Colorimetric / fluorescent dual-mode sensor as well as preparation method and application thereof in histamine detection

Abstract

The invention provides a colorimetric / fluorescent dual-mode sensor, a preparation method of the colorimetric / fluorescent dual-mode sensor and application of the colorimetric / fluorescent dual-mode sensor in histamine detection. The colorimetric / fluorescent dual-mode sensor comprises a horseradish peroxidase-loaded metal organic framework (HRP-AIE-MOF), hydrogen peroxide and a chromogenic substrate o-phenylenediamine (OPD), horse radish peroxidase (HRP) in the HRP-coated AIE-MOF can oxidize colorless OPD into yellow 2, 3-diaminophenazine (DAP), an ultraviolet absorption peak is generated at 425 nm, a strong fluorescence emission peak is generated at 555 nm, the HRP-coated AIE-MOF has a strong fluorescence emission peak at 450 nm, and the HRP-coated AIE-MOF and a fluorescence signal of the DAP can form a ratio fluorescence sensor. According to the invention, the excellent catalytic ability of natural enzyme and the luminescence characteristic of AIE-MOF are integrated in one system, and high-sensitivity histamine detection is realized.

Description

Technical Field

[0001] The present invention belongs to the field of biosensor technology, and in particular relates to a colorimetric / fluorescence dual-mode sensor, a preparation method thereof, and an application thereof in histamine detection. Background Art

[0002] Histamine (HA), a core biogenic amine mediator of type I hypersensitivity reactions, mediates vascular permeability changes and smooth muscle contraction by specifically binding to H1-H4 receptors. Acute exposure can trigger systemic allergic reactions, manifested by flushing, bronchospasm, hypotension, and tachycardia, making it a key pathological driver of anaphylactic shock. Chronic accumulation of histamine poses a potential carcinogenic risk, and the N-nitrosamine compounds formed by histamine and nitrite are classified as Group 2B carcinogens by the International Agency for Research on Cancer (IARC). Significant individual variability in diamine oxidase (DAO) metabolic capacity makes individuals with histamine intolerance highly sensitive to low-dose exposure. Current detection technologies face challenges in clinical application, including insufficient response speed, significant interference from complex matrices, and limited sensitivity. Developing highly specific, rapid-response, and trace histamine detection technologies suitable for complex biological samples would be of great medical value for the precise diagnosis and treatment of allergic diseases, food safety monitoring, and cancer prevention.

[0003] Natural enzymes, as environmentally friendly biocatalysts, possess high specificity and excellent catalytic efficiency. However, due to their poor stability under adverse reaction conditions such as excessive acidity, excessive alkali, high temperature, and organic solvents, as well as their short activity period and difficulty in recovery, these limitations significantly restrict their application in large-scale industry. Enzyme immobilization technology, as an advanced biocatalytic method, has garnered widespread attention in the fields of biochemistry and industry. This technology significantly improves the stability, reusability, and operability of enzymes by immobilizing enzymes on carriers to form stable enzyme-immobilized complexes. In particular, in sensor applications, immobilized enzymes can provide sustained and efficient catalytic performance, enhancing the sensitivity and long-term stability of sensors. Therefore, sensors based on immobilized enzymes not only overcome the shortcomings of natural enzymes, but also expand their application potential in environmental monitoring, food safety, and medical diagnosis.

[0004] Improving the anti-interference ability of histamine detection is a key requirement for current technological development. Although traditional fluorescence methods have high sensitivity, their commonly used fluorophores generally face the problem of aggregation-caused quenching (ACQ) in aggregated states or complex matrices, resulting in signal attenuation and unstable results. The core advantage of aggregation-induced emission metal-organic frameworks (AIE-MOFs) lies in their unique fluorescence properties: the AIE luminescent units fixed to the MOF skeleton exhibit an unconventional aggregation-induced emission (AIE) effect, that is, the fluorescence is significantly enhanced in the aggregated or solid state, effectively overcoming the ACQ effect and ensuring that the fluorescence signal has excellent intensity and stability in complex biological and food samples rich in interfering substances such as proteins and lipids. Therefore, the unique aggregation-induced emission properties of AIE-MOF materials give them good anti-fluorescence quenching ability and high signal stability in complex samples, which is of positive significance for improving the practical application performance of fluorescence-based histamine detection methods. Summary of the Invention

[0005] To address the above technical problems, the present invention provides a colorimetric / fluorescence dual-mode sensor, a preparation method thereof, and its application in histamine detection. The colorimetric / fluorescence dual-mode sensor can quantitatively detect histamine in real time / on-site with high sensitivity. The detection limits of the colorimetric method and the fluorescence method are 1.71 μM and 0.56 μM, respectively. The detection process is convenient, rapid, and low-cost.

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions: In the first aspect, the present invention provides a colorimetric / fluorescence dual-mode sensor, comprising a metal-organic framework (HRP@AIE-MOF) loaded with horseradish peroxidase, hydrogen peroxide, and a chromogenic substrate o-phenylenediamine (OPD); the metal-organic framework (HRP@AIE-MOF) loaded with horseradish peroxidase is a zirconium-based aggregation-induced emission metal-organic framework (AIE-MOF) material loaded with horseradish peroxidase (HRP), and the zirconium-based aggregation-induced emission metal-organic framework (AIE-MOF) is self-assembled by zirconium (Zr) metal ions and an organic ligand tetra-(carboxyphenyl)ethylene.

[0007] According to known technology, metal-organic frameworks (MOFs) are highly ordered, porous crystalline skeleton structures formed by self-assembly of organic ligands and transition metal ions through coordination. Unsaturated coordination sites of metal elements and amino, carboxyl and other groups of organic ligands still exist on their surfaces. By fixing enzymes on MOFs carriers, stable immobilized enzyme complexes can be formed, significantly improving the stability, reusability and operability of the enzymes. They can be particularly used in immobilized enzyme biosensors.

