CN115372297A - Aquatic product freshness detection method and kit - Google Patents
Aquatic product freshness detection method and kit Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/33—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using ultraviolet light
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
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Abstract
The invention belongs to the technical field of aquatic product freshness detection, and discloses an aquatic product freshness detection method and a kit. The invention uses the nucleic acid degradation product hypoxanthine in the aquatic products as a detection marker, and decomposes manganese dioxide by using the product after the enzyme reaction, so as to prevent the manganese dioxide from catalyzing the etching reaction of the gold nanorods, thereby achieving the detection purpose of identifying the freshness of the aquatic products. The detection method and the reagent kit provided by the invention have the advantages of short reaction time, high detection speed, strong specificity, high sensitivity, low detection limit, bright color output, visual naked eyes and the like, and are suitable for field real-time detection; particularly for aquatic products at the early stage of putrefaction, the detection method provided by the invention can sensitively and accurately judge the initial freshness of the aquatic products.
Description
Technical Field
The invention relates to the technical field of aquatic product freshness detection, in particular to an aquatic product freshness detection method and a kit.
Background
The aquatic product has high nutritive value, and fish meat and shrimp meat contain many nutritive components beneficial to human body, including protein, fatty acid, vitamins, etc. Under the combined action of enzymes and microorganisms, aquatic products are easy to deteriorate in the processes of storage and transportation, and the freshness of the aquatic products can be influenced by the treatment mode and temperature in the processes of storage and transportation. If the aquatic products are not fresh or polluted, the aquatic products usually cause illness and hospitalization, and can cause death in severe cases. Therefore, it is necessary to identify the freshness of aquatic products.
Generally speaking, there are two main methods for determining the freshness of a water product, one is a sensory assessment of freshness by a professional, and the other is a biochemical test of a target biomarker. The first method is rapid, but requires professional operation, depends to some extent on the level of perception of consumers, and is difficult to distinguish the difference in freshness of the aquatic products at the initial stage of spoilage. The second method is to detect biomarkers to identify the freshness of the aquatic product, such as total biogenic amines, total volatile bases, etc. Among them, an important marker affecting the freshness of aquatic products is the nucleotide and nucleoside metabolites produced by the degradation of Adenosine Triphosphate (ATP), which contains hypoxanthine, which is produced in the early stage of deterioration of aquatic products compared with other characteristic markers of freshness of aquatic products.
At present, the detection methods of hypoxanthine mainly comprise a high performance liquid chromatography, an electrochemical method and a fluorescence method, but the methods are expensive in instruments, complex in operation, incapable of realizing on-site real-time detection and difficult to popularize on a large scale. Compared with the detection technology, the colorimetric method has the advantages of simple and quick operation, easy identification of detection results by naked eyes, and flexibility and convenience for on-site real-time detection. However, when the colorimetric method is used for detecting hypoxanthine, the color output is single, and different observers read different color results, so that the freshness of the marine products cannot be accurately judged only by naked eyes; and the color development substance is easy to be photo-oxidized, the color output can be changed, and the detection result is unstable. Secondly, in the existing gold nanorod etching technology, the substances to be detected are usually promoted to be etched, and when the concentration of the substances to be detected is low, the substances are not easy to detect, so that the detection sensitivity is low, the detection limit is high, and especially for aquatic products at the early stage of corruption, the initial freshness of the aquatic products can not be accurately judged; the oxidant hydrogen peroxide is unstable and easy to decompose, and the output color is easy to be unstable, so that the detection result is unstable, and the detection error is caused.
Disclosure of Invention
The invention provides an aquatic product freshness detection method, which aims to solve the technical problems that detection errors are caused by single color output, the detection sensitivity is low, the detection limit is high, the initial freshness of aquatic products cannot be sensitively judged and the like in the prior art for aquatic product freshness detection.
In order to solve the technical problems, the invention adopts the following technical scheme:
the invention provides a method for detecting the freshness of aquatic products, which comprises the steps of decomposing hypoxanthine in aquatic products to be detected by Xanthine Oxidase (XOD) and reducing manganese dioxide (MnO) by a decomposition product 2 ) The nanoenzyme is manganese ion, and in acid solution, unreduced manganese dioxide nanoenzyme oxidizes 3,3', 5' -Tetramethylbenzidine (TMB) to generate diimine ion (TMB) 2+ ) The gold nanorods are etched by the diimine ion divalent ions, the freshness and the hypoxanthine content of aquatic products to be detected are different, the solution can present different colors after the etching is finished, the visual detection of the freshness of the aquatic products to be detected can be realized by the output of bright colors and the depth of the same color system, the ultraviolet absorption spectrum of the gold nanorods after the etching is finished is changed, and the freshness of the aquatic products to be detected can be accurately and quantitatively detected by an ultraviolet spectrophotometer.
