehyde content.

[0019] In a preferred embodiment, AIE-MOF@PCN-222 can oxidize colorless OPD to yellow DAP in the presence of hydrogen peroxide and under acidic conditions. Due to the ion activation effect in the buffer solution, AIE-MOF@PCN-222 can emit bright green fluorescence at 446 nm. After the addition of formaldehyde, formaldehyde inhibits the catalytic ability of AIE-MOF@PCN-222, resulting in a decrease in DAP content and a lighter solution color. As a colorimetric detection signal, AIE-MOF@PCN-222 can emit green fluorescence at 446 ± 10 nm, and DAP can emit yellow fluorescence at 560 ± 5 nm. The decrease in DAP content leads to a decrease in fluorescence intensity at 560 ± 5 nm and an increase in fluorescence intensity of AIE-MOF@PCN-222 at 446 ± 10 nm. The UV absorbance at 450 nm and the fluorescence intensity at 446 nm and 560 nm are measured by the colorimetric / fluorescence dual-mode nanoenzyme sensor. The ratio of fluorescence intensity at nm is linearly related to the concentration of the analyte, thus enabling the detection of formaldehyde concentration in the analyte. The conditions for ultraviolet-visible spectroscopy include: the observation range of ultraviolet-visible spectroscopy is 350-550 nm; the fluorescence excitation wavelength is 380-400 nm; and the fluorescence emission spectrum observation range is 410-650 nm.

[0020] The technical principle of this invention is as follows: In the presence of hydrogen peroxide, AIE-MOF@PCN-222 can catalyze the colorless OPD to turn into yellow DAP as a colorimetric detection signal. After the addition of formaldehyde, the catalytic ability of AIE-MOF@PCN-222 is inhibited, resulting in a decrease in DAP content and a lighter solution color. As a colorimetric detection signal, AIE-MOF@PCN-222 can emit green fluorescence at 446 ± 10 nm, and DAP can emit yellow fluorescence at 560 ± 5 nm. The decrease in DAP content will lead to a decrease in fluorescence intensity at 560 ± 5 nm, and an increase in fluorescence intensity of AIE-MOF@PCN-222 at 446 ± 10 nm. Based on the change in formaldehyde concentration, the content of yellow DAP in the sensor solution decreases, the solution color lightens, the fluorescence intensity of DAP decreases, and the fluorescence intensity of AIE-MOF@PCN-222 increases. Furthermore, the ratio of the ultraviolet absorption intensity of DAP and the fluorescence intensity of DAP and AIE-MOF@PCN-222 shows a linear relationship with the concentration of the analyte, thereby achieving quantitative detection of formaldehyde concentration in the analyte.

[0021] Advantages of this invention: 1. This invention uses the highly stable zirconium-based metal-organic framework PCN-222 as a carrier to synthesize a high-brightness aggregation-induced emission metal-organic framework AIE-MOF on its surface, solving the problems of difficulty in recovering free enzymes and low stability; 2. The colorimetric / fluorescence dual-mode nanozyme sensor constructed based on AIE-MOF@PCN-222 of this invention integrates the excellent catalytic ability of nanozymes with the luminescent properties of AIE-MOF into one system. It is simple in design, easy to operate, and has high selectivity and detection stability for formaldehyde, while reducing detection time and cost. 3. The formaldehyde detection method established in this invention has high detection sensitivity, with detection limits of 14.5 nM and 1.78 nM, respectively, which meet the relevant requirements of national standards, and has good anti-interference and stability. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0023] Figure 1These are SEM images and TEM images of PCN-222 in Embodiment 1 and AIE-MOF@PCN-222 in Embodiment 2 of the present invention.

[0024] Figure 2 This is the particle size distribution diagram of AIE-MOF@PCN-222 in Embodiment 2 of the present invention.

[0025] Figure 3 This is a verification diagram of the catalytic ability of AIE-MOF@PCN-222 in Example 2 of the present invention.

[0026] Figure 4 These are sunlight images of the colorimetric / fluorescence dual-mode nanozyme sensor solutions under different concentrations of formaldehyde in Example 4 of this invention.

