Microneedle patch containing alpha-naphthylamine carbon dots and application of microneedle patch in detection of nitrite by colorimetric-fluorescence dual-mode signals

Abstract

The invention discloses a microneedle patch containing alpha-naphthylamine carbon dots and application of the microneedle patch to detection of nitrite through colorimetric-fluorescence dual-mode signals, and belongs to the technical field of analytical chemistry. Alpha-naphthylamine is used as a carbon source and is prepared into the blue fluorescence alpha-naphthylamine carbon dots, the carbon dots have good color and fluorescence changes, and the blue fluorescence alpha-naphthylamine carbon dots are used as a color developing agent to be prepared into the microneedle patch so as to realize NO2-colorimetric-fluorescence dual-sensing application in food. The microneedle patch provided by the invention is used for carrying out nondestructive testing on a sample, whether nitrite exceeds the standard or not can be preliminarily judged through naked eyes, a colorimetric-fluorescence dual-mode signal is read through mobile phone photographing to obtain photographing data, and the content of nitrite in the sample can be accurately quantified after further processing; therefore, rapid and simple detection of nitrite in food is realized. Therefore, the invention has important application value and wide application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of analytical chemistry, and in particular to a microneedle patch containing α-naphthylamine carbon dots and application thereof in colorimetric-fluorescence dual-mode signal detection of nitrite. Background Art

[0002] Nitrite, a class of nitrogen-containing compounds, is a natural component found in water and food. Nitrite compounds can also be artificially added to food as food additives and preservatives, helping to enhance the flavor and color of meats, processed foods, and everyday products, including cheese, ham, and sausage. However, excessive nitrite intake can pose serious health risks. It reacts with hemoglobin to form methemoglobin, causing methemoglobinemia, which manifests as headaches, dizziness, nausea, and even death. Furthermore, long-term nitrite intake can react with amines in the body to form highly carcinogenic N-nitrosamines, increasing the risk of cancers such as colorectal and gastric cancer. Given concerns about food safety and public health, the development of sensitive and selective nitrite detection methods with direct readout signals is extremely necessary.

[0003] Traditional nitrite detection methods such as UV-visible spectroscopy, ion chromatography, capillary electrophoresis, chemiluminescence and electrochemical methods are highly sensitive and versatile, but their application in rapid on-site detection of food is still limited by complex and time-consuming pretreatment steps.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The purpose of the present invention is to provide a microneedle patch containing α-naphthylamine carbon dots and its application in colorimetric-fluorescence dual-mode signal detection of nitrite. The microneedle patch is used to perform non-destructive testing of samples, which does not require pretreatment of the samples. In addition, nitrite can be detected by reading the colorimetric-fluorescence dual-mode signal by taking a photo with a mobile phone, thereby performing rapid and accurate quantitative analysis of the samples.

[0006] The present invention is achieved in that:

[0007] In a first aspect, the present invention provides an α-naphthylamine carbon dot having an excitation wavelength of 335 nm and an emission wavelength of 440 nm.

[0008] In a second aspect, the present invention provides a method for preparing the above-mentioned α-naphthylamine carbon dots, which are prepared by carbonizing α-naphthylamine through microwave treatment, comprising the following steps: dissolving α-naphthylamine in an ethylene glycol solution, then subjecting the mixed solution to microwave treatment, and then purifying to obtain α-naphthylamine carbon dots.

[0009] In a third aspect, the present invention provides the use of the above-mentioned α-naphthylamine carbon dots in the colorimetric-fluorescence dual-mode signal detection of nitrite.

[0010] In a fourth aspect, the present invention provides a visualized microneedle patch for detecting nitrite, which comprises a hydrogel needle layer and a backing layer, wherein the backing layer contains α-naphthylamine carbon dots.

[0011] In a fifth aspect, the present invention provides a method for preparing the above-mentioned visualized microneedle patch, comprising:

[0012] Hyaluronic acid hydrogel is added to the mold, and after drying, concentration and photocuring, the hydrogel needle layer of the visualized microneedle patch is prepared; then a polyvinyl alcohol mixed hydrogel solution containing α-naphthylamine carbon dots is added to the mold, and the visualized microneedle patch is obtained after drying.

[0013] In the sixth aspect, the present invention provides an application of the above-mentioned visualized microneedle patch in the colorimetric-fluorescence dual-mode signal detection of nitrite, which quantitatively analyzes the nitrite in the sample by combining the color change and fluorescence intensity change of the microneedle patch with the RGB value of the color difference change read by mobile phone photography.

