Preparation method of SERS microneedle patch and application thereof in stroke risk monitoring

Abstract

The application discloses a preparation method of a SERS microneedle patch and application thereof in stroke risk monitoring, and belongs to the technical field of biological sensing, wherein the SERS microneedle patch comprises a SERS substrate and a porous microneedle patch, wherein the SERS substrate is composed of nanoparticles with a core-inner label molecule-shell structure and porous alumina. The SERS substrate is located at the bottom of the porous microneedle patch composed of a porous microneedle array, the porous microneedle patch is used for extracting tissue fluid and conveying the tissue fluid into the SERS substrate, and the SERS substrate is used for realizing quantitative detection of homocysteine. The application fuses surface enhanced Raman scattering technology and microneedle technology, and provides a solution for stroke risk monitoring. Through early discovery and timely treatment, the survival rate of stroke patients can be significantly improved.

Description

Technical Field

[0001] This invention belongs to the field of biosensing technology and relates to a method for preparing SERS microneedle patches and their application in stroke risk monitoring. Background Technology

[0002] Stroke is a life-threatening cerebrovascular disease with high incidence, recurrence, and mortality rates. It ranks third in incidence among human diseases worldwide and has become the leading cause of death and disability in adults. Homocysteine ​​(Hcy) is a metabolite of cystine and methionine. Under normal conditions, plasma Hcy concentration is approximately 5-15 μmol / L. A concentration >15 μmol / L is termed hyperhomocysteinemia. Hyperhomocysteinemia promotes atherosclerosis and thrombosis in susceptible animal models, and high Hcy levels are associated with early neurological deterioration in acute ischemic stroke. Current research confirms a close correlation between high Hcy levels and coronary heart disease and stroke. Higher Hcy concentrations indicate a higher risk of cardiovascular and cerebrovascular diseases.

[0003] Currently, imaging examinations are essential for determining stroke treatment plans. However, these examinations are generally conducted after admission, i.e., after the onset of symptoms. Time is extremely precious after the onset of symptoms; once the treatment window is missed, not only does the treatment effect decrease significantly, but the mortality rate also increases dramatically. Although rapid response channels and treatment centers for stroke have been established, the current medical practice of waiting until arterial stenosis or even cerebral infarction occurs before providing treatment is no longer sufficient to meet the medical needs. There is an urgent need for an early stroke monitoring method that reflects health conditions before physical signs of discomfort appear. However, a low-cost method that can accurately and efficiently monitor stroke risk remains a gap that urgently needs to be filled.

[0004] Currently, methods for detecting homocysteine ​​include high-performance liquid chromatography, mass spectrometry, cyclic enzymatic methods, immunoassay, and chemiluminescence detection, but none of these methods can provide real-time testing.

[0005] Currently, testing homocysteine ​​levels in the blood usually requires a blood sample, with the most common method being a blood draw. However, this process is time-consuming, involves a large incision, and requires a trip to the hospital for blood collection.

[0006] Hcy has a small scattering cross section and a very weak Raman signal, so direct SERS of Hcy cannot achieve good results. Summary of the Invention

[0007] To address the aforementioned problems, the present invention aims to integrate surface-enhanced Raman scattering (SERS) technology and microneedle technology to propose a solution for stroke monitoring; it can quantify the concentration of homocysteine ​​in serum in a minimally invasive manner, ultimately enabling the detection of stroke risk.

[0008] The technical solution of the present invention is: a method for preparing a SERS microneedle patch, wherein the SERS microneedle patch comprises a SERS substrate and a porous microneedle patch;

[0009] The SERS substrate is composed of nanoparticles with a core-internal standard molecule-shell structure and porous alumina. The porous microneedle patch includes several evenly distributed microneedle tips, and each microneedle tip has a hole.

[0010] The SERS substrate is located at the bottom of the porous microneedle array. The porous microneedle patch is used for minimally invasive and painless extraction of tissue fluid and delivery to the SERS substrate. The SERS substrate is used to achieve quantitative detection of homocysteine.

