Bioamine-responsive fluorescent color-changing hydrogel microneedle patch, and preparation method and application thereof

Abstract

The application discloses a kind of biogenic amine response fluorescent color-changing hydrogel microneedle patch, and preparation method and application thereof, belong to food detection technical field.The method for preparing biogenic amine response fluorescent color-changing microneedle patch of the application includes the following steps: (1) two fluorescent dyes are dissolved in dimethyl sulfoxide and ultrapure water respectively, after mixing according to proportion, obtain solution A that responds to biogenic amine;(2) solution A is added to hydrogel material, horseradish peroxidase (HRP), hydrogen peroxide (H2O2) to prepare mixed fluorescent hydrogel solution, which is dropped on the mold containing patch needle tip prepared;By drying, fluorescent color-changing hydrogel microneedle patch is prepared.The microneedle patch prepared by the method of the application is applied to rapid biogenic amine response, can respond to color in the storage process of fresh meat produced by biogenic amine, suitable for consumer on-site detection of fresh meat, so as to purchase healthy, high-quality food, with wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of food detection, in particular to a fluorescent color-changing hydrogel microneedle patch responsive to biogenic amines, and a preparation method and application thereof BACKGROUND

[0002] Fresh meat is one of the indispensable foods in human daily diet. Because fresh meat contains rich nutrients, it is easily contaminated by microorganisms during processing, storage, transportation and sales, leading to product spoilage, which not only reduces the nutritional value and health quality of meat, but also may endanger the health of consumers. The spoilage of fresh meat is mainly caused by the decomposition of nutrients due to enzymes, microorganisms and other factors in the meat, among which proteins are decomposed by tissue enzymes or microorganisms to produce biogenic amines. If the level of biogenic amines exceeds the allowable limit, it may cause serious health problems. Aromatic biogenic amines such as histamine, tyramine and phenylethylamine can cause abdominal cramps, low blood pressure and allergic symptoms. Putrescine, cadaverine and histamine have a synergistic effect, which can enhance the toxicity of histamine, leading to food poisoning and other health hazards. Since biogenic amine toxicity can cause health problems, it is of great significance to closely monitor the concentration of biogenic amines in the food industry to evaluate food quality.

[0003] Currently, methods for detecting biogenic amines in fresh meat food include high-performance liquid chromatography, spectroscopy, mass spectrometry, electrochemistry and electronic nose technology, however, these traditional methods require expensive analytical instruments and complex sample pretreatment processes, and are invasive and destructive methods, which are inconvenient to carry and cannot be detected on site, making them unable to perform in-situ rapid visual determination of the freshness of fresh meat. In order to ensure food quality and standards, it is very important to use portable analysis technology to determine the level of biogenic amines in fresh meat to detect its freshness, therefore, there is an urgent need to develop a method for determining biogenic amines in fresh meat and detecting the freshness of fresh meat. SUMMARY

[0004] In view of the problems and deficiencies of the prior art, the present application provides a preparation of a microneedle patch responsive to biogenic amines and its application in the freshness of fresh meat, the microneedle patch backing layer is loaded with a mixed fluorescent dye responsive to biogenic amines, which can realize rapid identification of biogenic amines in fresh meat and judge the freshness by visual observation. The microneedle patch of the present application can be used as a freshness indicator, which is simpler and more accurate in detecting the freshness of fresh meat.

[0005] The object of the present application is achieved by the following technical solution:

[0006] The first aspect of the present application is to provide a preparation method of a fluorescent color-changing hydrogel microneedle patch responsive to biogenic amines, the method comprising the following steps:

[0007] (1) Preparation of mixed fluorescent dye solution with specific response to biological amines: two or more fluorescent dyes are dissolved in dimethyl sulfoxide respectively, and two or more fluorescent dye solutions are obtained by diluting them with ultrapure water; two or more fluorescent dye solutions are mixed in a certain proportion to obtain solution A;

[0008] (2) Preparation of microneedle part of hydrogel microneedle patch: a photoinitiator is dissolved in ultrapure water, heated in a water bath and shaken to dissolve, to obtain solution B; then a certain amount of hydrogel material is taken, the solution B is mixed with the hydrogel material to obtain a hydrogel solution; the hydrogel solution is dropped on the microneedle mold, the mold is treated by vacuumizing, constant temperature drying, and then ultraviolet curing to prepare the hydrogel microneedle;

[0009] (3) Preparation of hydrogel microneedle patch with fluorescent color change in response to biological amines: the solution A is mixed with the hydrogel solution, and horseradish peroxidase (HRP) and hydrogen peroxide (H2O2) are added in turn to obtain a mixed fluorescent hydrogel solution with fluorescent color change in response to biological amines; the mixed fluorescent hydrogel solution is dropped on the mold containing the hydrogel microneedle, and then dried to obtain the hydrogel microneedle patch with fluorescent color change in response to biological amines.

[0010] In an embodiment, the fluorescent dyes in step (1) are two or more of BP Light, BP Flour, BODIPY, fluorescein isothiocyanate, rhodamine B (RhB), tetraiodofluorescein, amaranth, betacyanin and pyrene dye, preferably rhodamine B (RhB) and fluorescein isothiocyanate (FITC).

[0011] In an embodiment, the amount of fluorescent dye added in step (1) relative to dimethyl sulfoxide is 1.0-3.0 mg / 1.0 mL. Preferably, the amount of fluorescent dye added relative to dimethyl sulfoxide is 1.0 mg / 1.0 mL.

