阅读说明：本技术 一种挥发性盐基氮响应型化合物、便携式检测试纸及制备方法与应用 (Volatile basic nitrogen response type compound, portable detection test paper, preparation method and application ) 是由 吴水珠 刘欣 龙意 曾钫 于 2021-07-19 设计创作，主要内容包括：本发明公开了一种挥发性盐基氮响应型化合物、便携式检测试纸及制备方法与应用；本发明的挥发性盐基氮响应型化合物为3-(2-(4-(双(4-羟基苯基)氨基)苯乙烯基)-3,3-二甲基-3H-吲哚-1-盐-1-基)丙烷-1-磺酸盐；探针化合物的合成过程较简单,且化学性质较稳定。将探针化合物制备成便携式检测试纸后,试纸可对挥发性盐基氮物质做出灵敏的比色响应,随挥发性盐基氮物质的水平的增高,试纸颜色由浅紫红色逐渐变为蓝色。将该试纸用于实际的水产品鲜度检测时,该试纸可随水产品腐烂释放的TVB-N含量变化而发生颜色改变,从而对水产品的新鲜程度进行直观反映。(The invention discloses a volatile basic nitrogen response type compound, portable test paper, a preparation method and application; the volatile basic nitrogen response type compound is 3- (2- (4- (bis (4-hydroxyphenyl) amino) styryl) -3, 3-dimethyl-3H-indole-1-salt-1-yl) propane-1-sulfonate; the synthesis process of the probe compound is simple and the chemical property is stable. After the probe compound is prepared into portable test paper, the test paper can make sensitive colorimetric response to volatile basic nitrogen substances, and the color of the test paper is gradually changed from light purple to blue along with the increase of the level of the volatile basic nitrogen substances. When the test paper is used for detecting the freshness of actual aquatic products, the test paper can change the color along with the change of the content of TVB-N released by decay of the aquatic products, so that the freshness of the aquatic products is visually reflected.)

1. A volatile basic nitrogen-responsive compound, wherein the volatile basic nitrogen-responsive compound is 3- (2- (4- (bis (4-hydroxyphenyl) amino) styryl) -3, 3-dimethyl-3H-indol-1-yl) propane-1-sulfonate, having the following structural formula:

2. the method of claim 1, comprising the steps of:

adding 4- (bis (4-hydroxyphenyl) amino) benzaldehyde and 3- (2,3, 3-trimethyl-3H-indol-1-salt-yl) propane-1-sulfonate into absolute ethyl alcohol, then dropwise adding piperidine into the solution, heating and reacting under an inert gas atmosphere, controlling the temperature of the reaction solution to be 70-80 ℃, and reacting for 4-6 hours to obtain the volatile basic nitrogen response type compound.

3. The method according to claim 2, wherein the molar ratio of 4- (bis (4-hydroxyphenyl) amino) benzaldehyde to 3- (2,3, 3-trimethyl-3H-indol-1-ol-yl) propane-1-sulfonate is 1: 2-3.

4. The method according to claim 2, wherein the amount of the anhydrous ethanol added per mmol of the 4- (bis (4-hydroxyphenyl) amino) benzaldehyde is 15 to 25 mL; 150 μ L of piperidine per mmol of 4- (bis (4-hydroxyphenyl) amino) benzaldehyde; the inert gas atmosphere is that the system is vacuumized and filled with inert gas and repeated for at least 3 times; the inert gas is nitrogen.

5. The production process according to any one of claims 2 to 4, wherein the reacted solution is cooled to room temperature, the solvent is distilled off under reduced pressure, and then the solution is dissolved in methanol and added dropwise to anhydrous ether at 4 to 8 ℃ to precipitate a solid, which is then washed to obtain 3- (2- (4- (bis (4-hydroxyphenyl) amino) styryl) -3, 3-dimethyl-3H-indol-1-yl) propane-1-sulfonate as a purple black solid powder.

6. The method according to claim 5, wherein the washing is repeated at least three times with dehydrated ether in an amount of 40-50mL each time.

