A colorimetric fluorescent dual-signal ionic liquid micelle for freshness detection of aquatic products and preparation method thereof

Abstract

The present invention discloses a colorimetric fluorescent dual-signal ionic liquid micelle for detecting the freshness of aquatic products and a preparation method thereof. In order to develop a sensor tag and realize instant, non-invasive and rapid detection of aquatic products on sale, the present invention develops a proton-responsive ionic liquid ([P 66614 ]2[Fluo]) micelles; the gel sensor responds sensitively to amine substances and changes color obviously. Under naked eye conditions, the gel can be seen changing from orange-red to yellow-green under ultraviolet light. By utilizing the obvious fluorescent color response, the freshness of fish can be clearly and easily identified based on the color of the gel changing from red (fresh) to green (corruption). It has certain commercial potential in analyzing and detecting the freshness of aquatic products.

Description

Technical Field

[0001] The invention belongs to the field of analysis and detection and the field of material preparation, and relates to the preparation of fluorescein-based ionic liquid micelles, which are made into a gel form and applied to the rapid detection of volatile amines in actual samples, thereby realizing non-destructive and rapid visual monitoring of the freshness of aquatic products. Background Art

[0002] The assessment of food freshness is the most important issue in the food industry and the most concerned issue for consumers and suppliers. Unfresh food can cause diarrhea and other diseases, and in severe cases, acute poisoning. At the same time, the corruption of food can cause huge economic losses and the spread of malaria, leading to serious food safety issues. According to a report by the World Health Organization, about 600 million people get sick every year from eating unsafe food, which may cause 420,000 deaths. For aquatic products, the production of excessive biogenic amines means the beginning of food spoilage. Biogenic amines (BAs) are a class of low molecular weight compounds with amino groups. These biogenic amines are mainly produced by the decarboxylation of amino acids by microorganisms. A moderate amount of biogenic amines is beneficial to the normal physiological activities of the human body, while excessive intake can cause harm to human health. BAs are considered to be relevant indicators of food spoilage. BAs contain ammonia, trimethylamine, histamine, putrescine, cadaverine, etc., so these small molecular amine compounds are unfavorable.

[0003] There are many methods for detecting biogenic amines. However, traditional detection methods such as K value, total volatile basic nitrogen (TVB-N) and total viable count (TVC) analysis are time-consuming and irreversible for food, which greatly limits the application of this method in freshness detection. Current modern instrumental methods include HPLC, hair cell electrophoresis and biosensors, which face more complex pretreatment problems. Fluorescence sensors have received increasing attention in food freshness detection due to their advantages such as high sensitivity, fast response speed, convenient operation and convenient visualization. Since most fluorescent sensing fluorescent probes are hydrophobic, they are difficult to apply in aqueous media. Due to poor solubility, the detection sensitivity and selectivity of the probes in aqueous solutions are low. Therefore, improving the water solubility of the probe is also a hot topic. And most fluorescence assays rely on a single color change reaction, which is still not obvious enough to the naked eye, resulting in experimental errors and misjudgments of the content of biogenic amines in food. Therefore, there is an urgent need to develop ratiometric fluorescent probes for detecting food freshness.

[0004] As a new type of green solvent, ionic liquids (ILs) have good solubility for both organic and inorganic substances, and are non-flammable, have good thermal and chemical stability, and have extremely low vapor pressure. These advantages have greatly expanded the application field of ionic liquids. Because they are bio-friendly green solvents, they can also minimize the pollution of chemical reagents to the environment. Although ionic liquids are more commonly used in the field of green chemistry, they have functions such as gas enrichment and have certain applications in the analytical field. They are expected to be used in the detection of volatile biogenic amines in the analytical field to measure the freshness of food.

[0005] In order to design a probe with obvious color change and rapid real-time detection of the freshness of biogenic amine content, and apply it to food freshness detection, the present invention designs an amine-sensitive fluorescent ionic liquid probe micelle [P 66614 ]2[Fluo], combined with the internal reference Nile red fluorescent probe micelles for visual indication. At the same time, sodium alginate solution is used to make a convenient mixed packaging mode probe gel, which is used for the detection of the freshness of aquatic products during actual transportation or sales. Summary of the invention

[0006] The invention uses a micellar sensor of a highly sensitive and selective fluorescein-type ionic liquid fluorescent probe, and cooperates with an internal reference Nile red micelle to prepare a convenient device gel state sensor, thereby realizing rapid and real-time fluorescent dual signal detection of the freshness of aquatic products. The hybrid probe sensor has a highly sensitive response to amine substances, and is reversible and has certain anti-interference properties. After the hybrid probe is prepared into a gel and contacts biogenic amines (BAs), the color change of the fluorescent gel from orange-red to yellow-green can be observed under an ultraviolet lamp (254nm), and BAs can be detected quickly and sensitively.