[0012] In an embodiment, in step (1), the fluorescent dye is diluted with ultrapure water to a mass concentration of 1000.0-10 μg / mL. Preferably, the fluorescent dye is diluted with ultrapure water to a mass concentration of 1000.0 μg / mL, 500.0 μg / mL, 200.0 μg / mL, 100.0 μg / mL, 50.0 μg / mL, 20.0 μg / mL, 2.0 μg / mL, 1.0 μg / mL. Preferably, the fluorescent dye is diluted to a mass concentration of 200.0 μg / mL, 100.0 μg / mL, 50.0 μg / mL.

[0013] In an embodiment, the Rhodamine B (RhB) and fluorescein isothiocyanate (FITC) solution in step (1) is mixed in a ratio of 5:1-1:5, preferably 1:2-1:5, and more preferably 1:3.

[0014] In an embodiment, the photo initiator in step (2) is LAP, and the amount of LAP added to the ultrapure water is 0.05 g / 20 mL.

[0015] In an embodiment, the photo initiator LAP in step (2) is dissolved in ultrapure water under water bath heating conditions for a period of time, the water bath dissolving temperature is 40-55°C, and the dissolving time is 15-25 min; preferably, the water bath temperature is 45°C, and the water bath dissolving time is 15 min.

[0016] In an embodiment, the hydrogel material is one or more of chitin, methacrylated silk fibroin (SilMA), chitosan, sodium alginate, sodium hyaluronate, and methacrylic acid polymer; preferably, the hydrogel material is methacrylated silk fibroin (SilMA).

[0017] In an embodiment, the amount of hydrogel material added to solution A in step (2) is 0.05-0.10 g / 1.0 mL; preferably, the amount of hydrogel material added is 0.07 g / 1.0 mL.

[0018] In an embodiment, the amount of hydrogel solution added to the mold in step (2) is 200-300 μL; preferably, 250 μL.

[0019] In an embodiment, the vacuum treatment in step (2) is performed by using a vacuum drying oven at a temperature of 35-45°C, a vacuum of 1.0 MPa, and a vacuum time of not less than 6 minutes, and the process is repeated 3-6 times.

[0020] In an embodiment, the mold is concentrated and dried in step (2), which is performed by using a hot air drying oven at a temperature of 35-40°C for 5-6 h, then adding the hydrogel solution again for concentration and drying, and the process is repeated 2-3 times.

[0021] In an embodiment, the UV light curing in step (2) is performed for a period of time, the wavelength of the UV light is 254 nm, and the light curing time is 60-150 s; preferably, the light curing time is 90 s.

[0022] In an embodiment, the mass of the hydrogel material in the mixed fluorescent hydrogel solution in step (3) is 7 wt%.

[0023] In an embodiment, the total mass concentration of the dyes in the mixed fluorescent hydrogel solution in step (3) is 500.0-5 μg / mL. Specifically, 100.0 μg / mL, 50.0 μg / mL, 25.0 μg / mL are preferred.

[0024] In an embodiment, the mass concentration of HRP in step (3) is 0.01 g / mL, and the mass concentration of H2O2 is 100 mM / L.

[0025] In an embodiment, the dosing amount of HRP and H2O2 relative to the mixed fluorescent hydrogel solution in step (3) is 40 μL / mL.

[0026] In an embodiment, the mixed fluorescent hydrogel solution in step (3) is dropped onto the mold containing the hydrogel microneedle, and the dosing amount of the mixed fluorescent hydrogel solution is 100-200 μL.

[0027] In an embodiment, the drying of the hydrogel microneedle patch in step (3) is hot air drying, the drying temperature is 35-45°C, the drying time is 35-40 h, preferably the drying temperature is 37°C, and the drying time is 36 h.

[0028] In an embodiment, the mold used is a PDMS mold, the needle tip height is 1500 μm, the bottom diameter is 550 μm, the needle tip distance is 1200 μm, the number of arrays is 8*8, the patch size is 12*12 mm, and the groove depth is 2 mm.

[0029] In an embodiment, the method comprises the following steps:

[0030] (1) Preparation of a mixed fluorescent dye solution with specific response to biological amines:

[0031] 1.0 mg of RhB and FITC were dissolved in 1 mL of dimethyl sulfoxide, respectively, and diluted with ultrapure water to a mass concentration of 1000-10 μg / mL. Two fluorescent dye solutions of RhB and FITC with the same mass concentration were mixed in a certain volume ratio to obtain a mixed fluorescent dye solution with specific response to biological amines.

[0032] (2) Preparation of the microneedle part of the hydrogel microneedle patch:

[0033] Dissolve 0.05 g of photoinitiator LAP in 20 mL of ultrapure water, heat and dissolve in a 45℃ water bath for 15 min, shake several times during this period, obtain a 0.25% (w / v) photoinitiator LAP standard solution, then mix 0.7 g of SilMA with 1 mL of the photoinitiator LAP standard solution uniformly, stand at room temperature for 30-60 min to prepare a photocured hydrogel solution. Drop 200-300 μL of the hydrogel solution on the PDMS mold, and place it in a vacuum drying oven for multiple vacuum treatments, with the vacuum temperature controlled at 35-45℃, the vacuum degree is 1.0 MPa, the vacuum time is not less than 6 min, and the operation is repeated for 3-6 times. Absorb the excess hydrogel solution on the surface of the mold, place the PDMS mold in a hot air oven for drying and concentration, the temperature is 35-40℃, the time is 5-6 h, then drop the hydrogel solution again for concentration and drying, and the process is repeated for 2-3 times. Then take out the mold, irradiate with a handheld ultraviolet lamp with a wavelength of 254 nm for 60-150 s for photocuring, and the hydrogel microneedle is obtained.

