Preparation and application of fluorescent probe for rapidly and sensitively detecting food deterioration

Abstract

The invention relates to the technical field of fluorescent chemical sensors, and particularly discloses preparation and application of a fluorescent probe for rapidly and sensitively detecting food deterioration. The structural formula of the fluorescent probe is as shown in the following formula (FCHO). The fluorescent probe FCHO disclosed by the invention has the characteristics of simple design and quick response (lt; the histamine detection kit has the characteristics of high specificity, high detection sensitivity and the like, the histamine detection sensitivity is greatly improved through chemical and hydrogen bond interaction between FCHO and histamine, and the detection limit is as low as 51nM. In addition, FCHO and a portable paper-based device are successfully integrated together, a high-selectivity and visually-distinguishable recognition system is developed, with the increase of histamine concentration, the device can be observed by naked eyes and has obvious color conversion from light blue to bright green, and the device can be conveniently used for POCT (point-of-care testing). More importantly, the histamine level in the food deterioration process is subjected to in-situ imaging by using FCHO, so that the reliability of the FCHO in the aspect of directly monitoring the histamine distribution in the whole fish sample is proved. The work provides a new detection means for specifically and rapidly monitoring the histamine expression level in food rot. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the technical field of fluorescent chemical sensors, and in particular relates to a fluorescent probe for rapidly, sensitively and visually monitoring histamine in food spoilage, and a preparation method and application thereof. Background Art

[0002] Food safety is a core issue in the field of public health and is directly related to people's health and well-being. During the processing and storage of food, microbial amino acid decarboxylase can catalyze the decarboxylation of amino acids to form biogenic amines. Histamine is the most common biogenic amine in food and is a byproduct of microbial action during food processing, storage and transportation. The presence of high concentrations of histamine causes food to deteriorate and thus reduces the quality of food. Appropriate amounts of histamine play an important regulatory role in the human body, but excessive intake of histamine can cause adverse reactions such as headaches, vomiting, and diarrhea. More seriously, long-term or one-time large-dose intake of histamine may also cause respiratory distress or even death. Therefore, detecting whether food is spoiled or not is of great significance to human health and safety.

[0003] Traditional histamine detection methods such as liquid chromatography, gas chromatography, mass spectrometry and colorimetry, although they perform well in terms of stability and sensitivity, also expose a series of challenges in their application. First, these methods are usually very cumbersome and complicated in the sample pretreatment steps, and require multiple centrifugation, precipitation and filtration operations, which not only increases the time cost of sample processing, but also may cause the mixing of impurities, thereby affecting the accuracy of the analysis results. Secondly, these detection methods often rely on expensive professional instruments and equipment, and the entire detection cycle from sample preparation to final detection is often long. Furthermore, the excessive sample consumption generated during the histamine detection process is also a problem that cannot be ignored. Traditional detection methods are difficult to adapt to the needs of actual sample detection and point-of-care testing (POCT). Therefore, the establishment of a simple, fast and highly sensitive detection technology is an urgent problem that needs to be solved.

[0004] Fluorescence detection technology is a mature detection technology. Its main construction principle is based on fluorescence enhancement or quenching mediated by the target analyte. Its low equipment cost, simple operation and high sensitivity make it widely used in various fields, especially in trace analysis. At present, several fluorescent carbon dots, quantum dots, polymer nanoparticles, etc. have been reported for fluorescence detection of histamine levels in food. The above fluorescence detection systems also have certain limitations. The first is the solubility of the fluorescent system in aqueous solution, which directly affects the ability of the detection system to dissolve and diffuse in the sample, which may cause signal drift or unstable detection during the detection process and poor sensitivity. Secondly, the response time of the above system is long. Histamine is an active amine. Long-term exposure is prone to oxidation reaction, resulting in inaccurate detection. In addition, the preparation method of the reported detection system is complicated and requires multi-step synthesis. The preparation and detection costs are too high. Therefore, it is crucial to construct a fluorescent probe that is easy to synthesize and responds quickly in an aqueous system for the detection of histamine in food. Summary of the invention

[0005] In order to solve this problem, the present invention provides a fluorescent probe for rapid, sensitive and visual monitoring of histamine in food spoilage, as well as a preparation method and application. The probe has a rapid (<1s), high specificity and sensitive response to histamine. In addition, the probe is integrated with a paper-based chip to visually distinguish the histamine levels between fresh fish and spoiled fish. In addition, the probe also has the reliable ability to directly image the distribution of histamine in the whole spoiled fish in situ.