[0007] The present invention uses fluorescein ionic liquid probe micelle [P 66614 ]2[Fluo] was used as the indicator probe and Nile Red micelles were used as the internal reference probe. The two prepared and diluted probes were mixed in a ratio of 1:4 to detect 0-30.0ppm NH3. With NH3 concentration as the horizontal axis, the fluorescence ionic liquid probe micelles [P 66614 ]2[Fluo] fluorescence intensity is taken as the ordinate, and the fitted linear curve is used for quantitative analysis, giving the linear range of amine detection as 1.5ppm-17.7ppm and the minimum detection limit as 0.01ppm. Within the linear range, the color of the mixed probe solution can be observed to change from pink to orange-yellow and then to yellow-green under ultraviolet light.

[0008] Based on the potential of the probe to detect biogenic amines, the mixed probe solution was packaged into gel beads with sodium alginate solution. As the content of biogenic amines continued to increase, the gel color could change from orange-red (fresh) to yellow-green (corruption), indicating the change in the freshness of aquatic products. In order to verify the reliability of the experiment, the Conway microdiffusion method in the national standard method (GB 5009.228–2016) was used to measure the TVB-N content in actual samples.

[0009] The technical solution of the present invention is as follows:

[0010] A method for preparing colorimetric and fluorescent dual-signal ionic liquid micelles comprises the following steps:

[0011] (1) [P 66614 ]2[Fluo] solution was added dropwise to surfactant solution A to obtain [P 66614 ]2[Fluo] micelles; Nile Red solution was added dropwise to surfactant solution B to obtain Nile Red micelles;

[0012] Said [P 66614 ]2[Fluo] solution and Nile Red solution were prepared with DMF as solvent, and the concentration was 1×10 - 2 mol / L;

[0013] The "surfactant solution A" and "surfactant solution B" have no special meanings. They are marked as "A" and "B" only to distinguish the surfactant solutions used in different operation steps. Surfactant solution A and surfactant solution B are both prepared with water as solvent, and the concentration is 1×10 -4 mol / L;

[0014] The surfactant is selected from: Tween 20 (TW 20), lauramidopropyl betaine (LP), cetyltrimethylammonium bromide (CTAB) or sodium dodecylbenzene sulfonate (SDBS); preferably lauramidopropyl betaine (LP), the fluorescence intensity can be relatively small to achieve a high fluorescence intensity effect, and the fluorescence intensity of the prepared LP micelle solution can be stable for more than six days;

[0015] Said [P 66614 ]2The volume ratio of [Fluo] solution to surfactant solution A is 1:125;

[0016] The volume ratio of the Nile Red solution to the surfactant solution B is 1:250;

[0017] (2) In [P 66614]2[Fluo] micelles are added with hydrochloric acid, and the probe solution is obtained after diluting with pure water, and the obtained probe solution is mixed with Nile Red micelles to obtain the colorimetric fluorescence dual-signal ionic liquid micelles;

[0018] The concentration of the hydrochloric acid is 1×10 -1 mol / L, hydrochloric acid and [P 66614 ]2[Fluo] micelles were prepared at a volume ratio of 1:10 and then diluted with pure water to 4 times the original volume;

[0019] The volume ratio of the probe solution to Nile Red micelles is 1:4;

[0020] In the above preparation method, [P 66614 ]2[Fluo] is a fluorescein ionic liquid probe with the following structural formula:

[0021]

[0022] Nile Red is a Nile Red probe with the following structural formula:

[0023]

[0024] The invention also relates to the colorimetric fluorescent dual-signal ionic liquid micelles prepared by the preparation method.

[0025] The colorimetric fluorescence dual-signal ionic liquid micelle of the present invention can be applied to the detection of the freshness of aquatic products.

[0026] In order to evaluate [P 66614 ]2[Fluo] and Nile Red micelle mixed probe solution's response to amines, fluorescence titration of NH3 solution of different concentrations, specifically:

[0027] Ammonia water is added to the colorimetric fluorescence dual-signal ionic liquid micelles of the present invention. As the NH3 concentration increases (0-30.0ppm), the color of the solution changes from pink fluorescence to yellow-green fluorescence. The fluorescence intensity of the mixed probe is measured by a fluorescence meter, and the excitation wavelength used is 450nm, the emission wavelength is 512nm, and the slit width is 5×5. The fluorescence intensity gradually increases with the increase of NH3 concentration. After 19.9ppm of ammonia water is added, the change trend tends to be gentle. With the ammonia concentration as the horizontal coordinate and the fluorescence intensity as the vertical coordinate, linear fitting is performed to obtain a linear range of 1.5-17.7ppm and a detection limit of 0.01ppm.