[0034] (3) Preparation of a fluorescent color-changing hydrogel microneedle patch responsive to biological amines:

[0035] Take 500 μL of mixed fluorescent dyes with a mass concentration of 1000-10 μg / mL, the ratio of RhB and FITC in the mixed fluorescent dyes is 1:3, mix the mixed fluorescent dye solution with 420 μL of hydrogel solution with a mass fraction of 14 wt%, take 40 μL of HRP (0.01 g / mL) and H2O2 (100 mM) and add them to the mixed solution, mix uniformly to prepare a mixed fluorescent hydrogel microneedle patch solution. The final mixed fluorescent hydrogel solution has a mass concentration of RhB and FITC of 500-50 μg / mL, and the mass fraction of the hydrogel material is 7 wt%; take 100-200 μL of the mixed fluorescent hydrogel solution and drop it on the mold containing the hydrogel microneedle for hot air drying, the drying temperature is 35-45℃, the drying time is 35-40 h, then drop the mixed fluorescent hydrogel solution again, and perform hot air drying again, finally, the hydrogel microneedle patch is peeled off from the mold to obtain a fluorescent color-changing hydrogel microneedle patch responsive to biological amines.

[0036] In a second aspect of the present application, a fluorescent color-changing hydrogel microneedle patch responsive to biological amines prepared by the above-mentioned method is provided.

[0037] In a third aspect of the present application, the application provides the use of the hydrogel microneedle patch as described in the above scheme for fresh meat freshness detection, and the refrigeration conditions are 0℃ and 4℃, and the storage time is 10 days.

[0038] In an embodiment, the fresh meat includes chicken, pork and salmon.

[0039] The application also provides an application of the hydrogel microneedle patch with the above-mentioned biogenic amine response fluorescent color change in identifying and detecting biogenic amine.

[0040] The application also provides a method for detecting freshness of fresh meat, which comprises inserting the microneedle of the hydrogel microneedle patch with the above-mentioned biogenic amine response fluorescent color change into fresh meat, then taking out, and after the color is stable, taking a photo of the microneedle patch by using a smart phone, collecting RGB information, and obtaining G+B / (R+G+B) information; and using the G+B / (R+G+B) information to indicate the freshness of the fresh meat.

[0041] In an embodiment, when the G+B / (R+G+B) value is less than 0.60, the fresh meat belongs to an edible state; when the G+B / (R+G+B) value is between 0.60 and 0.65, the fresh meat belongs to a low-quality state about to deteriorate; and when the G+B / (R+G+B) value is greater than 0.65, the fresh meat belongs to a deteriorated state.

[0042] Compared with the prior art, the application has the following beneficial effects:

[0043] 1. The mixed fluorescent dye with specific response to biogenic amine in the application shows good fluorescent color response to biogenic amine, and the color of the biogenic amine response fluorescent dye can change from yellow to green with the increase of the concentration of biogenic amine, and the color change can be observed by vision.

[0044] 2. The biogenic amine response fluorescent color change hydrogel microneedle patch in the application uses a hydrophilic polymer material as a substrate, can quickly extract food tissue fluid, and makes the biogenic amine response fluorescent dye react with biogenic amine in food, so that the color of the biogenic amine response fluorescent dye changes from yellow to green.

[0045] 3. The application collects color information of the microneedle patch by using a smart phone, converts the color change into RGB information, analyzes the relationship between the freshness change of fresh meat caused by the biogenic amine index (BAI) and G+B / (R+G+B), converts the image into a remote RGB detection mode, so that the freshness of fresh meat during storage can be obtained simply and accurately by using a smart method.

[0046] 4. The biogenic amine response fluorescent color change hydrogel microneedle patch prepared in the application is suitable for various fresh meat foods such as chicken, pork and salmon, and has a wide application range.

[0047] 5. The biogenic amine response fluorescent color change hydrogel microneedle patch prepared in the application is safe and non-toxic in raw material, easy to prepare, pollution-free in the preparation process, has a wide application range, and can be used for industrial production.

[0048] Of course, it is not necessary to achieve all the technical effects mentioned above when implementing any product of the application. BRIEF DESCRIPTION OF DRAWINGS

[0049] Figure 1 Color response graph of mixed fluorescent dye solution with mass concentration of 100.0 μg / mL prepared for Example 1 reacting with biological amines.

[0050] Figure 2 Nonlinear curve fitting graph of G / R value of mixed fluorescent dye solution with mass concentration of 100.0 μg / mL prepared for Example 1 and histamine content.

[0051] Figure 3 Appearance morphology graph of microneedle patch of RhB and FITC mixed at a ratio of 1:3 with mass concentration of 100.0 μg / mL prepared for Example 1.

[0052] Figure 4 Microneedle part morphology graph of microneedle patch of RhB and FITC mixed at a ratio of 1:3 with mass concentration of 100.0 μg / mL prepared for Example 1.

[0053] Figure 5 Color response graph of mixed fluorescent dye solution with mass concentration of 50.0 μg / mL prepared for Example 2 reacting with biological amines.

[0054] Figure 6 Nonlinear curve fitting graph of G / R value of mixed fluorescent dye solution with mass concentration of 50.0 μg / mL prepared for Example 2 and histamine content.

[0055] Figure 7 Color response graph of mixed fluorescent dye solution with mass concentration of 25.0 μg / mL prepared for Example 3 reacting with biological amines.

[0056] Figure 8 Nonlinear curve fitting graph of G / R value of mixed fluorescent dye solution with mass concentration of 25.0 μg / mL prepared for Example 3 and histamine content.

[0057] Figure 9 Detection of chicken breast freshness under 0°C storage condition by fluorescent color-changing hydrogel microneedle patch responding to biological amines prepared for Example 1.

[0058] Figure 10 Detection of chicken breast freshness under 4°C storage condition by fluorescent color-changing hydrogel microneedle patch responding to biological amines prepared for Example 1.

