阅读说明：本技术 一种比率型荧光高分子水凝胶在海鲜新鲜度检测中的应用 (Application of ratio-type fluorescent polymer hydrogel in seafood freshness detection ) 是由 陈涛 刘浩 路伟 于 2020-04-22 设计创作，主要内容包括：本发明公开了一种比率型荧光高分子水凝胶在海鲜新鲜度检测中的应用,利用蓝色有机荧光团和红色发射中心构建具有自校正能力的比率型荧光高分子水凝胶探针,当遇到海鲜变质所释放的生物胺时,红色会被选择性淬灭,进而改变红、蓝荧光强度比值和水凝胶颜色,实现可视化检测；该水凝胶可用于海鲜产品新鲜度的常规快速检验或用于实时指示货架期内海鲜的新鲜度。利用本发明方法及检测管可实现海鲜新鲜度快速、实时的检测,且其检测成本低、操作简便。(The invention discloses an application of ratio-type fluorescent polymer hydrogel in seafood freshness detection, wherein a ratio-type fluorescent polymer hydrogel probe with self-correcting capability is constructed by utilizing a blue organic fluorophore and a red emission center, when biogenic amine released by seafood deterioration is encountered, red is selectively quenched, and then the ratio of red fluorescence intensity to blue fluorescence intensity and the color of the hydrogel are changed, so that visual detection is realized; the hydrogel can be used for conventional quick detection of the freshness of seafood products or used for indicating the freshness of seafood in real time during the shelf life. The method and the detection tube can realize the quick and real-time detection of the freshness of the seafood, and have low detection cost and simple and convenient operation.)

1. The application of the ratio-type fluorescent polymer hydrogel in seafood freshness detection is characterized in that the preparation method of the ratio-type fluorescent polymer hydrogel comprises the following steps:

(1) dissolving hydrogel monomer molecules, blue fluorescent molecules, pyridine carboxylic acid compounds, an initiator and a cross-linking agent in a mixed solution of deionized water and dimethyl sulfoxide, and dialyzing to remove the dimethyl sulfoxide to form uniform hydrogel pre-polymerization liquid;

(2) polymerizing the hydrogel pre-polymerization solution at-5-0 ℃ for 8-12 h, and then carrying out Eu³⁺Soaking in a salt solution for 1-10 min, taking out and washing with deionized water to obtain the ratio type fluorescent polymer hydrogel;

the blue fluorescent molecule has any one of the following structures:

the pyridine carboxylic acid compound and Eu³⁺The structure of the red fluorescent molecule is shown in any one of the following formulas:

2. the use according to claim 1, wherein the blue fluorescent molecule is prepared by a method comprising the steps of:

(a) reacting 4-bromo-1, 8-naphthalic anhydride with propylamine or allylamine at 75-85 ℃ for 6-10 h in the presence of ethanol, and then performing suction filtration to obtain an intermediate;

(b) reacting the intermediate, the substitute and potassium carbonate in the presence of dimethyl sulfoxide at 75-85 ℃ in an inert atmosphere for 10-14 h, sequentially adding water and dichloromethane for extraction after the reaction is finished, and finally performing rotary evaporation to obtain the blue fluorescent molecule;

the substitute is phenol, toluene or ethylbenzene.

3. The use according to claim 2, wherein in step (a), the molar ratio of 4-bromo-1, 8-naphthalenic anhydride to propylamine or allylamine is 1:1 to 7;

in the step (b), the molar ratio of the intermediate to the substituent is 1: 1-5.

4. The use according to claim 1, characterized in that the preparation method of pyridine carboxylic acids comprises the steps of:

(A) dissolving 6-aminopyridine-2-carboxylic acid methyl ester in a mixed solution of dichloromethane and triethylamine in an ice water bath protected by nitrogen, then dropwise adding the mixed solution of dichloromethane and acryloyl chloride or methacryloyl chloride, stirring for reacting for 2-4 h, carrying out rotary evaporation to obtain a crude product, and then carrying out chromatographic column purification by using normal hexane and ethyl acetate as eluents to obtain an intermediate product;

(B) and dissolving the intermediate product and sodium hydroxide in a mixed solution of tetrahydrofuran and water, stirring for 20-40 min, adding an excessive HCl solution, performing rotary evaporation to remove tetrahydrofuran, washing the rotary evaporated product with deionized water, and drying to obtain the pyridine carboxylic acid compound.

5. The use according to claim 4, wherein in the step (A), the molar ratio of the methyl 6-aminopyridine-2-carboxylate to the acryloyl chloride or the methacryloyl chloride is 1: 1.2-6, the volume ratio of the dichloromethane to the triethylamine in the mixed solution of the dichloromethane and the triethylamine is 4-8: 1, and the volume ratio of the n-hexane to the ethyl acetate is 4-6: 1;

in the step (B), the molar ratio of the intermediate product to sodium hydroxide is 1: 1.5-5, and the volume ratio of tetrahydrofuran to water in the mixed solution of tetrahydrofuran and water is 1: 1-3.