[0011] The preparation steps are as follows:

[0012] Step (1), Construction of the SERS substrate:

[0013] (1.1): Mix the prepared H2O, HAuCl4 and NaOH in a shaker and let it stand; add sodium sulfite solution to the standing mixture and shake continuously to obtain solution C;

[0014] (1.2): Mix the prepared H2O, CTAC, NPs, 4-MBA and AA to obtain solution D. Inject solution C into solution D and shake continuously.

[0015] After shaking, the solution was centrifuged, the supernatant was removed and discarded, the precipitate was retained, and CTAC was added to obtain a solution of nanoparticles with a core-internal standard molecule-shell structure.

[0016] (1.3): The nanoparticles with core-internal standard molecular-shell structure obtained by filtration of the solution of core-internal standard molecular-shell structured nanoparticles were assembled onto the prepared porous alumina surface to obtain the SERS substrate.

[0017] Step (2), Preparation of SERS microneedle patches:

[0018] (2.1): Under water bath conditions, the prepared polyethylene glycol 10000 and 2-methoxyethanol were mixed to obtain solution A;

[0019] (2.2): Take a clean magnetic rotor and put it into a brown glass bottle. Cut some tin foil and wrap it around the side of the glass bottle. Take glycidyl methacrylate and stir it.

[0020] Then add the prepared trimethylolpropane trimethacrylate, triethylene glycol dimethacrylate and 1-hydroxycyclohexylacetone, and heat in a water bath to obtain solution B;

[0021] Mix solutions A and B, stir in a water bath overnight, and store in a refrigerator;

[0022] (2.3): Take the above frozen mixture, heat it to a liquid state, and stir until homogeneous to obtain a mixed solution;

[0023] (2.4): Take out 500 μL of the mixed solution, inject it into the PDMS mold, place it in a vacuum drying oven and evacuate it. Then take out the mold and use a pipette to remove the air bubbles in the mold. Repeat this step twice.

[0024] The prepared SERS substrate was immersed in phthalaldehyde solution, then removed and air-dried.

[0025] The mixture in the mold is irradiated from the top with a UV lamp to cure the mixture at the bottom first. Then, the air-dried SERS substrate is placed horizontally on the surface of the mixture that is not completely cured on the top. The mixture is then irradiated from the top with a UV lamp to cure it completely, resulting in a SERS microneedle patch with polyethylene glycol 10000 not removed.

[0026] (2.5): Take out the SERS microneedle patch that has not had polyethylene glycol 10000 removed, put it into a mixture of ultrapure water and methanol for water bath to remove polyethylene glycol 10000, take it out after the water bath, seal and dry it to finally obtain the SERS microneedle patch.

[0027] Further, in step (1.1), the amount of H2O added is 15-20 mL, the amount of HAuCl4 added is 0.4-0.5 mL, the concentration of NaOH is 100 mM, and the amount added is 0.4-0.6 mL; preferably, the amount of H2O added is 17.1 mL, the amount of HAuCl4 added is 0.4 mL, and the amount of NaOH added is 0.5 mL;

[0028] The settling time is 2 to 3 minutes, preferably 2 minutes;

[0029] The sodium sulfite solution has a concentration of 10 mM and is added in an amount of 2-3 mL, preferably 2 mL.

[0030] Further, in step (1.2), the amounts of the reagents added are as follows: H2O 15-20 mL, 100 mM CTAC 1-2 mL, 10-fold concentrated NPs 0.2-0.5 mL, 10 mM CTAC 1.5-2 mL, 10 -61-2 mL of M 4-MBA, 0.2-0.5 mL of 100 mM AA; preferably, 15.2 mL of H2O, 1.8 mL of 100 mM CTAC, 0.2 mL of 10x concentrated NPs, 1.8 mL of 10 mM CTAC, 10 -6 0.8 mL of M 4-MBA, 0.2 mL of 100 mM AA;

[0031] The injection rate is 1 mL / min, and the shaking time is 2–3 h; preferably, the shaking time is 2 h.