[0006] To achieve the above object, the present invention discloses the following technical solution:

[0007] In the first aspect, a fluorescent probe for rapid, sensitive and visual monitoring of histamine is provided, which is denoted as FCHO and has the following molecular structure:

[0008] In a second aspect, a method for preparing the fluorescent probe FCHO is provided, which specifically comprises the following steps:

[0009] Fluorescein is mixed and dissolved with 15-crown ether-5, NaOH and chloroform, and then reacted in one step to obtain the target product, which is recorded as FCHO.

[0010] Thirdly, a reaction mechanism of a fluorescent probe FCHO for rapid, sensitive and visual monitoring of histamine is provided:

[0011] The present invention selects the aldehyde group in the fluorescent probe FCHO as the response part of histamine. In the process of FCHO detecting histamine, the aldehyde group is activated, and the paired electrons on the amino group are transferred to the oxygen atom, thereby forming an imine structure. At the same time, a hydrogen bond is formed between the nitrogen atom on the imidazole ring and the hydroxyl group on FCHO, and the π-π interaction between the imidazole on the histamine and the benzene ring on FCHO further promotes the fluorescence enhancement of FCHO. In vitro spectral property tests show that the probe FCHO has excellent specific response performance to histamine, the response time is less than 1s, and the detection limit of histamine reaches 51nM.

[0012] Fourthly, a fluorescent probe FCHO is provided for visually detecting the histamine content in food in POCT.

[0013] Specifically, the FCHO fluorescent probe can be combined with a paper-based device. Under the irradiation of ultraviolet light (365nm), the green fluorescence of the paper-based test area gradually increases with the increase of the histamine content in the food, thereby realizing visual differentiation and detection of the histamine content in the food.

[0014] In the fifth aspect, a fluorescent probe FCHO is provided for in situ imaging of fish deterioration sites.

[0015] Specifically, the fluorescent probe FCHO detected and imaged the histamine content in spoiled parts of fish in situ, confirming that the histamine content in spoiled parts was significantly higher than that in non-spoiled parts, thereby achieving accurate distinction of spoiled parts of food.

[0016] The fluorescent probe FCHO disclosed in the present invention has the characteristics of simple design and rapid response (<1s). The specific Schiff base reaction between FCHO and histamine greatly improves the sensitivity of histamine detection, and the detection limit is as low as 51nM. In addition, we have successfully integrated FCHO with a portable POCT test device to develop a highly selective and visually discernible recognition system. As the histamine concentration increases, there is a clear color transition from light blue to bright green. More importantly, we used FCHO for the first time to perform in situ imaging of histamine levels during food spoilage, thereby demonstrating its reliability in directly monitoring the distribution of histamine in whole fish samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to make the above and / or other objects, features, advantages and embodiments of the present invention more clearly understood, the accompanying drawings are described as follows:

[0018] Figure 1 This is the H NMR spectrum of the fluorescent probe FCHO.

[0019] Figure 2 This is the NMR carbon spectrum of the fluorescent probe FCHO.

[0020] Figure 3 This is the high-resolution mass spectrum of the fluorescent probe FCHO.

[0021] Figure 4 (A) is the UV-visible absorption spectrum of the histamine-specific fluorescent probe FCHO in response to histamine, (B) is the fluorescence emission spectrum, (C) is the reaction kinetics test, and (D) is a graph showing the concentration gradient changing with histamine concentration.

[0022] Figure 5 (A) is the selectivity test of the histamine-specific fluorescent probe FCHO for various biogenic amines and interfering ions and (B) the interference performance test. Among them, 0-21: blank, histamine, histidine, cadaverine, putrescine, spermine, tyramine, tryptamine, GSH, BSA, K + , Ca 2+ 、Zn 2+ Mg 2+ , Cu 2+ , HS - , S 2- 、SO3 2- 、SO4 2- 、CO3 2- 、NO2 - 、NO3 - .

[0023] Figure 6 (A) is the response mechanism diagram of the histamine-specific fluorescent probe FCHO to histamine, (B) is the liquid chromatogram before and after the reaction of the probe and histamine, and (C) is the nuclear magnetic resonance hydrogen spectrum.

[0024] Figure 7 (A) shows the change in fluorescence color of the FCHO probe loaded test strip under 365 nm ultraviolet light as histamine levels increase, and (B) the linear relationship between histamine concentration and color intensity. (C) shows the change in fluorescence color of the test strip impregnated with fish sample lysate at different storage temperatures and times. (D) shows the change in histamine measured by the fluorescent probe FCHO in fish sample lysate at different storage temperatures and times.