[0008] According to the solution of the present invention, the present invention uses an organic ligand tetra-(carboxyphenyl)ethylene with an AIE group and a zirconium (Zr) metal ion (Zr 4+ ) clusters self-assemble to form a metal-organic framework AIE-MOF material. Compared with other MOFs materials, the organic ligand tetra-(carboxyphenyl)ethylene used has aggregation-induced emission effect (AIE), which can turn on or enhance fluorescence in the aggregated state, effectively improving the luminescence efficiency of the metal-organic framework AIE-MOF material, and it has a denser, more rigid and more stable skeleton structure.

[0009] According to the solution of the present invention, the present invention adopts an organic ligand tetra-(carboxyphenyl)ethylene and a zirconium (Zr) metal ion (Zr 4+ ) clusters self-assembled into a metal-organic framework AIE-MOF material, horseradish peroxidase (HRP) was loaded on the AIE-MOF material, and the AIE-MOF material was mixed with hydrogen peroxide and a chromogenic substrate o-phenylenediamine (OPD) to prepare an immobilized enzyme biosensor, wherein horseradish peroxidase (HRP) can oxidize colorless o-phenylenediamine (OPD) to yellow 2,3-diaminophenazine (DAP), and produce an ultraviolet absorption peak at 425±25 nm and a strong fluorescence emission peak at 555±5 nm. The zirconium-based aggregation-induced emission metal-organic framework (AIE-MOF) in the horseradish peroxidase-loaded metal-organic framework (HRP@AIE-MOF) has a strong fluorescence emission peak at 450±10 nm, which can form a ratiometric fluorescence sensor with the fluorescence signal of 2,3-diaminophenazine (DAP).

[0010] As a preferred embodiment, in the horseradish peroxidase-loaded metal organic framework (HRP@AIE-MOF), the loading rate of horseradish peroxidase (HRP) is 85%-90%, and the particle size of the horseradish peroxidase-loaded metal organic framework (HRP@AIE-MOF) material is 150-300 nm.

[0011] In a second aspect, the present invention further provides a method for preparing the colorimetric / fluorescence dual-mode sensor as described above, comprising the following steps: Step (1), preparation of zirconium-based aggregation-induced emission metal-organic framework (AIE-MOF): dissolving zirconium metal salt and tetrakis-(carboxyphenyl)ethylene in N,N-dimethylformamide (DMF) solvent, then adding acetic acid and deionized water as regulators, performing a solvothermal reaction, and then washing, filtering, and drying the obtained solution to obtain a zirconium-based aggregation-induced emission metal-organic framework (AIE-MOF) powder material; Step (2), preparation of a metal organic framework loaded with horseradish peroxidase (HRP@AIE-MOF): dissolving the zirconium-based aggregation-induced emission metal organic framework (AIE-MOF) obtained in step (1) in deionized water, then adding crosslinking agents 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) and stirring for a period of time, then adding horseradish peroxidase (HRP) phosphate buffer and stirring for a period of time to perform a crosslinking reaction, and then washing, filtering, and drying the obtained solution to obtain a metal organic framework loaded with horseradish peroxidase (HRP@AIE-MOF) powder material; Step (3), preparation of a colorimetric / fluorescent dual-mode sensor: the phosphate buffer solution of the metal organic framework (HRP@AIE-MOF) loaded with horseradish peroxidase obtained in step (2) is stored at 4°C for future use, the colorimetric substrate o-phenylenediamine (OPD) is dissolved in anhydrous ethanol to prepare an OPD solution, a 30% mass concentration of hydrogen peroxide aqueous solution is diluted to prepare a hydrogen peroxide solution, and then the phosphate buffer solution of the metal organic framework (HRP@AIE-MOF) loaded with horseradish peroxidase, the OPD solution and the hydrogen peroxide solution are mixed, and the volume is fixed with deionized water, and the reaction is carried out for 15-30 minutes to obtain the colorimetric / fluorescent dual-mode sensor.

[0012] As a preferred embodiment, in step (1), the zirconium metal salt is selected from zirconium nitrate and / or zirconium tetrachloride, the mass ratio of the zirconium metal salt to tetra(carboxyphenyl)ethylene is 1:(1-5), preferably 1:(1-2), and the molar ratio of the zirconium metal salt to tetra(carboxyphenyl)ethylene is 1:(0.5-1); the temperature of the solvent thermal reaction is 100-120°C, the reaction time is 3-24h, the detergent used in washing is anhydrous ethanol, and the drying method adopts oven drying at a drying temperature of 60-75°C.

[0013] As a preferred embodiment, in step (2), the mass concentration of the zirconium-based aggregation-induced emission metal-organic framework (AIE-MOF) solution is 1-5 mg / mL, and the mass ratio of AIE-MOF, EDC and NHS is (1-10):1:1, preferably 5:1:1; the mass concentration of HRP in the phosphate buffer of the horseradish peroxidase (HRP) is 0.5-2 mg / mL, and the mass ratio of AIE-MOF and HRP is (0.5-2):1; both stirrings are carried out at room temperature, the stirring speed is 100-300 rpm, the stirring time for adding the cross-linking agents EDC and NHS is 1-2 h, and then the stirring time for adding the phosphate buffer of HRP is 8-24 h; the detergent used in washing is phosphate buffer, and the drying method adopts vacuum freeze drying.

