A ratiometric fluorescent sensor, its preparation method and application in histamine detection

Abstract

The application provides a ratio fluorescent sensor, a preparation method thereof and application thereof in histamine detection, and the ratio fluorescent sensor comprises a terbium metal organic framework (MR@Tb-MOF) loaded with methyl red dye; the methyl red dye in the MR@Tb-MOF can have a strong characteristic fluorescent emission peak at 390 nm, the terbium metal organic framework (Tb-MOF) in the MR@Tb-MOF has a strong characteristic fluorescent emission peak at 545 nm, and can form a ratio fluorescent sensor with the fluorescent signal of the methyl red (MR) dye. The application integrates the excellent fluorescent capacity of the Tb-MOF and the pH sensitive characteristics of the methyl red dye in one system, and realizes high-sensitivity histamine detection.

Description

Technical Field

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

[0002] Histamine (HA) is a widely distributed autoactive substance in living organisms, mainly synthesized and secreted by mast cells, basophils, and platelets. It participates in various physiological functions such as cell proliferation and differentiation, hematopoiesis, embryonic development, and wound healing, and is also a key mediator of inflammatory responses and immune damage. Abnormal changes in histamine concentration are closely related to the occurrence and development of various diseases, such as autoimmune diseases, tumors, and infectious diseases such as respiratory syncytial virus infection and chronic hepatitis. Accurate detection of histamine levels is of great significance for early disease diagnosis, pathological mechanism research, and treatment efficacy evaluation. Simultaneously, histamine is also a key biogenic amine indicator monitored in the food industry. As a "chemical fingerprint" of protein spoilage, it easily accumulates in high-protein foods such as fish and meat during spoilage. Excessive histamine intake can cause poisoning symptoms such as headache, allergies, vomiting, and even shock, seriously threatening food safety and public health. Current detection technologies face challenges in practical applications, including insufficient response speed, significant interference from complex matrices, and limited sensitivity. Therefore, there is an urgent practical need for rapid, accurate, and highly selective detection of histamine in biological samples and food, in fields such as clinical medicine and food safety supervision.

[0003] Fluorescence sensing technology has attracted widespread attention in the field of histamine detection due to its high sensitivity, fast response speed, ease of operation, and ability to achieve in-situ real-time detection. However, traditional single fluorescence sensors detect histamine based on changes in fluorescence intensity at a single wavelength, which is susceptible to environmental factors (such as temperature, pH value, and fluctuations in instrument light source) and sample matrix interference, resulting in limited accuracy and stability of detection results and making it difficult to adapt to histamine detection scenarios in complex sample systems.

[0004] The emergence of ratiometric fluorescence sensors offers an effective solution to the aforementioned problems. These sensors typically construct a dual-fluorophore system, detecting the intensity ratio of fluorescence signals at two different wavelengths to achieve quantitative analysis of the target analyte. Both fluorophores respond to the target analyte, histamine, but their response trends differ (e.g., one enhances fluorescence while the other quenches, or their fluorescence intensity changes at different rates and amplitudes). By capturing the changes in the fluorescence intensity ratio resulting from this differential response, the sensor can leverage the synergistic effect of the two signals to enhance the sensitivity of target analyte identification. Furthermore, it can utilize the consistency of the influence of environmental factors and matrix interference on the two fluorescence signals, allowing them to cancel each other out when calculating the intensity ratio, significantly improving the detection's anti-interference capability, accuracy, and stability. However, existing ratiometric fluorescence sensors generally have high detection limits, unable to detect histamine concentrations below 1 μM. Summary of the Invention

[0005] To address the above technical problems, this invention provides a ratiometric fluorescence sensor, its preparation method, and its application in histamine detection. This ratiometric fluorescence sensor can quantitatively detect histamine in real time / on-site, with high sensitivity, a detection limit of 3.34 ng / mL, and a convenient, rapid, and low-cost detection process.

