A colorimetric / fluorescence dual-mode sensor, its preparation method, and its application in histamine detection.

Abstract

This invention provides a colorimetric / fluorescence dual-mode sensor, its preparation method, and its application in histamine detection. The colorimetric / fluorescence dual-mode sensor comprises a metal-organic framework (HRP@AIE-MOF) loaded with horseradish peroxidase, hydrogen peroxide, and the chromogenic substrate o-phenylenediamine (OPD). The horseradish peroxidase (HRP) in the HRP@AIE-MOF oxidizes colorless OPD to yellow 2,3-diaminophenazine (DAP), producing a UV absorption peak at 425 nm and a strong fluorescence emission peak at 555 nm. The HRP@AIE-MOF also exhibits a strong fluorescence emission peak at 450 nm, which, together with the fluorescence signal of DAP, forms a ratiometric fluorescence sensor. This invention integrates the excellent catalytic ability of a natural enzyme with the luminescent properties of AIE-MOF into a single system, achieving highly sensitive histamine detection.

Description

Technical Field

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

[0002] Histamine (HA), a core biogenic amine mediator of type I hypersensitivity reactions, mediates altered vascular permeability and smooth muscle contraction through specific binding to H1-H4 receptors. Acute exposure can trigger systemic anaphylactic reactions, clinically manifested as skin flushing, bronchospasm, hypotension, and tachycardia, and is a key pathological driver of anaphylactic shock. Chronic accumulation poses a potential carcinogenic risk; N-nitrosamines formed from 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) metabolism leads to high sensitivity to low-dose exposure in histamine-intolerant individuals. Current detection technologies face challenges in clinical applications, including insufficient response speed, significant interference from complex matrices, and limited sensitivity. Developing highly specific, rapid-response trace histamine detection technologies applicable to complex biological samples is of significant medical value for precise diagnosis and treatment of allergic diseases, food safety monitoring, and tumor prevention.

[0003] Natural enzymes, as environmentally friendly biocatalysts, possess high specificity and excellent catalytic efficiency. However, their poor stability under adverse reaction conditions such as excessive acidity, alkalinity, high temperature, and organic solvents, coupled with short activity cycles and difficulties in recovery, significantly restricts their application in large-scale industry. Enzyme immobilization technology, as an advanced biocatalytic method, has gained widespread attention in the fields of biochemistry and industry. This technology significantly improves the stability, reusability, and operability of enzymes by immobilizing them on a support to form stable enzyme-immobilized complexes. Especially 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 fields such as environmental monitoring, food safety, and medical diagnostics.

[0004] Improving the anti-interference capability of histamine detection is a key requirement for current technological development. While traditional fluorescence methods offer high sensitivity, their commonly used fluorophores generally suffer from aggregation-caused quenching (ACQ) in aggregated states or complex matrices, leading to signal attenuation and result instability. The core advantage of aggregation-induced emission metal-organic frameworks (AIE-MOFs) lies in their unique fluorescence properties: the AIE luminescent units immobilized by the MOF framework exhibit an unconventional aggregation-induced emission (AIE) effect, meaning that fluorescence is significantly enhanced in aggregated or solid-state states, effectively overcoming the ACQ effect and ensuring excellent intensity and stability of fluorescence signals 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 endow them with good resistance to fluorescence quenching 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, this invention provides a colorimetric / fluorescence dual-mode sensor, its preparation method, and its application in histamine detection. This colorimetric / fluorescence dual-mode sensor can quantitatively detect histamine in real time / on-site, with high sensitivity. The detection limits for the colorimetric method and the fluorescence method are 1.71 μM and 0.56 μM, 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:

[0007] In a first aspect, the present invention provides a colorimetric / fluorescence dual-mode sensor comprising a horseradish peroxidase-loaded metal-organic framework (HRP@AIE-MOF), hydrogen peroxide, and a chromogenic substrate o-phenylenediamine (OPD); wherein the horseradish peroxidase-loaded metal-organic framework (HRP@AIE-MOF) 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 formed by the self-assembly of zirconium (Zr) metal ions and the organic ligand tetra-(carboxyphenyl)ethylene.

[0008] According to known technologies, metal-organic frameworks (MOFs) are highly ordered, porous crystalline framework structures formed by the self-assembly of organic ligands and transition metal ions through coordination. Their surfaces still contain unsaturated coordination sites of metal elements and amino and carboxyl groups of organic ligands. By immobilizing enzymes on MOFs, stable immobilized enzyme complexes can be formed, which significantly improves the stability, reusability, and operability of enzymes. In particular, they can be applied to immobilized enzyme biosensors.

[0009] According to the present invention, the organic ligand tetra-(carboxyphenyl)ethylene with an AIE group and zirconium (Zr) metal ions (Zr 4+ Clusters self-assemble to form metal-organic frameworks (AIE-MOF) materials. Compared with other MOF materials, the organic ligand tetra-(carboxyphenyl)ethylene has aggregation-induced emission (AIE) effect, which can turn on or enhance fluorescence in the aggregated state, effectively improving the luminescence efficiency of metal-organic framework AIE-MOF materials. Moreover, it has a denser, more rigid and more stable framework structure.