[0059] Figure 11 Detection of pork freshness under 0°C storage condition by fluorescent color-changing hydrogel microneedle patch responding to biological amines prepared for Example 1.

[0060] Figure 12The freshness of pork was detected by using the biogenic amine-responsive fluorescent color-changing hydrogel microneedle patch prepared in Example 1 under storage conditions at 4°C.

[0061] Figure 13 The freshness of salmon was detected by using the biogenic amine-responsive fluorescent color-changing hydrogel microneedle patch prepared in Example 1 under storage conditions at 0°C.

[0062] Figure 14 The freshness of salmon was detected by using the biogenic amine-responsive fluorescent color-changing hydrogel microneedle patch prepared in Example 1 under storage conditions at 4°C. Detailed Implementation

[0063] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0064] Unless otherwise specified, all reagents used in the embodiments of this invention can be purchased commercially.

[0065] Example 1: Preparation of biogenic amine-responsive fluorescent color-changing hydrogel microneedle patch

[0066] 1. Preparation of mixed fluorescent dye solutions:

[0067] (1) Preparation of 100 μg / mL and 200 μg / mL RhB and FITC solutions respectively: Dissolve 1 mg RhB in 1 mL of dimethyl sulfoxide and dilute it with ultrapure water to a mass concentration of 100 μg / mL and 200 μg / mL respectively; Dissolve 1 mg FITC in 1 mL of dimethyl sulfoxide and dilute it with ultrapure water to a mass concentration of 100 μg / mL and 200 μg / mL respectively.

[0068] (2) Preparation of mixed fluorescent dye solution, i.e., solution A:

[0069] The above-mentioned solutions of RhB and FITC fluorescent dyes with a mass concentration of 100.0 μg / mL were mixed in volume ratios of 1:0, 5:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:5, and 0:1 to obtain mixed fluorescent dye solutions that specifically respond to biogenic amines.

[0070] The above-mentioned solutions of RhB and FITC fluorescent dyes with a mass concentration of 200.0 μg / mL were mixed at a certain volume ratio to obtain a mixed fluorescent dye solution that specifically responds to biogenic amines.

[0071] 2. Preparation of hydrogel microneedle patch microneedle part:

[0072] (3) Preparation of a standard solution of the photoinitiator LAP, i.e. solution B: 0.05 g of the photoinitiator LAP was dissolved in 20 mL of ultrapure water, and heated to dissolve for 15 min in a water bath at 45°C, during which time the solution was shaken several times to obtain a 0.25% (w / v) standard solution of the photoinitiator LAP;

[0073] (4) Preparation of a SilMA hydrogel solution: 0.07 g of SilMA was mixed with 1 mL of the solution obtained in step (3) to obtain a uniform mixture, and the mixture was left to stand at room temperature for 30-60 min to obtain a photocured hydrogel solution;

[0074] (5) Preparation of a SilMA hydrogel microneedle: 200-300 μL of the solution obtained in step (4) was added to a PDMS mold, and the mold was placed in a vacuum drying oven for multiple vacuum treatments, with the vacuum temperature controlled at 35-45°C and the vacuum degree controlled at 1.0 MPa, and the vacuum time controlled at no less than 6 min, and this operation was repeated 3-6 times, then the excess hydrogel solution on the surface of the mold was removed, the PDMS mold was placed in a hot air oven for drying and concentration, with the temperature controlled at 35-40°C and the time controlled at 5-6 h, then the hydrogel solution was added again for drying and concentration, and this process was repeated 2-3 times. Then the mold was removed, and a handheld ultraviolet lamp with a wavelength of 254 nm was used for irradiation for photocuring for 60-150 s, to obtain a hydrogel microneedle.

[0075] 3. Preparation of a fluorescent color-changing hydrogel microneedle patch responsive to biological amines:

[0076] (6) Preparation of a mixed fluorescent hydrogel solution: 200 μg / mL of RhB and 200 μg / mL of FITC were mixed in a volume ratio of 1:3 to obtain a mixed fluorescent dye solution, and 500 μL of the mixed fluorescent dye solution was mixed with 420 μL of a hydrogel solution with a mass of 14 wt%, and 40 μL of HRP (0.01 g / mL) and 40 μL of H2O2 (100 mM) were added to the mixed solution, respectively, and mixed uniformly, so that the total mass concentration of RhB and FITC in the final mixed fluorescent dye hydrogel solution was 100 μg / mL, and the mass of the hydrogel material was 7 wt%.

[0077] (7) Preparation of a fluorescent color-changing hydrogel microneedle patch responsive to biological amines: 100-200 μL of the solution obtained in step (6) was added to the mold containing the hydrogel microneedle for hot air drying, with the drying temperature controlled at 35-45°C and the drying time controlled at 35-40 h, then the mixed fluorescent hydrogel solution was added again for hot air drying, and finally the hydrogel microneedle patch was peeled off the mold to obtain a fluorescent color-changing hydrogel microneedle patch responsive to biological amines, denoted as FITC / RhB@SilMA MN patch.

[0078] The prepared biogenic amine-responsive fluorescent color-changing hydrogel microneedle patch was tested.