[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 ratiometric fluorescence sensor comprising a dual-fluorophore system; said dual-fluorophore system is a terbium-based metal-organic framework (MR@Tb-MOF) loaded with methyl red (MR) dye, wherein the terbium-based metal-organic framework (Tb-MOF) in said MR@Tb-MOF is composed of terbium (Tb) metal ions (Tb 3+ The methyl red dye and the organic ligand 1,3,5-benzenetricarboxylic acid (H3BTC) are self-assembled to form a ratio fluorescence sensor. The methyl red dye has a strong characteristic fluorescence emission peak at 390 ± 10 nm, and the Tb-MOF has a strong characteristic fluorescence emission peak at 545 ± 5 nm. The fluorescence emission peaks of the two do not overlap significantly, thus constituting a ratio fluorescence sensor.

[0008] According to the present invention, the Tb-MOF has a regular three-dimensional porous structure, which provides abundant adsorption sites, enhances the loading stability of methyl red dye, and avoids luminescence quenching caused by fluorophore aggregation. The methyl red dye is loaded onto the surface and pores of the Tb-MOF through physical adsorption, forming a composite system MR@Tb-MOF. The MR@Tb-MOF material exhibits good dispersibility in aqueous media and retains the fluorescence properties of both the methyl red dye and the Tb-MOF.

[0009] According to the present invention, a metal-organic framework (Tb-MOF) material is formed by the self-assembly of the organic ligand 1,3,5-benzenetricarboxylic acid and terbium metal ion clusters. Compared with other MOF materials, it has the characteristics of long lifetime luminescence, sharp emission band, high quantum yield and good chemical stability, making it an ideal carrier for constructing ratiometric platforms. Methyl red, as a pH-sensitive dye, has strong fluorescence emission at a specific wavelength. Its emission peak is independent and distinguishable from the fluorescence emission peak of Tb-MOF. Its absorption peak highly overlaps with the emission spectrum of Tb-MOF, enabling efficient energy transfer. Furthermore, the regular three-dimensional porous structure enhances the coordination stability of the target analyte histamine and the loading capacity of methyl red dye, maintaining structural integrity in the liquid-phase detection system and exhibiting both high-sensitivity fluorescence response and excellent stability. This invention uses Tb-MOF as the core and loads the dye methyl red. It fully leverages the advantages of Tb-MOF's high specific surface area and the massive adsorption sites provided by its open channels to ensure efficient physical adsorption within the system. At the same time, it effectively avoids fluorescence quenching caused by fluorophore aggregation, which would weaken the signal. Under the premise of maintaining the integrity of the Tb-MOF channels, it achieves dual preservation of fluorescence signal in the detection system.

[0010] According to the present invention, a metal-organic framework (Tb-MOF) material is formed by the self-assembly of the organic ligand 1,3,5-benzenetricarboxylic acid and terbium metal ion clusters. This Tb-MOF material is then mixed with methyl red dye to prepare MR@Tb-MOF material. The Tb-MOF exhibits a strong characteristic fluorescence emission peak at 545 ± 5 nm, corresponding to Tb... 3+ of 5 D4→ 7 The F5 transition, with the methyl red dye exhibiting a strong characteristic fluorescence emission peak at 390 ± 10 nm, constitutes the ratiometric fluorescence sensor.

[0011] In a preferred embodiment, the MR@Tb-MOF material has a particle size of 650-750 nm. The ratiometric fluorescence sensor of this invention has a detection limit of 3.34 ng / mL for histamine, enabling real-time / on-site quantitative detection of histamine. This makes the detection process portable, rapid, and cost-effective, meeting the needs of food safety and clinical medical testing.

[0012] Secondly, the present invention also provides a method for preparing the ratio fluorescence sensor as described above, specifically including the following steps:

[0013] Step 1: Synthesis of Tb-MOF: Terbium metal salt and organic ligand 1,3,5-benzenetricarboxylic acid are dissolved in N,N-dimethylformamide (DMF) solvent, and then acetic acid is added as a regulator to carry out a solvothermal reaction. The resulting dispersion is then washed, filtered and dried to obtain Tb-MOF powder material.