[0032] The centrifugation conditions are 4000 r / min for 10–20 min; preferably, the time is 10 min.

[0033] The concentration of the added CTAC is 10 mM, and the amount added is 2 to 5 mL; preferably, the amount added is 2 mL.

[0034] Furthermore, in step (1.3), the obtained core-internal standard molecule-shell structured nanoparticles have a diameter of 100 nm, with an Ag shell and an Au core, and an internal standard molecule between the shell and the core, wherein the internal standard molecule is 4-MBA.

[0035] The porous alumina has a pore size of 80 nm.

[0036] Furthermore, in step (2.1), the mixture is prepared at a rate of 400 r / min under a water bath at 100°C;

[0037] The amount of polyethylene glycol 10000 added is 2-3g, and the amount of 2-methoxyethanol added is 10-12mL; preferably, the amount of polyethylene glycol 10000 added is 2g, and the amount of 2-methoxyethanol added is 10mL.

[0038] Further, in step (2.2), the amount of glycidyl methacrylate added is 2.5 to 5 mL; preferably, the amount of glycidyl methacrylate added is 2.5 mL, and the stirring condition is 400 r / min;

[0039] The amount of added trimethylolpropane trimethacrylate is 1.5-2 mL, the amount of added triethylene glycol dimethacrylate is 4-5 mL, and the amount of added 1-hydroxycyclohexylacetone is 25-30 mg; preferably, the amount of added trimethylolpropane trimethacrylate is 1.545 mL, the amount of added triethylene glycol dimethacrylate is 3.925 mL, and the amount of added 1-hydroxycyclohexylacetone is 25 mg.

[0040] Solution B is prepared by heating in a water bath at 65°C for 2-3 hours; preferably, solution B is prepared by heating in a water bath at 65°C for 3 hours.

[0041] The mixed solutions A and B were prepared under the following conditions: stirring in a water bath at 65°C at 400 rpm and stored in a refrigerator at 4°C.

[0042] Furthermore, in step (2.3), the stirring conditions are 400 r / min for 5 to 10 min; preferably, the stirring conditions are 400 r / min for 5 min.

[0043] The porous microneedle patch has a side length of 1cm and a main body thickness of 2mm. One side of the patch has a microneedle array consisting of 144 microneedles with a length of 500μm and a bottom diameter of 240μm. The micropore diameter of the porous microneedle is 2μm.

[0044] Furthermore, in step (2.4), the vacuum drying oven environment is 35°C, the vacuum pressure is 0.6 to 0.8 atmospheres, and the vacuum time is 15 to 20 minutes; preferably, the vacuum drying oven environment is 35°C, the vacuum pressure is 0.7 atmospheres, and the vacuum time is 15 minutes.

[0045] The concentration of the phthalaldehyde solution is 10. -4 M;

[0046] Irradiate with UV lamp for 15-20 seconds, place the SERS substrate, and then irradiate for 2-5 minutes; preferably, irradiate with UV lamp for 15 seconds, place the SERS substrate, and then irradiate for 2 minutes.

[0047] Furthermore, in step (2.5), the preparation conditions for the mixture are a water bath at 65°C for 12-24 hours, the amount of ultrapure water added is 5-7 mL, and the amount of methanol added is 5-6 mL; preferably, the preparation conditions for the mixture are a water bath at 65°C for 12 hours, the amount of ultrapure water added is 5 mL, and the amount of methanol added is 5 mL.

[0048] The sealed drying conditions are 50°C for 1-2 hours; preferably, the sealed drying conditions are 50°C for 1 hour.

[0049] Furthermore, an application of a prepared SERS microneedle patch in stroke risk monitoring was investigated.

[0050] Furthermore, the SERS microneedle patch extracts interstitial fluid and delivers it to the SERS substrate. During this process, homocysteine ​​undergoes a specific reaction with phthalaldehyde. The concentration of homocysteine ​​is calculated based on the detected SERS signal of the homocysteine-phthalaldehyde adduct to assess the risk of stroke.