6. The use according to claim 1, wherein the hydrogel monomer molecules are N-isopropylacrylamide, polyvinyl alcohol or N, N-dimethylacrylamide;

the initiator is potassium persulfate;

the cross-linking agent is N, N' -methylene bisacrylamide.

7. The use of claim 1, wherein the seafood is shrimp, crab, fish or shellfish.

8. The use according to any one of claims 1 to 7, wherein the ratio-type fluorescent polymer hydrogel is sealed with seafood, and the freshness of the seafood is judged according to the color change of the ratio-type fluorescent polymer hydrogel.

Technical Field

The invention relates to the technical field of seafood detection, in particular to application of ratio-type fluorescent polymer hydrogel in seafood freshness detection.

Background

If the seafood is improperly stored in the storage and transportation processes, the seafood can be rotten and deteriorated, which causes serious harm to the factory production and the health of consumers. Spoilage of seafood is mainly due to the activity of some microbial enzymes resulting in enzymatic decarboxylation of some specific amino acids, which occurs with the release of biogenic amines, especially cadaverine. Most of the existing methods for detecting freshness of seafood are mainly based on cadaverine concentration, for example, the high-pressure liquid chromatography is utilized for early J.T. style, the cadaverine is firstly extracted by a solvent, and then the concentration is calibrated by a chromatographic column, although the method has high resolution, the process is complex, and the equipment is expensive (Journal of Neural Transmission 1982, 53, 1-5); annapoorni later invented a polymer-based method in which aniline was dissolved and grown in situ on PET film, and its electrical resistance was changed by the extracted amine, but this method was susceptible to moisture interference and could not be used to detect seafood freshness in real time (Sensors and Actuators B2017, 240, 408-. Therefore, there is a need in the art to develop a detection device that is convenient and efficient and can detect the freshness of seafood in real time.

At present, an optical detection method gradually becomes a mainstream detection method in the field of food safety detection with the characteristics of high resolution, high selectivity, high identification degree and the like, for example, a pH response detection test paper is invented in Korean wealth and the like, and the freshness of fish is judged according to the color reaction of the pH response detection test paper and mucus on the surface of the fish body (CN 104297235A); jose m.garcia invented a polymer film based on trinitrobenzene derivatives, characterizing seafood freshness by its color change after reaction with cadaverine (chem.eur.j.2015,21, 8733-8736); tangben faith et al examined seafood freshness by using molecules that can be converted to molecules possessing aggregation-induced emission (AIE) effects under the action of cadaverine (ACS Sens.2016,1, 179-184).

However, these methods only have one kind of luminophor when performing visual detection, and the detection accuracy is easily affected by some factors, such as: photobleaching, stability in the surrounding microenvironment and light, etc.; and the change of fluorescence intensity is not easy to be detected by naked eyes in most detection processes, so that errors are easy to increase.

Disclosure of Invention

Aiming at the defects in the field, the invention provides the application of the ratio-type fluorescent polymer hydrogel in seafood freshness detection, a ratio-type fluorescent hydrogel probe with self-correcting capability is constructed by utilizing a blue organic fluorophore and a red emission center, the accurate, efficient and real-time visual detection of the seafood freshness can be realized, the operation is simple, and the detection cost is low.

The application of the ratio-type fluorescent polymer hydrogel in seafood freshness detection comprises the following steps:

(1) dissolving hydrogel monomer molecules, blue fluorescent molecules, pyridine carboxylic acid compounds, an initiator and a cross-linking agent in a mixed solution of deionized water and dimethyl sulfoxide (DMSO), and dialyzing to remove the dimethyl sulfoxide (DMSO) to form uniform hydrogel pre-polymerization liquid;

(2) polymerizing the hydrogel pre-polymerization solution at-5-0 ℃ for 8-12 h, and then carrying out Eu³⁺Soaking in a salt solution for 1-10 min, taking out and washing with deionized water to obtain the ratio type fluorescent polymer hydrogel;

the blue fluorescent molecule has any one of the following structures:

the pyridine carboxylic acid compound and Eu³⁺The structure of the red fluorescent molecule is shown in any one of the following formulas:

according to the invention, a blue organic fluorophore (blue fluorescent molecule) and a red luminescent center (red fluorescent molecule) responding to biogenic amine are introduced into hydrogel to construct a ratio type fluorescent hydrogel probe with self-correcting capability, when biogenic amine released by seafood deterioration is encountered, red is quenched, and further the ratio of the red fluorescence intensity to the blue fluorescence intensity and the color of the hydrogel are changed, so that visual detection is realized. The biogenic amine can be cadaverine, diethylamine, benzylamine, triethylamine and the like.

The blue organic fluorophore is a molecule capable of emitting blue fluorescence under ultraviolet irradiation, and has a relatively small response degree to biogenic amine.