The method specifically comprises the steps of mixing xanthine oxidase and manganese dioxide nanoenzyme solutions, adding the mixed solutions into an aquatic product to be detected, incubating for a certain time under a certain temperature condition, adding an acidic solution, adding a 3,3', 5' -tetramethylbenzidine solution after uniformly mixing, adding gold nanorods after fully and uniformly mixing again, incubating for a certain time again under a certain temperature condition, finishing etching the gold nanorods, keeping the color of the gold nanorods stable, and observing the freshness of the aquatic product to be detected through naked eyes or an ultraviolet visible spectrophotometer.
Further, incubating together at 37 ℃ for 10min; the mixture was incubated at 25 ℃ for another 5min.
Further, the acidic solution is a sulfuric acid solution.
Further, the enzyme activity of the xanthine oxidase is 2U/mL; the concentration of the manganese dioxide nano enzyme solution is 1mg/mL; the concentration of the 3,3', 5' -tetramethylbenzidine is 10mmol/L, and the concentration of the acidic solution is 5mol/L.
Adding chloroauric acid solution into hexadecyl ammonium bromide solution, uniformly stirring, adding sodium borohydride solution, stirring for several minutes until the color of the solution is changed from golden yellow to yellowish brown, stopping stirring to obtain seed solution, and standing at room temperature for later use; adding a silver nitrate solution into a cetyl ammonium bromide solution, uniformly mixing, adding a chloroauric acid solution, uniformly mixing, adding an ascorbic acid solution, uniformly mixing to obtain a solution which is changed from golden yellow to colorless, finally adding the seed solution, stirring to obtain a synthesized gold nanorod, and diluting with the cetyl ammonium bromide solution to obtain the gold nanorod solution containing the cetyl ammonium bromide solution.
Another purpose of the invention is to provide a kit for detecting the freshness of aquatic products, which comprises a Xanthine Oxidase (XOD) solution reagent with the enzyme activity of 2U/mL and manganese dioxide (MnO) with the enzyme activity of 1mg/mL 2 ) The kit comprises a nano enzyme solution reagent, a 5mol/L acidic solution reagent, a 10 mmol/L3, 3', 5' -Tetramethylbenzidine (TMB) solution reagent, a gold nanorod solution reagent containing a cetyl ammonium bromide (CTAB) solution and a standard colorimetric card.
Further, the acidic solution reagent is a sulfuric acid solution.
The preparation method of the standard colorimetric card comprises the steps of selecting a series of hypoxanthine solution samples with different concentrations to replace aquatic products to be detected, carrying out etching color development reaction on the gold nanorods by respectively adopting the detection method, shooting corresponding colors by using a camera after the color of the gold nanorods is stabilized, enabling a series of hypoxanthine samples with different concentrations to generate corresponding rainbow-like color changes, marking out the corresponding hypoxanthine concentrations, and carrying out color printing to prepare the standard colorimetric card for detecting the freshness of the aquatic products.
The preparation method of the gold nanorod solution reagent containing the hexadecyl ammonium bromide solution comprises the following steps,
1) The seed synthesis method comprises the steps of adding 0.5mmol/L chloroauric acid solution into hexadecyl ammonium bromide solution, stirring uniformly, adding iced sodium borohydride solution, stirring vigorously for several minutes until the color of the solution is changed from golden yellow to yellow brown, stopping stirring to obtain seed solution, and standing at room temperature for later use;
2) Adding 0.004mol/L silver nitrate solution into hexadecyl ammonium bromide solution for uniform mixing, adding 1mmol/L chloroauric acid solution, uniformly mixing, then adding 0.02758mol/L ascorbic acid solution, uniformly mixing until the solution turns colorless from golden yellow, finally adding the seed solution, fully stirring to obtain a synthesized gold nanorod, and standing overnight for later use;
3) And centrifuging the synthesized gold nanorods to remove supernatant, diluting, centrifuging again to remove supernatant, and diluting with 0.2mol/L hexadecylammonium bromide solution to obtain the gold nanorod solution reagent containing the hexadecylammonium bromide solution, wherein the concentration of the gold nanorod solution reagent is 2.5 times of that of the synthesized gold nanorods which are not centrifuged.