[0051] Surface-enhanced Raman scattering (SERS) technology has the characteristics of being fast, simple, capable of trace detection, and fingerprint recognition, and has been widely used in the detection of environmental pollutants, food and drinking water, chemical synthesis, and biomedicine.

[0052] Microneedling is an innovative biomedical engineering technique that uses tiny needle-like structures to penetrate the surface of a phantom for drug delivery, biomarker sampling, or vaccination. The key advantage of this technique is its minimally invasive nature, which can reduce pain and discomfort while improving the effectiveness and safety of treatment.

[0053] The beneficial effects of this invention are: 1. This invention integrates SERS technology and microneedle technology, proposing a highly efficient, accurate, and cost-effective solution for stroke monitoring; 2. This invention is easy to operate, and even non-professionals can quickly get started, making it promising for POCT and home testing; 3. This invention can be used in various minimally invasive testing scenarios, with good application prospects. It can detect not only Hcy, but also any substance that can produce SERS characteristic peaks. Only the substrate needs to be changed to detect it through the same process, making it highly universal and applicable to a wide range of scenarios; 4. This invention uses an auxiliary molecule, OPA, to enhance the Raman spectrum of Hcy. After the specific reaction between OPA and Hcy forms Hcy-OPA, the resulting Hcy-OPA adduct has a larger Raman scattering cross section than the original Hcy, and the -SH group of Hcy-OPA can be easily anchored on the surface of the SERS substrate, thus imparting high sensitivity. Attached Figure Description

[0054] Figure 1 This is a schematic diagram of the overall structure of the present invention;

[0055] Figure 2 This is a schematic diagram of the porous microneedle patch structure in this invention;

[0056] Figure 3 This is a schematic diagram of the SERS substrate structure in this invention;

[0057] Figure 4 This is a schematic diagram of the core-internal standard molecule-shell structure of the nanoparticles in the SERS substrate of this invention;

[0058] In the figure: 1. Nanoparticles with a core-internal standard molecule-shell structure; 2. Porous alumina; 3. Porous microneedle patch; 4. Microneedle tip; 5. Pore; 6. Ag shell; 7. Au core; 8. Internal standard molecule. Detailed Implementation

[0059] The specific technical solution of the present invention will be further described in detail below with reference to specific examples.

[0060] As shown in the figure, the present invention provides a method for preparing a SERS microneedle patch, wherein the SERS microneedle patch comprises two parts: a SERS substrate and a porous microneedle patch 3.

[0061] The SERS substrate is composed of core-internal standard molecule-shell structured nanoparticles 1 and porous alumina 2, and is located at the bottom of a porous microneedle patch 3 composed of a porous microneedle array.

[0062] The porous microneedle patch 3 includes a plurality of evenly distributed microneedle tips 4, and each microneedle tip 4 has a hole 5.

[0063] The porous microneedle patch 3 is used to extract tissue fluid and deliver it to the SERS substrate, which is used to achieve quantitative detection of homocysteine.

[0064] Furthermore, the nanoparticle 1 with the core-internal standard molecule-shell structure has a diameter of 100 nm, its outer shell is an Ag shell 6, its inner core is an Au core 7, and the inner core is an internal standard molecule 8 between the outer shell and the inner core. The internal standard molecule 8 is 4-MBA; the porous alumina 2 has a pore size of 80 nm.

[0065] Furthermore, the porous microneedle patch 3 has a side length of 1cm and a main body thickness of 2mm; one side of the patch has a microneedle array composed of 144 microneedles, with a microneedle length of 500μm and a microneedle bottom diameter of 240μm; the diameter of the pores 5 of the porous microneedle is 2μm.

[0066] Furthermore, the preparation steps are as follows:

[0067] I. Preparation steps of SERS substrate:

[0068] Step (1.1): Mix the prepared H2O, HAuCl4 and NaOH, place them in a shaker, shake rapidly and let stand;

[0069] Sodium sulfite solution was added to the settled mixture and shaken to obtain solution C;

[0070] Step (1.2): Mix H2O, CTAC, NPs, 4-MBA and AA to obtain solution D, inject solution C into solution D while shaking continuously;

[0071] After shaking, the solution was centrifuged, the supernatant was removed and discarded, leaving only the precipitate. CTAC was added to obtain a nanoparticle solution (a nanoparticle solution with a core-internal standard molecule-shell structure).