[0014] The present invention has the following beneficial effects:

[0015] The present invention uses α-naphthylamine as a carbon source to prepare blue fluorescent α-naphthylamine carbon dots, which have good color and fluorescence changes. It is used as a color developer to prepare a microneedle patch to achieve NO 2- Colorimetric-fluorescence dual sensing applications. The microneedle patch provided by this invention can be used to perform nondestructive testing on samples, allowing a preliminary visual assessment of nitrite levels. Using a mobile phone to capture the colorimetric-fluorescence dual-mode signal, the resulting data can be accurately quantified after further processing, enabling rapid and simple detection of nitrite in food. Therefore, this invention has significant application value and broad potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0017] Figure 1 This is a schematic diagram showing the principle of using α-naphthylamine carbon dots as a color developer to detect nitrite in the present invention;

[0018] Figure 2 This is the Raman spectroscopy analysis result of the α-naphthylamine carbon dots prepared in Example 1;

[0019] Figure 3 The Fourier transform infrared spectroscopy analysis results of the α-naphthylamine carbon dots prepared in Example 1;

[0020] Figure 4 The overall results of X-ray photoelectron spectroscopy (XPS) analysis of the α-naphthylamine carbon dots prepared in Example 1 are as follows;

[0021] Figure 5 The specific analysis results of the XPS analysis of α-naphthylamine carbon dots prepared in Example 1, wherein the leftmost is the C1s peak diagram; the middle is the N1s peak diagram; and the rightmost is the O1s peak diagram;

[0022] Figure 6 This is the fluorescence spectrum of α-naphthylamine carbon dots prepared in Example 1. The red line represents the excitation wavelength, the blue line represents the emission wavelength, and the inset is a photograph of NAP-CDs under sunlight and UV light.

[0023] Figure 7 The α-naphthylamine carbon dots with NO in Example 2 2- The detection results related to UV-visible spectra and fluorescence emission spectra with increasing concentrations from 0 to 300 μM;

[0024] Figure 8 The visualized microneedle patch prepared in Example 3;

[0025] Figure 9 The effect diagram of the visualized microneedle patch prepared in Example 3 under colorimetric and fluorescence detection;

[0026] Figure 10 This is the detection result of nitrite in the three samples in Example 4. DETAILED DESCRIPTION

[0027] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0028] Since existing nitrite detection methods in the food field have the defects of being unable to conduct non-destructive and rapid on-site detection and being complex and time-consuming, in order to overcome this problem, the present invention prepares a new microneedle patch. The microneedle patch mainly consists of two parts: a needle tip layer and a backing layer, wherein the backing layer contains α-naphthylamine carbon dots with good color and fluorescence changes.

[0029] In the present invention, α-naphthylamine carbon dots are blue fluorescent α-naphthylamine carbon dots (NAP-CDs) prepared by microwave method using α-naphthylamine as carbon source.

[0030] Specifically, the preparation method of the above-mentioned NAP-CDs is: dissolving α-naphthylamine (NAP) in an ethylene glycol solution, and then placing the mixed solution in a microwave oven to carbonize the NAP. The color of the solution changes from colorless to milky white, indicating that NAP-CDs have been formed; and then purifying the formed NAP-CDs to obtain NAP-CDs.

[0031] In some embodiments, the concentration of α-naphthylamine in the mixed solution is 2-3 mmol / L.

[0032] In some embodiments, the microwave treatment conditions are: power of 490W, time of 3 minutes.

[0033] In some embodiments, purification comprises dialysis using a dialysis bag with a molecular weight cut-off of 500 to 1000 Kd.

[0034] The NAP-CDs prepared by the above method have an excitation wavelength of 335 nm, an emission wavelength of 440 nm, and a quantum yield of 57.46%. It has been verified that the NAP-CDs of the present invention are based on the Griess reaction principle and can be applied to NO 2- Colorimetric-fluorescence dual-sensor signal detection, such as preparation for visual detection of NO 2- Microneedle patch.

[0035] The preparation method of the microneedle patch of the present invention is as follows:

[0036] S1. Completely dissolve hyaluronic acid (HAMA) in a phenyl (2,4,6-trimethylbenzoyl) phosphate lithium salt solution, and then remove bubbles by ultrasound after dissolution to obtain a HAMA hydrogel.