[0027] Figure 5 These are fluorescence images of the colorimetric / fluorescence dual-mode nanozyme sensor solutions under different concentrations of formaldehyde in Example 4 of this invention.

[0028] Figure 6 These are the UV-Vis and fluorescence emission spectra of the colorimetric / fluorescence dual-mode nanozyme sensor solutions under different concentrations of formaldehyde in Example 4 of this invention.

[0029] Figure 7 These are the colorimetric detection curves of the colorimetric / fluorescence dual-mode nanozyme sensor solutions under different concentrations of formaldehyde in Example 4 of this invention.

[0030] Figure 8 These are the fluorescence detection curves of the colorimetric / fluorescence dual-mode nanozyme sensor solutions under different concentrations of formaldehyde in Example 4 of this invention.

[0031] Figure 9 This is an analysis diagram from Example 2 of the present invention, which explores the selectivity and anti-interference properties of the colorimetric / fluorescence dual-mode nanozyme sensor. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0033] Unless otherwise specified, all raw materials used in the embodiments are commercially available conventional raw materials, and the technical means used are conventional means well known to those skilled in the art.

[0034] Example 1 This embodiment first provides a MOF material, the synthesis method of which includes the following steps: 70 mg ZrCl4, 50 mg tetracarboxyphenylporphyrin iron and 2.7 g benzoic acid were dissolved in 5 mL DMF and sonicated until completely dissolved.

[0035] Place the mixture in silicone oil at 120°C, turn on the magnetic stirrer and stir at 200 rpm for 30 min, then turn off the magnetic stirrer and keep heating at 120°C for 24 h.

[0036] After heating is complete, wait for the solution to cool to room temperature, then transfer the solution to equal portions into 50 mL centrifuge tubes, add 10 mL of anhydrous ethanol, and centrifuge at 12000 rpm, 4 ℃, for 20 min.

[0037] Remove the supernatant, add 10 mL of anhydrous ethanol, centrifuge, and repeat this step 3 times.

[0038] The resulting precipitate was placed in a 75 °C oven until completely dry. This yielded PCN-222 material.

[0039] PCN-222 was characterized. Figure 1 Figure 1a shows the SEM image of PCN-222. As can be seen from the figure, the synthesized PCN-222 has a rod-shaped structure and is uniform in size.

[0040] Example 2 This embodiment first provides an AIE-MOF@PCN-222 material, the synthesis method of which includes the following steps: Take 70 mg of PCN-222 into a 20 mL brown glass sample bottle, add 15 mL of ultrapure water, and sonicate for 1 h to completely disperse it, to obtain solution C.

[0041] 70 mg ZrCl4, 33 mg tetrakis[4-(3,5-dicarboxyphenyl)]tetraphenylethylene and 2.7 g benzoic acid were dissolved in 5 mL DMF and sonicated until completely dissolved. After complete dissolution, the solutions were mixed and sonicated for 30 min to obtain solution D.

[0042] Add solution D to solution C in one go, place it in silicone oil at 120°C, turn on the magnetic stirrer and stir at 200 rpm for 30 min, turn off the magnetic stirrer and keep heating at 120°C for 48 h.

[0043] After heating, the eggplant flask was removed from the silicone oil bath. After the solution cooled to room temperature, the solution was transferred equally into two 50 mL centrifuge tubes, 12.5 mL in each tube. Then, 10 mL of anhydrous ethanol was added to each tube, and the tubes were centrifuged at 12000 rpm and 4 °C for 20 min.

[0044] Remove the supernatant, add 10 mL of anhydrous ethanol, centrifuge, and repeat this step 3 times.

[0045] The resulting precipitate was placed in a 75 °C oven until completely dry. This yielded the AIE-MOF@PCN-222 material.