7. The method of making a portable test strip for volatile salt-based nitrogen-responsive compounds of claim 1, comprising the steps of:

dissolving the volatile basic nitrogen response type compound in a mixed solution of dimethyl sulfoxide and ethanol to prepare a mother solution; and then, fully soaking the filter paper in the mother solution, and drying by hot air to obtain the portable test paper.

8. The method according to claim 7, wherein the volume ratio of the dimethyl sulfoxide to the ethanol is 1: 9-11; the concentration of the volatile salt-based nitrogen-responsive compound in the mother solution is 400-500 mu M.

9. A portable test strip based on a volatile salt-based nitrogen-responsive compound prepared by the method of claim 7 or 8.

10. Use of the portable test strip based on volatile basic nitrogen-responsive compounds of claim 9 for detecting freshness of aquatic products.

Technical Field

The invention relates to the technical field of analysis and detection, in particular to a volatile basic nitrogen response type compound, portable detection test paper, a preparation method and application.

Background

With the continuous improvement of the living standard of people, the quality and the safety of food are more and more valued by the public, so that the quality and the safety of the food can be visually distinguished by consumers, and the detection test paper can be added into the food in the process of packaging the food. Therefore, the detection test paper responds to the characteristic substances released by the food in the storage process, so that the test paper generates color change, and the freshness or the putrefaction degree of the food can be known by visually observing the color change of the test paper. Therefore, consumers can visually judge the freshness of the food, the acquisition and purchase process of the consumers can be greatly facilitated, and the safety of the food is ensured.

Aquatic products such as fish, shrimp and crab are rich in high-quality protein, polyunsaturated fatty acid, various vitamins and mineral elements, and are delicious in taste and popular among consumers. Meanwhile, because the fish, the shrimp and the crab have high water activity, neutral pH value, low connective tissue content and the existence of autolytic enzyme, the fish, the shrimp and the crab are extremely rotten foods. In the process of decay of aquatic products such as fish, shrimp, crab and the like, various volatile and nonvolatile substances are generated by degradation due to the propagation of microorganisms and the action of self enzymes, wherein the content of volatile basic nitrogen (TVB-N) is widely used as an index for evaluating the freshness of the aquatic products. It is prepared from Trimethylamine (TMA), Dimethylamine (DMA) and ammonia (NH)₃) And the content level of the volatile nitrogen-containing compounds is closely related to the rotten degree of the aquatic products. According to the current national food sanitation standard GB2733-2015 national food safety standard fresh and frozen animal aquatic products, the measured value of TVB-N of seawater fish and shrimp products is less than or equal to 30mg/100g, and the measured value of TVB-N of fresh water fish and shrimp products is less than or equal to 20mg/100 g.

At present, methods for evaluating freshness of aquatic products mainly comprise a sensory evaluation method, a microbiological determination method and a physicochemical analysis method. The sensory evaluation method has subjectivity of different individual observation visual angles in the evaluation process, and the other two methods need more complicated sample pretreatment steps and instrument operation processes, have higher requirements on experimenters and are not suitable for mass real-time freshness judgment of products. In recent years, some novel detection methods are applied to detection of freshness of aquatic products, such as computer vision, chromatography, electronic tongue, electronic nose and other sensory bionic technologies. For example, patent CN105223240A provides a method for detecting freshness of crabs by using an electronic nose, which comprises collecting gas volatilized after a crab sample is placed for 30-50min, introducing the gas into a sensor array for detection and analysis to obtain a response characteristic value of the sample, calculating by using a built-in algorithm to obtain a signal-to-noise ratio characteristic value, and calculating by using an established freshness prediction model to obtain the freshness of crabs. For another example, patent CN101936912A provides a method and device for visually detecting freshness of fish based on smell, in which gas volatilized from a sample is first reacted with a porphyrin compound sensor, a pH indicator sensor, and a solvatochromic dye sensor, then a scanner collects color change images before and after the sensor reaction, and the freshness of fish is calculated and judged according to the relationship between the color change before and after the three sensors reaction and the gas concentration. It can be clearly seen that these methods still have the problems of high detection cost, complex data post-processing, and need of professional technicians and instruments, and are not suitable for a wide range of applications. Compared with the method, the compound indicator paper which responds to substances released in the process of the decay of the aquatic products shows great superiority, and the compound indicator paper can generate the most intuitive color change along with the change of the freshness of the aquatic products such as fishes, shrimps and the like, thereby guiding people to judge the freshness level of the aquatic products. For example, patent CN104297235A provides a method for detecting freshness of fish by using a mixed indicator (olfactory phenol red, olfactory cresol purple, olfactory thymol blue, neutral red, phenol red) test paper, which is light cyan when fish are fresh and blue black when fish are not fresh; however, the mixed indication test paper needs to take fish mucus to be placed on the test paper for color development to observe color change during testing, the operation is complex, and the detection effect is influenced by the quantity of mucus taken. Also as published paper Food Chemistry 2017, 218: 122- & ltSUB & gt 128- & gt, the purple sweet potato anthocyanin, the starch and the agar are compounded to form a film, so that the spoilage degree of meat can be visually monitored, but the indicator purple sweet potato anthocyanin used in the film belongs to natural dyes, and the problems of poor chemical stability, easy degradation by illumination, poor heat resistance, high purification cost and the like also exist in the indicator purple sweet potato anthocyanin.