[0010] According to the present invention, the present invention employs a mixture of the organic ligand tetra-(carboxyphenyl)ethylene and zirconium (Zr) metal ions (Zr 4+ A metal-organic framework (AIE-MOF) material formed by the self-assembly of horseradish peroxidase (HRP) clusters was loaded onto the AIE-MOF material. This AIE-MOF material was then mixed with hydrogen peroxide and the chromogenic substrate o-phenylenediamine (OPD) to prepare an immobilized enzyme biosensor. Horseradish peroxidase (HRP) can oxidize colorless o-phenylenediamine (OPD) to yellow 2,3-diaminophenazine (DAP), producing 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 AIE-MOF exhibits a strong fluorescence emission peak at 450 ± 10 nm, which, together with the fluorescence signal of 2,3-diaminophenazine (DAP), constitutes a ratiometric fluorescence sensor.

[0011] In a preferred embodiment, the horseradish peroxidase (HRP) loading rate in the horseradish peroxidase-loaded metal-organic framework (HRP@AIE-MOF) is 85%-90%, and the particle size of the horseradish peroxidase-loaded metal-organic framework (HRP@AIE-MOF) material is 150-300 nm.

[0012] Secondly, the present invention also provides a method for fabricating the colorimetric / fluorescence dual-mode sensor as described above, comprising the following steps:

[0013] Step (1), Preparation of zirconium-based aggregation-induced emission metal-organic framework (AIE-MOF): Zirconium metal salt and tetra-(carboxyphenyl)ethylene are dissolved in N,N-dimethylformamide (DMF) solvent, and then acetic acid and deionized water are added as regulators to carry out a solvothermal reaction. The obtained solution is then washed, filtered and dried to obtain zirconium-based aggregation-induced emission metal-organic framework (AIE-MOF) powder material.

[0014] Step (2), preparation of horseradish peroxidase-loaded metal-organic framework (HRP@AIE-MOF): The zirconium-based aggregation-induced emission metal-organic framework (AIE-MOF) obtained in step (1) is dissolved in deionized water, and then crosslinking agents 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) are added and stirred for a period of time. Then, horseradish peroxidase (HRP) phosphate buffer is added and stirred for a period of time to carry out the crosslinking reaction. Then, the obtained solution is washed, filtered and dried to obtain horseradish peroxidase-loaded metal-organic framework (HRP@AIE-MOF) powder material.

[0015] Step (3), preparation of the colorimetric / fluorescence dual-mode sensor: The phosphate buffer of the horseradish peroxidase-loaded metal-organic framework (HRP@AIE-MOF) obtained in step (2) is stored at 4°C for later use. The chromogenic substrate o-phenylenediamine (OPD) is dissolved in anhydrous ethanol to prepare an OPD solution. A 30% hydrogen peroxide aqueous solution is diluted to prepare a hydrogen peroxide solution. Then, the phosphate buffer, OPD solution and hydrogen peroxide solution of the horseradish peroxidase-loaded metal-organic framework (HRP@AIE-MOF) are mixed and diluted with deionized water. The reaction is carried out for 15-30 min to obtain the colorimetric / fluorescence dual-mode sensor.

[0016] In a preferred embodiment, in step (1), the zirconium metal salt is selected from zirconium nitrate and / or zirconium tetrachloride, the mass ratio of zirconium metal salt to tetra-(carboxyphenyl)ethylene is 1:(1-5), preferably 1:(1-2), and the molar ratio of zirconium metal salt to tetra-(carboxyphenyl)ethylene is 1:(0.5-1); the temperature of the solvothermal reaction is 100-120°C, the reaction time is 3-24h, the detergent used for washing is anhydrous ethanol, and the drying method is oven drying at a temperature of 60-75°C.

[0017] In 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 horseradish peroxidase (HRP) phosphate buffer is 0.5-2 mg / mL, and the mass ratio of AIE-MOF to HRP is (0.5-2):1; both stirring operations are carried out at room temperature, with a stirring speed of 100-300 rpm, and the stirring time for adding crosslinking agent EDC and NHS is 1-2 h, followed by the stirring time for adding HRP phosphate buffer is 8-24 h; the washing agent used is phosphate buffer, and the drying method is vacuum freeze-drying.

[0018] 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 OPD solution is 10 mM, and the molar concentration of hydrogen peroxide solution is 100 mM; 150 μL of phosphate buffer of horseradish peroxidase-loaded metal-organic framework (HRP@AIE-MOF), 60 μL of OPD solution and 15 μL of hydrogen peroxide solution are mixed, and the reaction system is brought to a final volume of 3 mL with deionized water.

[0019] Thirdly, the present invention also provides the application of the colorimetric / fluorescence dual-mode sensor as described above and / or the colorimetric / fluorescence dual-mode sensor prepared by the preparation method described above in histamine detection.

[0020] According to the present invention, the colorimetric / fluorescence dual-mode sensor prepared by the present invention can catalyze the colorless OPD to turn into yellow DAP. Under the condition 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, the fluorescence of HRP@AIE-MOF at 450 ± 10 nm and DAP at 555 ± 5 nm are used as fluorescence detection signals.

