CN114989188A - Fluorescent probe for detecting active amine and application thereof - Google Patents

Abstract

The invention provides a fluorescent probe for detecting amine, which has a chemical structural formula as follows:

. The fluorescent probe can detect active amine generated in a solution and in a biological decay process. The fluorescence of the fluorescent probe for detecting active amine provided by the invention is obviously enhanced after the active amine is added, and the result and the phenomenon thereof lay the theoretical foundation for the application of the test paper for rapidly detecting the food spoilage. Accordingly, the degree of food spoilage can be assessed by detecting active amines using the probes of the invention via fluorescence imaging techniques.

Description

A kind of fluorescent probe for detecting active amine and its application

technical field

The invention belongs to the technical field of analytical chemistry, and particularly relates to a fluorescent probe for detecting active amine and its application.

Background technique

Bioactive amines are the most important class of biogenic amines produced by bacterial decarboxylation of lysine in decaying animal tissues. They are usually found in rancid, high-protein foods such as meat and fish. Once formed, bioactive amines are released into the surrounding environment and cause unpleasant odors in the atmosphere. Bioactive primary diamines can easily enter the human body through ingestion, inhalation or skin contact and cause serious health hazards including food poisoning, respiratory injury and gastrointestinal damage. Therefore, in order to maintain food safety, it is necessary to develop appropriate techniques for the determination of biologically active amines in meat and fish in order to detect food spoilage.

Various analytical methods have been developed for the detection of biologically active amines, including gas chromatography-mass spectrometry (GC-MS), electrochemistry, spectrophotometry, and high performance liquid chromatography (HPLC). However, these methods are limited by cumbersome sample preparation, time-consuming procedures, and expensive instrumentation. The chromophore-based fluorescent probe has the characteristics of simple operation, high sensitivity and strong specificity, which is beneficial to the detection of biogenic amines. Researchers have proposed several different explanations for the interaction mechanism between chromophore and amine, including formation of Schiff base, deprotonation process, aminolysis reaction, nucleophilic addition, amine exchange and cyclization. These methods enrich the methods for monitoring biological diamines, however, these probes still have some limitations: a few work in the fluorescence "off" mode, the sensitivity is relatively low, and even false positive reactions; the design of the probes is based on biological The basicity and nucleophilicity of amines make the probes susceptible to interference from other amines; these structurally complex probes require a tedious preparation process. Therefore, there is an urgent need to develop small-molecule organic fluorescent probes with simple structures for rapid and highly selective detection of biological diamines in food.

SUMMARY OF THE INVENTION

In view of the problems in the prior art, the present invention provides a fluorescent probe for detecting biologically active amines, which has the characteristics of fast response speed and strong anti-interference ability.

Another object of the present invention is to provide a rapid detection of the above-mentioned fluorescent probe in the process of food spoilage.

In order to achieve the above objects, the present invention adopts the following technical solutions.

A fluorescent probe for the detection of amines, 2',7'-dichloro-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthine]-3',6'-diacetic acid Diester, referred to as CHW-Cl, its chemical structure is shown in formula (I):

Formula (I).

The amines are amine compounds other than amino acids. Preferably, the amine is selected from putrescine, cadaverine, spermine, spermidine, tyramine, phenethylamine, histamine or tryptamine; most preferably cadaverine.

The preparation method of the above-mentioned fluorescent probe comprises the following steps:

(1) 4-chlororesorcinol and phthalic anhydride are heated and reacted in peracetic acid. After the reaction is completed, the reaction mixture is added to ice water, filtered to obtain a precipitate, washed and dried to obtain 2′,7′-dichloromethane Fluorescein:

；

;

(2) 4-dimethylaminopyridine and pyridine are added to a suspension of 2′,7′-dichlorofluorescein in dichloromethane, acid chloride is added dropwise to carry out the reaction, and after the reaction is completed, separation and purification are performed to obtain a fluorescent probe:

。

.

In step (1), the temperature of described heating reaction is 90 ℃; Heating time is 24h.

In step (2), the separation and purification steps are as follows: the reaction mixture is diluted with water and then extracted with dichloromethane, the organic phase is washed with saturated ammonium chloride solution and saturated sodium chloride solution in turn, and the organic phase is dried with anhydrous magnesium sulfate , suction filtration, and remove the solvent under reduced pressure; dissolve the residue with a small amount of dichloromethane, add an equal volume of neutral silica gel powder to the residue, remove the solvent after adsorption, and fill the column with the obtained powder; the volume ratio is 10:4 The cyclohexane:ethyl acetate is the chromatographic liquid and passes through the silica gel column, and the chromatographic liquid is removed in vacuo to obtain the fluorescent probe.

An application of the above fluorescent probe in detecting active amines in solutions and foods.

The mechanism of the present invention is as follows:

Two acetate groups of 2',7'-dichloro-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthine]-3',6'-diacetic acid diester Attacked by an active amine, it is added and then eliminated to release a hydroxyl group, the five-membered ring is opened, and the π electrons are transferred to form a large conjugated system, resulting in strong fluorescence:

。

.