Aquatic products are extremely easy to decay due to high protein and moisture content, and the freshness level of the aquatic products not only affects the taste, but also carries a large number of pathogenic bacteria when the aquatic products decay and deteriorate, so that the human health is harmed, and therefore, the aquatic products are necessary to be quickly, conveniently and effectively monitored for freshness. Most of the widely researched food freshness detection methods at present need a complicated sample pretreatment process, expensive analytical instruments or professional data acquisition and analysis tools, and the response to the aquatic product spoilage process is not intuitive enough. Or the manufacturing process of the indicator paper is complicated, the detection is not simple and convenient enough, the adopted indicator has poor chemical stability, and the indicator is likely to degrade in the using process, loses the detection function and the like. Therefore, there is a need to develop a portable test paper of volatile basic nitrogen responsive compounds that can visually reflect the freshness of aquatic products, so that the portable test paper can be applied to the detection of the freshness of aquatic products with high sensitivity, convenience and low cost.

Disclosure of Invention

To address the above-discussed shortcomings and drawbacks of the prior art, it is a primary object of the present invention to provide a volatile basic nitrogen-responsive compound. The compound takes phenolic hydroxyl as a recognition group, and can realize the response to volatile basic nitrogen.

Another object of the present invention is to provide a method for preparing the volatile basic nitrogen-responsive compound and the portable test strip thereof.

The invention further aims to provide application of the portable test paper based on the volatile basic nitrogen-responsive compound in detection of freshness of aquatic products.

The purpose of the invention is realized by the following technical scheme:

a volatile basic nitrogen-responsive compound that is 3- (2- (4- (bis (4-hydroxyphenyl) amino) styryl) -3, 3-dimethyl-3H-indol-1-salt-1-yl) propane-1-sulfonate having the following structural formula:

the preparation method of the volatile salt-based nitrogen-responsive compound comprises the following steps:

adding 4- (bis (4-hydroxyphenyl) amino) benzaldehyde and 3- (2,3, 3-trimethyl-3H-indol-1-salt-yl) propane-1-sulfonate into absolute ethyl alcohol, then dropwise adding piperidine into the solution, heating and reacting under an inert gas atmosphere, controlling the temperature of the reaction solution to be 70-80 ℃, and reacting for 4-6 hours to obtain the volatile basic nitrogen response type compound.

Preferably, the molar ratio of 4- (bis (4-hydroxyphenyl) amino) benzaldehyde to 3- (2,3, 3-trimethyl-3H-indol-1-salt-yl) propane-1-sulfonate is 1: 2-3.

Preferably, the amount of absolute ethanol added per mmol of 4- (bis (4-hydroxyphenyl) amino) benzaldehyde is 15-25 mL.

Preferably, the piperidine is added in an amount of 100. mu.L per mmol of 4- (bis (4-hydroxyphenyl) amino) benzaldehyde.

Preferentially, the inert gas atmosphere is that the system is vacuumized and filled with inert gas, and the process is repeated for at least 3 times; the inert gas is nitrogen.