[0046] Characterization of AIE-MOF@PCN-222 Figure 1 1b and Figure 1 Image 1c shows the SEM and TEM images of AIE-MOF@PCN-222, respectively. The images reveal that the synthesized AIE-MOF@PCN-222 exhibits slight aggregation compared to PCN-222, and the surface of the rod-shaped PCN-222 is loaded with spherical AIE-MOF. Figure 2 (Dynamic light scattering DLS) shows that the particle size of AIE-MOF@PCN-222 is 3860 nm.

[0047] Verification Example 1 To verify the catalytic activity of AIE-MOF@PCN-222, 20 mg of AIE-MOF@PCN-222 obtained in Example 2 was dissolved in 10 mL of ultrapure water. Subsequently, 100 μL of AIE-MOF@PCN-222 solution, 100 μL of OPD solution, and 100 μL of hydrogen peroxide solution were taken and diluted to 2.5 mL with NaAc-HAc buffer solution at pH 3.8. After reacting for 20-30 min, the UV absorption intensity at 450 nm was recorded using a UV-Vis spectrophotometer, and the absorbance values ​​of AIE-MOF@PCN-222 and hydrogen peroxide, as well as the mixed solution of OPD and hydrogen peroxide, were measured at 450 nm.

[0048] As a control 1: PCN-222 was used instead of AIE-MOF@PCN-222, and all other conditions were the same. The reaction system was prepared and the absorbance at 652 nm was recorded.

[0049] As a control 2: no OPD solution was added to the reaction system, and all other conditions were the same. The absorbance at 652 nm was recorded.

[0050] As a control 3: This control is a blank control. AIE-MOF@PCN-222 was not added to the reaction system, and all other conditions were the same. The absorbance value at 652 nm was recorded.

[0051] like Figure 3 The results show that AIE-MOF@PCN-222 still retains high catalytic activity, and its catalytic activity comes from the synthesized PCN-222 nanozyme.

[0052] Example 3 This embodiment further provides a method for preparing a colorimetric / fluorescence dual-mode nanozyme sensor, specifically including the following steps: 20 mg of AIE-MOF@PCN-222 obtained in Example 2 was dissolved in 10 mL of ultrapure water. Then, 100 μL of AIE-MOF@PCN-222 solution, 100 μL of OPD solution, and 100 μL of hydrogen peroxide solution were taken and diluted to 2.5 mL with NaAc-HAc buffer at pH 3.8. The reaction was allowed to proceed for 20-30 min to obtain the colorimetric fluorescence dual-mode nanozyme sensor.

[0053] The OPD solution concentration was 35 mM, and the solvent was anhydrous ethanol; the hydrogen peroxide solution concentration was 10 mM.

[0054] Example 4 This embodiment provides a colorimetric / fluorescence dual-mode nanozyme detection method for formaldehyde, specifically including the following steps: 100 μL of formaldehyde solution of different concentrations was added to the colorimetric / fluorescence dual-mode nanozyme sensor prepared in Example 3 to form a 2.5 mL reaction system, and the color change of the solution was observed under sunlight and ultraviolet light.

[0055] Figure 4 These are daylight images of the colorimetric / fluorescence dual-mode nanozyme sensor solution under different formaldehyde concentrations. The images show that as the formaldehyde concentration increases, the solution color gradually changes from yellow to colorless.

[0056] Figure 5 These are fluorescence images of the colorimetric / fluorescence dual-mode nanozyme sensor solution at different formaldehyde concentrations. As can be seen from the images, the fluorescence of the solution changes from bright yellow to light green as the formaldehyde concentration increases.

[0057] The UV-Vis absorption spectrum and fluorescence emission spectrum were measured using a UV-Vis spectrophotometer and a fluorescence spectrophotometer. The observation range of the UV-Vis spectrum was 350-550 nm; the fluorescence excitation wavelength was 380-400 nm; and the observation range of the fluorescence emission spectrum was 410-650 nm.

[0058] Figure 6 These are the UV-Vis and fluorescence emission spectra of colorimetric / fluorescence dual-mode nanozyme sensor solutions under different concentrations of formaldehyde, among which... Figure 6 6a in the spectrum represents the ultraviolet-visible spectrum. Figure 6 6b in the figure represents the fluorescence emission spectrum.