The present invention has the following advantages:

The active amine fluorescent probe provided by the present invention belongs to the small molecule fluorescent probe. Currently, there are not many reports on the small molecule fluorescent probe recognized by the active amine, especially the fluorescent probe for rapidly detecting the active amine produced by biological spoilage. Rarely reported. In the active amine fluorescent probe provided by the present invention, when the active amine is added to the active amine fluorescent probe, the fluorescence is significantly enhanced, and the result and its phenomenon lay a theoretical foundation for the application of the rapid detection test paper for food spoilage detection.

Description of drawings

Fig. 1 is the hydrogen spectrum of probe CHW-Cl;

Fig. 2 is the carbon spectrum of probe CHW-Cl;

Figure 3 shows the titration experiment of the probe CHW-Cl and cadaverine (1,5-pentanediamine); the excitation wavelength is 480 nm; the concentration of the probe: 10 µM;

Figure 4 shows the kinetic experiment of the interaction between the probe CHW-Cl and cadaverine; the excitation wavelength is 430 nm; the concentration of the probe: 10 µM. Cadaverine concentration 275 µM, test time: 100 min;

Figure 5 shows the selectivity of the probe CHW-Cl in absolute ethanol; the excitation wavelength is 480 nm; the concentration of the probe is 10 µM, the concentration of the selective ion is 10 mM, and the concentration of the active amine (cadaverine) is 100 μM;

Fig. 6 is the photograph of the multi-segment of the same banana under different time periods, after the action of the probe CHW-Cl;

Figure 7 is a photo of the same batch of live loach that interacts with the dry test paper soaked in the CHW-Cl solution of the probe at different time periods after death.

Detailed ways

The present invention will be further described below with reference to the embodiments and the accompanying drawings, but the present invention is not limited by the following embodiments.

Example 1 Synthesis of Fluorescent Probes

Synthesis of 2',7'-dichloro-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthine]-3',6'-diacetic acid diester:

4-Chlororesorcinol (14.4 g, 99.6 mmol) and phthalic anhydride (7.3 g, 49.3 mmol) were dissolved in peracetic acid (50 mL) and the resulting solution was heated at 90 °C for 24 After cooling to room temperature, the reaction mixture was slowly added to 1 L of ice water and stirred. After filtration, the precipitate was washed with ice water and dried to obtain 2′,7′-dichlorofluorescein, which was directly put into the next step without purification;

4-Dimethylaminopyridine (15 μmol, 0.1 eq) and pyridine (0.33 mmol, 2.2 eq) were added to a mixture of 2′,7′-dichlorofluorescein (0.15 mmol, 1 eq) and dichloromethane (2.0 mL) Suspension; acid chloride (0.33 mmol, 2.2 eq) was added dropwise, and the resulting solution was stirred for 1 hour; diluted with water, extracted with dichloromethane, the organic phase was washed with saturated aqueous ammonium chloride and saturated brine, and the organic phase was washed with dried over anhydrous magnesium sulfate, suction filtered, and the solvent was removed under reduced pressure; the residue was fully dissolved with a small amount of dichloromethane, neutral silica gel powder approximately equal in volume to the residue was added, and after thorough stirring, the solvent was removed in vacuo to obtain a crude product -The powder of silica gel powder, which is then dry-column; purified by silica gel column chromatography (cyclohexane:ethyl acetate=10:4, volume ratio) to obtain the product, that is, the fluorescent probe;

Wherein, ¹ H NMR (500 MHz, DMSO-d ₆ ) δ 8.07 (d, J = 10.0 Hz, 1H), δ 7.83 (dt, J = 25.0 Hz, J =10.0 Hz, 2H), δ 7.59 (s, J = 0 Hz, 2H), δ 7.50 (d, J = 10.0Hz, 2H), δ 7.07 (s, J = 0 Hz, 2H), δ 2.36 (s, J =0, 6H);

¹³ C NMR (126 MHz, DMSO-D ₆ ) Δ 168.46, 151.69, 149.80, 148.70, 136.59,131.34, 129.05, 126.00, 125.86, 124.54, 122.26, 113.74, 40.33,40.00.00.6.6.6.6.6.6.6.6.6.6.6.8. , 39.50, 20.85.;

¹ H NMR is shown in Figure 1; ¹³ C NMR is shown in Figure 2.

Example 2 Response of fluorescent probes to different concentrations of cadaverine

Prepare 5 mL of anhydrous ethanol solution of cadaverine with a concentration of 1 mM and the fluorescent probe stock solution prepared in Example 1 with a concentration of 1 mM as spares. Probes were prepared at a concentration of 10 µM, interacted with different concentrations of cadaverine (0-275 µM), and were subjected to fluorescence detection (λ _ex = 480 nm), the fluorescence intensity in each system was calculated, and the fluorescence intensity and cadaverine concentration were established. standard curve line. As shown in Figure 3, within the tested concentration range, with the increase of the cadaverine concentration, the fluorescence intensity of the reaction system gradually increased linearly, and after calculation, the detection limit LOD was 52.26nM.