[0028] The colorimetric fluorescent dual-signal ionic liquid micelles of the present invention respond significantly to biogenic amines, but hardly respond to amino acids and urea, and thus can be used for specific detection of amines.

[0029] The present invention also relates to a gel bead, which is [P 66614 ]2[Fluo] ionic liquid micelles and Nile Red micelles mixed probes were made into the form of gel beads. The specific preparation method is as follows:

[0030] 2 g of sodium alginate was dissolved in 80 ml of water, stirred for 2 h, allowed to stand for 0.5 h, and bubbles were removed to obtain a translucent sodium alginate gel; 30 g of sodium alginate gel was mixed with 0.1143 g of lauramide propyl betaine and 10 mL of pure water to obtain a sodium alginate gel mixture; two portions of the sodium alginate gel mixture were prepared respectively and marked as "A" and "B";

[0031] 160 μL of Nile Red solution (1×10 -2 M, DMF solution), stirred for 2 h to obtain Nile Red micellar gel solution; 320 μL [P 66614 ]2[Fluo] solution (1×10 -2 M, DMF solution), stirred for 2 h to obtain [P 66614 ]2[Fluo] micellar gel solution;

[0032] Take 10g[P 66614 ]2[Fluo] micellar gel solution was diluted 4 times with pure water and 500 μL HCL (1×10 -1 mol / L), and then mixed with Nile Red micellar gel solution at a volume ratio of 1:4 to obtain a light pink mixed gel solution;

[0033] The mixed gel solution was added to a calcium chloride solution (0.5 mol / L) under stirring. After 2 min, the beads were taken out, rinsed with pure water, and stored at 4°C for subsequent experiments.

[0034] The gel beads prepared as above can be used to detect biogenic amines in actual environments. The freshness of aquatic products can be judged based on the color changes of the gel beads: the orange-red state is fresh, the khaki state is between the critical value, and the yellow-green state is rotten and inedible.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] (1) [P 66614 ]2[Fluo] Fluorescein ionic liquid has better visible fluorescence effect than raw materials. The synthesis method is simple and atom-economical. The synthesized [P 66614]2[Fluo] Fluorescein ionic liquid has good thermal stability and low volatility and is suitable for long-term storage. The green solvent of the present invention has a simple atomic structure and does not cause much pollution to the environment.

[0037] (2) The prepared [P 66614 ]2[Fluo] ionic liquid micelles and NR micelles have good water solubility, which overcomes the defect that the probe cannot be dissolved in water and improves the sensitivity of measuring dissolved amines in water. The mixture of the two micelle probes increases the color change span and reduces the difficulty of naked eye identification.

[0038] (3) The detection of amines is also more sensitive, and the quantitative detection limit of NH3 within the linear range can reach 0.01ppm.

[0039] (4) The hybrid probe of the present invention can be made into a gel bead state, which can achieve a rapid response to the fluorescent dual signals of biogenic amines and can be used to detect the freshness of aquatic products at different storage temperatures, providing a new strategy for detecting the freshness of actual commercial samples during sales. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 For the present invention [P 66614 ]2[Fluo] encapsulated with four surfactants under sunlight and ultraviolet light (245nm) (a), (b) [P 66614 ]2[Fluo]Fluorescence intensity before and after 6 days using four surfactants.

[0041] Figure 2 Fluorescence photographs of the two mixed optimized micelles and two single micelles of the present invention under 254 nm and 365 nm ultraviolet light before and after adding 35.0 ppm of ammonia.

[0042] Figure 3 The fluorescence spectra of the mixed probe solution of the present invention when different concentrations of ammonia (0-30.0 ppm) are added and the corresponding color change diagrams.

[0043] Figure 4 It is the linear curve of the mixed probe solution of the present invention. The excitation wavelength is 450nm, the emission wavelength is 512nm, and the linear range is 1.5ppm-17.7ppm.

[0044] Figure 5 This is a fluorescence spectrum diagram of the mixed probe solution of the present invention in response to organic amines and amino acids in water (25.0 ppm).