[0014] As a preferred embodiment, in step (3), the mass concentration of HRP@AIE-MOF in the phosphate buffer of the horseradish peroxidase-loaded metal organic framework (HRP@AIE-MOF) is 0.5-1 mg / mL, the molar concentration of the OPD solution is 10 mM, and the molar concentration of the hydrogen peroxide solution is 100 mM; 150 μL of the phosphate buffer of the horseradish peroxidase-loaded metal organic framework (HRP@AIE-MOF), 60 μL of the OPD solution and 15 μL of the hydrogen peroxide solution are mixed, and the reaction system is diluted to 3 mL with deionized water.

[0015] In a third aspect, the present invention further provides a use of the colorimetric / fluorescence dual-mode sensor as described above and / or the colorimetric / fluorescence dual-mode sensor prepared by the preparation method as described above in the detection of histamine.

[0016] According to the scheme of the present invention, the colorimetric / fluorescence dual-mode sensor prepared by the present invention, HRP@AIE-MOF can catalyze the colorless OPD into yellow DAP. In the presence of histamine, the pH value of the solution increases, inhibiting the catalytic ability of HRP@AIE-MOF, resulting in a decrease in DAP content and a lighter color of the solution. As colorimetric detection signals, the fluorescence of HRP@AIE-MOF at 450±10 nm and DAP at 555±5 nm are used as fluorescence detection signals.

[0017] According to the solution of the present invention, the pH value of the colorimetric / fluorescence dual-mode sensor prepared by the present invention is affected by the concentration of the analyte histamine, which in turn affects the catalytic ability of HRP@AIE-MOF, resulting in changes in the colorimetric signal and fluorescence intensity, thereby achieving quantitative detection of the concentration of histamine in the analyte. The colorimetric / fluorescence dual-mode sensor of the present invention has detection limits of 1.71 μM and 0.56 μM for histamine, respectively, and can accurately detect histamine in a concentration range of 1-2000 μM. It can quantitatively detect histamine in real time / on-site, making the detection process portable, rapid and low-cost, meeting the detection requirements of food safety.

[0018] In a fourth aspect, the present invention further provides a method for detecting histamine using the colorimetric / fluorescence dual-mode sensor as described above, comprising the following steps: Step S1: The colorimetric / fluorescence dual-mode sensor is mixed with a series of histamine solutions of different concentrations in proportion to prepare a reaction system. After reacting at 25-30°C for 15-30 minutes, spectral detection is performed using a UV-visible spectrophotometer and a fluorescence spectrometer. A calibration curve is drawn based on the changes in UV absorption at 425 nm and the changes in fluorescence intensity at 450 nm and 555 nm of the colorimetric / fluorescence dual-mode sensor, with the logarithm of the histamine concentration as the abscissa and the ratio of the UV peak value at 425 nm to the fluorescence intensity peak values ​​at 450 nm and 555 nm of the colorimetric / fluorescence dual-mode sensor as the ordinate; Step S2: The colorimetric / fluorescence dual-mode sensor is mixed with the histamine solution to be tested in proportion to form a reaction system. After reacting at 25-30°C for 15-30 minutes, spectral detection is performed using a UV-visible spectrophotometer and a fluorescence spectrometer. The histamine concentration in the sample is calculated based on the changes in UV absorption at 425 nm and fluorescence intensity at 450 nm and 555 nm of the colorimetric / fluorescence dual-mode sensor according to the calibration curve obtained in step S1, thereby achieving the detection of histamine.

[0019] As a preferred embodiment, the preparation method of the reaction system of step S1 and step S2 includes: adding 150 μL of HRP@AIE-MOF solution, 30 μL of histamine solution to be tested, 60 μL of OPD solution and 15 μL of hydrogen peroxide solution, and diluting the volume to 3 mL with deionized water to prepare a 3 mL reaction system; the histamine solution uses ultrapure water as a solvent, the OPD solution concentration is 10 mM, and the solvent is anhydrous ethanol; the concentration of the hydrogen peroxide solution is 100 mM.

[0020] As a preferred embodiment, a UV-visible spectrophotometer and a fluorescence spectrometer are used for spectrum detection, and the absorption value A of the UV absorption peak at 425 nm is recorded. 425 , fluorescence intensity F at 450 nm 450 , fluorescence intensity F at 555 nm 555 , calculate F 555 / F 450 The fluorescence intensity ratio value is obtained by using the ultraviolet absorption peak A 425 and the fluorescence intensity ratio F 555 / F 450A calibration curve is obtained based on the functional relationship between the colorimetric and histamine concentrations, and the concentration of the histamine to be measured is calculated using the calibration curve. In the absence of histamine, the colorimetric / fluorescence dual-mode sensor appears yellow and emits yellow fluorescence. As the histamine concentration increases, the ultraviolet absorption intensity at 425 nm decreases, and the color changes from yellow to colorless; the fluorescence intensity at 450 nm increases, and the fluorescence intensity at 555 nm decreases, and the fluorescence color changes from yellow to blue, thereby realizing colorimetric / fluorescence dual-mode detection of histamine content.

[0021] As a preferred embodiment, HRP@AIE-MOF can catalyze the conversion of colorless OPD into yellow DAP. After the addition of histamine, the pH value of the solution increases, which inhibits the catalytic ability of HRP@AIE-MOF, resulting in a decrease in the DAP content and a lighter color of the solution. As a colorimetric detection signal, HRP@AIE-MOF can emit blue fluorescence at 450 ± 10 nm, and DAP can emit yellow fluorescence at 555 ± 5 nm. A decrease in DAP content will cause a decrease in the fluorescence intensity at 555 ± 5 nm, and an increase in the fluorescence intensity of HRP@AIE-MOF at 450 ± 10 nm. The ratio of the ultraviolet absorption value at 425 nm and the fluorescence intensity at 450 nm and 555 nm of the colorimetric / fluorescence dual-mode sensor is linearly related to the concentration of the detected substance, thereby realizing the detection of the concentration of histamine in the test substance. The conditions of the ultraviolet-visible spectrum include: the observation range of the ultraviolet-visible spectrum is 350-600 nm; the fluorescence excitation wavelength is 365 nm, and the fluorescence emission spectrum observation range was 380-650 nm.