[0037] In some embodiments, the mass volume ratio of hyaluronic acid to phenyl (2,4,6-trimethylbenzoyl) lithium phosphate solution in the HAMA hydrogel is 15% to 20%, and the concentration of phenyl (2,4,6-trimethylbenzoyl) lithium phosphate in the HAMA hydrogel is 0.3% to 0.4% (g / mL).

[0038] S2. HAMA hydrogel was added to the mold, centrifuged to remove the foam, and then dried, concentrated, and photocured to prepare the hydrogel needle layer of the visualized microneedle patch.

[0039] In some embodiments, after the HAMA hydrogel is added to the mold, the centrifugation conditions are: centrifugation at a speed of 12,000 r / min for 5 minutes; the drying and concentration conditions are: temperature 37°C, time 6 to 8 hours; and the light curing conditions are: irradiation under a 365nm ultraviolet lamp for 150 to 180 seconds.

[0040] S3. Add polyvinyl alcohol (PVA) to the NAP-CDs solution, and then add p-aminobenzenesulfonic acid (Sa) and hydrochloric acid (HCl) to obtain a PVA mixed hydrogel solution.

[0041] In some embodiments, the PVA mixed hydrogel solution has a PVA concentration of 20% (g / mL), a Sa concentration of 1.5 mM, and a hydrochloric acid concentration of 0.9 mM.

[0042] S4. The mixed hydrogel solution containing NAP-CDs was added to the mold, and after drying, a visualized microneedle patch (NAP-CDs / HAMA / PVA) was obtained.

[0043] In some embodiments, the drying conditions of the mixed hydrogel are: temperature of 37° C. and time of 24 to 36 hours.

[0044] The above preparation method can be used to obtain NAP-CDs / HAMA / PVA visualized microneedle patch. The needle tip of the microneedle patch exhibits strong mechanical strength and water absorption properties, easily penetrates meat food and can transport liquid (nitrite) to the backing layer within 12 minutes. The NAP-CDs in the backing layer and NO 2- The reaction shows color change and fluorescence intensity change, such as Figure 1 As shown, the nitrite in the sample is quantitatively analyzed by color change and fluorescence intensity change combined with naked eye observation and / or mobile phone photography to read the RGB value of the color difference change.

[0045] Based on this, the present invention can also provide a method for detecting nitrite content in food, which comprises: directly inserting the above-mentioned microneedle patch into a sample, transmitting liquid through the needle layer, and allowing nitrite to flow into the needle layer along with the liquid, reacting with the color developer NAP-CDs, and showing color changes and fluorescence intensity changes. The nitrite content in the sample can be judged based on the color changes and fluorescence intensity changes: when the nitrite content in the food exceeds the national standard of 400 μM, the color of the microneedle patch changes to orange-red, and the excess can be clearly identified by the naked eye; the RGB value of the color difference change is read by taking a photo with a mobile phone, and then substituting it into a linear equation to obtain an accurate nitrite content value.

[0046] In some embodiments, the detection objects of the above detection method include food, preferably meat, meat products and pickles, and can also be extended to other samples that require rapid detection, such as ham, luncheon meat, well water, and dairy products.

[0047] The microneedle patch and nitrite content detection method provided by the present invention do not require sample pretreatment, and the detection method is simple and easy to operate. The method provided by the present invention can achieve rapid detection of nitrite in food.

[0048] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0049] Example 1

[0050] This embodiment is a method for preparing a NAP-CDs solution, and the specific steps are as follows:

[0051] (1) In a 50 mL conical flask, dissolve NAP in 13 mL of ethylene glycol solution to a final concentration of 2.0 mmol / L.

[0052] (2) The mixture was treated in a 490 W household microwave oven for 3 min to achieve carbonization of the NAP. After 3 min of carbonization, the color of the solution changed from colorless to milky white, indicating that NAP-CDs had been formed.

[0053] (3) The formed NAP-CDs were dialyzed for 12 h using a dialysis bag with a molecular weight of 500 Kd to remove ethylene glycol and unreacted NAP.

[0054] (4) The purified NAP-CDs were stored at 4°C for further characterization and use.

[0055] Characterization results:

[0056] Raman spectroscopy Figure 2 As shown, it shows that the synthesized NAP-CDs have a peak at 1364 cm -1 (D belt) and 1595cm -1 There are two obvious Raman peaks at (G band), which indicates that NAP-CDs have good crystallinity and graphite-like structure.