The detection principle diagram of the detection method and the kit provided by the invention is shown in figure 1, and the detection principle is as follows: the detection aim of identifying the freshness of the aquatic products is achieved by using the nucleic acid degradation product hypoxanthine in the aquatic products as a detection marker and decomposing manganese dioxide by using a product after an enzyme reaction to prevent the manganese dioxide from catalyzing an etching reaction of gold nanorods. Specifically, the decayed aquatic product contains a certain amount of hypoxanthine (Hx), the higher the decay degree is, the higher the content of hypoxanthine is, the hypoxanthine can generate Uric Acid (UA) and hydrogen peroxide under the action of Xanthine Oxidase (XOD), and both the uric acid and the hydrogen peroxide can decompose manganese dioxide nanoenzyme. While manganese dioxide has oxidase-like activity, the remaining undecomposed manganese dioxide nanoenzyme can oxidize 3,3', 5' -Tetramethylbenzidine (TMB) to TMB under acidic condition 2+ ，TMB 2+ Etching gold nanorods (AuNRs) with adjustable longitudinal Localized Surface Plasmon Resonance (LSPR) optical characteristics, and changing the length of the gold nanorods by etchingThe length-diameter ratio of the nano-gold rod can be changed (the nano-gold rod is in TMB) 2+ Transmission electron micrographs before and after etching are shown in fig. 2), so that different colors are displayed, the un-etched gold nanorods are green, the gold nanorods are shortened from length to length along with the increase of the etching degree, the colors are green, blue, bluish-purple, purple and pink in sequence, and the colors are obviously different even in the same color system, so that the etching degrees of the gold nanorods are different along with the difference of the corruption degrees of corrupt aquatic products, and the corresponding rainbow-like colors are displayed to realize the identification of the freshness of the aquatic products.
Compared with the related technology, the detection method and the kit provided by the invention have the following beneficial technical effects:
most of the existing colorimetric techniques output a single color, e.g., only by resolution of TMB in the related art + Or TMB 2+ The detection purpose is achieved by only depending on the shade of the single color, and the reading results of different observers can be different; and the color developing substance is easy to change under the influence of photooxidation, so that the read color is inaccurate, and the detection result has errors. The invention adopts a multicolor colorimetric analysis method, the color of the detection phenomenon is rainbow-like (green, blue, purple and pink), the color is easy to distinguish, the detection result is easy to distinguish by naked eyes, and an observer can easily and accurately judge the freshness of the aquatic product to be detected; and because the chromogenic substance is not easy to be oxidized by light, the color output is not easy to change, and the detection result is more stable and reliable. Therefore, the detection method and the kit provided by the invention have the advantages that the detection color is distinct and distinguishable, the output is stable, and the freshness of the aquatic product can be easily and accurately identified by naked eyes.
Secondly, for the aquatic products in the early stage of putrefaction, the method provided by the invention can sensitively judge the initial freshness of the aquatic products. The hypoxanthine to be detected prevents the gold nanorods from being etched, so that the aim of detecting the freshness of aquatic products is fulfilled. The content of hypoxanthine in aquatic products in the early stage of decay is low, the content of manganese dioxide nanoenzyme which is not decomposed is high, the etching degree of the gold nanorod is high on the contrary, the color can be in a pink color system, the contrast with the green color of the initial gold nanorod is more obvious, the visual contrast is higher, and the visual contrast is easier to distinguish by naked eyes; along with the gradual increase of the putrefaction degree of aquatic products, the etching degree of the gold nanorods is gradually weakened, and the system color sequentially presents purple, blue and green. In the existing gold nanorod etching technology, the etching of the gold nanorods is promoted by the substances to be detected, so that the etching degree of the gold nanorods is lower when the concentration of the substances to be detected is lower, and therefore when the concentration of the substances to be detected is lower, the color change of a system is not obvious and is not easy to detect, the detection result is insensitive, the detection limit is higher, and the freshness detection of the aquatic product at the early stage of putrefaction cannot be accurately realized. In addition, the detection result of the detection method provided by the invention is not easily interfered by other interferents in marine products, and the detection specificity is strong. Finally, the manganese dioxide nanoenzyme in the detection system can be stably placed at room temperature and is not easy to decompose, and light shielding is not needed, so that inaccurate detection results caused by wrong output colors due to decomposition of the oxidant are avoided. In conclusion, the detection method and the kit provided by the invention utilize the nucleic acid degradation product hypoxanthine in the aquatic products as a detection marker, and prevent the manganese dioxide from catalyzing the etching reaction of the gold nanorods, thereby achieving the detection purpose of distinguishing the freshness of the aquatic products. The detection limit is lower, the detection range is wider, the detection result is more accurate, the detection specificity is stronger, particularly, for aquatic products in the early decay stage, the freshness of the aquatic products can be more accurately distinguished by naked eyes, and the detection sensitivity is higher.
