Disclosure of Invention
The invention aims to solve the technical problem of providing a preparation method and application of a ratio type fluorescence response sensing tag for salmon freshness, wherein the sensing tag not only has the function of qualitatively detecting salmon freshness according to different fluorescence color changes, but also can realize quantitative analysis of salmon freshness markers TVB-N in a packaging bag without sample pretreatment, has high sensitivity, is accurate and reliable, can be used for intelligent packaging for salmon freshness detection, and is used for nondestructive real-time detection of salmon freshness.
The technical scheme of the invention is as follows:
a preparation method of a ratio type fluorescence response sensing label for salmon freshness comprises the following specific steps:
(1) Preparation of tetraphenyl ethylene/polymethacrylic acid solution
Weighing 0.2g of polymethacrylic acid, completely dissolving the polymethacrylic acid in 10mL of tetrahydrofuran, adding 2mg of tetraphenyl ethylene, oscillating, standing at room temperature for 12 hours after dissolving, and preparing tetraphenyl ethylene/polymethacrylic acid solution for later use;
(2) Preparation of rhodamine B solution
Weighing 3mg of rhodamine B, adding 10mL of absolute ethyl alcohol, performing ultrasonic auxiliary dissolution, and preparing rhodamine B solution for later use after complete dissolution;
(3) Preparation of freshness sensor tags
Soaking the filter paper strip in rhodamine B solution for 10min, uniformly adsorbing the filter paper, taking out, and placing the filter paper strip in a 37 ℃ oven for drying for 30min; soaking the label coated with rhodamine B in a tetraphenyl ethylene/polymethyl methacrylate solution, immediately taking out the label after uniform dyeing, placing the label in a 37 ℃ oven for drying for 30min, and cutting to obtain the ratio type fluorescence response sensing label for the freshness of salmon.
Further, the cut label size was 2cm×2cm.
An application of a ratio-based fluorescence response sensing tag in detecting salmon freshness comprises the following steps:
(1) Production of standard colorimetric labels for sensor labels
Placing 30g of fresh salmon blocks in a tray, sealing the tray filled with the fish samples by using a preservative film, and sticking a ratio type fluorescence response sensing tag for the freshness of the salmon on a packaging bag positioned on the top of the sample to be tested in the package without directly contacting with the fish; extracting color difference L, a and b values of sensing tag freshness grade critical point response information under 365nm ultraviolet light excitation as characteristic values according to TVB-N values of salmon under different freshness degrees, and manufacturing a standard colorimetric tag of the sensing tag according to Lab values of the freshness grade critical point;
(2) A ratio type fluorescence response sensing label for the freshness of salmon is used as an indication part to be stuck on a packaging bag positioned at the top of the salmon in a package, and is not in direct contact with the salmon; and (3) irradiating the sensing label by 365nm ultraviolet light, comparing the color with a standard colorimetric label, judging the freshness of salmon in the storage stage, and finishing real-time freshness detection of salmon.
Further, according to the TVB-N values of salmon under different freshness degrees, standard colorimetric labels of the sensing labels are manufactured, wherein the TVB-N values are respectively selected from mg/100g, 6mg/100g, 7mg/100g, 9mg/100g, 13mg/100g, 15mg/100g, 17mg/100g, 20mg/100g, 25mg/100g, 31mg/100g and 34mg/100g.
Further, when the freshness of salmon in the storage stage is judged, the TVB-N value is less than 15mg/100g and is fresh, the TVB-N value is more than 25mg/100g and is more than or equal to 15mg/100g, and the TVB-N value is more than or equal to 25mg/100g and is putrefaction.
Further, under 365nm ultraviolet light irradiation, when the color of the ratio type fluorescence response sensing label is a pink fluorescence area in the standard colorimetric label, the salmon is fresh; the ratio-based fluorescence indicates sub-freshness when the color of the ratio-based fluorescence response sensor tag shows a blue-violet fluorescence region in the standard colorimetric tag; the ratio-based fluorescence indicates freshness when the ratio-based fluorescence response sensor tag color reveals a dark blue fluorescence region in the standard colorimetric tag.