Preferably, the reacted solution is cooled to room temperature, the solvent is distilled off under reduced pressure, and then the solution is dissolved in methanol and added dropwise into anhydrous ether at 4 to 8 ℃ to precipitate a solid, which is washed to obtain a purple black solid powder of 3- (2- (4- (bis (4-hydroxyphenyl) amino) styryl) -3, 3-dimethyl-3H-indol-1-yl) propane-1-sulfonate.

Further preferably, in step 2), the washing is repeated at least three times with anhydrous ether in an amount of 40-50mL each time.

The preparation method of the portable test paper based on the volatile basic nitrogen-responsive compound comprises the following steps:

dissolving the volatile basic nitrogen response type compound in a mixed solution of dimethyl sulfoxide and ethanol to prepare a mother solution; and then, fully soaking the filter paper in the mother solution, and drying by hot air to obtain the portable test paper.

Preferentially, the volume ratio of the dimethyl sulfoxide to the ethanol is 1: 9-11;

preferably, the concentration of the volatile basic nitrogen-responsive compound in the mother liquor is 400-500. mu.M.

The portable test paper based on the volatile basic nitrogen response type compound prepared by the preparation method is provided.

The portable test paper based on the volatile basic nitrogen response type compound is applied to detection of freshness of aquatic products.

The compound obtained by the invention is 3- (2- (4- (bis (4-hydroxyphenyl) amino) styryl) -3, 3-dimethyl-3H-indole-1-salt-1-yl) propane-1-sulfonate (TPA-2OH-Y), and the molecular formula is C₃₃H₃₂N₂O₅S, relative molecular mass 568.2032. TPA-2OH-Y is purple black solid powder, is slightly soluble in water, and is easily soluble in organic solvents such as dimethyl sulfoxide, N-dimethylformamide, N-dimethylacetamide, etc. The compound has good light stability and chemical stability, and is nontoxic.

With the increase of the content of volatile basic nitrogen, the phenolic hydroxyl group on TPA-2OH-Y is deprotonated, so that the electron pushing capacity is enhanced, the electron mobility is increased, the electron cloud density on a benzene ring is changed, the Intramolecular Charge Transfer (ICT) effect from triphenylamine to an indole sulfonate part is enhanced, and the red shift of the absorption spectrum of the compound and the change of the solution color from light purple red to blue are caused. Therefore, the compound can be applied to the application of responding to volatile basic nitrogen released in the process of decay of aquatic products in the field of food safety.

The compounds provided by the invention have the following outstanding advantages compared with the prior art:

(1) the invention provides a portable test paper based on a volatile basic nitrogen response type compound, which is placed or packaged together with aquatic products or pasted on the inner side of a transparent package of the aquatic products, and can respond to the change of the concentration of volatile basic nitrogen released by the decay of the aquatic products, and the color change of the test paper from light purple red to blue can be observed by naked eyes, so that a product transporter or a consumer can be guided to judge the freshness of the products. The detection process is non-destructive to the food or packaging.

(2) The volatile basic nitrogen responsive compound has the advantages of simple preparation process, stable chemical property, sensitive response to volatile basic nitrogen, and suitability for detection of volatile basic nitrogen released in the decay process of aquatic products.

(3) The compound is prepared into portable qualitative detection test paper, the process is simple, the detection cost is low, the use is visual and convenient, and the compound has good application potential in the field of detection of freshness of aquatic products.

Drawings

FIG. 1 is a schematic synthesis scheme of a volatile basic nitrogen-responsive compound of the present invention;

FIG. 2 is a NMR spectrum of 3- (2- (4- (bis (4-hydroxyphenyl) amino) styryl) -3, 3-dimethyl-3H-indol-1-yl salt-1-yl) propane-1-sulfonate (TPA-2OH-Y) in example 1;

FIG. 3 is a mass spectrum m/z [ TPA-2OH-Y + H ] of 3- (2- (4- (bis (4-hydroxyphenyl) amino) styryl) -3, 3-dimethyl-3H-indol-1-yl salt-1-yl) propane-1-sulfonate in example 1]⁺；

Fig. 4 is an example of the detection test paper for detecting the freshness of the aquatic metapenaeus ensis: a relationship graph of volatile basic nitrogen value (TVB-N) and standing time after the metapenaeus ensis is placed for 0, 12, 18, 24 and 36 hours at room temperature; in the drawing a, a metapenaeus ensis sample is placed for 0h of test paper at room temperature; b, placing the metapenaeus ensis sample in the test paper for 18h at room temperature; and c, placing the metapenaeus ensis sample on the test paper for 24 hours at room temperature.