Drawings
FIG. 1 is a schematic diagram of the detection provided by the present invention;
FIG. 2 shows the gold nanorods provided by the present invention in TMB 2+ Transmission electron micrographs before (left) and after (low concentration in the middle and high concentration in the right) etching;
FIG. 3 is a diagram of a standard color chart provided by the present invention;
FIG. 4 is a graph of the ultraviolet-visible absorption spectra of etched gold nanorods after decomposing manganese dioxide with hypoxanthine metabolites of different concentrations according to the present invention;
FIG. 5 is a flow chart of the present invention for detecting the freshness of an aquatic product;
FIG. 6 is a histogram of the specific identification color of the present invention (A) and a histogram of the UV absorption peak of the present invention (B).
Detailed Description
The invention discloses a method and a kit for detecting freshness of an aquatic product, and a person skilled in the art can use the contents for reference and appropriately improve process parameters for realization. It is specifically noted that all such substitutions and modifications will be apparent to those skilled in the art and are intended to be included within the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The present invention will be described in further detail below with reference to specific embodiments in order to enable those skilled in the art to better understand the present invention.
The preparation method of the solution used in the examples of the present invention is as follows:
preparation of xanthine oxidase solution: adding xanthine oxidase XOD into potassium phosphate buffer solution to dissolve the xanthine oxidase XOD into mother solution with enzyme activity of 500U/mL, and then diluting with water to obtain the xanthine oxidase XOD;
preparing a manganese dioxide nano enzyme solution: dissolving 10mg of manganese dioxide powder in 10mL of water, and performing ultrasonic treatment for three minutes to uniformly disperse the manganese dioxide powder;
preparation of 3,3', 5' -Tetramethylbenzidine (TMB) solution: dissolving TMB powder in dimethyl sulfoxide solution to obtain the solution;
preparation of sulfuric acid solution: diluted with 98.3% of analytically pure sulfuric acid;
it should be noted that the acidic solution described in the present invention can also be selected from other acidic solutions commonly used in the art, and the sulfuric acid solution used in the present invention should not limit the present invention.
Example 1 preparation of gold nanorods containing hexadecylammonium bromide solution
1) Synthesizing seeds, namely adding 0.5mL of chloroauric acid (0.5 mmol/L) solution into a three-necked bottle containing 0.5mL of cetylammonium bromide (CTAB) solution, uniformly stirring, adding 0.06mL of iced sodium borohydride solution, violently stirring for several minutes until the color of the solution is changed from golden yellow to yellowish brown, stopping stirring, standing at room temperature of 25 ℃, and waiting for subsequent use;
2) Growing, adding 0.5mL of silver nitrate (0.004 mol/L) solution into 25mL of hexadecylammonium bromide (CTAB) solution, uniformly mixing, adding 25mL of chloroauric acid (1 mmol/L) solution, uniformly mixing, then adding 1mL of ascorbic acid (0.02758 mol/L) solution, uniformly mixing until the solution is colorless from golden yellow, finally adding 60 mu L of the seed solution synthesized in the step 1), fully stirring to obtain a synthesized gold nanorod, and standing overnight for use;
3) And (3) centrifuging 25mL of the synthesized gold nanorod at 7500 r/min for 15min, removing supernatant, diluting to 25mL, centrifuging at 7500 r/min for 15min again, removing supernatant, and diluting to 10mL by using 0.2mol/L cetyl ammonium bromide (CTAB) solution to obtain the gold nanorod containing the cetyl ammonium bromide solution. The gold nanorods containing the hexadecyl ammonium bromide solution are prepared by adopting the method in the embodiment unless specially stated.
Example 2 visual inspection of aquatic product freshness
Cleaning the surface of a fish sample, removing scales, fish skin, fish bones and viscera, keeping the fish part, mashing the fish sample into paste by using a mortar, weighing 1g of the sample, adding 10mL of deionized water, shaking and mixing uniformly, carrying out ultrasonic treatment for 15min, carrying out post-centrifugation, filtering by using a 0.45 mu m filter membrane, and collecting filtrate to prepare a sample to be detected for later use.
Adding 50 mu L of a sample to be detected into a 50 mu L centrifuge tube containing a xanthine oxidase solution with 2U/mL enzyme activity and a 10 mu L manganese dioxide solution, uniformly mixing, incubating for 10min at 37 ℃, then adding 2 mu L of a sulfuric acid solution with the concentration of 5mol/L into the centrifuge tube, uniformly mixing, adding 5 mu L of a TMB solution with the concentration of 10mmol/L, uniformly mixing, adding, fully and uniformly mixing, adding 80 mu L of the gold nanorod containing CTAB prepared in the example 1, uniformly mixing, incubating for 5min again at room temperature and 25 ℃, after the etching of the gold nanorod is finished, the color is stable and unchanged, an observer can output the color and shade, and the freshness of the fish sample can be easily observed by naked eyes.