[0021] According to the present invention, the colorimetric / fluorescence dual-mode sensor prepared by the present invention has its pH value affected by the concentration of histamine in the analyte, which in turn affects the catalytic ability of HRP@AIE-MOF, leading to changes in the colorimetric signal and fluorescence intensity. This enables quantitative detection of the concentration of histamine in the analyte. The detection limits of the colorimetric / fluorescence dual-mode sensor of the present invention for histamine are 1.71 μM and 0.56 μM, respectively, and it can accurately detect histamine in the concentration range of 1-2000 μM. It can perform real-time / on-site quantitative detection of histamine, making the detection process portable, rapid, and low-cost, thus meeting the detection requirements for food safety.

[0022] Fourthly, the present invention also provides a method for detecting histamine using the colorimetric / fluorescence dual-mode sensor as described above, comprising the following steps:

[0023] Step S1: The colorimetric / fluorescence dual-mode sensor is mixed with a series of histamine solutions of different concentrations in a certain proportion to prepare a reaction system. After reacting at 25-30 ℃ for 15-30 min, the spectrophotometer and fluorescence spectrometer are used for spectral detection. The changes in ultraviolet absorption at 425 nm and fluorescence intensity at 450 nm and 555 nm of the colorimetric / fluorescence dual-mode sensor are used as the abscissa, and the ratio of the ultraviolet peak at 425 nm and the fluorescence intensity peak at 450 nm and 555 nm of the colorimetric / fluorescence dual-mode sensor is used as the ordinate to plot the calibration curves.

[0024] Step S2: Mix the colorimetric / fluorescence dual-mode sensor with the histamine solution to be tested in a certain proportion to prepare a reaction system. After reacting at 25-30 °C for 15-30 min, perform spectral detection using a UV-Vis spectrophotometer and a fluorescence spectrometer. Calculate the histamine concentration in the sample based on the calibration curve obtained in step S1 by observing the UV absorption change at 425 nm and the fluorescence intensity changes at 450 nm and 555 nm of the colorimetric / fluorescence dual-mode sensor.

[0025] As a preferred embodiment, the preparation method of the reaction system in steps S1 and 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 making up to 3 mL with deionized water to prepare a 3 mL reaction system; the histamine solution uses ultrapure water as solvent, the OPD solution concentration is 10 mM and the solvent is anhydrous ethanol; the hydrogen peroxide solution concentration is 100 mM.

[0026] 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 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 ratio of fluorescence intensity is obtained by using the ratio of the ultraviolet absorption peak A. 425 The ratio of fluorescence intensity to numerical value F 555 / F 450A calibration curve is obtained by analyzing the functional relationship between histamine concentration and the concentration of histamine. The concentration of histamine to be tested is calculated using the calibration curve. The colorimetric / fluorescence dual-mode sensor exhibits a yellow color and emits yellow fluorescence in the absence of histamine. As the concentration of histamine 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. This enables the colorimetric / fluorescence dual-mode detection of histamine content.

[0027] In a preferred embodiment, HRP@AIE-MOF catalyzes the conversion of colorless OPD to yellow DAP. Upon addition of histamine, the pH of the solution increases, inhibiting the catalytic ability of HRP@AIE-MOF, leading to a decrease in DAP content and a lighter solution color. As a colorimetric detection signal, HRP@AIE-MOF emits blue fluorescence at 450 ± 10 nm, while DAP emits yellow fluorescence at 555 ± 5 nm. A decrease in DAP content results in a decrease in fluorescence intensity at 555 ± 5 nm and an increase in fluorescence intensity of HRP@AIE-MOF at 450 ± 10 nm. The ratio of the UV absorbance at 425 nm to the fluorescence intensity at 450 nm and 555 nm, obtained from the colorimetric / fluorescence dual-mode sensor, exhibits a linear relationship with the concentration of the analyte, thus enabling the detection of histamine concentration in the analyte. The UV-Vis spectral conditions include: an observation range of 350-600 nm; and a fluorescence excitation wavelength of 365 nm. nm, the fluorescence emission spectrum observation range is 380-650 nm.

[0028] The technical principle of this invention is as follows: HRP@AIE-MOF can catalyze the conversion of colorless OPD into yellow DAP. Upon addition of histamine, the pH of the solution increases, inhibiting the catalytic ability of HRP@AIE-MOF, leading to a decrease in DAP content and a lighter solution color. As a colorimetric detection signal, HRP@AIE-MOF emits blue fluorescence at 450 ± 10 nm, while DAP emits yellow fluorescence at 555 ± 5 nm. A decrease in DAP content results in a decrease in fluorescence intensity at 555 ± 5 nm, while the fluorescence intensity of HRP@AIE-MOF at 450 ± 10 nm increases. Based on the change in the concentration of histamine, the yellow DAP content in the sensor solution decreases, the solution color lightens, and simultaneously, the fluorescence intensity of DAP decreases while the fluorescence intensity of HRP@AIE-MOF increases. Furthermore, the ratio of the UV absorption intensity of DAP and the fluorescence intensity of DAP to HRP@AIE-MOF shows a linear relationship with the concentration of the analyte, thereby achieving quantitative detection of histamine concentration in the analyte.