[0014] Step 2: Synthesis of terbium-based metal-organic framework (MR@Tb-MOF) loaded with methyl red dye: The Tb-MOF obtained in Step 1 was dispersed in deionized water, and then methyl red solution was added. The mixture was sonicated to form a uniform dispersion, and then stirred at room temperature in the dark for a period of time. The obtained dispersion was washed, filtered, and dried to obtain MR@Tb-MOF powder material.

[0015] Step 3: Preparation of ratio fluorescence sensor: Disperse the MR@Tb-MOF obtained in step 2 in an aqueous medium to obtain the ratio fluorescence sensor.

[0016] In a preferred embodiment, in step 1, the terbium metal salt is selected from terbium nitrate hexahydrate (Tb(NO3)3·6H2O), the molar ratio of the terbium metal salt to the organic ligand 1,3,5-benzenetricarboxylic acid is 1:1; the solvothermal reaction temperature is 100-120°C, the reaction time is 1-3 h, the washing agent used 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 Tb-MOF dispersed in deionized water is 1-5 mg / mL, and the mass ratio of Tb-MOF to dye MR is 1:(0.75-2), preferably 1:1; the stirring time in the dark is 2-4 h, the stirring speed is 100-300 rpm, the detergent used for washing is deionized water, and the drying method is oven drying at a temperature of 60-75℃.

[0018] In a preferred embodiment, in step 3, the MR@Tb-MOF in the ratiometric fluorescence sensor is dispersed in deionized water at a concentration of 0.5-1 mg / mL.

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

[0020] According to the present invention, the ratiometric fluorescence sensor prepared by the present invention is based on the specific interaction mechanism between dual fluorophores and histamine. When histamine is present, the pH value of the dispersion increases, and the fluorescence intensity of methyl red dye and Tb-MOF is synchronously quenched with a difference in response sensitivity. This difference serves as the fluorescence detection signal, and the fluorescence intensity ratio (F) is used to detect the fluorescence intensity. 390 / F 545 The regular changes in histamine levels enable quantitative detection of histamine.

[0021] According to the present invention, the ratiometric fluorescence sensor prepared by the present invention has a pH value of the sensor dispersion that is affected by the concentration of histamine in the analyte, resulting in a change in fluorescence intensity, thereby realizing the quantitative detection of the concentration of histamine in the analyte. The ratiometric fluorescence sensor of the present invention has a detection limit of 3.34 ng / mL for histamine and can accurately detect histamine in the concentration range of 5-200 ng / mL; it can quantitatively detect histamine in real time / on-site, making the detection process portable, fast and low cost, meeting the detection requirements for food safety.

[0022] Fourthly, the present invention also provides a method for detecting histamine using a ratio fluorescence sensor as described above, specifically comprising the following steps:

[0023] Step S1: Mix the ratio fluorescence 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 20-25 min, perform spectral detection using a fluorescence spectrometer. Plot a calibration curve by using the fluorescence intensity changes of the ratio fluorescence sensor at 390 nm and 545 nm, with the histamine concentration as the abscissa and the ratio of the fluorescence intensity peaks of the ratio fluorescence sensor at 390 nm and 545 nm as the ordinate.

[0024] Step S2: Mix the ratio fluorescence sensor with the histamine solution to be tested in a certain proportion to prepare a reaction system. After reacting at 25-30℃ for 20-25 min, perform spectral detection using a fluorescence spectrometer. Calculate the histamine concentration in the sample based on the fluorescence intensity changes of the ratio fluorescence sensor at 390 nm and 545 nm, according to the calibration curve obtained in step S1, thereby achieving the detection of histamine.

[0025] As a preferred embodiment, the preparation method of the reaction system in steps S1 and S2 includes: adding 600 μL of MR@Tb-MOF dispersion and 600 μL of histamine solution to prepare a 1.2 mL reaction system; the histamine solution uses ultrapure water as the solvent, the mass concentration of the MR@Tb-MOF dispersion is 1 mg / mL, and the dispersant is deionized water.