The putrefaction degree of the fish to be detected is increased from small to large, the final solution displays pink, purple, blue and green colors in sequence, and each color system is different in color depth, if the freshness of the fish to be detected is higher, the etching degree of the gold nanorods is higher, the etched solution becomes pure pink, and the color difference with the green color of the original gold nanorods is larger, so that the method has more advantages for detecting marine products at the early stage of putrefaction. With the reduction of the freshness of the fish to be detected, more and more purple systems are mixed in the pink system of the final solution until the pink system is completely purple, and other color systems are analogized in turn, if the putrefaction degree of the fish to be detected is large, the color of the final solution may be blue-green or even green. In the embodiment, the content of the hypoxanthine in the sample to be detected can also be accurately and quantitatively analyzed by adopting an ultraviolet-visible spectrophotometer.
EXAMPLE 3 preparation of the kit
A Xanthine Oxidase (XOD) solution reagent bottle A with the enzyme activity of 2U/mL and manganese dioxide (MnO) of 1mg/mL are arranged in the kit body 2 ) A solution reagent bottle B, a 5mol/L sulfuric acid solution reagent bottle C, a 10 mmol/L3, 3', 5' -Tetramethylbenzidine (TMB) solution reagent bottle D, a gold nanorod solution reagent bottle E containing hexadecylammonium bromide (CTAB) solution, a standard colorimetric card F and a 0.6mL centrifuge tube G. The preparation of the solutions in the respective reagent bottles was carried out according to the method of the present invention.
The preparation method of the standard colorimetric card described in this embodiment is as follows:
50 mu L of xanthine oxidase solution with the enzyme activity of 2U/mL and 10 mu L of manganese dioxide solution are put into a 0.6mL centrifuge tube, 50 mu L of hypoxanthine sample with different contents are respectively added, after uniform mixing, incubation is carried out for 10min under the condition of 37 ℃, then 2 mu L of sulfuric acid solution with the concentration of 5mol/L is added into the centrifuge tube, after uniform mixing, 5 mu L of TMB solution with the concentration of 10mmol/L is added, after full uniform mixing, 80 mu L of prepared gold nanorod with CTAB is added, uniform mixing is carried out, incubation is carried out for 10min under the environment of room temperature of 25 ℃, the hypoxanthine sample with different contents enables the final solution to generate corresponding color output, a camera is used for shooting the color change of the centrifuge tube after reaction, then the corresponding hypoxanthine concentration is marked, finally color printing is carried out, the standard colorimetric card for detecting the freshness of aquatic products is prepared, and the color printing picture of the standard colorimetric card is shown in figure 3. The standard colorimetric card of fig. 3 shows rainbow-like color distribution, and because a color picture cannot be displayed, the colors in fig. 3 are displayed in a text description mode, and the colors which can be achieved by a final system by the hypoxanthine with different concentrations are shown in the text description of fig. 3, so that the color depth change can be seen by naked eyes even in the same color system. It should be noted that the color presented in the system shown in fig. 3 is limited after all by using the text description, and is not as accurate and intuitive as a color chart, and an observer can easily and accurately distinguish the freshness of the seafood to be tested from a colored standard color chart.
The gold nanorods are etched by hypoxanthine samples with different contents in different degrees, and the ultraviolet absorption spectrum of the gold nanorods also changes correspondingly, as shown in fig. 4. Therefore, the method can accurately and quantitatively analyze the content of the hypoxanthine in the sample to be detected by adopting an ultraviolet-visible spectrophotometer.