Further, Δe is calculated by extracting Lab values and substituting them into a color difference value formula, and Δe values of a certain period of time are substituted into a fitting formula to calculate:
fitting formula is y=1.559x+26.883, linear correlation coefficient R2 =0.993, x=Δe value, and the fitting equation calculates the result as the period TVB-N value.
According to the invention, rhodamine B is used as a reference dye, a ratio type fluorescent probe with sensing responsiveness and aggregation-induced emission characteristic is constructed by using a polymethyl methacrylate and tetraphenyl ethylene composite material, and the ratio type fluorescent probe is coated on filter paper in a coating superposition mode to prepare a freshness sensing label with fluorescence responsiveness, and compared with the traditional fish freshness indicating label which depends on single color fluorescence shade change indication, the freshness sensing label has the beneficial effects that:
when the prepared ratio type fluorescence response sensing label is used for detecting the freshness of salmon, the freshness of the salmon can be qualitatively and quantitatively monitored through the change of fluorescence response information, and the ratio type fluorescence response sensing label has the dual functions of qualitatively and quantitatively detecting the freshness of salmon. In the quantitative analysis, quantitative analysis of the salmon freshness marker TVB-N in the packaging bag can be realized without sample pretreatment; in qualitative analysis, the pink color in the standard colorimetric label represents freshness of salmon, the blue-violet fluorescence represents sub-freshness, the deep blue fluorescence represents non-freshness, the fluorescence color among the freshness grades is clear, and the detection result has high sensitivity, accuracy and reliability.
The ratio type fluorescence response indication label can perform non-contact and nondestructive freshness monitoring on salmon under different storage duration and temperature, and provides accurate and effective freshness information for manufacturers and retailers in time; the method is used for detecting and packaging the freshness of the Sanwen, and can be directly used for nondestructive real-time detection of the freshness of the Sanwen in transportation or sales.
Detailed Description
Example 1
(1) Preparation of tetraphenyl ethylene/polymethacrylic acid solution:
weighing 0.2g of polymethacrylic acid, completely dissolving the polymethacrylic acid in 10mL of tetrahydrofuran, adding 2mg of tetraphenyl ethylene, oscillating, standing at room temperature for 12 hours after dissolving, and preparing tetraphenyl ethylene/polymethacrylic acid solution for later use;
(2) Preparation of rhodamine B solution:
weighing 3mg of rhodamine B, adding 10mL of absolute ethyl alcohol, performing ultrasonic auxiliary dissolution, and preparing rhodamine B solution for later use after complete dissolution;
(3) Preparation of freshness sensor tag:
soaking the filter paper strip in rhodamine B solution for 10min, uniformly adsorbing the filter paper, taking out, and placing the filter paper strip in a 37 ℃ oven for drying for 30min; soaking the label coated with rhodamine B in a tetraphenyl ethylene/polymethyl methacrylate solution, immediately taking out the label after uniform dyeing, placing the label in a 37 ℃ oven for drying for 30min, cutting the label into square blocks with the length multiplied by the width of 2cm multiplied by 2cm, obtaining the ratio type fluorescence response sensing label for the freshness of salmon, and collecting the label for later use.