[0022] The technical principle of the present invention is that HRP@AIE-MOF can catalyze the conversion of colorless OPD to yellow DAP. After the addition of histamine, the pH value of the solution increases, inhibiting the catalytic ability of HRP@AIE-MOF, resulting in a decrease in DAP content and a lighter solution color. As a colorimetric detection signal, HRP@AIE-MOF can emit blue fluorescence at 450 ± 10 nm, and DAP can emit yellow fluorescence at 555 ± 5 nm. A decrease in DAP content will cause the fluorescence intensity at 555 ± 5 nm to decrease, while the fluorescence intensity of HRP@AIE-MOF at 450 ± 10 nm to increase. According to the concentration of the analyte, histamine, the yellow DAP content in the sensor solution decreases, the solution color becomes lighter, and the fluorescence intensity of DAP decreases, while the fluorescence intensity of HRP@AIE-MOF increases. The DAP ultraviolet absorption intensity and the ratio of the fluorescence intensities of DAP and HRP@AIE-MOF show a linear relationship with the concentration of the analyte, thereby achieving quantitative detection of the concentration of histamine in the analyte.

[0023] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention uses a highly bright aggregation-induced emission metal-organic framework (AIE-MOF) as a carrier to immobilize horseradish peroxidase (HRP) on its surface, solving the problems of difficult recovery and low stability of free enzymes. 2. The colorimetric / fluorescence dual-mode sensor constructed based on HRP@AIE-MOF in this invention integrates the excellent catalytic ability of natural enzymes with the luminescent properties of AIE-MOF into a single system. It has a simple design and is easy to operate. It also has high selectivity and detection stability for histamine, while reducing detection time and cost. 3. The histamine detection method established in the present invention has high detection sensitivity. The detection limits of colorimetry and fluorescence are 1.71 μM and 0.56 μM, respectively, which meet the relevant requirements of national standards and have good anti-interference and stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the present invention or the prior art, a brief introduction is given below to 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 any creative work.

[0025] Figure 1 TEM images of AIE-MOF in Example 1 and HRP@AIE-MOF in Example 2 of the present invention.

[0026] Figure 2 This is the particle size distribution diagram of AIE-MOF in Inventive Example 1 and HRP@AIE-MOF in Example 2.

[0027] Figure 3 This is a diagram for verifying the catalytic ability of HRP@AIE-MOF in Verification Example 1 of the present invention.

[0028] Figure 4 3 are daylight images of the colorimetric / fluorescence dual-mode sensor solution at different histamine concentrations in Example 4 of the present invention.

[0029] Figure 5 1 and 2 are fluorescence images of the colorimetric / fluorescence dual-mode sensor solution at different histamine concentrations in Example 4 of the present invention.

[0030] Figure 6 1 and 2 are the UV-visible spectra and fluorescence emission spectra of the colorimetric / fluorescence dual-mode sensor solution at different histamine concentrations in Example 4 of the present invention.

[0031] Figure 7The UV-visible absorption peak and colorimetric detection curve of the colorimetric / fluorescence dual-mode sensor solution at different histamine concentrations in Example 4 of the present invention are shown in FIG. Figure 8 1 is the fluorescence intensity ratio and fluorescence detection curve of the colorimetric / fluorescence dual-mode sensor solution at different histamine concentrations in Example 4 of the present invention.

[0032] Figure 9 This is a selectivity analysis diagram of the colorimetric / fluorescence dual-mode sensor in Verification Example 2 of the present invention.

[0033] Figure 10 This is an analysis diagram of the anti-interference performance of the colorimetric / fluorescence dual-mode sensor in Verification Example 3 of the present invention. DETAILED DESCRIPTION To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0034] Unless otherwise specified, the various 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.

[0035] Example 1: This embodiment first provides an AIE-MOF material, and its synthesis method includes the following steps: 23 mg of ZrCl4 and 23 mg of tetrakis-(carboxyphenyl)ethylene were dissolved in 6.95 mL of DMF and sonicated until completely dissolved.

[0036] To the above DMF solution, 1 mL of acetic acid and 0.05 mL of deionized water were added as regulators.

[0037] Stir for 30 min, turn off the magnetic stirrer, and heat at 120 °C for 3 h.

[0038] After heating, the solution was cooled to room temperature and then transferred equally into 50 mL centrifuge tubes. 10 mL of anhydrous ethanol was added and the tubes were centrifuged at 12,000 rpm, 4 °C for 20 min.

[0039] The supernatant was removed, 10 mL of anhydrous ethanol was added, and the mixture was centrifuged. This step was repeated three times.

[0040] The final precipitate was placed in an oven at 75°C until completely dried to obtain the AIE-MOF material.

[0041] Characterization of AIE-MOF, Figure 1 Figure 1a is the TEM image of AIE-MOF. It can be seen from the figure that the synthesized AIE-MOF has a flower-like structure and is uniform in size.

[0042] Example 2: This embodiment further provides a method for synthesizing HRP@AIE-MOF, which specifically includes the following steps: 20 mg of AIE-MOF was placed in a 20 mL brown glass vial, 5 mL of ultrapure water was added, and the mixture was sonicated for 1 h to completely disperse it. 10 mg each of the crosslinking agents EDC and NHS was added to the above solution, and the mixture was stirred at 200 rpm for 1.5 h to obtain Solution C.