Example 4 identification of Fish freshness Using kit
1) Sample pretreatment: cleaning the surface of a fish sample, removing scales, fish skin, fish bones and viscera, keeping the fish part, mashing into paste by using a mortar, weighing 1g of sample, adding 10mL of deionized water, shaking and uniformly mixing, carrying out ultrasonic treatment for 15min, centrifuging, filtering by using a 0.45 mu m filter membrane, and collecting filtrate for later use;
2) Taking 50 mu L of the filtrate sample, adding the filtrate sample into a 0.6mL centrifuge tube, sequentially taking 50 mu L of xanthine oxidase in a reagent bottle A in the kit prepared in the example 3 and 10 mu L of manganese dioxide solution in a reagent bottle B, adding the xanthine oxidase and the manganese dioxide solution into the centrifuge tube, shaking, uniformly mixing, and incubating at 37 ℃ for 10min;
3) Adding 2 mu L of sulfuric acid solution in the reagent bottle C into a centrifuge tube, and uniformly mixing by shaking;
4) Adding 5 mu L of TMB solution in the reagent bottle D into a centrifuge tube, shaking and uniformly mixing, and changing the color of the solution in the centrifuge tube from light brown to yellow;
5) Adding 80 mu L of gold nanorod solution in a reagent bottle E into a centrifuge tube, fully shaking and uniformly mixing, and etching the gold nanorods after 5min, wherein the color is stable and unchanged;
6) The color of the solution in the centrifuge tube is compared with the standard colorimetric card F (figure 3) prepared in the embodiment 3 by naked eyes, the freshness of the fish can be accurately determined through color comparison, the concentration range of the hypoxanthine in the fish can be judged, the content of the hypoxanthine in the fish to be detected can be accurately and quantitatively analyzed by adopting an ultraviolet visible spectrophotometer, and the detection limit of the hypoxanthine can be found to be as low as 0.378 mu mol/L through multiple tests.
Example 5 identification of freshness of shrimp meat Using the kit
1) Sample pretreatment: cleaning the surface of a taken shrimp sample, removing shrimp shells, shrimp heads, shrimp feet and shrimp threads, keeping the shrimp part, mashing into a paste by using a mortar, weighing 1g of sample, adding 10mL of deionized water, shaking and uniformly mixing, carrying out ultrasonic treatment for 15min, centrifuging, filtering by using a 0.45-micrometer filter membrane, and collecting filtrate for later use;
2) Taking 50 mu L of the filtrate sample, adding the filtrate sample into a 0.6mL centrifuge tube, sequentially taking 50 mu L of xanthine oxidase in a reagent bottle A in the kit prepared in the example 3 and 10 mu L of manganese dioxide solution in a reagent bottle B, adding the xanthine oxidase and the manganese dioxide solution into the centrifuge tube, shaking, uniformly mixing, and incubating at 37 ℃ for 10min;
3) Adding 2 mu L of sulfuric acid solution in the reagent bottle C into a centrifuge tube, and shaking and uniformly mixing;
4) Adding 5 mu L of TMB solution in the reagent bottle D into a centrifuge tube, shaking and uniformly mixing, and changing the color of the solution in the centrifuge tube from light brown to yellow;
5) Adding 80 mu L of gold nanorod solution in a reagent bottle E into a centrifuge tube, fully shaking and uniformly mixing, and etching the gold nanorods after 5min, wherein the color is stable and unchanged;
6) The color of the solution in the centrifuge tube is compared with the standard colorimetric card F (shown in figure 3) prepared in example 3 by naked eyes, the concentration range of the hypoxanthine in the shrimp meat is determined, the freshness of the shrimp meat is determined, and the content of the hypoxanthine in the shrimp meat to be detected can be accurately and quantitatively analyzed by adopting an ultraviolet visible spectrophotometer.
The invention uses the nucleic acid degradation product hypoxanthine in the aquatic products as a detection marker, and decomposes manganese dioxide by using the product after the enzyme reaction, so as to prevent the manganese dioxide from catalyzing the etching reaction of the gold nanorods, thereby achieving the detection purpose of identifying the freshness of the aquatic products. The method has the advantages of simple and quick operation, short reaction time, high detection speed, high sensitivity, low detection limit (the detection limit of hypoxanthine is as low as 0.378 mu mol/L), wider detection range, bright color output and visual naked eyes, and is suitable for field real-time detection. Particularly for aquatic products at the early stage of putrefaction, the detection method or the kit provided by the invention can sensitively and accurately judge the initial freshness.
Effect example 1 discrimination of specificity Using kit
1) Preparation of interferent solution reagents: the interference product comprises Histamine (Histamine), glucose (Glucose), sucrose (Sucrose), KCl, caCl 2 、NaCl、MgCl 2 、ZnCl 2 The above solids were weighed and dissolved separately to a final concentration of 0.5mmol/L.