Example 3 Response kinetics of fluorescent probes to saturated cadaverine

Prepare 5 mL of anhydrous ethanol solution with a concentration of 1 mM cadaverine and a fluorescent probe stock solution for the detection of active amines according to the present invention with a concentration of 1 mM as backup. Prepare solutions of probe and cadaverine at the following concentrations: probe 10 µM; cadaverine concentration: 275 µM. Fluorescence detection (λ _ex = 480 nm, ) was carried out, and the test was performed every 5 min for 100 min. The fluorescence intensity of each system was calculated over time, and a standard curve of fluorescence intensity and action time was established. As shown in Fig. 4, after about 90 minutes of reaction, the fluorescence intensity of the reaction system reached a saturated state.

Example 4 Selectivity of fluorescent probes for different ions

Prepare 5 mL of absolute ethanol solutions of various selective samples with a concentration of 10 mM and the fluorescent probe stock solution prepared in Example 1 with a concentration of 1 mM as spares. Add 20 μL of probe stock solution and 20 μL of each selective sample solution, shake well and perform fluorescence detection (λ _ex = 480 nm), and establish a matrix diagram of fluorescence intensity and each selective sample. The results are shown in Figure 5, where , the ions added by 1-18 are respectively: hydrogen peroxide, aniline, ethylenediamine, putrescine, cadaverine, n-propylamine, formaldehyde, isoleucine, sodium bisulfite, histidine, serine, phenylalanine acid, cysteine, leucine, glycine, arginine, lysine and proline. After 60 minutes, the fluorescence emission spectrum changes of the solution were detected. From Figure 5, it can be found that most of the amino acids and oxidizing substances had almost no effect on the fluorescence of the compound fluorescein-Cl, while the fluorescence of ethylenediamine, putrescine, cadaverine, and n-propylamine had little effect. Addition significantly enhanced the fluorescence of the compound fluorescein-Cl.

Application Example 1 Interaction of fluorescent probes with bananas with different degrees of decay

The fluorescent probe stock solution for detecting active amines according to the present invention with a concentration of 50 μM was prepared as a spare. Take a whole fresh banana, peel off the outer skin, take out four small pieces, put them into four sample bottles respectively, add 1 mL of probe mother solution after 0 h, 5 h and 24 h in turn, cover the lid and leave it for fifteen minutes, placed in a dark box UV analyzer to observe the fluorescence brightness, and photographed and recorded, as shown in Figure 6.

Application Example 2 Interaction between fluorescent probes and loach with different degrees of decay

The fluorescent probe mother solution for detecting active amines of the present invention with a concentration of 50 μM is prepared, and four equal-sized filter paper strips are cut with qualitative filter paper (quickly), soaked in the probe mother solution, and dried for use. Take four well-grown live loaches, kill them, and put them into four clean conical flasks with stoppers. After 0 h, 5 h and 24 h, hang the treated filter paper strips in turn, and close the stopper. After standing for 15 min, the fluorescence brightness was observed in a dark box UV analyzer, and photographed and recorded, as shown in Figure 7.

Claims (8)

1. A fluorescent probe for detecting amine, the chemical structural formula of which is shown in formula (I):

Formula (I). 2 . The fluorescent probe according to claim 1 , wherein the amine is an amine compound other than amino acids. 3 . 3. The fluorescent probe according to claim 1, wherein the amine is selected from putrescine, cadaverine, spermine, spermidine, tyramine, phenethylamine, histamine or tryptamine. 4. the preparation method of the fluorescent probe of claim 1, is characterized in that, comprises the following steps: (1) 4-chlororesorcinol and phthalic anhydride are heated and reacted in peracetic acid. After the reaction is completed, the reaction mixture is added to ice water, filtered to obtain a precipitate, washed and dried to obtain 2′,7′-dichloromethane Fluorescein:

; (2) 4-dimethylaminopyridine and pyridine are added to a suspension of 2′,7′-dichlorofluorescein in dichloromethane, acid chloride is added dropwise to carry out the reaction, and after the reaction is completed, separation and purification are performed to obtain a fluorescent probe:

. 5. preparation method according to claim 4, is characterized in that, in step (1), the temperature of described heating reaction is 90 ℃; The heating time is 24h. 6 . The preparation method according to claim 4 , wherein in step (2), the separation and purification steps are as follows: the reaction mixture is diluted with water and then extracted with dichloromethane, and the organic phase is sequentially mixed with saturated ammonium chloride solution. 7 . Wash with saturated sodium chloride solution, dry the organic phase with anhydrous magnesium sulfate, filter with suction, and remove the solvent under reduced pressure; dissolve the residue with a small amount of dichloromethane, add an equal volume of neutral silica gel powder to the residue, and remove after adsorption solvent, the obtained powder was packed into the column; the cyclohexane:ethyl acetate with a volume ratio of 10:4 was used as the chromatographic solution to pass through a silica gel column, and the chromatographic solution was removed in vacuo to obtain a fluorescent probe. 7. The application of the fluorescent probe as claimed in claim 1 in detecting active amines in solutions and foods. 8. The use according to claim 7, wherein the active amine is selected from putrescine, cadaverine, spermine, spermidine, tyramine, phenethylamine, histamine or tryptamine.

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