[0043] Take 10 mg of HRP in a 15 mL centrifuge tube and add 5 mL of phosphate buffer to obtain solution D. The above solution D was added to solution C at one time, and stirred at 200 rpm for 1.5 h to obtain solution E.

[0044] After stirring, transfer solution E equally into two 50 mL centrifuge tubes, 10 mL of solution in each tube, and then add 10 mL of phosphate buffer solution to each tube. Centrifuge at 12000 rpm, 4 ℃, for 20 min.

[0045] The supernatant was discarded, 10 mL of phosphate buffer solution was added, and the mixture was centrifuged. This step was repeated three times.

[0046] The final precipitate was placed in a freeze dryer and freeze-dried for 3 days to obtain HRP@AIE-MOF material.

[0047] HRP@AIE-MOF was characterized by Figure 1 In Figure 1b, it can be seen that the prepared HRP@AIE-MOF is aggregated compared with AIE-MOF, and the thorn-like protrusions on the surface are not obvious, confirming that HRP is fixed on AIE-MOF. Figure 2 It can be seen that the particle size of HRP@AIE-MOF is significantly increased, which further confirms that HRP@AIE-MOF is successfully synthesized.

[0048] Verification Example 1: To verify the catalytic activity of HRP@AIE-MOF, all the HRP@AIE-MOF obtained in Example 2 was dissolved in 20 mL of phosphate buffer (1X). Subsequently, 15 μL of HRP@AIE-MOF solution, 60 μL of TMB solution, and 15 μL of hydrogen peroxide solution were taken and diluted to 3 mL with NaAc-HAc buffer (pH 4.0). After 15-30 minutes of reaction, the UV absorbance at 652 nm was recorded using a UV-visible spectrophotometer. HRP was substituted for HRP@AIE-MOF, and all other conditions were the same. The reaction system was set up and the absorbance at 652 nm was recorded as a control. Simultaneously, 15 μL of the AIE-MOF solution was taken, 60 μL of TMB solution and 15 μL of hydrogen peroxide solution were added, and the volume was diluted to 3 mL with NaAc-HAc buffer (pH 4.0). After 15-30 minutes of reaction, the UV absorbance at 652 nm was recorded using a UV-visible spectrophotometer. The absorbance at 652 nm of the AIE-MOF, TMB, hydrogen peroxide, and the mixed solution of TMB and hydrogen peroxide was also measured.

[0049] like Figure 3 It shows that HRP@AIE-MOF still retains high catalytic activity, and its catalytic activity comes from the HRP immobilized on the surface.

[0050] Example 3: This embodiment further provides a method for preparing a colorimetric / fluorescence dual-mode sensor, which specifically includes the following steps: The entire amount of HRP@AIE-MOF obtained in Example 2 was dissolved in 20 mL of 1X phosphate buffer. Subsequently, 150 μL of the HRP@AIE-MOF solution, 60 μL of the OPD solution, and 15 μL of the hydrogen peroxide solution were added to the final volume of 3 mL with deionized water. The reaction was allowed to proceed for 15-30 minutes to obtain a colorimetric / fluorescent dual-mode sensor.

[0051] The concentration of the OPD solution is 10 mM, the solvent is anhydrous ethanol, and the concentration of the hydrogen peroxide solution is 100 mM.

[0052] Example 4: This embodiment provides a colorimetric / fluorescent dual-mode detection method for detecting histamine, which specifically includes the following steps: 30 μL of histamine solutions of different concentrations to be tested were added to the colorimetric / fluorescence dual-mode 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.

[0053] Figure 4 The following are daylight images of the colorimetric / fluorescent dual-mode sensor solution at different histamine concentrations. As can be seen from the image, the color of the solution gradually changes from yellow to colorless as the histamine concentration increases.

[0054] Figure 5The fluorescence images of the colorimetric / fluorescence dual-mode sensor solution at different histamine concentrations are shown in Figure 2. As can be seen from the figure, the fluorescence of the solution changes from bright yellow to light blue as the histamine concentration increases.

[0055] The UV-visible absorption spectrum and fluorescence emission spectrum were measured using a UV-visible spectrophotometer and a fluorescence spectrophotometer. The observation range of the UV-visible spectrum was 350-600 nm; the fluorescence excitation wavelength was 360 nm, and the observation range of the fluorescence emission spectrum was 380-650 nm.

[0056] Figure 6 are the UV-visible spectra and fluorescence emission spectra of the colorimetric / fluorescent dual-mode sensor solution at different histamine concentrations, where Figure 6 6a in is the UV-visible spectrum, Figure 6 6b in FIG is the fluorescence emission spectrum.

[0057] The functional relationship between different histamine concentration values ​​of the solution and the UV-visible absorption peak value and the fluorescence emission peak value is used to obtain the histamine concentration value corresponding to the UV-visible absorption peak value and the fluorescence emission peak value in the solution.

[0058] Figure 7 is the UV-visible absorption value and colorimetric detection curve of the colorimetric / fluorescence dual-mode sensor solution under different histamine concentrations, where Figure 7 7a in the figure is the relationship between histamine concentration and UV-visible absorption peak. Figure 7 Figure 7b is the colorimetric detection curve of the colorimetric / fluorescence dual-mode sensor solution, where the x-axis is the logarithmic value of the histamine concentration and the y-axis is the UV-visible absorption peak.