[0045] Figure 6 The present invention uses the mixed probe gel as an intelligent label to monitor the freshness of shrimps under different storage conditions and determine the TVB-N content in the shrimps. DETAILED DESCRIPTION

[0046] In order to better understand the present invention, the content of the present invention is further explained below in conjunction with examples, but the content of the present invention is not limited to the following examples, and the following content should not be understood as limiting the scope of protection requested by the claims of the present invention.

[0047] The chemical reagents and solvents used in the examples were of analytical grade.

[0048] The excitation wavelength for fluorescence measurement was 450 nm, and the maximum emission wavelength was 512 nm.

[0049] Example 1: Ionic liquid mixed fluorescent micelle probe is used for the detection of biogenic amines, and the specific steps include:

[0050] (1)[P 66614 ]2[Fluo] Optimization of the hybrid probe conditions of ionic liquid micelles and NR micelles

[0051] Select different surfactants to optimize [P 66614 ]2[Fluo] micelles. The four different surfactants were SDBS, CTAB, TW 20 and LP. Taking LP as an example, three 40 mL LP aqueous solutions were prepared, with LP contents of 5×10 - 4 mol / L and 2.5×10 -3 mol / L,5×10 -3 Then add 320 μL [P 66614 ]2[Fluo](1×10 -2 mol / L, DMF), stirred for half an hour for future use. The other three surfactants were also prepared in the same amount and method. Figure 1 (a) shows the difference between sunlight and ultraviolet light under different surfactant wrapping conditions. The results show that the fluorescence is brightest when wrapped with LP. Figure 1 (b) shows that the fluorescence intensity was measured on the first and sixth days of synthesizing micelles. The results showed that LP encapsulated [P 66614 ]2[Fluo] ionic liquid, the fluorescence intensity of micelles is also the best. The above shows that [P 66614 ]2[Fluo] The preparation of ionic liquid micelles is suitable for encapsulation with LP.

[0052] (2) Prepare [P 66614 ]2[Fluo] and Nile Red to 1×10 -2 mol / L. Two samples containing LP (1×10 -4Then, 320 μL of [P 66614 ]2[Fluo] solution and 160μL Nile Red (1×10 -2 mol / L), stir for half an hour and it can be used. 66614 ]2[Fluo] solution was diluted 4 times, and then acid was added to adjust the pH to about 2.7. Then the two probe solutions were mixed in a ratio of 1:4. 35.0ppm ammonia was added for comparison. Then the LP concentration was (1.5×10 -4 mol / L) of [P 66614 ]2[Fluo] micellar solution, other quantities are the same as above. Figure 2 It means that [P 66614 ]2[Fluo] ionic liquid micelles responded to ammonia water, and the color changed from light yellow to green. Nile Red micelles did not respond to ammonia water. The color change was more obvious after the two probes were mixed. When the LP concentration was 1×10 -4 mol / L, the mixing effect achieves the best effect.

[0053] (3) According to the above optimization results, the fluorescence spectra of the mixed probe solution with different concentrations of ammonia (0-30.0ppm) and the corresponding color change diagrams were obtained. The concentrations of ammonia water were 1.5ppm, 3.0ppm, 4.5ppm, 6.3ppm, 8.1ppm, 9.7ppm, 10.5ppm, 11.7ppm, 12.9ppm, 14.7ppm, 15.9ppm, 17.7ppm, 19.9ppm, 23.0ppm, 25.0ppm, 27.0ppm, and 30.0ppm. Figure 3 The results show that as the concentration of ammonia increases, the fluorescence intensity increases and the color of the solution changes from pink to yellow-green. A linear fit is performed with ammonia concentration as the horizontal axis and fluorescence intensity as the vertical axis. Figure 4 As shown, the ammonia concentration is 1.5-17.7ppm (R 2 =0.996) has a good linear relationship, and the linear formula is y=251.73x-544.35. The detection limit (LOD) of ammonia is 0.01ppm. These results show that the dual-signal mixed probe solution has good sensing performance for ammonia and has great application potential in food freshness.

[0054] (4) According to the ratio of the above mixed probes, a mixed probe solution with a concentration of 25.0 ppm of interfering substances (cadaverine, histamine, putrescine, ethylenediamine, triethylamine, histidine, L-cysteine, phenylalanine, phenylalanine, alanine, glutamic acid, glutamic acid, tryptophan, urea) was prepared. The fluorescence characteristics of the solution were recorded using a fluorometer. The results are shown in Figure 2. Figure 5 As shown, the mixed probe solution responds significantly to biogenic amines, but hardly responds to amino acids and urea.