[0059] The functional relationship between different formaldehyde concentrations in the solution and the values ​​of UV-Vis absorption peak and fluorescence emission peak is used to obtain the formaldehyde concentration values ​​corresponding to the values ​​of UV-Vis absorption peak and fluorescence emission peak in the solution.

[0060] Figure 7 These are colorimetric detection curves of colorimetric / fluorescence dual-mode nanozyme sensor solutions at different formaldehyde concentrations, where the x-axis represents the formaldehyde concentration and the y-axis represents the UV-Vis absorption peak.

[0061] The ultraviolet-visible absorption peak value is: A 450 The values ​​were obtained by monitoring the UV-Vis absorption peak around 450±5 nm in multiple parallel experiments and calculating the average value.

[0062] The functional relationship between the UV-Vis absorption peak and formaldehyde concentration is: Y = -0.0076X + 0.5291 (R 2 =0.9922), where Y represents the UV-Vis absorption peak at 450 nm, and X represents the formaldehyde concentration. Therefore, the A value can be determined by measuring the colorimetric / fluorescence dual-mode nanozyme sensor containing an unknown concentration of formaldehyde. 450 The formaldehyde concentration can be calculated using the above formula to achieve quantitative analysis of formaldehyde.

[0063] According to the formula 3σ / S, where σ is the standard deviation of the blank response value and S is the slope of the detection curve, the detection limit of this colorimetric / fluorescence dual-mode nanozyme sensor for formaldehyde detection is calculated to be 14.5 nM.

[0064] Figure 8 These are fluorescence detection curves of colorimetric / fluorescence dual-mode nanozyme sensor solutions under different formaldehyde concentrations, where the x-axis represents the formaldehyde concentration and the y-axis represents the fluorescence intensity ratio.

[0065] The fluorescence intensity ratio is calculated using the following method: R = F 446 / F 560 .

[0066] F 446 The fluorescence intensity value at 446 nm in the fluorescence spectrum corresponding to the sensor solution; F 560 : The fluorescence intensity value at 560 nm in the fluorescence spectrum corresponding to the sensor solution; F 446 F 560 The fluorescence intensity values ​​near 446 nm and 560 nm were obtained by monitoring multiple parallel experiments and calculating the average value.

[0067] The functional relationship between the fluorescence intensity ratio and the formaldehyde concentration is: Y = 0.0272X + 0.5292 (R 2 =0.9921), where Y represents the ratio of the fluorescence intensity peak at 446 nm to the fluorescence intensity peak at 560 nm, and X represents the formaldehyde concentration. Therefore, the F-value of a colorimetric / fluorescence dual-mode nanozyme sensor containing an unknown concentration of formaldehyde can be measured. 446 / F 560 The formaldehyde concentration can be calculated using the above formula to achieve quantitative analysis of formaldehyde.

[0068] According to the formula 3σ / S, where σ is the standard deviation of the blank response value and S is the slope of the detection curve, the detection limit of this colorimetric / fluorescence dual-mode nanozyme sensor for formaldehyde detection by fluorescence method can be calculated to be 1.78 nM.

[0069] Example 5 This embodiment provides a colorimetric / fluorescence dual-mode detection method for formaldehyde, specifically including: Step (1): Referring to Example 4, obtain the fluorescence detection curve and colorimetric detection curve of the colorimetric / fluorescence dual-mode nanozyme sensor solution, as well as the functional relationship between the UV-Vis absorption peak and the formaldehyde concentration, and the functional relationship between the fluorescence intensity ratio and the formaldehyde concentration; Step (2): Add 100 μL of formaldehyde solution (concentration unknown, prepared by diluting 10 mM formaldehyde solution several times) to the colorimetric / fluorescence dual-mode nanozyme sensor prepared in Example 3 to make a 2.5 mL reaction system. Use a UV-Vis spectrophotometer and a fluorescence spectrometer for spectral detection. Calculate the formaldehyde concentration in the sample based on the detection curve and function relationship obtained in step (1) by observing the UV absorption change at 450 nm and the fluorescence intensity changes at 446 nm and 560 nm of the colorimetric / fluorescence dual-mode nanozyme sensor. The average value is 550 μM.