[0059] The UV-visible absorption peak is: A 425 The UV-visible absorption peak near 425±5 nm was monitored by multiple parallel experiments and the average value was calculated to obtain like Figure 7 As shown in 7b, the functional relationship between the UV-visible absorption peak and the histamine concentration is: Y = -0.4464X + 1.674 (R 2 =0.9956), where Y represents the UV-visible absorption peak at 425 nm and X represents the logarithmic value of histamine concentration. 425 , the histamine concentration is calculated by the above formula to achieve quantitative analysis of histamine.

[0060] The detection limit was calculated using the formula 3σ / S, where σ is the standard deviation of the blank response and S is the slope of the detection curve. This linear relationship revealed a detection limit of 1.71 μM for histamine detection using this colorimetric / fluorescence dual-mode sensor.

[0061] Figure 8 is the fluorescence intensity value and fluorescence detection curve of the colorimetric / fluorescence dual-mode sensor solution under different concentrations of histamine, where Figure 8 8a in is the relationship between histamine concentration and fluorescence intensity value, Figure 8 Figure 8b is the fluorescence detection curve of the colorimetric / fluorescence dual-mode sensor solution, where the x-axis is the logarithmic value of the histamine concentration and the y-axis is the fluorescence intensity ratio value. The numerical calculation method of the fluorescence intensity ratio is: R = F 555 / F 450 .

[0062] F 555 : Fluorescence intensity value at 555 nm in the fluorescence spectrum of the sensor solution; F 450 : Fluorescence intensity value at 450 nm in the fluorescence spectrum of the sensor solution; F 555 , F 450 The fluorescence intensity values ​​near 555 nm and 450 nm were monitored by multiple parallel experiments and the average value was calculated.

[0063] like Figure 8 As shown in 8b, the functional relationship between the fluorescence emission peak and the histamine concentration is: Y = -7.2388X + 21.7544 (R 2 =0.9893), where Y represents the ratio of the peak fluorescence intensity at 555 nm to the peak fluorescence intensity at 450 nm, and X represents the logarithmic value of the histamine concentration. 555 / F 450 , the histamine concentration is calculated by the above formula to achieve quantitative analysis of histamine.

[0064] The detection limit was calculated using the formula 3σ / S, where σ is the standard deviation of the blank response and S is the slope of the detection curve. This linear relationship revealed a detection limit of 0.56 μM for histamine detection using this colorimetric / fluorescence dual-mode sensor.

[0065] Example 5 This embodiment provides a colorimetric / fluorescence dual-mode detection method for detecting histamine, specifically comprising: Step (1): Referring to Example 4, obtain the fluorescence detection curve and colorimetric detection curve of the colorimetric / fluorescence dual-mode sensor solution, as well as the functional relationship between the UV-visible absorption peak and the histamine concentration, and the functional relationship between the fluorescence emission peak and the histamine concentration; Step (2): The histamine solution to be tested (the concentration is unknown, prepared by diluting a 1000 μM histamine solution several times) was added to the colorimetric / fluorescence dual-mode sensor prepared in Example 3 to prepare a 3 mL reaction system. Spectral detection was performed using a UV-visible spectrophotometer and a fluorescence spectrometer. The histamine concentration in the sample was calculated based on the detection curve and functional relationship obtained in step (1) by observing the changes in UV absorption at 425 nm and the changes in fluorescence intensity at 450 nm and 555 nm of the colorimetric / fluorescence dual-mode sensor. The average value was 312 μM.

[0066] Example 6 This embodiment provides a colorimetric / fluorescence dual-mode detection method for detecting histamine, specifically comprising: Step (1): Referring to Example 4, obtain the fluorescence detection curve and colorimetric detection curve of the colorimetric / fluorescence dual-mode sensor solution, as well as the functional relationship between the UV-visible absorption peak and the histamine concentration, and the functional relationship between the fluorescence emission peak and the histamine concentration; Step (2): The histamine solution to be tested (the concentration is known, 300 μM histamine solution) was added to the colorimetric / fluorescence dual-mode sensor prepared in Example 3 to prepare a 3 mL reaction system. Spectral detection was performed using a UV-visible spectrophotometer and a fluorescence spectrometer. The histamine concentration in the sample was calculated based on the detection curve and functional relationship obtained in step (1) by observing the changes in UV absorption at 425 nm and the changes in fluorescence intensity at 450 nm and 555 nm of the colorimetric / fluorescence dual-mode sensor. The average value was 303 μM.

[0067] Verification Example 2: A colorimetric / fluorescent dual-mode sensor was prepared according to Example 3, and Ca 2+ 、Cl - 、CO3 2- , plasma, precursor amino acids such as histidine and lysine, and biogenic amines such as cadaverine and putrescine were used as interfering ions to explore the selectivity of the colorimetric / fluorescence dual-mode sensor in detecting histamine. The concentration of the interfering ions was 100 times that of the histamine solution. After reacting at 25-30°C for 15-30 minutes, spectral detection was performed using a UV-visible spectrophotometer and a fluorescence spectrometer. The changes in UV absorption at 425 nm and the changes in fluorescence intensity at 450 nm and 555 nm of the colorimetric / fluorescence dual-mode sensor were observed.

[0068] Figure 9 9a in the figure is a selective analysis in colorimetric mode. Figure 9Figure 9b is the selectivity 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 sensor change significantly only in the presence of biogenic amines such as histamine and cadaverine. Neither ions nor precursor amino acids can cause changes in the sensor, indicating that the sensor is also effective for other biogenic amines, but has no detection effect on various precursor amino acids.

[0069] Verification Example 3: A colorimetric / fluorescent dual-mode sensor was prepared according to Example 3, and Ca 2+ 、Cl - 、CO3 2- , plasma, histidine, lysine and other precursor amino acids as interfering ions, and explore the anti-interference ability of the colorimetric / fluorescence dual-mode sensor in detecting histamine. The concentration of the interfering ions was 100 times that of the histamine solution. After reacting at 25-30 °C for 15-30 min, spectral detection was performed using a UV-visible spectrophotometer and a fluorescence spectrometer. The colorimetric / fluorescence dual-mode sensor was detected by the changes in UV absorption at 425 nm and the changes in fluorescence intensity at 450 nm and 555 nm.