[0057] Fourier transform infrared spectroscopy (FT-IR) Figure 3 As shown, it is displayed at 3384cm -1 There is a broad peak at 1625cm, corresponding to the stretching vibration of OH and NH bonds. -1 The absorption peak at 1458 cm corresponds to C=C vibration. -1The absorption peak at 1084 cm-1 is attributed to the in-plane CH bending vibration of the aromatic ring. In addition, a peak at 1084 cm-1 is observed, which is attributed to the stretching vibration of CO and CN. A peak at about 772 cm-1 is also observed. -1 These findings confirm the existence of OH, NH, C=C, CN, and COC bonds in NAP-CDs.

[0058] XPS analysis results are as follows Figure 4 As shown in Figure 2, it shows that the main components of NAP-CDs are C, N and O. The detailed XPS spectrum of C1s (e.g. Figure 5 The main characteristic peak at 284.18 eV is attributed to the strong CC / C=C bond, which indicates the main feature of carbonization. The peak at 285.88 eV originates from the C-OH bond, while the peak at 288.18 eV corresponds to the CO / CN bond. Figure 5 The binding energy peaks of O1s (shown in the middle) are located at 398.18eV, 398.88eV and 399.78eV, which can be attributed to NH, CN and N-(C)3 groups, respectively. Figure 5 (shown on the right), three pairs of peaks are located at 531.28, 532.28, and 533.18 eV, corresponding to C═O, COC, and COH groups, respectively. High-resolution XPS spectra of C1s, N1s, and O1s indicate that the NAP-CDs are composed of C═C, CO / CN / NH, and COC bonds, consistent with the FT-IR results, indicating that the NAP-CDs were successfully prepared.

[0059] Figure 6 Figure 3 is the fluorescence spectrum of NAP-CDs, from which we can see that the excitation wavelength of NAP-CDs is 335 nm and the emission wavelength is 440 nm. According to the detection, the quantum yield of NAP-CDs is 57.46% and the fluorescence lifetime is 19.34 ns.

[0060] Example 2

[0061] This example verifies that the NAP-CDs prepared in Example 1 2- The effect of colorimetric-fluorescence dual sensing, the results are as follows Figure 7 As shown: Figure 7 (a) is NAP-CDs with NO 2- UV-visible spectra with increasing concentrations from 0 to 300 μM; (b) ΔA520nm and NO 2- (c) is the relationship between ΔA520 nm and NO in the range of 0 to 100 μM. 2- (d) The linear relationship between NAP-CDs and NO 2-Fluorescence emission spectra with increasing concentrations from 0 to 1000 μM; (e) is (F0-F) / F0 and NO 2- The relationship between the concentration of NO and the concentration of NO in the range of 0 to 60 μM is shown in Figure 2. 2- Linear relationship with concentration.

[0062] In colorimetric sensing, ΔA520nm is related to NO 2- The linear relationship was good in the concentration range of 0 to 100 μM (R 2 =0.998), the linear equation was Y=0.0117X+0.00297, and the LOD was 0.047 μM.

[0063] In fluorescence sensing, ΔF / F0 and NO 2- The linear relationship was good in the range of 0 to 60 μM (R 2 =0.985), the linear equation was Y=0.00729X+0.02506, and the LOD was 0.032 μM.

[0064] Example 3

[0065] This embodiment is a visual microneedle patch and its preparation method, and the specific steps are as follows:

[0066] 1. Preparation of HAMA Hydrogel Solution

[0067] (1) 0.06 g of lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP) was dissolved in 20 mL of ultrapure water as a photoinitiator and shaken at 45° C. for 30 min to obtain a 0.3% (mass-to-volume) LAP solution.

[0068] (2) 0.15 g of HAMA was added to 1 mL of LAP solution and placed at room temperature for 0.5 h to completely dissolve it, thereby obtaining a 15% (mass-to-volume) HAMA hydrogel solution.

[0069] (3) Ultrasonicate the HAMA hydrogel solution for 5 minutes to remove bubbles and set aside.

[0070] 2. Preparation of the Needle Layer of the Microneedle Patch

[0071] (1) 250 mL (15% mass volume ratio) of HAMA hydrogel solution was added to the PDMS mold, and then centrifuged at 12000 rpm for 5 min to remove foam.