[0079] The color response test was performed on the mixed fluorescent dye solution prepared in this example, which has a specific response to biogenic amines. The test method is as follows: 100.0 μg / mL of RhB and FITC solutions were mixed in a ratio of 1:0, 5:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:5, and 0:1 to obtain a mixed fluorescent dye solution with a specific response to biogenic amines. 0-100 μL of 1.0 mg / mL histamine solution was mixed with the above-mentioned nine ratios of mixed fluorescent dye solution to make the final reaction system have a histamine mass concentration of 0, 10, 30, 50, 70, and 100 μg / mL. The color of the solution system was photographed using a mobile phone. Figure 1 The color response graph of the mixed fluorescent dye solution prepared by mixing RhB and FITC solutions with a mass concentration of 100.0 μg / mL in a ratio of 1:0, 5:1, 3:1, 2:1, 1:1, 1:2, 1:3, and 1:5 and reacting with biogenic amines is shown in FIG. 2. Figure 1 It can be seen that the mixed fluorescent dye solution prepared by mixing RhB and FITC solutions in a ratio of 1:2, 1:3, and 1:5 has a good color change effect when reacting with biogenic amines. The 1:3 ratio has the best effect, with the most color-changing levels, and can more accurately indicate the concentration of biogenic amines.

[0080] The specific color-changing effect is shown in Table 1.

[0081] Table 1 Biogenic amine response results of 100 μg / mL mixed fluorescent dye hydrogel solution with different RhB and FITC usage ratios

[0082]

[0083] The RGB analysis was performed on the mixed fluorescent dye solution prepared in this example, which has a specific response to biogenic amines. The test method is as follows: the RGB values of the color response graph of the mixed fluorescent dye solution obtained in step (3) above and reacting with biogenic amines were extracted using the mobile phone application software "Color call". Figure 2 The non-linear curve fitting graph of the G / R value of the mixed fluorescent dye solution with a mass concentration of 100.0 μg / mL and the histamine content is shown in FIG. 4. Figure 2 It can be seen that the non-linear curve fitting R 2 is 0.9972, which has the best color change effect.

[0084] The appearance morphology of the bioamine-responsive fluorescent color-changing hydrogel microneedle patch prepared in this embodiment was photographed. The testing method was as follows: the microneedle patch was picked up with tweezers, and a mobile phone was used to take a photo of it under appropriate light and background, Figure 3 For

[0085] The appearance morphology of the bioamine-responsive fluorescent color-changing hydrogel microneedle patch prepared in this embodiment was photographed. The testing method was as follows: the microneedle patch was picked up with tweezers, and a mobile phone was used to take a photo of it under appropriate light and background,

[0086] The appearance morphology of the bioamine-responsive fluorescent color-changing hydrogel microneedle patch prepared in this embodiment was photographed. The testing method was as follows: the microneedle patch was picked up with tweezers, and a mobile phone was used to take a photo of it under appropriate light and background, Figure 4 The appearance morphology of the bioamine-responsive fluorescent color-changing hydrogel microneedle patch prepared in this embodiment was photographed. The testing method was as follows: the microneedle patch was picked up with tweezers, and a mobile phone was used to take a photo of it under appropriate light and background,

[0087] Example 2

[0088] (1) 50.0 μg / mL, 100 μg / mL RhB, FITC solutions were respectively prepared: 1 mg of RhB was dissolved in 1 mL of dimethyl sulfoxide, and ultrapure water was used to dilute it to a mass concentration of 50 μg / mL and 100 μg / mL; 1 mg of FITC was dissolved in 1 mL of dimethyl sulfoxide, and ultrapure water was used to dilute it to a mass concentration of 50 μg / mL and 100 μg / mL.

[0089] (2)-(5) are the same as steps (2)-(5) in Example 1.

[0090] (6) Preparation of mixed fluorescent hydrogel solution: 100 μg / mL of RhB and 100 μg / mL of FITC were mixed in a volume ratio of 1:3 to prepare a mixed fluorescent dye solution, 500 μL of the mixed fluorescent dye solution was mixed with 420 μL of a hydrogel solution with a mass of 14 wt%, 40 μL of HRP (0.01 g / mL) and 40 μL of H2O2 (100 mM) were respectively added to the mixed solution, and mixed uniformly, so that the total mass concentration of RhB and FITC in the final mixed fluorescent hydrogel solution was 50 μg / mL, and the mass of the hydrogel material was 7 wt%.

[0091] (7) The same as step (7) in Example 1.

[0092] The color response test was performed on the mixed fluorescent dye solution prepared in this embodiment which had specific response to bioamines, and the testing method was the same as step (8) in Example 1,Figure 5 The color response diagrams show the reaction of mixed fluorescent dye solutions with biogenic amines obtained by mixing RhB and FITC solutions with a mass concentration of 50.0 μg / mL prepared in Example 2 at ratios of 1:0, 5:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:5, and 0:1. Figure 5 It can be seen that the mixed fluorescent dye solutions obtained by mixing RhB and FITC solution in ratios of 1:2, 1:3, and 1:5 show good color changes when reacting with biogenic amines, with the 1:5 ratio showing the best color change. RGB analysis was performed on the mixed fluorescent dye solutions prepared in this example that exhibit specific responses to biogenic amines, using the same method as step (9) in Example 1. Figure 6 The nonlinear curve fitting plot of its G / R value versus histamine content is obtained from... Figure 6 It can be seen that the nonlinear curve fitting R obtained by mixing RhB and FITC solution in a 1:5 ratio is... 2 The optimal color change effect was 0.9998. The biogenic amine-responsive fluorescent color-changing hydrogel microneedle patch prepared in this example was tested, and its appearance morphology and needle tip morphology were photographed. The testing method was the same as steps (10) to (11) in Example 1.

[0093] The specific color-changing effects are shown in Table 2.

[0094] Table 2. Biogenic amine response results in 50 μg / mL mixed fluorescent dye hydrogel solutions with different RhB to FITC ratios.