[0029] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0030] 1. This invention uses aggregation-induced emission metal-organic framework AIE-MOF with high brightness as a carrier to immobilize horseradish peroxidase (HRP) on its surface, which solves the problems of difficulty in recovering free enzymes and low stability.

[0031] 2. The colorimetric / fluorescence dual-mode sensor constructed based on HRP@AIE-MOF integrates the excellent catalytic ability of natural enzymes 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 histamine, while reducing detection time and cost.

[0032] 3. The histamine detection method established in this invention has high detection sensitivity, with detection limits of 1.71 μM and 0.56 μM for colorimetric and fluorescence methods, respectively, which meet the relevant requirements of national standards, and also has good anti-interference and stability. Attached Figure Description

[0033] 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.

[0034] Figure 1 These are TEM images of AIE-MOF in Embodiment 1 and HRP@AIE-MOF in Embodiment 2 of the present invention.

[0035] Figure 2 These are particle size distribution diagrams of AIE-MOF in Example 1 and HRP@AIE-MOF in Example 2.

[0036] Figure 3 This is a verification diagram of the catalytic ability of HRP@AIE-MOF in Verification Example 1 of this invention.

[0037] Figure 4 These are daylight images of the colorimetric / fluorescence dual-mode sensor solutions with different concentrations of histamine in Example 4 of this invention.

[0038] Figure 5 These are fluorescence images of the colorimetric / fluorescence dual-mode sensor solutions at different concentrations of histamine in Example 4 of this invention.

[0039] Figure 6 These are the UV-Vis and fluorescence emission spectra of the colorimetric / fluorescence dual-mode sensor solutions with different concentrations of histamine in Example 4 of this invention.

[0040] Figure 7This refers to the UV-Vis absorption peak and colorimetric detection curve of the colorimetric / fluorescence dual-mode sensor solution at different concentrations of histamine in Example 4 of this invention.

[0041] Figure 8 This is the fluorescence intensity ratio and fluorescence detection curve of the colorimetric / fluorescence dual-mode sensor solution under different concentrations of histamine in Example 4 of the present invention.

[0042] Figure 9 This is the selective analysis diagram of the colorimetric / fluorescence dual-mode sensor explored in Verification Example 2 of this invention.

[0043] Figure 10 This is the anti-interference analysis diagram of the colorimetric / fluorescence dual-mode sensor explored in Verification Example 3 of this invention. Detailed Implementation

[0044] 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.

[0045] 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.

[0046] Example 1:

[0047] This embodiment first provides an AIE-MOF material, the synthesis method of which includes the following steps:

[0048] Dissolve 23 mg ZrCl4 and 23 mg tetra-(carboxyphenyl)ethylene in 6.95 mL DMF and sonicate until completely dissolved.

[0049] Add 1 mL of acetic acid and 0.05 mL of deionized water to the above DMF solution as a conditioner.

[0050] Stir for 30 minutes, then turn off the magnetic stirrer and heat at 120 °C for 3 hours.

[0051] 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.

[0052] Remove the supernatant, add 10 mL of anhydrous ethanol, centrifuge, and repeat this step 3 times.

[0053] The resulting precipitate was placed in a 75 °C oven until completely dry. This yielded the AIE-MOF material.

[0054] Characterizing AIE-MOF, Figure 1 1a in the figure is a TEM image of AIE-MOF. As can be seen from the figure, the synthesized AIE-MOF has a flower-like structure and is uniform in size.

[0055] Example 2:

[0056] This embodiment further provides a method for synthesizing HRP@AIE-MOF, which specifically includes the following steps:

[0057] Take 20 mg of AIE-MOF and place it in a 20 mL brown glass sample bottle. Add 5 mL of ultrapure water and sonicate for 1 h to disperse it completely. Add 10 mg each of crosslinking agent EDC and NHS to the above solution and stir at 200 rpm for 1.5 h to obtain solution C.

[0058] Take 10 mg of HRP into a 15 mL centrifuge tube, add 5 mL of phosphate buffer, and obtain solution D.

[0059] The above solution D was added to solution C in one go, and the mixture was stirred at 200 rpm for 1.5 h to obtain solution E.

[0060] After stirring, transfer solution E into two equal portions of 50 mL centrifuge tubes, 10 mL in each tube, and add 10 mL of phosphate buffer solution to each tube. Centrifuge at 12000 rpm, 4 ℃, for 20 min.

[0061] Remove the supernatant, add 10 mL of phosphate buffer solution, centrifuge, and repeat this step 3 times.

[0062] The final precipitate was placed in a freeze dryer and freeze-dried for 3 days to obtain HRP@AIE-MOF material.

[0063] HRP@AIE-MOF was characterized by... Figure 1 As shown in 1b, compared to AIE-MOF, the prepared HRP@AIE-MOF aggregates, and the spike-like protrusions on the surface are less obvious, confirming that HRP is immobilized on AIE-MOF. Furthermore, from... Figure 2 The results show that the particle size of HRP@AIE-MOF has increased significantly, further confirming the successful synthesis of HRP@AIE-MOF.