[0070] Figure 10 10a in the figure is a selective analysis in colorimetric mode. Figure 10 Figure 10b shows the selective analysis in fluorescence mode. As can be seen from the figure, there is no significant difference in the effect of the histamine solution mixed with interfering ions and the histamine solution alone on the ultraviolet absorbance and fluorescence intensity of the colorimetric / fluorescence dual-mode sensor, indicating that the sensor has good anti-interference ability and can be used for histamine detection in complex matrices.

[0071] 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 embodied 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 illustrative and non-restrictive, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations that come within the meaning and range of equivalents of the claims be embraced therein.

Claims

1. A colorimetric / fluorescence dual-mode sensor, characterized in that: The invention comprises a metal organic framework (HRP@AIE-MOF) loaded with horseradish peroxidase, hydrogen peroxide and a color developing substrate o-phenylenediamine (OPD); the metal organic framework (HRP@AIE-MOF) loaded with horseradish peroxidase is a zirconium-based aggregation-induced emission metal organic framework (AIE-MOF) material loaded with horseradish peroxidase (HRP), and the zirconium-based aggregation-induced emission metal organic framework (AIE-MOF) is self-assembled by zirconium (Zr) metal ions and an organic ligand tetra-(carboxyphenyl)ethylene; the horseradish peroxidase (HRP) in the metal organic framework (HRP@AIE-MOF) loaded with horseradish peroxidase can oxidize the colorless color developing substrate o-phenylenediamine (OPD) to yellow 2,3-diaminophenazine (DAP), and generate an ultraviolet absorption peak at 425±25 nm and a yellow 2,3-diaminophenazine (DAP) at 555±5 The zirconium-based aggregation-induced emission metal-organic framework (AIE-MOF) in the horseradish peroxidase-loaded metal-organic framework (HRP@AIE-MOF) has a strong fluorescence emission peak at 450 ± 10 nm, which can form a ratiometric fluorescence sensor with the fluorescence signal of 2,3-diaminophenazine (DAP).

2. A colorimetric / fluorescence dual-mode sensor according to claim 1, characterized in that: In the horseradish peroxidase-loaded metal organic framework (HRP@AIE-MOF), the loading rate of horseradish peroxidase (HRP) is 85%-90%, and the particle size of the horseradish peroxidase-loaded metal organic framework (HRP@AIE-MOF) material is 150-300 nm.

3. A method for preparing a colorimetric / fluorescence dual-mode sensor according to claim 1 or 2, characterized in that: The steps include: Step (1), preparation of zirconium-based aggregation-induced emission metal-organic framework (AIE-MOF): dissolving zirconium metal salt and tetrakis-(carboxyphenyl)ethylene in N,N-dimethylformamide (DMF) solvent, then adding acetic acid and deionized water as regulators, performing a solvothermal reaction, and then washing, filtering, and drying the obtained solution to obtain a zirconium-based aggregation-induced emission metal-organic framework (AIE-MOF) powder material; Step (2), preparation of a metal organic framework loaded with horseradish peroxidase (HRP@AIE-MOF): dissolving the zirconium-based aggregation-induced emission metal organic framework (AIE-MOF) obtained in step (1) in deionized water, then adding crosslinking agents 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) and stirring for a period of time, then adding horseradish peroxidase (HRP) phosphate buffer and stirring for a period of time to perform a crosslinking reaction, and then washing, filtering, and drying the obtained solution to obtain a metal organic framework loaded with horseradish peroxidase (HRP@AIE-MOF) powder material; Step (3), preparation of a colorimetric / fluorescent dual-mode sensor: the phosphate buffer solution of the metal organic framework (HRP@AIE-MOF) loaded with horseradish peroxidase obtained in step (2) is stored at 4°C for future use, the colorimetric substrate o-phenylenediamine (OPD) is dissolved in anhydrous ethanol to prepare an OPD solution, a 30% mass concentration of hydrogen peroxide aqueous solution is diluted to prepare a hydrogen peroxide solution, and then the phosphate buffer solution of the metal organic framework (HRP@AIE-MOF) loaded with horseradish peroxidase, the OPD solution and the hydrogen peroxide solution are mixed, and the volume is fixed with deionized water, and the reaction is carried out for 15-30 minutes to obtain the colorimetric / fluorescent dual-mode sensor.

4. The method for preparing the colorimetric / fluorescence dual-mode sensor according to claim 3, wherein: In step (1), the zirconium metal salt is selected from zirconium nitrate and / or zirconium tetrachloride, the mass ratio of the zirconium metal salt to tetra(carboxyphenyl)ethylene is 1:(1-5), preferably 1:(1-2), and the molar ratio of the zirconium metal salt to tetra(carboxyphenyl)ethylene is 1:(0.5-1); the temperature of the solvent thermal reaction is 100-120°C, the reaction time is 3-24h, the detergent used in washing is anhydrous ethanol, and the drying method adopts oven drying at a drying temperature of 60-75°C.

5. The preparation method according to claim 3, characterized in that In step (2), the mass concentration of the zirconium-based aggregation-induced emission metal-organic framework (AIE-MOF) solution is 1-5 mg / mL, and the mass ratio of AIE-MOF, EDC and NHS is (1-10):1:1, preferably 5:1:1; the mass concentration of HRP in the phosphate buffer of the horseradish peroxidase (HRP) is 0.5-2 mg / mL, and the mass ratio of AIE-MOF and HRP is (0.5~2):1; both stirrings are carried out at room temperature, the stirring speed is 100-300 rpm, the stirring time for adding the cross-linking agents EDC and NHS is 1-2 hours, and then the stirring time for adding the phosphate buffer of HRP is 8-24 hours; the detergent used during washing is phosphate buffer, and the drying method adopts vacuum freeze drying.