[0095]

[0096] Example 3

[0097] (1) Preparation of 25 μg / mL and 50 μg / mL RhB and FITC solutions respectively: Dissolve 1 mg RhB in 1 mL of dimethyl sulfoxide and dilute it with ultrapure water to a mass concentration of 25 μg / mL and 50 μg / mL respectively; Dissolve 1 mg FITC in 1 mL of dimethyl sulfoxide and dilute it with ultrapure water to a mass concentration of 25 μg / mL and 50 μg / mL respectively.

[0098] (2) to (5) are the same as steps (2) to (5) in Example 1.

[0099] (6) Preparation of mixed fluorescent hydrogel solution: 50 μg / mL of RhB and 50 μg / mL of FITC were mixed at a volume ratio of 1:3 to prepare a mixed fluorescent dye solution. 500 μL of the mixed fluorescent dye solution was mixed with 420 μL of a hydrogel solution with a mass of 14 wt%, and 40 μL of HRP (0.01 g / mL) and 40 μL of H2O2 (100 mM) were added to the mixed solution, respectively, and mixed uniformly, so that the total mass concentration of RhB and FITC in the final mixed fluorescent hydrogel solution was 25 μg / mL, and the mass of the hydrogel material was 7 wt%.

[0100] (7) The same as step (7) in Example 1.

[0101] The color response test of the mixed fluorescent dye solution prepared in this example and having a specific response to biological amines was performed, and the test method was the same as that in step (8) in Example 1. Figure 7 The color response graph of the mixed fluorescent dye solution prepared in Example 3 and having a mass concentration of 25.0 μg / mL of RhB and FITC, mixed at a ratio of 1:0, 5:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:5, and 0:1, and reacted with biological amines. From Figure 7 It can be seen that the mixed fluorescent dye solution obtained by mixing RhB and FITC solution at a ratio of 1:2, 1:3, and 1:5 has a good color change effect when reacted with biological amines, among which 1:2 is the best, has more obvious change levels, and each layer has a narrow concentration range and is more sensitive. The RGB analysis of the mixed fluorescent dye solution prepared in this example and having a specific response to biological amines was performed, and the test method was the same as that in step (9) in Example 1. Figure 8 The nonlinear curve fitting graph of the G / R value and the histamine content, from Figure 8 It can be seen that the nonlinear curve fitting R 2 was 0.9940, and the color change effect was the best. The fluorescent color-changing hydrogel microneedle patch prepared in this example and having a response to biological amines was tested, and the apparent morphology and needle tip morphology were photographed, and the test method was the same as steps (10)-(11) in Example 1.

[0102] The specific color change effect is shown in Table 3.

[0103] Table 3 Biological amine response results of 25 μg / mL mixed fluorescent dye hydrogel solution with different RhB and FITC usage ratios

[0104]

[0105] Example 4

[0106] (1) Preparation of tetraiodofluorescein and FITC solutions: 1 mg of tetraiodofluorescein was dissolved in 1 mL of dimethyl sulfoxide, and then diluted with ultrapure water to a mass concentration of 100 μg / mL and 200 μg / mL. 1 mg of FITC was dissolved in 1 mL of dimethyl sulfoxide, and then diluted with ultrapure water to a mass concentration of 100 μg / mL and 200 μg / mL.

[0107] (2)-(5) are the same as steps (2)-(5) in Example 1.

[0108] (6) Preparation of a mixed fluorescent hydrogel solution: 200 μg / mL of tetraiodofluorescein and 200 μg / mL of FITC were mixed at a volume ratio of 1:3 to prepare a mixed fluorescent dye solution. 500 μL of the mixed fluorescent dye solution was mixed with 420 μL of a hydrogel solution with a mass fraction of 14 wt%, and then 40 μL of HRP (0.01 g / mL) and H2O2 (100 mM) were added to the mixed solution, respectively, and mixed uniformly. The final mixed fluorescent hydrogel solution had a mass concentration of 100 μg / mL of tetraiodofluorescein and FITC, and a mass fraction of 7 wt% of hydrogel material.

[0109] (7) The same as step (7) in Example 1.

[0110] Example 5

[0111] (1) Preparation of amaranth and FITC solutions: 1 mg of amaranth was dissolved in 1 mL of dimethyl sulfoxide, and then diluted with ultrapure water to a mass concentration of 100 μg / mL and 200 μg / mL. 1 mg of FITC was dissolved in 1 mL of dimethyl sulfoxide, and then diluted with ultrapure water to a mass concentration of 100 μg / mL and 200 μg / mL.

[0112] (2)-(5) are the same as steps (2)-(5) in Example 1.

[0113] (6) Preparation of a mixed fluorescent hydrogel solution: 200 μg / mL of amaranth and 200 μg / mL of FITC were mixed at a volume ratio of 1:3 to prepare a mixed fluorescent dye solution. 500 μL of the mixed fluorescent dye solution was mixed with 420 μL of a hydrogel solution with a mass fraction of 14 wt%, and then 40 μL of HRP (0.01 g / mL) and H2O2 (100 mM) were added to the mixed solution, respectively, and mixed uniformly. The final mixed fluorescent hydrogel solution had a mass concentration of 100 μg / mL of amaranth and FITC, and a mass fraction of 7 wt% of hydrogel material.

[0114] (7) The same as step (7) in Example 1.

[0115] Example 6

[0116] (1) Preparation of betacyanin and FITC solution: 1 mg of betacyanin was dissolved in 1 mL of dimethyl sulfoxide, and then diluted with ultrapure water to a mass concentration of 100 μg / mL and 200 μg / mL; 1 mg of FITC was dissolved in 1 mL of dimethyl sulfoxide, and then diluted with ultrapure water to a mass concentration of 100 μg / mL and 200 μg / mL.

[0117] (2)-(5) are the same as steps (2)-(5) in Example 1.