[0064] Verification Example 1:

[0065] To verify the catalytic activity of HRP@AIE-MOF, all of the HRP@AIE-MOF obtained in Example 2 was dissolved in 20 mL of phosphate buffer (1X).

[0066] 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 solution at pH 4.0. After reacting for 15–30 min, the UV absorption intensity at 652 nm was recorded using a UV-Vis spectrophotometer. HRP was used instead of HRP@AIE-MOF under the same conditions, and the absorbance at 652 nm was recorded as a control. Simultaneously, 15 μL of 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 solution at pH 4.0. After reacting for 15–30 min, the UV absorption intensity at 652 nm was recorded using a UV-Vis spectrophotometer, and the absorbance at 652 nm was measured for AIE-MOF, TMB, hydrogen peroxide, and a mixture of TMB and hydrogen peroxide.

[0067] like Figure 3 The results show that HRP@AIE-MOF still retains high catalytic activity, and its catalytic activity comes from the HRP immobilized on the surface.

[0068] Example 3:

[0069] This embodiment further provides a method for fabricating a colorimetric / fluorescence dual-mode sensor, specifically including the following steps:

[0070] All the HRP@AIE-MOF obtained in Example 2 was dissolved in 20 mL of phosphate buffer (1X). Then, 150 μL of HRP@AIE-MOF solution, 60 μL of OPD solution and 15 μL of hydrogen peroxide solution were taken and diluted to 3 mL with deionized water. The reaction was allowed to proceed for 15-30 min to obtain the colorimetric / fluorescence dual-mode sensor.

[0071] The OPD solution concentration was 10 mM, and the solvent was anhydrous ethanol; the hydrogen peroxide solution concentration was 100 mM.

[0072] Example 4:

[0073] This embodiment provides a colorimetric / fluorescence dual-mode detection method for histamine, specifically including the following steps:

[0074] Add 30 μL of histamine solution of different concentrations to the colorimetric / fluorescence dual-mode sensor prepared in Example 3 to make a 3 mL reaction system, and observe the color change of the solution under sunlight and ultraviolet light.

[0075] Figure 4 These are daylight images of colorimetric / fluorescence dual-mode sensor solutions with different histamine concentrations. As can be seen from the images, the solution color gradually changes from yellow to colorless as the histamine concentration increases.

[0076] Figure 5 These are fluorescence images of the colorimetric / fluorescence dual-mode sensor solution at different histamine concentrations. As can be seen from the images, the fluorescence of the solution changes from bright yellow to light blue as the histamine concentration increases.

[0077] 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 350-600 nm; the fluorescence excitation wavelength was 360 nm, and the observation range of the fluorescence emission spectrum was 380-650 nm.

[0078] Figure 6 These are the UV-Vis and fluorescence emission spectra of the colorimetric / fluorescence dual-mode sensor solutions at different concentrations of histamine, among which... Figure 6 6a in the spectrum represents the ultraviolet-visible spectrum. Figure 6 6b in the figure represents the fluorescence emission spectrum.

[0079] The functional relationship between different histamine concentrations in the solution and the values ​​of UV-Vis absorption peak and fluorescence emission peak yields the histamine concentration values ​​corresponding to the values ​​of UV-Vis absorption peak and fluorescence emission peak in the solution.

[0080] Figure 7 These are the UV-Vis absorbance values ​​and colorimetric detection curves of a colorimetric / fluorescence dual-mode sensor solution at different concentrations of histamine. Figure 7 7a in the figure represents the relationship between histamine concentration and the UV-Vis absorption peak. Figure 7 7b in the figure represents the colorimetric detection curve of the colorimetric / fluorescence dual-mode sensor solution. The x-axis represents the logarithm of the histamine concentration, and the y-axis represents the UV-Vis absorption peak.

[0081] The ultraviolet-visible absorption peak value is: A 425 The UV-Vis absorption peak around 425±5 nm was obtained by monitoring multiple parallel experiments and calculating the average value.

[0082] like Figure 7 As shown in 7b, the functional relationship between the UV-Vis absorption peak and histamine concentration is: Y = -0.4464X + 1.674 (R 2=0.9956), where Y represents the UV-Vis absorption peak at 425 nm, and X represents the logarithm of the histamine concentration. Therefore, the A value corresponding to a colorimetric / fluorescence dual-mode sensor containing an unknown concentration of histamine can be measured. 425 The histamine concentration can be calculated using the above formula to achieve quantitative analysis of histamine.

[0083] 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 sensor for histamine detection is 1.71 μM, calculated using the above linear relationship.

[0084] Figure 8 These are the fluorescence intensity values ​​and fluorescence detection curves of a colorimetric / fluorescence dual-mode sensor solution at different concentrations of histamine. Figure 8 8a in the figure represents the relationship between histamine concentration and fluorescence intensity value. Figure 8 Figure 8b shows the fluorescence detection curve of the colorimetric / fluorescence dual-mode sensor solution. The x-axis represents the logarithm of histamine concentration, and the y-axis represents the fluorescence intensity ratio.