[0072] Step (1.3): The nanoparticles 1 with core-internal standard molecular-shell structure obtained by filtration of the nanoparticle solution are assembled onto the surface of the prepared porous alumina 2 to obtain the SERS substrate.

[0073] Further, in step (1.1), the amount of H2O added is 15-20 mL, the amount of HAuCl4 added is 0.4-0.5 mL, the concentration of NaOH is 100 mM, and the amount added is 0.4-0.6 mL; preferably, the amount of H2O added is 17.1 mL, the amount of HAuCl4 added is 0.4 mL, the concentration of NaOH is 100 mM, and the amount added is 0.5 mL;

[0074] The settling time is 2 to 3 minutes, preferably 2 minutes;

[0075] The sodium sulfite solution has a concentration of 10 mM and is added in an amount of 2-3 mL, preferably 2 mL.

[0076] Further, in step (1.2), the amounts of the reagents added are as follows: H2O 15-20 mL, 100 mM CTAC 1-2 mL, 10-fold concentrated NPs 0.2-0.5 mL, 10 mM CTAC 1.5-2 mL, 10 -6 1-2 mL of 4-MBA (M), 0.2-0.5 mL of 100 mM AA; preferably, 15.2 mL of H2O, 1.8 mL of 100 mM CTAC, 0.2 mL of 10x concentrated NPs, 1.8 mL of 10 mM CTAC, 10 -6 0.8 mL of M 4-MBA, 0.2 mL of 100 mM AA;

[0077] The injection rate is 1 mL / min, and the shaking time is 2–3 h; preferably, the shaking time is 2 h.

[0078] The centrifugation conditions are 4000 r / min for 10–20 min; preferably, the time is 10 min.

[0079] The concentration of the added CTAC is 10 mM, and the amount added is 2 to 5 mL; preferably, the amount added is 2 mL.

[0080] Furthermore, in step (1.3), the obtained core-internal standard molecule-shell structured nanoparticle 1 has a diameter of 100 nm, its outer shell is an Ag shell, its inner core is an Au core, and the inner standard molecule 8 is between the outer shell and the inner core. The inner standard molecule 8 is 4-MBA.

[0081] The porous alumina 2 has a pore size of 80 nm.

[0082] Further steps in the preparation of SERS microneedle patches:

[0083] Step (2.1): Under water bath conditions, polyethylene glycol 10000 and 2-methoxyethanol were mixed to obtain solution A;

[0084] Step (2.2): Take a clean magnetic rotor, put it into a brown glass bottle, cut some tin foil, wrap it around the side of the glass bottle, and start stirring with glycidyl methacrylate.

[0085] Add trimethylolpropane trimethacrylate, triethylene glycol dimethacrylate and 1-hydroxycyclohexylacetone, mix and heat in a water bath to obtain solution B;

[0086] Mix solutions A and B, stir in a water bath overnight, and store in a refrigerator;

[0087] Step (2.3): Take the above frozen mixture, heat it to a liquid state, and stir it evenly to obtain a mixed solution;

[0088] Step (2.4): Take out 500 μL of the mixed solution and inject it into the PDMS mold, then put it into the vacuum drying oven for vacuum drying. Then take out the mold and use a pipette to remove air bubbles. Repeat this step twice.

[0089] The prepared SERS substrate was immersed in o-phthalaldehyde solution (OPA solution), then removed and air-dried.

[0090] The mixture in the mold is irradiated from the top with a UV lamp to cure the mixture at the bottom first. Then, the air-dried SERS substrate is placed horizontally on the surface of the mixture that is not completely cured on top. The mixture is then irradiated from the top with a UV lamp to completely cure it, thus obtaining a SERS microneedle patch with polyethylene glycol 10000 not removed.