[0072] (2) The solution in the PDMS mold was concentrated and dried in a constant temperature forced air drying oven at 37°C for 6 h, and then the HAMA solution was added again.

[0073] (3) Repeat the above steps three times to allow the hydrogel to fill the tip microcavity.

[0074] (4) Excess surface hydrogel solution was removed and the hydrogel in the tip was irradiated with a handheld UV lamp (365 nm) for 180 s to obtain a photocured MN needle layer.

[0075] 3. Microneedle Patch Preparation

[0076] (1) 1.0 g of PVA was added to 5 mL of the NAP-CDs solution obtained in Example 1 and magnetically stirred at 60° C. for 1 h to ensure complete dissolution, thereby obtaining a 20% (mass-volume ratio) NAP-CDs / PVA hydrogel solution.

[0077] (2) Add Sa and HCl to the above solution, with final concentrations of 1.5 mmol / L and 0.9 mmol / L, respectively.

[0078] (3) The mixed hydrogel solution (300 mL) was dropped onto a mold equipped with a pre-prepared MN tip, and the mold was dried at 37 °C for 24 h.

[0079] (4) The mold was peeled off to obtain the colorimetric and fluorescent hydrogel NAP-CDs / HAMA / PVA visualized microneedle patch, e.g. Figure 8 As shown, Figure 8 (a) and (b) are photos of the appearance structure; (c) is a detailed panoramic view of the microneedle; (d) is a fluorescence image of the microneedle patch; (e) and (f) are SEM images of the microneedle patch; (g) is the mechanical properties test results of the microneedle patch.

[0080] Figure 9 The colorimetric and fluorescence intensity related index detection of NAP-CDs / HAMA / PVA visual microneedle patch, (a) NAP-CDs / HAMA / PVA visual microneedle patch with NO 2- The colorimetric and fluorescence intensity changes of the concentration from 0 to 1000 μM, (b) is the R / (R+G+B) value and NO 2- Concentration linear relationship; (c) (F0-F) / F0 value and NO 2- Concentration linear relationship.

[0081] Under natural light, with NO 2- With the increase of concentration, the color of the microneedle patch changed from colorless to orange-red, and the R / (R+B+G) value of the backing layer was similar to that of NO 2- There is a good linear relationship (R 2 =0.988), the linear equation was Y=0.000218X+0.34936, and the LOD was 3.16 μM.

[0082] Under ultraviolet light, with the 2- As the concentration increases, the fluorescence intensity of the microneedle patch decreases, and the ΔF / F0 fluorescence value of the backing layer is consistent with that of NO 2- There is a good linear relationship (R 2 =0.997), the linear equation was Y=0.00058X+0.00342, and the LOD was 3.87 μM.

[0083] Example 4

[0084] According to national standards, the nitrite content in cured meat products, marinated meat products, and smoked and grilled meat products cannot exceed 30 mg / kg, or 400 μM. When the nitrite concentration of the microneedle patch of the present invention exceeds 400 μM, it can be clearly identified as exceeding the standard by the naked eye.

[0085] This example verifies the effectiveness of the microneedle patch prepared in Example 3 in detecting the concentration of nitrite in food, as follows:

[0086] The newly developed dual-model NAP / HAMA / PVA@MN patch demonstrated satisfactory sensitivity and selectivity, and its applicability was evaluated in real samples. Nitrite recovery was investigated by adding different concentrations of 0, 150, and 400 μM nitrite to pickled radish, sausage, and cured meat samples, according to the linear range.

[0087] When the amount of nitrite added to the pickled radish, sausage, and cured meat samples was 0 μM, the NAP / HAMA / PVA@MN patches appeared white. When the concentration of nitrite added to the pickled radish, sausage, and cured meat samples increased to 150 μM and 400 μM, respectively, the NAP / HAMA / PVA@MN patches turned light orange at 150 μM and orange-red at 400 μM. Furthermore, the fluorescence intensity of the NAP / HAMA / PVA@MN patches in the pickled radish, sausage, and cured meat samples decreased with increasing nitrite concentration.

[0088] Test results such as Figure 10 As shown in the figure, (a) is the recovery rate test result of nitrite spiked in pickled radish; (b) is the recovery rate test result of nitrite spiked in sausage; (c) is the recovery rate test result of nitrite spiked in bacon.