[0025] Figure 8 (A) is the fluorescence photograph of fresh fish slices after incubation with probe FCHO (FCHO group) and after incubation with probe FCHO after adding histamine (FCHO+histamine group), (B) is the fluorescence intensity graph of fresh fish slices under different treatment conditions in Figure A, (C) is the fluorescence photograph of spoiled fish slices after incubation with probe FCHO (FCHO group) and after incubation without adding probe FCHO (Control group), (D) is the fluorescence intensity graph of spoiled fish slices under different treatment conditions in Figure C.

[0026] Fig. 9 (A) is a flow chart of the designed in situ imaging of histamine distribution in the whole body of the spoiled fish, (B) is the fluorescence (FCHO), bright field (Bright field) and merged (Merged) photos of the spoiled crucian carp sprayed with the probe FCHO solution (10μM), (C) is the average fluorescence intensity of the fish back (Back) and abdomen (Belly), and (D) is the fluorescence intensity distribution along the red line in Figure A. DETAILED DESCRIPTION

[0027] The present invention is described in detail below, specifically in conjunction with the drawings of the present invention specification, to provide a clearer and more complete description of the embodiments of the present invention, without limiting the contents thereof. The embodiments described in the present invention are only a part, not all, of the embodiments, which are all within the protection scope of the present invention.

[0028] Example 1

[0029] [Reference Figure 1 The synthetic route shown is to synthesize the fluorescent probe FCHO]

[0030] Briefly, fluorescein (5.0 g) was dissolved in 10 mL of methanol. The solution was cooled to 0°C in an ice bath, and 20 mL of 50% NaOH aqueous solution and 60 μL of 15-crown ether-5 were added. After stirring for 10 minutes, the solution was returned to room temperature. 20 mL of chloroform was added dropwise, and the reaction solution was heated to reflux at 55°C for 12 hours and then cooled to room temperature. An excess of 10 M sulfuric acid was added under ice bath conditions. The solid-liquid mixture was filtered to obtain a dark purple filter cake. It was purified by flash column chromatography and spin dried to obtain the fluorescent probe FCHO. The probe was confirmed by nuclear magnetic resonance and mass spectrometry. Figure 1 is the H NMR spectrum, Figure 2 is the carbon NMR spectrum, Figure 3 High-resolution mass spectrometry.

[0031] Example 2

[0032] [In vitro optical response of FCHO to histamine]

[0033] When detecting histamine, the final concentration of FCHO in the aqueous solution is 10 μM (containing 1% DMSO), the final concentration of histamine is 40 μM, and the total volume is maintained at 100 μL. Before the test, the above reaction system is continuously shaken and incubated at 25°C for an appropriate time. The specific experimental results are as follows:

[0034] (1) Figure 4As shown in (A) and (B), we tested the response of the probe FCHO to histamine. After the addition of histamine, the absorbance of FCHO at 460nm increased significantly, and a new absorbance peak appeared around 500nm, indicating that new substances were produced after the interaction between histamine and FCHO. With a laser of 460nm wavelength as the excitation wavelength, the fluorescence signal of FCHO at 530nm was also significantly enhanced (about 40 times), corresponding to green fluorescence.

[0035] (2) Figure 4 As shown in (C), we tested the change of the fluorescence intensity ratio F / F0 after the reaction of FCHO and histamine over time. F0 and F represent the fluorescence intensity of the reaction system at 530nm before and after the addition of histamine, respectively. The kinetic test was performed at 25℃, and the results showed that FCHO and histamine can respond instantly (<1s).

[0036] (3) Figure 4 (D) shows the concentration gradient test of histamine. Specifically, the FCHO concentration is 10 μM (1% DMSO), different concentrations of histamine (0-40 μM) are added, and then reacted at 25°C to obtain the concentration gradient response spectrum (E x =460nm) and response curve. The results showed that the fluorescence intensity of FCHO after the reaction with histamine gradually increased with the increase of histamine concentration, and showed a good linear relationship in the concentration range of histamine 1-40μM, and the detection limit was as low as 51nM.