(4) Production of standard colorimetric labels for producing sensor labels
Detecting the TVB-N content at intervals at the constant temperature of 4 ℃, photographing the phenomenon under 365nm ultraviolet lamp irradiation by using a photographing device when the TVB-N content is 5mg/100g, 6mg/100g, 7mg/100g, 9mg/100g, 13mg/100g, 15mg/100g, 17mg/100g, 20mg/100g, 25mg/100g, 31mg/100g and 34mg/100g, extracting L, a and b values of sensing label freshness grade critical point response information under 365nm ultraviolet excitation by using Photoshop software as characteristic values, and manufacturing a standard colorimetric label of the sensing label according to Lab values of freshness grade critical points, as shown in figure 1;
(5) Production of standard colorimetric labels for sensor labels
Placing 30g of fresh salmon blocks in a tray, sealing the tray filled with the fish samples by using a preservative film, and sticking a ratio type fluorescence response sensing label for the freshness of the salmon and a standard colorimetric label of the sensing label on a packaging bag positioned at the top of the sample to be tested in the package without directly contacting with the fish;
and determining a colorimetric region corresponding to the freshness of salmon by adopting TVB-N, wherein the TVB-N content gradually rises along with the extension of the storage time of the fish sample. The selection criteria of the critical point of the invention are: the freshness grade of the sensing label is selected to be fresh with TVB-N value smaller than 15mg/100g, qualified with TVB-N value larger than or equal to 15mg/100g and spoilage with TVB-N value larger than or equal to 25mg/100g respectively. ( The national standard prescribes that when the content of TVB-N is lower than 15mg/100g, the freshness grade of the fish is a good grade. When the content of TVB-N is 15mg/100g-30mg/100g, the fresh grade of the fish is qualified. When the TVB-N content is higher than 30mg/100g, the fish sample becomes putrefactive. Whereas internationally a TVB-N content of more than 25mg/100g is generally considered to be a food product entering the spoilage stage. )
(6) Determination of salmon freshness
a. The sensitivity of salmon in the storage stage can be primarily judged by irradiating the sensing label with 365nm ultraviolet light and comparing the color with the standard colorimetric label;
under 365nm ultraviolet light irradiation, when the color of the ratio type fluorescence response sensing label is a pink fluorescence area in the standard colorimetric label, the salmon is fresh; the ratio-based fluorescence indicates sub-freshness when the color of the ratio-based fluorescence response sensor tag shows a blue-violet fluorescence region in the standard colorimetric tag; the ratio-based fluorescence indicates freshness when the ratio-based fluorescence response sensor tag color reveals a dark blue fluorescence region in the standard colorimetric tag.
b. Delta E is calculated by extracting Lab values of each time period and substituting the Lab values into a color difference value formula, and an x-value fitting formula for substituting the delta E values of a certain time period is calculated, wherein the fitting formula is y=1.559x+26.883, and the linear correlation coefficient R2 =0.993. The y value is the TVB-N value of the obtained time, and the accurate detection of the salmon freshness can be completed by comparing the y value with the TVB-N value judgment level.
The color difference value is calculated as
Wherein Δl=l-L0 *；Δa*＝a*-a0 *；Δb*＝b*-b0 *
Wherein: l is the sample brightness value; l (L)0 * Is a control brightness value; b is the sample yellowness/blueness value; b0 * Is a control yellow/blue value; a is the sampleRed/green value; a, a0 * Is a control red/green value.
1. Salmon freshness correlation detection
Placing fresh salmon meat serving as a sample to be tested in a tray, sealing the tray filled with the fish meat sample by using a preservative film, and sticking the ratio type fluorescence response sensing label manufactured in the embodiment 1 of the invention at a sample headspace in a package without directly contacting with the fish meat; the standard colorimetric label manufactured in example 1 of the present invention was used when determining the color change of salmon.
(1) Salmon freshness determination for different times
Determination of volatile basic Nitrogen (TVB-N):
the detection was carried out by the micro-diffusion method prescribed in GB 5009.228-2016 "determination of volatile basic Nitrogen in food safety national Standard food". Weighing 5g of fish meat, adding 50mL of deionized water, mincing with a meat mincer, standing, filtering, and standing filtrate for later use. Uniformly coating the edge of a diffusion dish by using prepared water-soluble glue, adding 1mL of 20g/L boric acid solution and 1 drop of bromocresol green-methyl red mixed indicator into the inner chamber of the dish, adding 1mL of filtrate into the outer chamber of the dish, rapidly covering a frosted glass cover, only leaving a gap capable of being inserted into a gun head, rapidly adding 1mL of saturated potassium carbonate solution from the gap, immediately pushing the glass cover horizontally, regulating to be sealed, rotating on a table until the mixture is fully and uniformly mixed, placing the mixture in a 37 ℃ incubator for culturing for 2 hours, taking out the mixture, airing the mixture until the mixture is at room temperature, uncovering the mixture, titrating the mixture to mauve by using 0.01 mol/L hydrochloric acid standard solution, and recording the volume of consumed hydrochloric acid. Samples for each time period were repeatedly assayed 3 times and averaged for the recording analysis.