[0118] (6) Preparation of mixed fluorescent hydrogel solution: 200 μg / mL of betacyanin and 200 μg / mL of FITC were mixed at a volume ratio of 1:3 to prepare a mixed fluorescent dye solution, 500 μL of the mixed fluorescent dye solution was mixed with 420 μL of a hydrogel solution with a mass of 14 wt%, 40 μL of HRP (0.01 g / mL) and H2O2 (100 mM) were added to the mixed solution respectively, and then mixed uniformly, so that the mass concentration of betacyanin and FITC in the final mixed fluorescent hydrogel solution was 100 μg / mL, and the mass of the hydrogel material was 7 wt%.

[0119] Example 7 Application of hydrogel microneedle patch to detect freshness of chicken

[0120] The fluorescent color-changing hydrogel microneedle patch for detecting biological amines prepared in Example 1 was used to detect fresh chicken breast packaged with preservative film under the conditions of 0°C and 4°C. The content of biological amines (putrescine, cadaverine, histamine and tyramine) was detected according to the method of Yu Dawei et al. (Yu Dawei. Study on the effect of chitosan film on the quality of chilled grass carp fillets [D]. Jiangnan University, 2019.), and the biological amine index (BAI) of fresh meat is usually less than 20 mg / kg, which is fresh and edible; the BAI is between 20-50 mg / kg, which is low quality; and the BAI is greater than 50 mg / kg, which is spoiled meat and not edible.

[0121] The changes of the contents of putrescine (Put), cadaverine (Cad), histamine (His), tyramine (Tyr) and biological amine index (BAI) in chicken breast stored at 0°C for 10 days are shown in Table 1. Figure 9 As shown in Table 1, the BAI was 14.77 mg / kg on the 4th day, and the chicken breast was edible fresh meat from day 0 to day 4, and the microneedle patch was yellow at this time. The BAI was 42.08 mg / kg on the 6th day, and the chicken breast was low quality fresh meat from day 5 to day 6, and the microneedle patch was yellow-green at this time. The BAI was 62.24 mg / kg on the 7th day, and the chicken breast was spoiled meat after this time, and the microneedle patch was light green at this time.

[0122] The changes of the contents of putrescine (Put), cadaverine (Cad), histamine (His), tyramine (Tyr) and biological amine index (BAI) in chicken breast stored at 4°C for 10 days are shown in Table 2.Figure 10 As shown, the BAI is 13.99 mg / kg on the first day, which is edible fresh meat, and the microneedle patch is light yellow with a yellow-greenish color. The BAI is 26.72 mg / kg on the second day and 46.84 mg / kg on the fourth day, which is low-quality fresh meat, and the microneedle patch is gray-green. On the fifth day, the BAI is 59.52 mg / kg, and after that, the chicken breast is spoiled meat, and the microneedle patch is light green. Therefore, the fluorescent color-changing hydrogel microneedle patch responsive to biological amines can indicate the freshness of fresh chicken breast meat by color change.

[0123] By combining with a smart phone and photo color RGB analysis, visual and accurate freshness evaluation can be easily achieved, as shown in Figure 9 and Figure 10 As shown, when the value of G+B / (R+G+B) is less than 0.60, the fresh meat is edible, when the value of G+B / (R+G+B) is between 0.60 and 0.65, the fresh meat is low-quality and will be spoiled, and when the value of G+B / (R+G+B) is greater than 0.65, the fresh meat is spoiled. Therefore, the fluorescent color-changing hydrogel microneedle patch responsive to biological amines can further accurately detect the freshness of fresh meat during storage by the color of the microneedle patch.

[0124] Example 8 Application of hydrogel microneedle patch to detect the freshness of pork

[0125] Test process: same as Example 7. Take pork as food sample, insert the microneedle patch into the tissue of the pork sample, place it in a culture dish sealed with plastic wrap, and store it at 0°C and 4°C for 10 days.

[0126] Results: the microneedle patch ( Figure 11 ) is yellow at the beginning on the first day, the color of the microneedle patch becomes lighter and has a tendency to turn green on the third day, the microneedle patch is yellow-green on the sixth day, and the microneedle patch is gray-green on the seventh day. The green color of the microneedle patch gradually deepens from the seventh day to the tenth day. The microneedle patch ( Figure 12 ) is yellow on the first day, the yellow color gradually fades from the first day to the second day, and the microneedle patch is gray-green on the second day. The color of the microneedle patch starts to turn green on the third day, and the color of the microneedle patch is green from the third day to the tenth day, and the color gradually deepens with the extension of storage time. At the same time, the color change of the FITC / RhB@SilMA MN patch can be quantitatively described by G+R / (R+G+B), as shown in Figure 11 As shown, the value of G+R / (R+G+B) of the microneedle patch is less than 0.60 from the first day to the second day at 0°C, the value of G+R / (R+G+B) is in the range of 0.60 to 0.65 from the third day to the sixth day, and the value of G+R / (R+G+B) is greater than 0.65 from the seventh day to the tenth day. As shown inFigure 12 It can be seen that the G+R / (R+G+B) value of the microneedle patch G+R / (R+G+B) value is less than 0.60 at 0 day; the G+R / (R+G+B) value is in the range of 0.60 to 0.65 at 1 to 3 days, and the G+R / (R+G+B) value is greater than 0.65 after 4 days.