[0085] The fluorescence intensity ratio is calculated using the following method: R = F 555 / F 450 .

[0086] F 555 : The fluorescence intensity value at 555 nm in the fluorescence spectrum corresponding to the sensor solution;

[0087] F 450 : The fluorescence intensity value at 450 nm in the fluorescence spectrum corresponding to the sensor solution;

[0088] F 555 F 450 The fluorescence intensity values ​​near 555 nm and 450 nm were obtained by monitoring multiple parallel experiments and calculating the average value.

[0089] 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 fluorescence intensity peak at 555 nm to the fluorescence intensity peak at 450 nm, and X represents the logarithm of the histamine concentration. Therefore, the F-value of a colorimetric / fluorescence dual-mode sensor containing an unknown concentration of histamine can be measured. 555 / F 450 The histamine concentration can be calculated using the above formula to achieve quantitative analysis of histamine.

[0090] 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 sensor for histamine detection by fluorescence method is calculated to be 0.56 μM.

[0091] Example 5

[0092] This embodiment provides a colorimetric / fluorescence dual-mode detection method for histamine, specifically including:

[0093] 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-Vis absorption peak and histamine concentration, and the functional relationship between the fluorescence emission peak and histamine concentration;

[0094] Step (2): Add the histamine solution to be tested (concentration unknown, prepared by diluting 1000 μM histamine solution several times) to the colorimetric / fluorescence dual-mode sensor prepared in Example 3 to make a 3 mL reaction system. Use a UV-Vis spectrophotometer and a fluorescence spectrometer for spectral detection. Calculate the histamine concentration in the sample based on the detection curve and function relationship obtained in step (1) by observing the UV absorption change at 425 nm and the fluorescence intensity changes at 450 nm and 555 nm of the colorimetric / fluorescence dual-mode sensor. The average value is 312 μM.

[0095] Example 6

[0096] This embodiment provides a colorimetric / fluorescence dual-mode detection method for histamine, specifically including:

[0097] 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-Vis absorption peak and histamine concentration, and the functional relationship between the fluorescence emission peak and histamine concentration;

[0098] Step (2): Add the histamine solution to be tested (concentration known, 300 μM histamine solution) to the colorimetric / fluorescence dual-mode sensor prepared in Example 3 to make a 3 mL reaction system. Use a UV-Vis spectrophotometer and a fluorescence spectrometer for spectral detection. Calculate the histamine concentration in the sample based on the detection curve and function relationship obtained in step (1) by the UV absorption change at 425 nm and the fluorescence intensity change at 450 nm and 555 nm of the colorimetric / fluorescence dual-mode sensor. The average value is 303 μM.

[0099] Verification Example 2:

[0100] A colorimetric / fluorescence dual-mode sensor was prepared according to Example 3, using Ca... 2+ Cl- CO3 2- The selective detection of histamine by a colorimetric / fluorescence dual-mode sensor was investigated using interfering ions such as histidine, lysine, and other precursor amino acids, as well as biogenic amines such as cadaverine and putrescine. The concentration of the interfering ions was 100 times that of the histamine solution. After reacting at 25-30℃ for 15-30 min, the spectroscopic detection was performed using a UV-Vis spectrophotometer and a fluorescence spectrometer. The changes in UV absorption at 425 nm and fluorescence intensity at 450 nm and 555 nm were observed by the colorimetric / fluorescence dual-mode sensor.

[0101] Figure 9 9a in the text refers to the selective analysis of the colorimetric mode. Figure 9 Figure 9b shows the selective analysis of the fluorescence mode. As can be seen from the figure, the UV absorbance and fluorescence intensity of the colorimetric / fluorescence dual-mode sensor change significantly only in the presence of histamine and biogenic amines such as cadaverine. Ions and precursor amino acids do not 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.

[0102] Verification Example 3:

[0103] A colorimetric / fluorescence dual-mode sensor was prepared according to Example 3, using Ca... 2+ Cl - CO3 2- Using histidine, lysine, and other precursor amino acids as interfering ions, the anti-interference ability of a colorimetric / fluorescence dual-mode sensor for detecting histamine was investigated. 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-Vis spectrophotometer and a fluorescence spectrometer. The changes in UV absorption at 425 nm and fluorescence intensity at 450 nm and 555 nm were observed by the colorimetric / fluorescence dual-mode sensor.

[0104] Figure 10 10a in the text refers to the selective analysis of the colorimetric mode. Figure 10 Figure 10b shows the selective analysis of the fluorescence mode. As can be seen from the figure, there is no significant difference in the effect of histamine solution mixed with interfering ions and histamine solution alone on the ultraviolet absorption value and fluorescence intensity of this colorimetric / fluorescence dual-mode sensor. This indicates that the sensor has good anti-interference ability and can be used for histamine detection in complex matrices.