[0037] (4) The selectivity and interference performance of the probe FCHO were tested. Specifically, 20 common biogenic amines and other interfering ions were selected as interfering substances, including 0-21: blank, histamine, histidine, cadaverine, putrescine, spermine, tyramine, tryptamine, GSH, BSA, K + , Ca 2+ 、Zn 2+ Mg 2+ , Cu 2+ , HS - , S 2- 、SO3 2- 、SO4 2- 、CO3 2- 、NO2 - 、NO3 - In the aqueous solution of FCHO (10 μM, 1% DMSO), various interfering substances were added in turn, and the fluorescence emission spectra after the response were measured respectively (E x =460nm). The results are as follows Figure 5As shown in (A), only when histamine was added did the fluorescence enhancement (about 42 times) appear. In addition, histidine showed a fluorescence enhancement of about 12 times. Since the concentration of histidine in spoiled food is significantly lower than that of histamine, its interference can be ignored. Figure 5 As shown in (B), when histamine coexists with other interfering substances, the fluorescence intensity of FCHO also shows similar changes, indicating that fluorescence spectroscopy can quantitatively detect histamine while maintaining good selectivity and anti-interference ability.

[0038] Example 3

[0039] [Mechanism verification of FCHO's response to histamine]

[0040] The reaction mechanism of FCHO to histamine proposed by the present invention is as follows Figure 6 (A) As shown. In the presence of histamine, the aldehyde group on FCHO is activated, and the paired electrons on the amino group are transferred to the oxygen atom, thereby forming an imine structure. At the same time, the nitrogen atom on the imidazole ring forms a hydrogen bond with the hydroxyl group on FCHO, combined with the π-π interaction between the imidazole on histamine and the benzene ring on FCHO, which further promotes the fluorescence enhancement of FCHO. We then studied the detection mechanism in detail. Figure 6 As shown in (B), histamine was first added to the probe FCHO solution for liquid chromatography and mass spectrometry testing. Compared with the pure probe and histamine, a new peak appeared at t = 11.61min, and its corresponding mass-to-charge ratio m / z = 454.13736, which is highly consistent with the molecular weight of the hypothesized Schiff base product. In order to further confirm that the aldehyde group and the amino group generate imine, we also performed nuclear magnetic hydrogen spectrum analysis on each substance before and after the reaction. The results are shown in Figure 6 As shown in (C), there is a characteristic peak at 10.15 ppm, indicating that the aldehyde group is indeed introduced into FCHO. After reacting with histamine, the signal corresponding to the aldehyde group disappears, further proving the formation of the imine structure. These results clearly support the specific reaction between the aldehyde group on FCHO and the amino group on histamine to form a Schiff base structure.

[0041] Example 4

[0042] [ Point-of-care testing (POCT) based on paper-based devices]

[0043] After confirming that the fluorescent probe FCHO can detect histamine quickly, sensitively and specifically, we further evaluated its potential applications, especially as a portable and highly sensitive paper-based device for POCT. We prepared a circular test area with a diameter of 5 mm on the paper surface by wax printing technology, and added 2 μL of FCHO solution (10 μM) to the test area and dried it to prepare the paper-based test device. Figure 7As shown in (A), we added different concentrations of histamine to the paper-based test area containing the probe FCHO. Under ultraviolet irradiation (365nm), as the histamine content increased, the color of the test device changed from light blue to bright green. Figure 7 As shown in (B), the color signal was converted into grayscale using Image J, and there was a good linear relationship between histamine and color intensity in the range of 0-2 mM (R 2 =0.9881), which makes it possible to quantitatively detect histamine in POCT.

[0044] Histamine is synthesized and accumulated in food samples during processing and storage, and is an important parameter for evaluating food quality. Therefore, in order to accurately evaluate the performance of paper-based devices, we used the developed paper-based devices to test the changing levels of histamine in real samples. Specifically, fish samples of the same weight were stored at three different temperatures, -20°C, 4°C, and 25°C, for 1, 3, and 5 days. Then, fish lysates were extracted from the samples, and the histamine content was monitored using paper-based test devices. Figure 7 As shown in (C), as the storage time increased from 0 days to 5 days, the color of the test paper changed from light blue to green, indicating that histamine release was enhanced. In addition, at higher temperatures, an increase in green was observed, also showing the same histamine release curve. In order to verify the reliability of the results, we also used our probe solution to measure the histamine in the above fish lysate. Figure 7 As shown in (D), at 4°C and 25°C, the histamine concentration increased significantly. The histamine content of the sample stored at low temperature (-20°C) changed more slowly than the other two samples, and it can be kept fresh for a long time. These findings not only verify the reliability of the paper-based test device used in the probe, but also emphasize the great application potential of this sensor platform in food quality monitoring.