The tray was placed at a constant temperature of 4℃and the TVB-N content was measured at intervals of 5mg/100g, 6mg/100g, 7mg/100g, 9mg/100g, 13mg/100g, 15mg/100g, 17mg/100g, 20mg/100g, 25mg/100g, 31mg/100g and 34mg/100g, and when the TVB-N content was measured, the image was taken by using an image-taking device to take an image of the phenomenon irradiated with 365nm ultraviolet lamp, the law between the change in fluorescence color and the spoilage of fish meat was observed, and a change chart of the fluorescence color response of the sensor tag was obtained as shown in FIG. 1.
FIG. 1 is a graph showing the response of salmon stored under refrigeration to changes in fluorescent color of a sensor tag. As can be seen from the graph, when the TVB-N content of salmon is 5mg/100g, 6mg/100g, 7mg/100g, 9mg/100g and 13mg/100g, the fluorescence color of the sensing tag sequentially transits in pink, pink and blue powder color intervals, and the fluorescence color of the sensing tag is close to pink in a standard colorimetric tag, so that the salmon is fresh, and the salmon meat is a top grade. When the TVB-N content is 15mg/100g, 17mg/100g and 20mg/100g, the fluorescence color of the sensing tag sequentially transits between a blue purple color interval and a blue color interval, and at the moment, the fluorescence color of the sensing tag is close to the blue purple color in the standard colorimetric tag, which indicates that salmon is fresher. When the TVB-N content of salmon is 25mg/100g, 31mg/100g and 34mg/100g, the fluorescence color of the sensing tag is dark blue, and the fluorescence color of the sensing tag is close to the dark blue in the standard colorimetric tag, so that the salmon is putrefactive. Therefore, whether salmon is fresh or not can be judged primarily and qualitatively through the change of the fluorescent color of the ratio-type fluorescent response sensing tag.
Fig. 2 is a conceptual diagram of a freshness sensor tag design, the sensor tag comprising a sensor portion and a color card. The sensing part is used for indicating the freshness change of salmon, and the salmon is stuck to the headspace of a sample in the package and is not contacted with the sample when in use; the color chart served as a reference control. Under the ultraviolet mode, comparing with the color part of the colorimetric card, and when the fluorescent color of the sensing part is pink, the pink is fresh, which indicates that the fish meat is fresh; when the fluorescence color of the indication label sensing part changes to blue-violet, the fish meat is sub-fresh; with increasing storage time, the sensor tag appeared dark blue, which is stale, indicating spoilage of fish. When the sensing label is used for detecting the freshness of salmon, the fluorescence response information of the sensing part is compared with the color of the colorimetric card in an ultraviolet mode, so that the freshness of the salmon can be rapidly and accurately analyzed, and meanwhile, an ultraviolet light source can be realized by means of a simple ultraviolet flashlight and an ultraviolet lamp.
(2) Fitting curve production
In the detection process, in order to further accurately analyze the freshness of salmon, quantitative analysis is performed on a salmon freshness marker TVB-N, and further linear fitting is performed by taking the TVB-N value as an abscissa and taking a chromatic aberration value (delta E) of a Photoshop software extraction tag as an ordinate, so that a linear fitting relation diagram of the delta E value of the TVB-N content within the range of 15.45-25.02mg/100g to the TVB-N content can be obtained, as shown in figure 3.