[0026] In a preferred embodiment, a fluorescence spectrometer was used for spectral detection, and the fluorescence intensity F at 390 nm was recorded. 390 Fluorescence intensity F at 545 nm 545 Calculate F 390 / F 545 The ratio of fluorescence intensity is used to obtain the fluorescence intensity ratio value, and the fluorescence intensity ratio value F is used to obtain the fluorescence intensity ratio value. 390 / F 545A 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 ratio fluorescence sensor appears green and emits green fluorescence in the absence of histamine. As the concentration of histamine increases, the fluorescence intensity at 390 nm and at 545 nm both decrease, and the fluorescence color changes from green to light green, thus realizing the detection of histamine content in ratio fluorescence mode.

[0027] In a preferred embodiment, the addition of histamine increases the pH of the dispersion. Histamine can disrupt the conjugated π system of methyl red dye, leading to a significant decrease in its fluorescence emission efficiency and a drop in fluorescence intensity at 390 ± 10 nm. Histamine can also react with Tb in Tb-MOF. 3+ The empty orbitals form a coordination effect, hindering Tb 3+ Energy transfer processes between organic ligands inhibit Tb 3+ The characteristic fluorescence emission of Tb-MOF causes a decrease in fluorescence intensity at 545 ± 5 nm. The ratio of fluorescence intensity at 390 nm and 545 nm by the ratiometric fluorescence sensor shows a linear relationship with the concentration of the analyte, thereby enabling the detection of histamine concentration in the analyte. The fluorescence spectrometer conditions include: fluorescence excitation wavelength of 310-330 nm and fluorescence emission spectrum observation range of 330-650 nm.

[0028] The technical principle of this invention is as follows: Methyl red dye with a carboxyl group (-COOH) can undergo a specific proton transfer interaction with the amino group (-NH2) in histamine molecules, disrupting the conjugated π system of the methyl red dye, leading to a significant decrease in its fluorescence emission efficiency. The fluorescence intensity at 390 ± 10 nm is significantly quenched with increasing histamine concentration. According to the scheme of this invention, histamine, as a nitrogen-containing organic molecule, has amino and imino groups that can interact with Tb in Tb-MOF. 3+ The empty orbitals form coordination interactions, and histamine molecules occupy porous structural sites in Tb-MOF, hindering Tb 3+ Energy transfer processes between organic ligands inhibit Tb 3+ The characteristic fluorescence emission of the Tb-MOF causes the fluorescence intensity at 545 ± 5 nm to decrease synchronously with increasing histamine concentration. Due to the difference in the response sensitivity of the two fluorophores to histamine, the ratio of their fluorescence intensities (F...)... 390 / F 545 The fluorescence intensity decreases systematically with changes in histamine concentration, providing a stable signal basis for quantitative detection. By establishing a linear correlation between the ratio of fluorescence intensity and histamine concentration, the quantitative detection of histamine concentration in the analyte can be achieved.

[0029] Advantages of this invention:

[0030] 1. This invention uses terbium-based metal-organic frameworks (Tb-MOF) with good chemical stability as a carrier to physically adsorb the pH-sensitive dye methyl red, constructing a dual-response ratio fluorescence system, which amplifies the variation range of the ratio signal and can meet the detection requirements of low-concentration histamine.

[0031] 2. The ratiometric fluorescence sensor based on MR@Tb-MOF of this invention integrates the excellent fluorescence ability of terbium-based metal-organic framework Tb-MOF with the pH-sensitive properties of methyl red dye into one system. It is simple to 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 a detection limit of 3.34 ng / mL, meeting 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 SEM images of Tb-MOF in Embodiment 1 and MR@Tb-MOF in Embodiment 2 of the present invention.

[0035] Figure 2 This is the particle size distribution diagram of MR@Tb-MOF in Embodiment 2 of the present invention.

[0036] Figure 3 This is a fluorescence performance verification diagram of MR@Tb-MOF in Example 2 of the present invention.

[0037] Figure 4 These are daylight images of the ratio fluorescence sensor dispersions under different concentrations of histamine in Example 4 of this invention.

[0038] Figure 5 These are fluorescence images of the ratio fluorescence sensor dispersions at different histamine concentrations in Example 4 of this invention.

[0039] Figure 6 These are the fluorescence emission spectra of the ratio fluorescence sensor dispersions under different concentrations of histamine in Example 4 of this invention.