[0105] 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. An application of a colorimetric / fluorescence dual-mode sensor in histamine detection, characterized in that, The colorimetric / fluorescence dual-mode sensor comprises a horseradish peroxidase-loaded metal-organic framework (HRP@AIE-MOF), hydrogen peroxide, and the chromogenic substrate o-phenylenediamine (OPD). The horseradish peroxidase-loaded HRP@AIE-MOF is a zirconium-based aggregation-induced emission (AIE-MOF) material loaded with horseradish peroxidase (HRP). The zirconium-based AIE-MOF is formed by the self-assembly of zirconium (Zr) metal ions and the organic ligand tetra-(carboxyphenyl)ethylene. The horseradish peroxidase (HRP) in the HRP@AIE-MOF can oxidize the colorless chromogenic substrate o-phenylenediamine (OPD) to a yellow 2,3-diaminophenazine (DAP), producing a UV absorption peak at 425 ± 25 nm and a 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. HRP@AIE-MOF catalyzes the conversion of colorless OPD into yellow DAP. Upon the addition of histamine, the pH of the solution increases, inhibiting the catalytic activity of HRP@AIE-MOF, leading to a decrease in DAP content and a lighter solution color. As a colorimetric detection signal, HRP@AIE-MOF emits blue fluorescence at 450 ± 10 nm, while DAP emits yellow fluorescence at 555 ± 5 nm. A decrease in DAP content results in a decrease in fluorescence intensity at 555 ± 5 nm, while the fluorescence intensity of HRP@AIE-MOF at 450 ± 10 nm increases. Based on the change in histamine concentration, the yellow DAP content in the sensor solution decreases, the solution color lightens, and the fluorescence intensity of DAP decreases while the fluorescence intensity of HRP@AIE-MOF increases. The linear relationship between the UV absorption intensity of DAP and the ratio of the fluorescence intensities of DAP and HRP@AIE-MOF with the concentration of the analyte allows for the quantitative detection of histamine concentration in the analyte.

2. The application according to claim 1, characterized in that, In the horseradish peroxidase-loaded metal-organic framework HRP@AIE-MOF, the horseradish peroxidase (HRP) loading rate is 85%-90%, and the particle size of the horseradish peroxidase-loaded metal-organic framework HRP@AIE-MOF material is 150-300 nm.

3. The application according to claim 1 or 2, characterized in that, The fabrication method of the colorimetric / fluorescence dual-mode sensor includes the following steps: Step (1), Preparation of zirconium-based aggregation-induced emission metal-organic framework AIE-MOF: Zirconium metal salt and tetra-(carboxyphenyl)ethylene are dissolved in N,N-dimethylformamide DMF solvent, and then acetic acid and deionized water are added as regulators to carry out a solvothermal reaction. The obtained solution is then washed, filtered and dried to obtain zirconium-based aggregation-induced emission metal-organic framework AIE-MOF powder material. Step (2), preparation of horseradish peroxidase-loaded metal-organic framework HRP@AIE-MOF: The zirconium-based aggregation-induced emission metal-organic framework AIE-MOF obtained in step (1) was dissolved in deionized water, and then crosslinking agents 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride EDC and N-hydroxysuccinimide NHS were added and stirred for a period of time. Then horseradish peroxidase HRP phosphate buffer was added and stirred for a period of time to carry out the crosslinking reaction. Then the obtained solution was washed, filtered and dried to obtain horseradish peroxidase-loaded metal-organic framework HRP@AIE-MOF powder material. Step (3), preparation of the colorimetric / fluorescence dual-mode sensor: The phosphate buffer of the horseradish peroxidase-loaded metal-organic framework HRP@AIE-MOF obtained in step (2) is stored at 4°C for later use. The chromogenic substrate o-phenylenediamine OPD is dissolved in anhydrous ethanol to prepare an OPD solution. A 30% hydrogen peroxide aqueous solution is diluted to prepare a hydrogen peroxide solution. Then, the phosphate buffer of the horseradish peroxidase-loaded metal-organic framework HRP@AIE-MOF, the OPD solution and the hydrogen peroxide solution are mixed and diluted with deionized water. The reaction is carried out for 15-30 min to obtain the colorimetric / fluorescence dual-mode sensor.

4. The application according to claim 3, characterized in that, In step (1), the zirconium metal salt is selected from zirconium nitrate and / or zirconium tetrachloride, the mass ratio of zirconium metal salt to tetra-(carboxyphenyl)ethylene is 1:(1-5), and the molar ratio of zirconium metal salt to tetra-(carboxyphenyl)ethylene is 1:(0.5-1); the temperature of the solvothermal reaction is 100-120°C, the reaction time is 3-24h, the washing agent used is anhydrous ethanol, and the drying method is oven drying at a temperature of 60-75°C.

5. The application 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; the mass concentration of HRP in the horseradish peroxidase (HRP) phosphate buffer is 0.5-2 mg / mL, and the mass ratio of AIE-MOF to HRP is (0.5-2):1; both stirring operations are carried out at room temperature, with a stirring speed of 100-300 rpm, and the stirring time for adding crosslinking agent EDC and NHS is 1-2 h, followed by the stirring time for adding HRP phosphate buffer is 8-24 h; the washing agent used is phosphate buffer, and the drying method is vacuum freeze-drying.