[0045] Example 5

[0046] [ Visualizing spoilage sites in food by in situ imaging]

[0047] As the degree of spoilage in food increases, the histamine content will also increase significantly. By in situ imaging of the histamine content level, we can obtain the spatiotemporal information of food spoilage and effectively distinguish the spoiled parts of the food. Our probe FCHO can identify and detect histamine in situ through chemical bonds and hydrogen bond interactions. In this way, it can monitor and track the changes of histamine during food processing and storage in real time. First, the probe FCHO solution (10μM) was sprayed on fresh crucian carp slices, and the fluorescence intensity was recorded with a laser confocal microscope after 10s. Figure 8As shown in (A) and (B), in the untreated control group (Control), we observed very weak green fluorescence, while in the histamine pretreatment group (FCHO+histamine), bright green fluorescence appeared, indicating that the fluorescence change was consistent with the change in histamine content. Next, we used FCHO to image the degree of corruption of crucian carp samples. Figure 8 As shown in (C) and (D), after being incubated with FCHO solution (10 μM) for 10 s, the fluorescence image intensity of the spoiled fish fillets was higher than that of the untreated fish fillets.

[0048] Finally, we evaluated the ability of this probe to directly distinguish the degree of food spoilage by imaging histamine in whole spoiled fish. Fig. 9 As shown in (A), we sprayed FCHO solution (10 μM) on the surface of spoiled fish, incubated for 10 s, and then observed the fluorescence distribution under UV light (365 nm). Fig. 9 As shown in (B) and (C), under ultraviolet light, the fish's abdomen showed obvious green fluorescence, while the fluorescence on the fish's back was weaker, indicating that the fish's abdomen was more rotten than its back. Fig. 9 As shown in (D), it can be noted that even in the belly of the fish, the fluorescence distribution is different. Therefore, our probe demonstrated for the first time that we can directly distinguish the degree of food decay in different parts of the food under naked eye observation.

[0049] The materials, methods and embodiments described herein are exemplary only and not restrictive. A person skilled in the art may refer to the contents of this article and appropriately replace and / or modify the process parameters. However, it should be particularly noted that all similar replacements and / or modifications are obvious to a person skilled in the art and do not deviate from the spirit of the present invention or exceed the scope defined by the appended claims. Therefore, they are all considered to be included in the present invention.

Claims

1. Preparation and application of a fluorescent probe for rapid and sensitive detection of food spoilage, characterized in that: The fluorescent probe includes a core fluorophore composed of fluorescein and a recognition group composed of an aldehyde group and a hydroxyl group, and its structural formula is as follows:

2. The fluorescent probe for rapid and sensitive detection of food spoilage according to claim 1, characterized in that: The response time of the fluorescent probe is less than 1 s, and the detection limit is 51 nM.

3. The design and preparation method of a fluorescent probe for rapid and sensitive detection of food spoilage according to claim 1, characterized in that: The preparation scheme is as follows: Fluorescein was mixed and dissolved with 15-crown ether-5, NaOH and chloroform and heated overnight. After the reaction was completed, an excess of 10M sulfuric acid was added under ice bath conditions to neutralize the reaction. The solid-liquid mixture was filtered to obtain a dark purple filter cake. FCHO was obtained by purification by flash column chromatography and spin-dried.

4. A method for detecting histamine in vitro using the fluorescent probe prepared by the preparation method according to claim 3, characterized in that: The final concentration of FCHO in the detection system was 10 μM (1% DMSO), the final concentration of histamine and other interfering substances was 40 μM, the reaction temperature was 25° C., and the reaction time was <1 s.

5. A method for instant testing of histamine using a fluorescent probe prepared by the preparation method according to claim 3 in combination with a paper-based device, characterized in that: The method comprises the following steps: adding histamine solutions of different concentrations to the paper-based test area containing the probe FCHO, and after the reaction, observing the change of the fluorescence color of the paper-based test area under ultraviolet irradiation (365nm). The fish samples of the same weight are stored at three different temperatures of -20°C, 4°C and 25°C for 1, 3 and 5 days respectively. Then, the fish lysate is extracted from the sample, and the histamine content is monitored using a paper-based test device.

6. A method for using the fluorescent probe prepared by the preparation method according to claim 3 to visualize in situ imaging of histamine in spoiled fish, characterized in that: The following steps are involved: The probe FCHO solution (10 μM) was sprayed on fresh / corrupted crucian carp slices, and the fluorescence photos and intensity were recorded using a laser confocal microscope after 10 seconds. The FCHO solution (10 μM) was sprayed on the surface of the spoiled fish, incubated for 10 seconds, and then the fluorescence distribution of the probe on the fish surface was observed with an ultraviolet lamp (365 nm) to determine the spoiled part of the fish.

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