FIG. 3 is a graph of a linear fit of ΔE values to TVB-N content over a range of 15.45-25.02mg/100g TVB-N content. It can be seen from the graph that the ΔE value and the TVB-N content have a good linear correlation. The resulting linear fit equation is y=1.559x+26.883, the linear correlation coefficient R2 =0.993. Through the equation, consumers can judge the freshness of salmon through the fluorescence color change of the sensing tag, and can calculate the TVB-N content by acquiring the delta E value through collecting the fluorescence color change information of the freshness sensing tag, so that the freshness grade of salmon is accurately judged.
FIG. 4 is a scanning electron microscope image at 50,000 magnification, where (a) is a blank filter, (B) is a blank rhodamine B stained filter, and (c) is a sensor label. It can be seen from the figures that fig. 4 (a), (b) and (c) each show a different microstructure. Fig. 4 (a) shows a fiber-like structure aligned. In comparison with fig. 4 (a), when the surface of the filter paper is coated with rhodamine B, fig. 4 (B) shows a staggered fibrous structure, and oval particles can be seen between the pores of the fibrous structure, probably because rhodamine B is immobilized on the blank filter paper. The surface structure of fig. 4 (c) changed greatly, and the fibrous structure disappeared after the filter paper substrate was coated with tetraphenyl ethylene/polymethyl methacrylate/rhodamine B, and the surface became smoother and smoother, which also demonstrated that the tetraphenyl ethylene/polymethyl methacrylate/rhodamine B was successfully coated on the blank filter paper substrate.
Fig. 5 shows a practical application of the sensor tag to salmon monitoring. Fresh salmon is placed in a PET box with a cover, a sensing tag is stuck to the top space in the box, and the cover is fastened. During the storage of salmon, fluorescent signals of the indicator tag were collected to obtain fig. 5 (a), (b) and (c), respectively. And judging the freshness grade of salmon according to the fluorescence color of the indication label. Fig. 5 (a) the sensor tag is pink fluorescent, indicating that salmon is fresh. Fig. 5 (b) the sensor tag was blue-violet fluorescent, indicating that salmon was secondary fresh. FIG. 5 (c) shows the sensor tag as dark blue fluorescent, indicating salmon spoilage. To further verify the accuracy of the indication results, the actual contents of TVB-N of salmon in FIGS. 5 (a), (b) and (c) were measured according to the national standard method, and the measured actual values were 6.72.+ -. 0.54, 15.31.+ -. 1.59 and 26.67.+ -. 1.44mg/100g, respectively. The proving sensing label can indicate the freshness of salmon. When the TVB-N content is 15-25mg/100g, the TVB-N content can be further calculated by extracting the delta E value of the sensing tag and combining a linear fitting equation. Calculated TVB-N values of FIGS. 5 (b) and (c) were 17.26mg/100g and 25.27mg/100g, respectively. The calculated value of the TVB-N is subjected to difference significance analysis with the actual value measured according to the national standard method, and the result shows that the difference is not significant, so that the sensing tag can quantitatively analyze the TVB-N content.
2. Influence of freshness indicating tags on sensitivity under different conditions
Comparative examples 1-8 rhodamine B solution was formulated: weighing rhodamine B, adding absolute ethyl alcohol, performing ultrasonic auxiliary dissolution, and preparing rhodamine B solution for later use after complete dissolution.