[0089] Table 1 shows the test results statistics:

[0090] Table 1 Test results of three kinds of food

[0091]

[0092] According to the above results, the NO in pickled radish, sausage and bacon was successfully detected by combining the color change and fluorescence intensity change of the microneedle patch with the RGB value of the color difference change read by mobile phone photography. 2- Quantitative analysis was performed, and the recoveries were 81.1% to 116.24% with RSDs not exceeding 2.56%.

[0093] In summary, the present invention realizes the NO 2- The colorimetric-fluorescence dual-mode detection has high anti-interference ability, and the mutual verification of the two methods improves the accuracy and reliability of the results. The successful application of NAP-CDs hydrogel microneedles provides a reference for the detection of NO in food. 2- It provides a reliable technical means for non-destructive monitoring.

[0094] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. An α-naphthylamine carbon dot, characterized in that Its excitation wavelength is 335nm and its emission wavelength is 440nm.

2. The method for preparing α-naphthylamine carbon dots according to claim 1, wherein: The α-naphthylamine carbon dots are prepared by carbonizing α-naphthylamine through microwave treatment, comprising the following steps: dissolving α-naphthylamine in an ethylene glycol solution, then subjecting the mixed solution to microwave treatment, and then purifying the mixture to obtain the α-naphthylamine carbon dots; Preferably, the concentration of α-naphthylamine in the mixed solution is 2 to 3 mmol / L; Preferably, the microwave treatment conditions are: power 490w, time 3min; Preferably, the purification comprises dialysis, and the molecular weight cut-off of the dialysis bag used for dialysis is 500-1000Kd.

3. Use of the α-naphthylamine carbon dots as claimed in claim 1 in colorimetric-fluorescence dual-mode signal detection of nitrite.

4. A visual microneedle patch for detecting nitrite, characterized in that: The invention comprises a hydrogel needle layer and a backing layer, wherein the backing layer contains the α-naphthylamine carbon dots.

5. The method for preparing a visualized microneedle patch according to claim 4, wherein: include: The hyaluronic acid hydrogel is added into the mold, and the hydrogel needle layer of the visualized microneedle patch is prepared through drying, concentration and light curing; Then, the polyvinyl alcohol mixed hydrogel solution containing the α-naphthylamine carbon dots is added into the mold, and the visualized microneedle patch is obtained after drying.

6. The preparation method according to claim 5, characterized in that The preparation method of the hyaluronic acid hydrogel comprises: completely dissolving hyaluronic acid in a phenyl (2,4,6-trimethylbenzoyl) phosphate lithium salt solution to obtain the hyaluronic acid hydrogel; Preferably, the mass volume ratio of hyaluronic acid to phenyl (2,4,6-trimethylbenzoyl) lithium phosphate solution in the hyaluronic acid hydrogel is 15% to 20% (g / mL), and the concentration of phenyl (2,4,6-trimethylbenzoyl) lithium phosphate in the hyaluronic acid hydrogel is 0.3% to 0.4% (g / mL).

7. The preparation method according to claim 5, characterized in that The conditions for drying and concentrating the hyaluronic acid hydrogel after adding it into the mold are: temperature 37° C., time 6 to 8 hours; the conditions for light curing are: irradiation under a 365 nm ultraviolet lamp for 180 seconds.

8. The preparation method according to claim 5, characterized in that The preparation method of the polyvinyl alcohol mixed hydrogel comprises: adding polyvinyl alcohol to an α-naphthylamine carbon dot solution, and then adding p-aminobenzenesulfonic acid and hydrochloric acid to obtain the polyvinyl alcohol mixed hydrogel solution; Preferably, the concentration of polyvinyl alcohol in the polyvinyl alcohol mixed hydrogel solution is 20% (g / mL), the concentration of p-aminobenzenesulfonic acid is 1.5 mM, and the concentration of hydrochloric acid is 0.9 mM; Preferably, the drying conditions for adding the mixed hydrogel are: temperature of 37° C. and time of 24 to 36 hours.

9. Use of the visualized microneedle patch according to claim 4 or the visualized microneedle patch obtained by the preparation method according to any one of claims 5 to 8 in colorimetric-fluorescence dual-mode signal detection of nitrite, characterized in that: The nitrite in the sample is quantitatively analyzed by combining the color change and fluorescence intensity change of the microneedle patch with naked eye observation and / or mobile phone photography to read the RGB value of the color difference change.

10. Use according to claim 9, characterized in that The sample includes food; preferably, the sample is meat, meat products and pickles.

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