Detailed Description

The following description of the embodiments of the present invention is provided in connection with the accompanying drawings and examples, but the invention is not limited thereto. It is noted that the processes described below, if not specifically described in detail, are all realizable or understandable by those skilled in the art with reference to the prior art. The reagents or apparatus used are not indicated to the manufacturer, and are considered to be conventional products available through commercial purchase.

The synthetic route of the volatile salt-based nitrogen-responsive compound of the present invention is shown in fig. 1.

Example 1

Adding 305.11mg (1mmol) of 4- (bis (4-hydroxyphenyl) amino) benzaldehyde and 562.22mg (2mmol) of 3- (2,3, 3-trimethyl-3H-indol-1-salt-yl) propane-1-sulfonate into 15mL of anhydrous ethanol, then dropwise adding 100 mu L of piperidine into the solution, vacuumizing the system, filling nitrogen and repeating for 3 times, heating to 70 ℃, stirring and reacting for 4 hours, cooling the reacted solution to room temperature, removing the solvent through reduced pressure distillation, dissolving in methanol, dropwise adding into 8 ℃ anhydrous ether to separate out a solid, and repeatedly washing with the anhydrous ether for three times, wherein the dosage of the anhydrous ether is 40mL each time, so that the 3- (2- (4- (bis (4-hydroxyphenyl) amino) styryl) -3, 3-dimethyl-3H-indol-1-salt-1-yl) propane-1-one-membered wine is obtained Sulfonate 300mg (yield: 52.80%).

The obtained product was characterized by nuclear magnetic resonance hydrogen spectroscopy:¹H NMR(600MHz,DMSO-d₆) δ 9.67(s,2H),8.32(d, J ═ 15.7Hz,1H),8.09(d, J ═ 8.2Hz,2H),7.85(d, J ═ 8.0Hz,1H),7.79(d, J ═ 7.4Hz,1H),7.59 to 7.52(m,2H),7.50(t, J ═ 7.4Hz,1H),7.12(d, J ═ 8.5Hz,4H),6.84(d, J ═ 8.5Hz,4H),6.64(d, J ═ 8.8Hz,2H),4.78 to 4.62(m,2H),2.67 to 2.58(m,2H),2.11(s,2H),1.75(s, 6H). The NMR spectrum is shown in FIG. 2.

Further validation was performed by high resolution mass spectrometry testing: MS (ESI) M/z 569.2105[ M + H ]]⁺(ii) a The mass spectrum is shown in FIG. 3. The synthesized product can be determined as a target product through nuclear magnetism and mass spectrum analysis.

Example 2

Adding 305.11mg (1mmol) of 4- (bis (4-hydroxyphenyl) amino) benzaldehyde and 702.78mg (2.5mmol) of 3- (2,3, 3-trimethyl-3H-indol-1-salt-yl) propane-1-sulfonate into 20mL of anhydrous ethanol, then dropwise adding 125 mu L of piperidine into the solution, vacuumizing the system, filling nitrogen and repeating for 3 times, heating to 75 ℃, stirring for 5 hours, cooling the reacted solution to room temperature, removing the solvent by reduced pressure distillation, dissolving in methanol, dropwise adding into 6 ℃ of anhydrous ether to separate out a solid, and repeating for three times by using anhydrous ether, wherein the amount of the anhydrous ether is 45mL each time, so as to obtain 3- (2- (4- (bis (4-hydroxyphenyl) amino) styryl) -3, 3-dimethyl-3H-indol-1-salt-1-yl) propane-1-one-carboxylic acid Sulfonate 315mg (yield: 55.44%).

The characterization of the compound obtained in this example is identical to the characterization result in example 1.