[0055] (5) Monitoring of shrimp freshness using hybrid probe micellar gel bead sensor

[0056] Dissolve 2g of sodium alginate in 80ml of water, stir for 2 hours, let stand for half an hour, remove bubbles, and obtain a translucent sodium alginate gel. Mix 30g of sodium alginate gel with 0.1143g of LP and 10mL of pure water, and prepare two portions, add 160μL of Nile red solution and 320μL of [P 66614 ]2[Fluo] in DMF solution (1×10 -2 After stirring, 500 μL of HCl solution (0.1 mol / L) was added to 10 g [P 66614 ]2[Fluo] micellar gel solution diluted to 40g. Then mix the Nile Red micellar gel solution with the diluted [P 66614 ]2[Fluo] micellar gel solution was mixed in a ratio of 4:1, and 1 mL of the mixed gel solution was pipetted into 5 mL of calcium chloride solution (0.5 mol / L). After 2 minutes, the beads were taken out, rinsed with pure water, and stored at 4°C for subsequent experiments.

[0057] The finished gel beads were placed in a culture dish containing shrimps, which were sealed and stored in the culture dishes at three temperatures: 25°C, 4°C, and -20°C. The freshness of the shrimps was observed at 0, 1, 2, and 3 days. The gel beads were observed after 25 minutes. The culture dish was placed in a UV dark box (254nm). The actual results are shown below. Figure 6 The results showed that the increase of biogenic amines caused the gel beads to change from orange-red to yellow-green. The measurement results of TVB-N showed the same trend as the gel.

Claims

1. A method for preparing colorimetric fluorescent dual-signal ionic liquid micelles, characterized in that: The steps include: (1) [P 66614 ]2[Fluo] solution was added dropwise to surfactant solution A to obtain [P 66614 ]2[Fluo] micelles; Nile Red solution was added dropwise to surfactant solution B to obtain Nile Red micelles; The surfactant is selected from: Tween 20, lauramidopropyl betaine, cetyltrimethylammonium bromide or sodium dodecylbenzene sulfonate; (2) In [P 66614 ]2[Fluo] micelles are added with hydrochloric acid, and the probe solution is obtained after diluting with pure water, and the obtained probe solution is mixed with Nile Red micelles to obtain the colorimetric fluorescence dual-signal ionic liquid micelles; In the above preparation method, [P 66614 ]2[Fluo] is a fluorescein ionic liquid probe with the following structural formula: Nile Red is a Nile Red probe with the following structural formula:

2. The method for preparing the colorimetric fluorescent dual-signal ionic liquid micelles according to claim 1, characterized in that: In step (1), the [P 66614 ]2[Fluo] solution and Nile Red solution were prepared with DMF as solvent, and the concentration was 1×10 -2 mol / L.

3. The method for preparing the colorimetric fluorescent dual-signal ionic liquid micelles according to claim 1, characterized in that: In step (1), surfactant solution A and surfactant solution B are prepared with water as solvent, and the concentration is 1×10 -4 mol / L.

4. The method for preparing the colorimetric fluorescent dual-signal ionic liquid micelles according to claim 1, characterized in that: In step (1), the [P 66614 ]2The volume ratio of [Fluo] solution to surfactant solution A is 1:

125.

5. The method for preparing the colorimetric fluorescent dual-signal ionic liquid micelles according to claim 1, characterized in that: In step (1), the volume ratio of the Nile Red solution to the surfactant solution B is 1:

250.

6. The method for preparing the colorimetric fluorescent dual-signal ionic liquid micelles according to claim 1, characterized in that: In step (2), the concentration of hydrochloric acid is 1×10 -1 mol / L, hydrochloric acid and [P 66614 ]2[Fluo] micelles were prepared at a volume ratio of 1:10 and then diluted with pure water to 4 times the original volume.

7. The method for preparing the colorimetric fluorescent dual-signal ionic liquid micelles according to claim 1, characterized in that: In step (2), the volume ratio of the probe solution to the Nile Red micelles is 1:

4.

8. The colorimetric fluorescent dual-signal ionic liquid micelles prepared by the preparation method according to any one of claims 1 to 7.

9. A gel bead, characterized in that [P 66614 ]2[Fluo] ionic liquid micelles and Nile Red micelles mixed probes were made into the form of gel beads; Among them, [P 66614 ]2[Fluo] is a fluorescein ionic liquid probe with the following structural formula: Nile Red is a Nile Red probe with the following structural formula:

10. Use of the colorimetric fluorescent dual-signal ionic liquid micelles as claimed in claim 8 or the gel beads as claimed in claim 9 in the detection of freshness of aquatic products.

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