6. The preparation method according to claim 3, characterized in that In step (3), the mass concentration of HRP@AIE-MOF in the phosphate buffer of the metal organic framework (HRP@AIE-MOF) loaded with horseradish peroxidase is 0.5-1 mg / mL, the molar concentration of the OPD solution is 10 mM, and the molar concentration of the hydrogen peroxide solution is 100 mM; 150 μL of the phosphate buffer of the metal organic framework (HRP@AIE-MOF) loaded with horseradish peroxidase, 60 μL of the OPD solution and 15 μL of the hydrogen peroxide solution are mixed, and the reaction system is diluted to 3 mL with deionized water.

7. Use of the colorimetric / fluorescence dual-mode sensor according to claim 1 or 2 and / or the colorimetric / fluorescence dual-mode sensor obtained by the preparation method according to any one of claims 3 to 6 in the detection of histamine.

8. The use according to claim 7, characterized in that The detection limits of the colorimetric and fluorescence assays were 1.71 μM and 0.56 μM, respectively.

9. A method for detecting histamine using a colorimetric / fluorescence dual-mode sensor according to claim 1 or 2, characterized in that: The steps include: Step S1: The colorimetric / fluorescence dual-mode sensor is mixed with a series of histamine solutions of different concentrations in proportion to prepare a reaction system. After reacting at 25-30° C. for 10-20 minutes, spectral detection is performed using a UV-visible spectrophotometer and a fluorescence spectrometer. A calibration curve is plotted based on the changes in UV absorption at 425 nm and the changes in fluorescence intensity at 450 nm and 555 nm of the colorimetric / fluorescence dual-mode sensor, with the logarithm of the histamine concentration as the abscissa and the ratio of the UV peak value at 425 nm to the fluorescence intensity peak values ​​at 450 nm and 555 nm of the colorimetric / fluorescence dual-mode sensor as the ordinate; Step S2: The colorimetric / fluorescence dual-mode sensor is mixed with the histamine solution to be tested in proportion to form a reaction system. After reacting at 25-30° C. for 10-20 minutes, spectral detection is performed using a UV-visible spectrophotometer and a fluorescence spectrometer. The histamine concentration in the sample is calculated based on the changes in UV absorption at 425 nm and fluorescence intensity at 450 nm and 555 nm of the colorimetric / fluorescence dual-mode sensor according to the calibration curve obtained in step S1, thereby achieving the detection of histamine.

10. The method for detecting histamine using a colorimetric / fluorescence dual-mode sensor according to claim 9, wherein: The preparation method of the reaction system of step S1 and step S2 includes: adding 150 μL of HRP@AIE-MOF solution, 30 μL of histamine solution to be tested, 60 μL of OPD solution and 15 μL of hydrogen peroxide solution, and diluting the volume to 3 mL with water to prepare a 3 mL reaction system; the histamine solution uses ultrapure water as a solvent, the OPD solution concentration is 10 mM, and the solvent is anhydrous ethanol; the hydrogen peroxide solution concentration is 100 mM.

11. The method for detecting histamine using a colorimetric / fluorescence dual-mode sensor according to claim 9, wherein: The spectrum was detected by UV-visible spectrophotometer and fluorescence spectrometer, and the absorption value A of the UV absorption peak at 425 nm was recorded. 425 , fluorescence intensity F at 450 nm 450 , fluorescence intensity F at 555 nm 555 , calculate F 555 / F 450 The fluorescence intensity ratio value is obtained by using the ultraviolet absorption peak A 425 and the fluorescence intensity ratio F 555 / F 450 A calibration curve is obtained based on the functional relationship between the colorimetric and histamine concentrations, and the concentration of the histamine to be measured is calculated using the calibration curve. In the absence of histamine, the colorimetric / fluorescence dual-mode sensor appears yellow and emits yellow fluorescence. As the histamine concentration increases, the ultraviolet absorption intensity at 425 nm decreases, and the color changes from yellow to colorless; the fluorescence intensity at 450 nm increases, and the fluorescence intensity at 555 nm decreases, and the fluorescence color changes from yellow to blue, thereby realizing colorimetric / fluorescence dual-mode detection of histamine content.

12. The method for detecting histamine using a colorimetric / fluorescence dual-mode sensor according to claim 9, wherein: HRP@AIE-MOF can catalyze the conversion of colorless OPD into yellow DAP. After the addition of histamine, the pH value of the solution increases, inhibiting the catalytic ability of HRP@AIE-MOF, resulting in a decrease in the DAP content and a lighter color of the solution. As colorimetric detection signals, HRP@AIE-MOF can emit blue fluorescence at 450±10 nm, and DAP can emit yellow fluorescence at 555±5 nm. A decrease in DAP content will lead to a decrease in the fluorescence intensity at 555±5 nm, while the fluorescence intensity of HRP@AIE-MOF at 450±10 nm increases. The colorimetric / fluorescence dual-mode sensor shows a linear relationship between the UV absorbance value at 425 nm and the fluorescence intensity at 450 nm and 555 nm, thereby realizing the detection of the histamine concentration in the analyte. The UV-visible spectral conditions include: the UV-visible spectral observation range is 350-600 nm; the fluorescence excitation wavelength is 365 nm, and the fluorescence emission spectrum observation range is 380-650 nm.

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