[0040] Figure 7 These are the fluorescence detection curves of the ratio fluorescence sensor dispersions under different concentrations of histamine in Example 4 of this invention.

[0041] Figure 8 This is the selective analysis diagram of the ratio fluorescence sensor explored in Verification Example 2 of this invention.

[0042] Figure 9 This is the anti-interference analysis diagram of the ratio fluorescence sensor explored in Verification Example 2 of this invention. Detailed Implementation

[0043] 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.

[0044] 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.

[0045] Example 1

[0046] This embodiment first provides a Tb-MOF material, the synthesis method of which includes the following steps:

[0047] 45.3 mg of terbium nitrate hexahydrate and 21.0 mg of 1,3,5-benzenetricarboxylic acid were dissolved in 20 mL of DMF and sonicated until completely dissolved.

[0048] Add 1 mL of acetic acid to the above DMF solution as a regulator.

[0049] Place the mixture in silicone oil at 120°C, turn on the magnetic stirrer and stir at 200 rpm for 30 minutes, then turn off the magnetic stirrer and keep heating at 120°C for 2 hours.

[0050] After heating is complete, wait for the mixture to cool to room temperature, then transfer the mixture into equal portions to 50 mL centrifuge tubes, add 10 mL of anhydrous ethanol, and centrifuge at 12000 rpm, 4℃, for 20 min.

[0051] Remove the supernatant, add 10 mL of anhydrous ethanol, centrifuge, and repeat this step 3 times.

[0052] The resulting precipitate was placed in a 75°C oven until completely dry, yielding the Tb-MOF material.

[0053] Tb-MOF was characterized. Figure 1 Figure 1a shows the SEM image of Tb-MOF. As can be seen from the figure, the synthesized Tb-MOF has a rod-shaped structure with uniform size.

[0054] Example 2

[0055] This embodiment provides an MR@Tb-MOF material, the synthesis method of which includes the following steps:

[0056] Take 10 mg of Tb-MOF into a 20 mL brown glass sample bottle, add 10 mL of ultrapure water, and sonicate for 1 h to completely disperse it, thus obtaining a uniform dispersion C.

[0057] Take 10 mg of MR into a 20 mL brown glass sample bottle, add 10 mL of ethanol, and sonicate until completely dissolved. After complete dissolution, obtain solution D.

[0058] Add the above solution D to the dispersion C all at once, turn on the magnetic stirrer and stir at 200 rpm, keep away from light, stir for 3 h, then turn off the magnetic stirrer to obtain a uniform dispersion E.

[0059] After stirring, transfer the dispersion E into two equal portions of 50 mL centrifuge tubes, 10 mL of solution in each tube, and then add 10 mL of deionized water to each tube. Centrifuge at 12000 rpm, 4℃, for 20 min.

[0060] Remove the supernatant, add 10 mL of deionized water, and centrifuge. Repeat this step 3 times.

[0061] The resulting precipitate was placed in a 75°C oven until completely dry, yielding the MR@Tb-MOF material.

[0062] The MR@Tb-MOF was characterized. Figure 1 Figure 1b shows the SEM image of MR@Tb-MOF. As can be seen from the image, compared to Tb-MOF, the synthesized MR@Tb-MOF surface exhibits aggregation due to the loading of MR dyes, indicating that MR neutralizes the electrostatic interactions between Tb-MOF components. Figure 2 (Dynamic light scattering DLS) shows that the particle size of MR@Tb-MOF is 650-750 nm.

[0063] Verification Example 1

[0064] To verify the fluorescence properties of MR@Tb-MOF, 10 mg of MR@Tb-MOF obtained in Example 2 was dispersed in 10 mL of ultrapure water.

[0065] Then, 600 μL of MR@Tb-MOF dispersion was taken and diluted to 1.2 mL with deionized water. After standing for 20-25 min, the fluorescence intensity at 390 ± 10 nm and 545 ± 5 nm was recorded using a fluorescence spectrophotometer.

[0066] As a control 1: Tb-MOF was used instead of MR@Tb-MOF, and all other conditions were the same. The reaction system was prepared and the fluorescence intensity at 545 nm was recorded.