6. The application 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 OPD solution is 10 mM, and the molar concentration of hydrogen peroxide solution is 100 mM. 150 μL of phosphate buffer of the metal-organic framework HRP@AIE-MOF loaded with horseradish peroxidase, 60 μL of OPD solution and 15 μL of hydrogen peroxide solution are mixed, and the reaction system is brought to a final volume of 3 mL with deionized water.

7. The application according to claim 4, characterized in that, The mass ratio of the zirconium metal salt to tetra-(carboxyphenyl)ethylene is 1:(1-2).

8. The application according to claim 5, characterized in that, The mass ratio of AIE-MOF, EDC, and NHS is 5:1:

1.

9. The application according to claim 1, characterized in that, The detection limits for colorimetric and fluorescence methods were 1.71 μM and 0.56 μM, respectively.

10. A method for detecting histamine using a colorimetric / fluorescence dual-mode sensor, characterized in that, Includes the following steps: Step S1: Mix the colorimetric / fluorescence dual-mode sensor with a series of histamine solutions of different concentrations in a certain proportion to prepare a reaction system. After reacting at 25-30℃ for 10-20 min, perform spectral detection using a UV-Vis spectrophotometer and a fluorescence spectrometer. Plot calibration curves by using the changes in UV absorption at 425 nm and fluorescence intensity at 450 nm and 555 nm of the colorimetric / fluorescence dual-mode sensor as the abscissa and the ratio of the UV peak at 425 nm and the fluorescence intensity peak at 450 nm and 555 nm of the colorimetric / fluorescence dual-mode sensor as the ordinate. Step S2: Mix the colorimetric / fluorescence dual-mode sensor with the histamine solution to be tested in a certain proportion to prepare a reaction system. After reacting at 25-30℃ for 10-20 min, perform spectral detection using a UV-Vis spectrophotometer and a fluorescence spectrometer. Calculate the histamine concentration in the sample based on the calibration curve obtained in step S1 by observing the UV absorption change at 425 nm and the fluorescence intensity changes at 450 nm and 555 nm of the colorimetric / fluorescence dual-mode sensor. The colorimetric / fluorescence dual-mode sensor comprises a horseradish peroxidase-loaded metal-organic framework (HRP@AIE-MOF), hydrogen peroxide, and the chromogenic substrate o-phenylenediamine (OPD). The horseradish peroxidase-loaded HRP@AIE-MOF is a zirconium-based aggregation-induced emission (AIE-MOF) material loaded with horseradish peroxidase (HRP). The zirconium-based AIE-MOF is formed by the self-assembly of zirconium (Zr) metal ions and the organic ligand tetra-(carboxyphenyl)ethylene. The horseradish peroxidase (HRP) in the HRP@AIE-MOF can oxidize the colorless chromogenic substrate o-phenylenediamine (OPD) to a yellow 2,3-diaminophenazine (DAP), producing a UV absorption peak at 425±25 nm and a 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. HRP@AIE-MOF catalyzes the conversion of colorless OPD to yellow DAP. Upon addition of histamine, the pH of the solution increases, inhibiting the catalytic activity of HRP@AIE-MOF, leading to a decrease in DAP content and a lighter solution color. As a colorimetric detection signal, HRP@AIE-MOF emits blue fluorescence at 450 ± 10 nm, while DAP emits yellow fluorescence at 555 ± 5 nm. A decrease in DAP content results in a decrease in fluorescence intensity at 555 ± 5 nm and an increase in fluorescence intensity of HRP@AIE-MOF at 450 ± 10 nm. The ratio of the UV absorbance at 425 nm to the fluorescence intensity at 450 nm and 555 nm, obtained through a colorimetric / fluorescence dual-mode sensor, exhibits a linear relationship with the concentration of the analyte, thus enabling the detection of histamine concentration in the analyte.

11. The method for detecting histamine using a colorimetric / fluorescence dual-mode sensor according to claim 10, characterized in that, The preparation method of the reaction system in steps S1 and 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 making up to 3 mL with water to prepare a 3 mL reaction system; the histamine solution uses ultrapure water as solvent, the OPD solution concentration is 10 mM and the solvent is anhydrous ethanol; the hydrogen peroxide solution concentration is 100 mM.

12. The method for detecting histamine using a colorimetric / fluorescence dual-mode sensor according to claim 10, 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 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 ratio of fluorescence intensity is obtained by using the ratio of the ultraviolet absorption peak A. 425 The ratio of fluorescence intensity to numerical value F 555 / F 450 A calibration curve is obtained by analyzing the functional relationship between histamine concentration and the concentration of histamine. The concentration of histamine to be tested is calculated using the calibration curve. The colorimetric / fluorescence dual-mode sensor exhibits a yellow color and emits yellow fluorescence in the absence of histamine. As the concentration of histamine 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. This enables the colorimetric / fluorescence dual-mode detection of histamine content.

13. The method for detecting histamine using a colorimetric / fluorescence dual-mode sensor according to claim 10, characterized in that, The conditions for ultraviolet-visible spectroscopy include: the observation range of ultraviolet-visible spectroscopy is 350-600 nm; the fluorescence excitation wavelength is 365 nm; and the observation range of fluorescence emission spectroscopy is 380-650 nm.

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