2) Taking 50 mu L of each solution reagent and 50 mu L of hypoxanthine solution with the concentration of 0.5mmol/L, respectively adding into a 0.6mL centrifuge tube, sequentially adding 50 mu L of xanthine oxidase in a reagent bottle A and 10 mu L of manganese dioxide solution in a reagent bottle B in the kit prepared in the example 3 into each centrifuge tube, shaking, uniformly mixing, and incubating at 37 ℃ for 10min;
3) Adding 2 mu L of sulfuric acid solution in the reagent bottle C into a centrifuge tube, and uniformly mixing by shaking;
4) Adding 5 mu L of TMB solution in the reagent bottle D into a centrifuge tube, shaking and uniformly mixing, and changing the color of the solution in the centrifuge tube from light brown to yellow;
5) Adding 80 mu L of gold nanorod solution in a reagent bottle E into a centrifuge tube, fully shaking and uniformly mixing, and etching the gold nanorods after 5min, wherein the color is stable and unchanged;
6) The color of the solution in each centrifuge tube was compared with the standard color chart F (fig. 3) prepared in example 3 by naked eyes to obtain the etching degree and the color of the gold nanorods, and the ultraviolet absorption of the gold nanorods was accurately quantified by using an ultraviolet-visible spectrophotometer.
Specific effect identification result: FIG. 6A shows histamine, glucose, sucrose, KCl, caCl 2 、NaCl、MgCl 2 、ZnCl 2 The color of the system is etched to be stable in light pink (the color is as shown in the left eight centrifuge tubes in FIG. 6A), the color of the hypoxanthine group system is green (the color is as shown in the rightmost centrifuge tube in FIG. 6A), and the XOD enzyme pairs in the kit are shownThe hypoxanthine is reacted, the decomposition product can reduce the manganese dioxide nano-sheets into manganese ions, the manganese dioxide nano-enzyme is prevented from further catalyzing and etching the gold nano-rods, and the color of the system is green. Histamine, glucose, sucrose, KCl, caCl 2 、NaCl、MgCl 2 、ZnCl 2 Products for decomposing manganese dioxide nano-sheets are not generated, manganese dioxide is hardly consumed, the gold nano-rods are catalyzed and etched by the manganese dioxide, and the system is pink. Therefore, the detection method provided by the invention only has good specificity to the biomarker hypoxanthine, and simultaneously means that even if the interferent appears in the marker to be detected, the detection result is not interfered.
Secondly, ultraviolet spectrum measurement is carried out on the solution after etching is finished by using an ultraviolet spectrophotometer, and after the hypoxanthine reaction is found, a graph 6B shows that the ultraviolet absorption of the gold nanorods is about 2 (such as the rightmost column in the graph 6B), the gold nanorods are close to the extranatal absorption peak value when the gold nanorods are not etched initially, and histamine, glucose, sucrose, KCl, caCl 2 、NaCl、MgCl 2 、ZnCl 2 After the reaction, each system measures the ultraviolet absorption spectrum, the ultraviolet absorption peak value of the ultraviolet absorption spectrum is about 0.25 (as shown in the left eight columns of fig. 6B), the ultraviolet absorption peak values are compared with a histogram (as shown in fig. 6B), and the difference between the interference object and the ultraviolet absorption peak value of the hypoxanthine is obvious, so that the detection method provided by the invention has good specificity on the detection of the hypoxanthine, and the interference object does not interfere the detection result.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and amendments can be made without departing from the principle of the present invention, and these modifications and amendments should also be considered as the protection scope of the present invention.
Claims (10)
1. A method for detecting the freshness of aquatic products is characterized by comprising the following steps: the xanthine oxidase decomposes hypoxanthine in an aquatic product to be detected, the decomposition product reduces manganese dioxide nanoenzyme into manganese ions, the manganese dioxide nanoenzyme which is not reduced in an acid solution oxidizes 3,3', 5' -tetramethylbenzidine to generate diimine ions, the diimine ions etch the gold nanorods, the aquatic product to be detected has different freshness and hypoxanthine content, the etched solution can show different colors, visual detection of the freshness of the aquatic product to be detected can be realized through the output of bright colors and the shade of the same color system, the ultraviolet absorption spectrum of the etched gold nanorods is changed, and the aquatic product to be detected can be accurately and quantitatively detected through an ultraviolet spectrophotometer.
2. The aquatic product freshness detection method according to claim 1, characterized in that: mixing xanthine oxidase and a manganese dioxide nanoenzyme solution, adding an aquatic product to be detected, incubating for a certain time under a certain temperature condition, adding an acidic solution after mixing uniformly, adding a 3,3', 5' -tetramethylbenzidine solution after mixing uniformly, adding gold nanorods after sufficiently mixing uniformly again, incubating for a certain time again under a certain temperature condition, finishing the stable etching color of the gold nanorods, and detecting the freshness of the aquatic product to be detected by naked eyes or an ultraviolet visible spectrophotometer.
3. The aquatic product freshness detection method according to claim 2, characterized in that: incubating at 37 deg.C for 10min; the cells were incubated at 25 ℃ for another 5min.