[0067] As a control 2: MR was used instead of MR@Tb-MOF, and all other conditions were the same. The reaction system was prepared and the fluorescence intensity at 390 nm was recorded.

[0068] like Figure 3 The results showed that Tb-MOF exhibited a characteristic fluorescence emission peak at 545 nm, MR dye exhibited a characteristic fluorescence emission peak at 390 nm, and MR@Tb-MOF exhibited obvious fluorescence emission peaks at both 390 nm and 545 nm, with stable fluorescence intensity. This demonstrates that the two fluorophores successfully coexisted and maintained their respective fluorescence characteristics, indicating that the composite system has excellent fluorescence performance.

[0069] Example 3

[0070] This embodiment further provides a method for fabricating a ratiometric fluorescence sensor, specifically including the following steps:

[0071] 10 mg of the MR@Tb-MOF obtained in Example 2 was dispersed in 10 mL of ultrapure water. Then, 600 μL of the MR@Tb-MOF dispersion was taken to obtain the ratiometric fluorescence sensor.

[0072] Example 4

[0073] This embodiment provides a ratio fluorescence sensor detection method for histamine, which specifically includes the following steps:

[0074] Add 600 μL of histamine solution of different concentrations to the ratio fluorescence sensor prepared in Example 3 to make a 1.2 mL reaction system, and observe the color change of the solution under sunlight and ultraviolet light.

[0075] Figure 4 These are daylight images of the dispersion of a ratio fluorescence sensor at different histamine concentrations. The images show that as the histamine concentration increases, the color of the dispersion gradually changes from orange to yellow.

[0076] Figure 5 These are fluorescence images of the ratiometric fluorescence sensor dispersion at different histamine concentrations. As can be seen from the images, the fluorescence of the dispersion changes from green to light green as the histamine concentration increases.

[0077] Fluorescence emission spectrometry was performed using a fluorescence spectrophotometer. The fluorescence excitation wavelength was 310-330 nm, and the fluorescence emission spectrum observation range was 330-650 nm.

[0078] Figure 6 These are the fluorescence emission spectra of the ratio fluorescence sensor dispersions at different concentrations of histamine.

[0079] The functional relationship between different histamine concentration values ​​of the dispersion and the fluorescence emission peak value yields the histamine concentration values ​​corresponding to the fluorescence emission peak values ​​in the dispersion.

[0080] Figure 7 These are fluorescence detection curves of the ratio fluorescence sensor dispersion at different histamine concentrations, where the x-axis represents the histamine concentration and the y-axis represents the fluorescence intensity ratio.

[0081] The fluorescence intensity ratio is calculated using the following method: R = F 390 / F 545 .

[0082] F 390 The fluorescence intensity value at 390 nm in the fluorescence spectrum corresponding to the sensor dispersion;

[0083] F 545 : The fluorescence intensity value at 545 nm in the fluorescence spectrum corresponding to the sensor dispersion;

[0084] F 390 F 545 The fluorescence intensity values ​​near 390 nm and 545 nm were obtained by monitoring multiple parallel experiments and calculating the average value.

[0085] The functional relationship between the fluorescence intensity ratio and histamine concentration is: Y = -0.00181X + 0.984(R) 2 =0.995), where Y represents the ratio of the fluorescence intensity peak at 390 nm to the fluorescence intensity peak at 545 nm, and X represents the histamine concentration. Therefore, the F-value of the ratio fluorescence sensor corresponding to an unknown concentration of histamine can be measured. 390 / F 545 The histamine concentration can be calculated using the above formula to achieve quantitative analysis of histamine.

[0086] The detection limit is 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. Based on this linear relationship, the detection limit for histamine by this ratiometric fluorescence sensor is calculated to be 3.34 ng / mL.