[0070] Example 6 This embodiment provides a colorimetric / fluorescence dual-mode detection method for formaldehyde, specifically including: Step (1): Referring to Example 4, obtain the fluorescence detection curve and colorimetric detection curve of the colorimetric / fluorescence dual-mode nanozyme sensor solution, as well as the functional relationship between the UV-Vis absorption peak and the formaldehyde concentration, and the functional relationship between the fluorescence emission peak and the formaldehyde concentration; Step (2): Add 100 μL of the formaldehyde solution to be tested (concentration known, 300 μM formaldehyde solution) to the colorimetric / fluorescence dual-mode nanozyme sensor prepared in Example 3 to make a 2.5 mL reaction system. Use a UV-Vis spectrophotometer and a fluorescence spectrometer for spectral detection. Calculate the formaldehyde concentration in the sample based on the detection curve and function relationship obtained in step (1) by observing the UV absorption change at 450 nm and the fluorescence intensity changes at 446 nm and 560 nm of the colorimetric / fluorescence dual-mode nanozyme sensor. The average value is 298 μM.

[0071] Verification Example 2 A colorimetric / fluorescence dual-mode nanozyme sensor was prepared according to Example 3, using Na... 2+ Mg 2+ K + Ca 2+ His, Suc, Glu, and Arg were used as interfering substances to investigate the selectivity and anti-interference ability of a colorimetric / fluorescence dual-mode nanozyme sensor in detecting formaldehyde. The concentration of the interfering substances was 100 times that of the formaldehyde solution. After reacting at 25-30℃ for 20-30 min, the spectral detection was performed using a UV-Vis spectrophotometer and a fluorescence spectrometer. The changes in UV absorption at 450 nm and fluorescence intensity at 446 nm and 560 nm of the colorimetric / fluorescence dual-mode nanozyme sensor were observed.

[0072] Figure 9 Figure 9a shows the selectivity and anti-interference performance of the colorimetric mode, and Figure 9b shows the selectivity and anti-interference performance of the fluorescence mode. As can be seen from the figure, the UV absorption value and fluorescence intensity of the colorimetric / fluorescence dual-mode nanozyme sensor change significantly only in the presence of formaldehyde, indicating that the sensor has good selectivity and anti-interference performance for formaldehyde detection.

[0073] 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 invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

Claims

1. A colorimetric / fluorescence dual-mode nanozyme sensor, characterized in that, This invention comprises an AIE metal-organic framework nanozyme (AIE-MOF@PCN-222) supported on aggregation-induced emission metal-organic framework (AIE-MOF) material, hydrogen peroxide, and the chromogenic substrate o-phenylenediamine (OPD). The AIE-MOF@PCN-222 is a zirconium-based metal-organic framework nanozyme (PCN-222) supported on AIE-MOF. The AIE-MOF is formed by the self-assembly of zirconium metal ions and the organic ligand tetrakis[4-(3,5-dicarboxyphenyl)]tetraphenylethylene, and the PCN-222 is formed by the self-assembly of zirconium metal ions and tetracarboxyphenylporphyrin iron. The PCN-222 nanozyme in AIE-MOF@PCN-222 can oxidize the colorless chromogenic substrate o-phenylenediamine (OPD) to a yellow 2,3-diaminophenazine (DAP), producing a UV absorption peak at 450±25 nm and a strong fluorescence emission peak at 560±5 nm. The AIE-MOF exhibits a fluorescence emission peak at 446±10 nm. It has a strong fluorescence emission peak at nm, which can be combined with the fluorescence signal of 2,3-diaminophenazine DAP to form a ratiometric fluorescence sensor.