4. A method of detecting freshness of an aquatic product according to claim 2, wherein: the acidic solution is a sulfuric acid solution.
5. A method of detecting the freshness of an aquatic product according to claim 1 or 2, characterized in that: the enzyme activity of the xanthine oxidase is 2U/mL; the concentration of the manganese dioxide nano enzyme solution is 1mg/mL; the concentration of the 3,3', 5' -tetramethyl benzidine is 10mmol/L, and the concentration of the acid solution is 5mol/L.
6. A method of detecting the freshness of an aquatic product according to claim 1 or 2, characterized in that: adding chloroauric acid solution into hexadecyl ammonium bromide solution, uniformly stirring, adding sodium borohydride solution, stirring for several minutes until the color of the solution is changed from golden yellow to yellow brown, and stopping stirring to obtain seed solution; adding a silver nitrate solution into a cetyl ammonium bromide solution, uniformly mixing, adding a chloroauric acid solution, uniformly mixing, adding an ascorbic acid solution, uniformly mixing to obtain a solution which is changed from golden yellow to colorless, finally adding the seed solution, stirring to obtain a synthesized gold nanorod, and diluting with the cetyl ammonium bromide solution to obtain the gold nanorod containing the cetyl ammonium bromide solution.
7. A kit for detecting freshness of aquatic products is characterized in that: the reagent comprises a xanthine oxidase solution reagent with the enzyme activity of 2U/mL, a manganese dioxide nano enzyme solution reagent with the enzyme activity of 1mg/mL, a 3,3', 5' -tetramethylbenzidine solution reagent with the enzyme activity of 10mmol/L, an acidic solution reagent with the enzyme activity of 5mol/L, a gold nanorod solution reagent containing a hexadecylammonium bromide solution, and a standard colorimetric card.
8. The kit of claim 7, wherein: the acidic solution reagent is a sulfuric acid solution.
9. The kit of claim 7, wherein: the preparation method of the standard colorimetric card comprises the steps of selecting a series of hypoxanthine solution samples with different concentrations to replace aquatic products to be detected, carrying out etching color development reaction on the gold nanorods by respectively adopting the detection method according to claim 2, shooting corresponding colors by using a camera after the colors of the gold nanorods are stable after etching is completed, enabling the hypoxanthine samples with the different concentrations to generate corresponding rainbow-like color changes, marking the corresponding hypoxanthine concentrations, and preparing the standard colorimetric card for detecting the freshness of the aquatic products after printing.
10. The kit of claim 7, wherein: the preparation method of the gold nanorod solution reagent containing the hexadecyl ammonium bromide solution comprises the following steps,
1) The seed synthesis method comprises the steps of adding 0.5mmol/L chloroauric acid solution into hexadecyl ammonium bromide solution, stirring uniformly, adding iced sodium borohydride solution, stirring vigorously for several minutes until the color of the solution is changed from golden yellow to yellow brown, stopping stirring to obtain seed solution, and standing at room temperature for later use;
2) Adding 0.004mol/L silver nitrate solution into hexadecyl ammonium bromide solution, uniformly mixing, adding 1mmol/L chloroauric acid solution, uniformly mixing, then adding 0.02758mol/L ascorbic acid solution, uniformly mixing until the solution turns colorless from golden yellow, finally adding the seed solution, fully stirring to obtain a synthesized gold nanorod, and standing overnight for later use;
3) And centrifuging the synthesized gold nanorods to remove supernatant, diluting, centrifuging again to remove supernatant, and diluting with 0.2mol/L hexadecyl ammonium bromide solution to obtain the gold nanorod solution reagent containing the hexadecyl ammonium bromide solution.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116769486A (en) * | 2023-06-25 | 2023-09-19 | 中国海洋大学 | Up-conversion fluorescence sensor and preparation method and application thereof |
| CN117417976A (en) * | 2023-09-13 | 2024-01-19 | 中国海洋大学 | Method for rapidly detecting early freshness of aquatic product based on cooperation of UV and nano enzyme |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116769486A (en) * | 2023-06-25 | 2023-09-19 | 中国海洋大学 | Up-conversion fluorescence sensor and preparation method and application thereof |
| CN117417976A (en) * | 2023-09-13 | 2024-01-19 | 中国海洋大学 | Method for rapidly detecting early freshness of aquatic product based on cooperation of UV and nano enzyme |
| CN117417976B (en) * | 2023-09-13 | 2024-05-24 | 中国海洋大学 | Method for rapidly detecting early freshness of aquatic product based on cooperation of UV and nano enzyme |
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