[0087] Example 5

[0088] This embodiment provides a ratio fluorescence sensor detection method for histamine, specifically including:

[0089] Step (1): Referring to Example 4, obtain the fluorescence detection curve of the ratio fluorescence sensor dispersion and the functional relationship between the fluorescence intensity ratio and histamine concentration;

[0090] Step (2): Add 600 μL of the histamine solution to be tested (concentration unknown, prepared by diluting 1 mg / mL histamine solution several times) to the ratio fluorescence sensor prepared in Example 3 to make a 1.2 mL reaction system. Perform spectral detection using a fluorescence spectrometer. Calculate the histamine concentration in the sample based on the fluorescence intensity changes of the ratio fluorescence sensor at 390 nm and 545 nm, according to the detection curve and functional relationship obtained in step (1). The average value is 168 ng / mL.

[0091] Example 6

[0092] This embodiment provides a ratio fluorescence sensor detection method for histamine, specifically including:

[0093] Step (1): Referring to Example 4, obtain the fluorescence detection curve of the ratiometric fluorescence sensor dispersion and the functional relationship between the fluorescence emission peak and histamine concentration;

[0094] Step (2): Add 600 μL of the histamine solution to be tested (concentration known, 200 ng / mL histamine solution) to the ratio fluorescence sensor prepared in Example 3 to make a 1.2 mL reaction system. Perform spectral detection using a fluorescence spectrometer. Calculate the histamine concentration in the sample based on the fluorescence intensity changes of the ratio fluorescence sensor at 390 nm and 545 nm, according to the detection curve and functional relationship obtained in step (1). The average value is 189 ng / mL.

[0095] Verification Example 2

[0096] A ratiometric fluorescence sensor was prepared according to Example 3, and K was selected. + Ca 2+ Na + Mg 2+ Cl - Zn 2+ Plasma, histidine, lysine, tyrosine and other precursor amino acids, glucose, L-ascorbic acid and other food matrices were used as control substances to explore the selectivity of ratio fluorescence sensor in detecting histamine. The concentration of the control substances was 100 times that of the histamine solution. After reacting at 25-30℃ for 20-25 min, fluorescence spectrometry was used to detect the fluorescence intensity changes at 390 nm and 545 nm by ratio fluorescence sensor.

[0097] Figure 8The figure shows the selectivity analysis of the ratiometric fluorescence sensor. As can be seen from the figure, the fluorescence intensity of the ratiometric fluorescence sensor changes significantly only in the presence of histamine. Ions, food matrix, and precursor amino acids do not cause changes in the ratiometric fluorescence sensor, indicating that the ratiometric fluorescence sensor has good selectivity for histamine detection.

[0098] Verification Example 3

[0099] A ratiometric fluorescence sensor was prepared according to Example 3, and K was selected. + Ca 2+ Na + Mg 2+ Cl - Zn 2+ Plasma, histidine, lysine, tyrosine and other precursor amino acids, glucose, L-ascorbic acid and other food matrices were used as interfering substances to investigate the anti-interference ability of ratio fluorescence sensor in detecting histamine. The concentration of the interfering substances was 100 times that of the histamine solution. After reacting at 25-30℃ for 20-25 min, fluorescence spectrometry was used for spectral detection. The fluorescence intensity changes at 390 nm and 545 nm were observed by ratio fluorescence sensor.

[0100] Figure 9 The figure shows the anti-interference analysis of the ratio fluorescence sensor. As can be seen from the figure, there is no significant difference in the effect of histamine solution mixed with interfering substances and histamine solution alone on the fluorescence intensity of the ratio fluorescence sensor, indicating that the ratio fluorescence sensor has good anti-interference ability and can be used for histamine detection in complex matrices.

[0101] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

Claims

1. A ratio fluorescence sensor, characterized in that, The system comprises a dual-fluorophore system; the dual-fluorophore system is a terbium-based metal-organic framework MR@Tb-MOF loaded with methyl red dye. The terbium-based metal-organic framework Tb-MOF in MR@Tb-MOF is formed by the self-assembly of terbium metal ions and the organic ligand 1,3,5-benzenetricarboxylic acid. The methyl red dye has a characteristic fluorescence emission peak at 390 ± 10 nm, and the Tb-MOF has a characteristic fluorescence emission peak at 545 ± 5 nm. The fluorescence emission peaks of the two do not overlap significantly, thus constituting a ratiometric fluorescence sensor.