2. The colorimetric / fluorescence dual-mode nanozyme sensor according to claim 1, characterized in that, The AIE-MOF@PCN-222 is composed of a MOF-on-MOF structure, with the core being the metal-organic framework nanozyme PCN-222, which has peroxidase properties, and the surface loaded with AIE-MOF. It retains the peroxidase properties of PCN-222 and has the fluorescence properties of AIE-MOF. The particle size of the AIE-MOF@PCN-222 material is 3860 nm.

3. A method for preparing a colorimetric / fluorescence dual-mode nanozyme sensor as described in claim 1 or 2, characterized in that, Includes the following steps: Step 1: Synthesis of metal-organic framework nanoenzyme PCN-222: Zirconium metal salt and tetracarboxyphenylporphyrin iron were dissolved in N,N-dimethylformamide (DMF) solvent, and then benzoic acid was added as a regulator to carry out a solvothermal reaction. The resulting solution was then washed, filtered, and dried to obtain zirconium-based metal-organic framework nanoenzyme PCN-222 powder material. Step 2: Synthesis of AIE metal-organic framework nanozyme AIE-MOF@PCN-222: PCN-222 obtained in Step 1, zirconium metal salt and tetrakis[4-(3,5-dicarboxyphenyl)]tetraphenylethylene were dissolved in DMF solvent, and then benzoic acid was added as a regulator to carry out a solvothermal reaction. The resulting solution was then washed, filtered and dried to obtain AIE-MOF@PCN-222 powder material. Step 3: Preparation of colorimetric / fluorescence dual-mode nanozyme sensor: The AIE-MOF@PCN-222 obtained in step 2 is dispersed in an aqueous medium and mixed with the chromogenic substrate OPD and hydrogen peroxide in a phosphate buffer solution to obtain the colorimetric / fluorescence dual-mode nanozyme sensor.

4. The method for preparing a colorimetric / fluorescence dual-mode nanozyme sensor according to claim 3, characterized in that, In step 1, the zirconium metal salt is selected from zirconium tetrachloride, and the mass ratio of zirconium metal salt to tetracarboxyphenylporphyrin iron is 1:(0.5-1); the solvothermal reaction temperature is 120°C, the reaction time is 24-48h, the washing agent used is anhydrous ethanol, and the drying method is oven drying at a temperature of 60-75°C.

5. The method for preparing a colorimetric / fluorescence dual-mode nanozyme sensor according to claim 3, characterized in that, In step 2, the zirconium metal salt is selected from zirconium tetrachloride, and the mass ratio of zirconium metal salt to tetra[4-(3,5-dicarboxyphenyl)]tetraphenylethylene is 1:(0.3-0.7); the solvothermal reaction temperature is 120°C, the reaction time is 24-48h, the washing agent used is anhydrous ethanol, and the drying method is oven drying at a temperature of 60-75°C.

6. The preparation method according to claim 3, characterized in that, In step 3, the OPD concentration in the colorimetric / fluorescence dual-mode nanozyme sensor is 1.4±0.1mM, the hydrogen peroxide concentration is 0.4±0.1mM, and the mass concentration of AIE-MOF@PCN-222 is 0.04-0.08mg / mL.

7. The application of a colorimetric / fluorescence dual-mode nanozyme sensor as described in claim 1 or 2 and / or a colorimetric / fluorescence dual-mode nanozyme sensor prepared by any one of claims 3-6 in formaldehyde detection.

8. The application according to claim 7, characterized in that, The detection limits for colorimetric and fluorescence methods are 14.5 nM and 1.78 nM, respectively.