[0127] In combination with the analysis of the change of the BAI content of pork, when the BAI is less than 20 mg / kg, the pork is edible, the color of the microneedle patch under the conditions of 0℃ and 4℃ is yellow, and the corresponding G+R / (R+G+B) value is <0.60; when the BAI is in the range of 20 to 50 mg / kg, the pork is low-quality meat, the color of the microneedle patch under the conditions of 0℃ and 4℃ is light yellow and has a trend of changing to green, and the corresponding G+R / (R+G+B) value is in the range of 0.60 to 0.65; when the color of the microneedle patch under the conditions of 0℃ and 4℃ is green, the pork BAI is greater than 50 mg / kg, which is spoiled meat, and the corresponding G+R / (R+G+B) value is greater than 0.65. That is, when the color of the FITC / RhB@SilMA MN patch is yellow, it indicates that the pork is relatively fresh and edible at this time; when the microneedle patch is light yellow and partially green, the pork is no longer fresh and belongs to low-quality meat; when the color of the microneedle patch is green, the pork has been spoiled, and the freshness of the pork during storage can be accurately detected by the color change of the microneedle patch.

[0128] Example 9 Application of Hydrogel Microneedle Patch to Detect Freshness of Salmon

[0129] Test process: same as Example 7. Take salmon as a food sample, insert the microneedle patch into the tissue of the salmon sample, place it in a culture dish sealed with plastic wrap, and store it at 0℃ and 4℃ for 10 days.

[0130] Results: The microneedle patch stored at 0℃ ( Figure 13 ) and 4℃ ( Figure 14The BAI content of salmon treated with the FITC / RhB@SilMA MN patch showed a growth trend with time. At the beginning of storage at 0℃, the fish quality was fresh and the BAI content was less than 20 mg / kg. The G+R / (R+G+B) value of the micro-needle patch changed little and was less than 0.60, and the color of the micro-needle patch was yellow. When stored for 3 to 7 days, the BAI content increased sharply to 20-50 mg / kg, and the salmon was in a low-quality stage. The color of the micro-needle patch gradually changed to yellow-green. On the 8th day, the BAI content exceeded 50 mg / kg, and the salmon had begun to spoil. The color of the micro-needle patch changed to green, and the G+R / (R+G+B) value was greater than 0.65. The BAI content of salmon stored at 4℃ reached the limit of 20 mg / kg and 50 mg / kg on the 1st day and the 4th day, respectively. The micro-needle patch was gray-yellow on the 1st to 3rd day, and the salmon was in a low-quality stage. After the 4th day, the micro-needle patch turned green, and the salmon had spoiled. The G+R / (R+G+B) value of the micro-needle patch was greater than 0.65. The color change of the micro-needle patch and the G+R / (R+G+B) value were consistent with the freshness determined by the BAI content standard in the salmon sample, showing the potential of the FITC / RhB@SilMA MN patch for salmon freshness detection.

[0131] It can be understood that the above only describes the embodiments of the present application, and does not limit the protection scope of the present application. Any equivalent structure or equivalent process transformation, or direct or indirect application in other related technical fields, is also included in the patent protection scope of the present application.

Claims

1. A method for preparing a biogenic amine-responsive fluorescent color-changing hydrogel microneedle patch, characterized in that, Includes the following steps: (1) Preparation of a mixed fluorescent dye solution that specifically responds to biogenic amines: Dissolve the fluorescent dyes Rhodamine B and fluorescein isothiocyanate in dimethyl sulfoxide, and dilute them with ultrapure water to obtain a fluorescent dye solution. Solution A is obtained by mixing fluorescent dye solutions; (2) Preparation of the microneedle part of the hydrogel microneedle patch: Dissolve the photoinitiator in ultrapure water and mix well to obtain solution B; mix solution B with hydrogel material to obtain hydrogel solution; then drop the hydrogel solution onto the microneedle mold, vacuum process, dry at constant temperature, and then cure with ultraviolet light to obtain hydrogel microneedles. (3) Preparation of biogenic amine-responsive fluorescent color-changing hydrogel microneedle patch: Mix solution A with hydrogel solution, add horseradish peroxidase and hydrogen peroxide in sequence, mix evenly to obtain mixed fluorescent hydrogel solution; then drop the mixed fluorescent hydrogel solution onto a mold containing hydrogel microneedles, dry and peel off the mold to obtain biogenic amine-responsive fluorescent color-changing hydrogel microneedle patch. In step (1), the volume ratio of Rhodamine B solution to fluorescein isothiocyanate solution is 1:

3. The hydrogel material is methacrylamide silk fibroin; In step (3), the total mass concentration of fluorescent dye in the mixed fluorescent hydrogel solution is 100 µg / mL, and the mass of hydrogel material in the mixed fluorescent hydrogel solution is 7 wt%. The mass concentration of horseradish peroxidase was 0.01 g / mL, the mass concentration of hydrogen peroxide was 100 mM / L, and the dosage of horseradish peroxidase and hydrogen peroxide relative to the mixed fluorescent hydrogel solution was 40 µL / mL.

2. The biogenic amine-responsive fluorescently changing hydrogel microneedle patch prepared by the method of claim 1.

3. The application of the biogenic amine-responsive fluorescently changing hydrogel microneedle patch of claim 2 in the identification and detection of biogenic amines.

4. A method for detecting the freshness of raw meat, characterized in that, The method involves inserting the microneedles of the biogenic amine-responsive fluorescent color-changing hydrogel microneedle patch of claim 2 into fresh meat, then removing it, and after the color stabilizes, taking a picture of the microneedle patch with a smartphone to collect RGB information and obtain G+B / (R+G+B) information; and using the G+B / (R+G+B) information to indicate the freshness of the fresh meat.

5. The method according to claim 4, characterized in that, When the G+B / (R+G+B) value is less than 0.60, the fresh meat is edible; when the G+B / (R+G+B) value is between 0.60 and 0.65, the fresh meat is of low quality and about to spoil; when the G+B / (R+G+B) value is greater than 0.65, the fresh meat is spoiled.

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