(1) Sensitivity parallel test of freshness indicating labels prepared from different substrates
Comparative example 1
Example 1 was used as comparative example 1. Namely, filter paper is selected as a substrate, the concentration of rhodamine B serving as a reference dye is 0.3g/L, and the mass ratio of tetraphenyl ethylene to polymethacrylic acid is 1:100, so that the freshness indicating label is prepared
Comparative example 2
The polyamide film is selected as a base material, the concentration of rhodamine B serving as a reference dye is 0.3g/L, and the mass ratio of tetraphenyl ethylene to polymethacrylic acid is 1:100 to prepare the freshness indicating label
(2) Sensitivity parallel test of sensing labels prepared by reference dye rhodamine B solutions with different concentrations
Comparative example 3
Filter paper is selected as a substrate, the concentration of rhodamine B serving as a reference dye is 0.1g/L, and the mass ratio of tetraphenyl ethylene to polymethacrylic acid is 1:100 to prepare the freshness indicating label
Comparative example 4
Example 1 was used as comparative example 4. Filter paper is selected as a substrate, the concentration of rhodamine B serving as a reference dye is 0.3g/L, and the mass ratio of tetraphenyl ethylene to polymethacrylic acid is 1:100 to prepare the freshness indicating label
Comparative example 5
Filter paper is selected as a substrate, the concentration of a reference dye rhodamine B is 0.6g/L, and the mass ratio of tetraphenyl ethylene to polymethacrylic acid is 1:100 made freshness indicating label
(3) Sensitivity parallel test of freshness sensing labels prepared by tetraphenyl ethylene/polymethacrylic acid coating solutions with different proportions
Comparative example 6
Example 1 was used as comparative example 6. The filter paper is selected as a substrate, the concentration of the reference dye rhodamine B is 0.3g/L, and the mass ratio of the tetraphenyl ethylene to the polymethacrylic acid is 1:100, so that the freshness indicating label is prepared.
Comparative example 7
The filter paper is selected as a substrate, the concentration of the reference dye rhodamine B is 0.3g/L, and the mass ratio of the tetraphenyl ethylene to the polymethacrylic acid is 1:40, so that the freshness indicating label is prepared.
Comparative example 8
The filter paper is selected as a substrate, the concentration of the reference dye rhodamine B is 0.3g/L, and the mass ratio of the tetraphenyl ethylene to the polymethacrylic acid is 1:20, so that the freshness indicating label is prepared.
Examples and comparative examples 1-comparative example 8 determination of sensitivity
Placing fresh salmon in a PET box with a cover, sticking a sensing label at the top space in the box, fastening the cover, and collecting an initial image of the sensing label under ultraviolet irradiation. Along with the extension of the salmon storage time, the change of the fluorescence color of the sensing label is observed until the color is changed to dark blue and no change is generated, namely, the actual TVB-N content is higher than 31mg/100g, and an image is acquired. RGB values of the image and the initial image are extracted, and the sensitivity S is calculated with reference to the following formula.
Sensitivity S calculation formula:
wherein R is0 、G0 、B0 R is the characteristic value of the initial sensing labelX 、GX 、BX Is a characteristic value when the fluorescence color of the sensing label is kept unchanged.
The sensitivity of freshness indicating labels prepared from different substrates was obtained from comparative example 1 (example 1) and comparative example 2. As can be seen from fig. 6, the sensitivity of the sensor tag prepared from the filter paper substrate was high, up to 56%. The indicator labels based on polyamide films were relatively low at 39%.
FIG. 7 is the sensitivity of sensor labels prepared with reference dye rhodamine B solutions at different concentrations for comparative example 3, comparative example 4 (example 1) and comparative example 5. The sensitivity of the sensor tag prepared at a rhodamine B concentration of 0.1g/L was 15%. The sensitivity of the sensor label prepared at a concentration of 0.3g/L was 56%. The sensitivity of the sensor tag prepared at a concentration of 0.6g/L was 40%.
FIG. 8 is the sensitivity of freshness sensor labels prepared from the tetraphenyl ethylene/polymethacrylic acid coating solutions of comparative example 6 (example 1), comparative example 7 and comparative example 8 in different ratios. When the mass ratio of the tetraphenyl ethylene to the polymethacrylic acid is 1:100, the sensitivity of the sensing tag is 56%. When the mass ratio of the coating liquid is 1:40, the sensitivity of the sensing label is 30%. When the mass ratio of the coating liquid is 1:10, the sensitivity of the sensing label is 25.68%.
The above is only a specific embodiment of the present invention, and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.