9. A method for detecting formaldehyde using a colorimetric / fluorescence dual-mode nanozyme sensor as described in claim 1 or 2, characterized in that, Includes the following steps: Step S1: The colorimetric / fluorescence dual-mode nanozyme sensor is mixed with a series of formaldehyde solutions of different concentrations in a certain proportion to prepare a reaction system. After reacting at 25-30℃ for 20-30 min, the spectral detection is performed using a UV-Vis spectrophotometer and a fluorescence spectrometer. The changes in UV absorption at 450 nm and fluorescence intensity at 446 nm and 560 nm of the colorimetric / fluorescence dual-mode nanozyme sensor are used as the basis for plotting calibration curves with the formaldehyde concentration as the abscissa and the ratio of the UV peak at 450 nm and the fluorescence intensity peak at 446 nm and 560 nm as the ordinate. Step S2: Mix the colorimetric / fluorescence dual-mode nanozyme sensor with the formaldehyde solution to be tested in a certain proportion to prepare a reaction system. After reacting at 25-30℃ for 20-30 min, perform spectral detection using a UV-Vis spectrophotometer and a fluorescence spectrometer. Calculate the formaldehyde concentration in the sample based on the changes in UV absorption at 450 nm and fluorescence intensity at 446 nm and 560 nm of the colorimetric / fluorescence dual-mode nanozyme sensor, according to the calibration curve obtained in step S1, thus achieving the detection of formaldehyde.

10. The method for detecting formaldehyde using a colorimetric / fluorescence dual-mode nanozyme sensor according to claim 9, characterized in that, The preparation method of the reaction system in steps S1 and S2 includes: adding 100 μL of AIE-MOF@PCN-222 solution, 100 μL of formaldehyde solution, 100 μL of OPD solution and 100 μL of hydrogen peroxide solution, and adjusting the volume to 2.5 mL with acetate-sodium acetate buffer to prepare a 2.5 mL reaction system; the formaldehyde solution uses ultrapure water as solvent, the OPD solution concentration is 35 mM and the solvent is anhydrous ethanol; the hydrogen peroxide solution concentration is 10 mM.

11. The method for detecting formaldehyde using a colorimetric / fluorescence dual-mode nanozyme sensor according to claim 9, characterized in that, Spectroscopic detection was performed using a UV-Vis spectrophotometer and a fluorescence spectrometer, and the absorbance value A at the UV absorption peak of 450 nm was recorded. 450 Fluorescence intensity F at 446 nm 446 Fluorescence intensity F at 560 nm 560 Calculate F 446 / F 560 The ratio of fluorescence intensity is obtained by using the ratio of the ultraviolet absorption peak A. 450 The ratio of fluorescence intensity to numerical value F 446 / F 560 A calibration curve is obtained by analyzing the functional relationship between formaldehyde concentration and the formaldehyde concentration. The concentration of formaldehyde to be tested is calculated using the calibration curve. The colorimetric / fluorescence dual-mode nanoenzyme sensor exhibits a yellow color and emits yellow fluorescence in the absence of formaldehyde. As the formaldehyde concentration increases, the ultraviolet absorption intensity at 450 nm decreases, and the color changes from yellow to colorless. The fluorescence intensity at 446 nm increases, and the fluorescence intensity at 560 nm decreases, and the fluorescence color changes from yellow to light green. This enables the colorimetric / fluorescence dual-mode detection of formaldehyde content.

12. The method for detecting formaldehyde using a colorimetric / fluorescence dual-mode nanozyme sensor according to claim 9, characterized in that, AIE-MOF@PCN-222 catalyzes the conversion of colorless OPD to yellow DAP. Upon the addition of formaldehyde, the formaldehyde inhibits the catalytic ability of AIE-MOF@PCN-222, leading to a decrease in DAP content and a lighter solution color. As a colorimetric detection signal, AIE-MOF@PCN-222 emits green fluorescence at 446 ± 10 nm, while DAP emits yellow fluorescence at 560 ± 5 nm. A decrease in DAP content results in a decrease in fluorescence intensity at 560 ± 5 nm, while the fluorescence intensity of AIE-MOF@PCN-222 at 446 ± 10 nm increases. The ratio of the UV absorbance at 450 nm to the fluorescence intensity at 446 nm and 560 nm, as measured by the colorimetric / fluorescence dual-mode nanozyme sensor, exhibits a linear relationship with the concentration of the analyte, thus enabling the detection of formaldehyde concentration in the analyte. The UV-Vis spectral conditions include an observation range of 350-550 nm. nm; the fluorescence excitation wavelength is 380-400 nm, and the fluorescence emission spectrum observation range is 410-650 nm.

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