Example 3

Adding 305.11mg (1mmol) of 4- (bis (4-hydroxyphenyl) amino) benzaldehyde and 844.33mg (3mmol) of 3- (2,3, 3-trimethyl-3H-indol-1-salt-yl) propane-1-sulfonate into 25mL of anhydrous ethanol, dropwise adding 150 mu L of piperidine into the solution, vacuumizing the system, filling nitrogen and repeating for 3 times, heating to 80 ℃, stirring and reacting for 6 hours, cooling the reacted solution to room temperature, distilling under reduced pressure to remove the solvent, dissolving in methanol, dropwise adding into 4 ℃ anhydrous ether to separate out a solid, and repeatedly washing with the anhydrous ether for three times, wherein the dosage of the anhydrous ether is 50mL each time, so that the 3- (2- (4- (bis (4-hydroxyphenyl) amino) styryl) -3, 3-dimethyl-3H-indol-1-salt-1-yl) propane-1-one-carboxylic acid-containing organic solvent is obtained Sulfonate salt 340mg (yield: 59.84%).

The characterization of the compound obtained in this example is identical to the characterization result in example 1.

Example 4

Preparation of portable test paper based on volatile basic nitrogen-responsive compounds:

2.27mg of solid 3- (2- (4- (bis (4-hydroxyphenyl) amino) styryl) -3, 3-dimethyl-3H-indol-1-salt-1-yl) propane-1-sulfonate was dissolved in 10mL of a solution of dimethyl sulfoxide ethanol (1:9) to prepare a mother liquor with a concentration of 400. mu.M; filter paper (cut to 2x2 cm)²) Soaking in the mother liquor for 30 minutes, taking out and drying by hot air to obtain the portable test paper based on the volatile basic nitrogen response type compound.

Example 5

Preparation of portable test paper based on volatile basic nitrogen-responsive compounds:

2.81mg of solid 3- (2- (4- (bis (4-hydroxyphenyl) amino) styryl) -3, 3-dimethyl-3H-indol-1-ol salt-1-yl) propane-1-sulfonate was dissolved in 11mL of a solution of dimethyl sulfoxide ethanol (1:10) to prepare a mother liquor with a concentration of 450. mu.M; filter paper (cut to 2x2 cm)²) Soaking in the mother liquor for 30 minutes, taking out and drying by hot air to obtain the portable test paper based on the volatile basic nitrogen response type compound.

Example 6

Preparation of portable test paper based on volatile basic nitrogen-responsive compounds:

3.41mg of solid 3- (2- (4- (bis (4-hydroxyphenyl) amino) styryl) -3, 3-dimethyl-3H-indol-1-salt-1-yl) propane-1-sulfonate was dissolved in 12mL of a solution of dimethyl sulfoxide, ethanol (1:11) to prepare a mother liquor with a concentration of 500. mu.M; filter paper (cut to 2x2 cm)²) Soaking in the mother liquor for 30 minutes, taking out and drying by hot air to obtain the portable test paper based on the volatile basic nitrogen response type compound.

Example 7

The application of the portable detection test paper in detecting the freshness of the aquatic metapenaeus ensis is as follows:

taking the metapenaeus ensis as a representative sample for detecting the freshness of the aquatic product, storing the metapenaeus ensis sample in a transparent plastic container, and simultaneously placing the prepared portable detection test paper in the container. The metapenaeus ensis sample and the test paper are naturally placed at room temperature (25 +/-3 ℃) for 0, 12, 18, 24 and 36 hours, the volatile basic nitrogen (TVB-N) value of the metapenaeus ensis sample is tested by a Kjeldahl apparatus, and meanwhile, the color change process of the test paper is photographed and recorded. The method for measuring TVB-N by using the Kjeldahl apparatus is carried out by referring to an automatic Kjeldahl apparatus method recommended in national food safety standard GB 5009.228-2016 (determination of volatile basic nitrogen in food). The experimental result is shown in fig. 4, when the metapenaeus ensis is rotten to the deterioration critical point, that is, the TVB-N value reaches 30mg/100g, it can be observed that the color of the test paper changes from light purple to blue purple, and after the TVB-N value exceeds the critical point, the color of the test paper can be observed to deepen and change into blue, which indicates that the test paper can perform sensitive detection on the rotting process of the metapenaeus ensis.

The above examples are preferred embodiments of the present invention, but the present invention is not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and they are included in the scope of the present invention.