2. A ratio fluorescence sensor according to claim 1, characterized in that, The particle size of the MR@Tb-MOF material is 650-750 nm.

3. A method for fabricating a ratiometric fluorescence sensor as described in claim 1 or 2, characterized in that, Includes the following steps: Step 1: Synthesis of Tb-MOF: Terbium metal salt and organic ligand 1,3,5-benzenetricarboxylic acid are dissolved in N,N-dimethylformamide solvent, and then acetic acid is added as a regulator to carry out a solvothermal reaction. The resulting dispersion is then washed, filtered, and dried to obtain Tb-MOF powder material. The terbium metal salt is selected from terbium nitrate hexahydrate, and the molar ratio of terbium metal salt to organic ligand 1,3,5-benzenetricarboxylic acid is 1:

1. The temperature of the solvothermal reaction is 100-120°C. Step 2: Synthesis of MR@Tb-MOF: The Tb-MOF obtained in Step 1 was dispersed in deionized water, and then methyl red solution was added. The mixture was sonicated to form a uniform dispersion, and then stirred at room temperature in the dark for a period of time. The resulting dispersion was washed, filtered, and dried to obtain MR@Tb-MOF powder material. The mass concentration of Tb-MOF dispersed in deionized water was 1-5 mg / mL, and the mass ratio of Tb-MOF to dye MR was 1:(0.75-2). Step 3: Preparation of ratio fluorescence sensor: Disperse the MR@Tb-MOF obtained in step 2 in an aqueous medium to obtain the ratio fluorescence sensor.

4. The preparation method according to claim 3, characterized in that, In step 1, the solvothermal reaction time is 1-3 hours, the washing agent used is anhydrous ethanol, and the drying method is oven drying at a temperature of 60-75℃.

5. The preparation method according to claim 3, characterized in that, In step 2, the stirring time in the dark is 2-4 hours, the stirring speed is 100-300 rpm, the detergent used for washing is deionized water, and the drying method is oven drying at a temperature of 60-75℃.

6. The preparation method according to claim 3, characterized in that, In step 3, the ratiometric fluorescence sensor contains MR@Tb-MOF dispersed in deionized water at a concentration of 0.5-1 mg / mL.

7. The application of a ratio fluorescence sensor as described in claim 1 or 2 and / or a ratio fluorescence sensor prepared by any one of claims 3-6 in histamine detection.

8. The application according to claim 7, characterized in that, The detection limit of the ratiometric fluorescence sensor is 3.34 ng / mL.

9. A method for detecting histamine using a ratiometric fluorescence sensor as described in claim 1 or 2, characterized in that, Includes the following steps: Step S1: Mix the ratio fluorescence 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 20-25 min, perform spectral detection using a fluorescence spectrometer. Plot a calibration curve by using the fluorescence intensity changes of the ratio fluorescence sensor at 390 nm and 545 nm, with the histamine concentration as the abscissa and the ratio of the fluorescence intensity peaks of the ratio fluorescence sensor at 390 nm and 545 nm as the ordinate. Step S2: Mix the ratio fluorescence sensor with the histamine solution to be tested in a certain proportion to prepare a reaction system. After reacting at 25-30℃ for 20-25 min, perform spectral detection using a fluorescence spectrometer. Calculate the histamine concentration in the sample based on the fluorescence intensity changes of the ratio fluorescence sensor at 390 nm and 545 nm, according to the calibration curve obtained in step S1, thereby achieving the detection of histamine.

10. The method for detecting histamine using a ratiometric fluorescence sensor according to claim 9, characterized in that, The preparation method of the reaction system in steps S1 and S2 includes: adding 600 μL of MR@Tb-MOF dispersion and 600 μL of histamine solution to prepare a 1.2 mL reaction system; the histamine solution uses ultrapure water as solvent, the mass concentration of the MR@Tb-MOF dispersion is 1 mg / mL, and the dispersant is deionized water.

11. The method for detecting histamine using a ratiometric fluorescence sensor according to claim 9, characterized in that, The conditions for a fluorescence spectrometer include: fluorescence excitation wavelength of 310-330 nm and fluorescence emission spectrum observation range of 330-650 nm.

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