Preferably, the preparation method of the blue fluorescent molecule comprises the following steps:

(a) reacting 4-bromo-1, 8-naphthalic anhydride with propylamine or allylamine at 75-85 ℃ for 6-10 h in the presence of ethanol, and then performing suction filtration to obtain an intermediate;

(b) reacting the intermediate, the substitute and potassium carbonate in the presence of dimethyl sulfoxide at 75-85 ℃ in an inert atmosphere for 10-14 h, sequentially adding water and dichloromethane for extraction after the reaction is finished, and finally performing rotary evaporation to obtain the blue fluorescent molecule;

the substitute is phenol, toluene or ethylbenzene.

The inert atmosphere may be a rare gas or nitrogen atmosphere.

Further preferably, in the step (a), the molar ratio of the 4-bromo-1, 8-naphthalic anhydride to the propylamine or allylamine is 1: 1-7;

in the step (b), the molar ratio of the intermediate to the substituent is 1: 1-5.

The red luminescence center is a molecule capable of emitting red fluorescence under the irradiation of ultraviolet light, and has a large response degree to biogenic amine.

Preferably, the preparation method of the pyridine carboxylic acid compounds comprises the following steps:

(A) dissolving 6-aminopyridine-2-carboxylic acid methyl ester in a mixed solution of dichloromethane and triethylamine in an ice water bath protected by nitrogen, then dropwise adding the mixed solution of dichloromethane and acryloyl chloride or methacryloyl chloride, stirring for reacting for 2-4 h, carrying out rotary evaporation to obtain a crude product, and then carrying out chromatographic column purification by using normal hexane and ethyl acetate as eluents to obtain an intermediate product;

(B) and dissolving the intermediate product and sodium hydroxide in a mixed solution of Tetrahydrofuran (THF) and water, stirring for 20-40 min, adding an excessive HCl solution, performing rotary evaporation to remove the THF, washing the rotary evaporated product with deionized water, and drying to obtain the pyridine carboxylic acid compound.

Further preferably, in the step (a), the molar ratio of the methyl 6-aminopyridine-2-carboxylate to acryloyl chloride or methacryloyl chloride is 1: 1.2-6, the volume ratio of dichloromethane to triethylamine in the mixed solution of dichloromethane and triethylamine is 4-8: 1, and the volume ratio of n-hexane to ethyl acetate is 4-6: 1;

in the step (B), the molar ratio of the intermediate product to sodium hydroxide is 1: 1.5-5, and the volume ratio of tetrahydrofuran to water in the mixed solution of tetrahydrofuran and water is 1: 1-3.

Preferably, the hydrogel monomer molecule is N-isopropylacrylamide, polyvinyl alcohol or N, N-dimethylacrylamide;

the initiator is potassium persulfate;

the cross-linking agent is N, N' -methylene bisacrylamide.

Preferably, the seafood is shrimps, crabs, fish or shellfish.

In a preferred embodiment, the application specifically comprises: and sealing the ratio-type fluorescent polymer hydrogel and the seafood, and judging the freshness of the seafood according to the color change of the ratio-type fluorescent polymer hydrogel.

Compared with the prior art, the invention has the main advantages that: the invention adopts the ratio type fluorescent hydrogel probe, can enrich the color change, has more accurate detection result, does not need expensive detection equipment, can judge the freshness of the seafood by naked eyes, has the characteristics of accuracy, high efficiency and real-time detection, and has low detection cost.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The following examples are conducted under conditions not specified, usually according to conventional conditions, or according to conditions recommended by the manufacturer.

The inventor provides a technical scheme of the invention through long-term research and a large amount of practice, mainly constructs a ratio type fluorescent hydrogel probe with responsiveness to cadaverine released by seafood deterioration according to the colorimetry principle of color compounding, and realizes the visual real-time detection in the seafood deterioration process.

Specifically, the ratio-type fluorescent hydrogel probe with self-correcting capability is constructed by using a blue organic fluorophore and a pyridine carboxylic acid-europium red emission center, blue is used as a reference color, red is used as a response color, and under the action of cadaverine released in the deterioration process of seafood, the red is gradually quenched, so that the ratio of the fluorescence intensity of the red to the fluorescence intensity of the blue and the color of the hydrogel are changed, and visual detection is realized.

The color comparison card can be prepared by the following method: sealing the prepared ratio type fluorescent hydrogel with fresh shrimps, crabs, fishes and the like, sealing at room temperature or 4 ℃ under a refrigeration condition, and photographing at intervals to obtain a color comparison card with the color of the gel changing along with time; and simultaneously, determining the content of TVB-N at different time according to a half-trace nitrogen determination method in GB/T5009.44-2003 'analytical method for hygienic standards of meat and meat products' to determine the freshness of the meat at corresponding time, thereby obtaining the color comparison cards with different gel colors and corresponding seafood freshness.