[0091] Step (2.5): Take out the SERS microneedle patch that has not had polyethylene glycol 10000 removed, put it into a mixture of ultrapure water and methanol for water bath to remove polyethylene glycol 10000, take it out after the water bath is completed, seal and dry it to finally obtain the SERS microneedle patch.

[0092] Furthermore, in step (2.1), the mixture preparation conditions are as follows: mixing at a rate of 400 r / min under a water bath at 100°C;

[0093] The amount of polyethylene glycol 10000 added is 2-3g, and the amount of 2-methoxyethanol added is 10-12mL; preferably, the amount of polyethylene glycol 10000 added is 2g, and the amount of 2-methoxyethanol added is 10mL.

[0094] Further, in step (2.2), the amount of glycidyl methacrylate added is 2.5 to 5 mL; preferably, the amount of glycidyl methacrylate added is 2.5 mL, and the stirring condition is 400 r / min;

[0095] The amount of added trimethylolpropane trimethacrylate is 1.5-2 mL, the amount of added triethylene glycol dimethacrylate is 4-5 mL, and the amount of added 1-hydroxycyclohexylacetone is 25-30 mg; preferably, the amount of added trimethylolpropane trimethacrylate is 1.545 mL, the amount of added triethylene glycol dimethacrylate is 3.925 mL, and the amount of added 1-hydroxycyclohexylacetone is 25 mg.

[0096] Solution B is prepared by heating in a water bath at 65°C for 2-3 hours; preferably, solution B is prepared by heating in a water bath at 65°C for 3 hours.

[0097] The mixed solutions A and B were prepared under the following conditions: stirring in a water bath at 65°C at 400 rpm and stored in a refrigerator at 4°C.

[0098] Furthermore, in step (2.3), the stirring conditions are 400 r / min for 5 to 10 min; preferably, the stirring conditions are 400 r / min for 5 min.

[0099] The porous microneedle patch 3 has a side length of 1cm and a main body thickness of 2mm; one side of the patch has a microneedle array consisting of 144 microneedles, with a microneedle length of 500μm and a microneedle bottom diameter of 240μm; the micropore diameter of the porous microneedle is 2μm.

[0100] Furthermore, in step (2.4), the vacuum drying oven environment is 35°C, the vacuum pressure is 0.6 to 0.8 atmospheres, and the vacuum time is 15 to 20 minutes; preferably, the vacuum drying oven environment is 35°C, the vacuum pressure is 0.7 atmospheres, and the vacuum time is 15 minutes.

[0101] The concentration of the phthalaldehyde solution is 10. -4 M;

[0102] Irradiate with UV lamp for 15-20 seconds, place the SERS substrate, and then irradiate for 2-5 minutes; preferably, irradiate with UV lamp for 15 seconds, place the SERS substrate, and then irradiate for 2 minutes.

[0103] Furthermore, in step (2.5), the preparation conditions for the mixture are a water bath at 65°C for 12-24 hours, the amount of ultrapure water added is 5-7 mL, and the amount of methanol added is 5-6 mL; preferably, the preparation conditions for the mixture are a water bath at 65°C for 12 hours, the amount of ultrapure water added is 5 mL, and the amount of methanol added is 5 mL.

[0104] The sealed drying conditions are 50°C for 1-2 hours; preferably, the sealed drying conditions are 50°C for 1 hour.

[0105] This invention provides a SERS microneedle patch, which is prepared using any of the preparation methods described above.

[0106] This invention provides an application of the above-mentioned SERS microneedle patch, which is used for stroke risk monitoring.

[0107] Furthermore, in stroke risk monitoring, interstitial fluid is extracted through SERS microneedle patches and delivered to the SERS substrate. During this process, homocysteine ​​undergoes a specific reaction with phthalaldehyde. The concentration of homocysteine ​​is calculated based on the detected SERS signal of the homocysteine-phthalaldehyde adduct to assess the risk of stroke.

[0108] Example 1

[0109] Preparation of SERS microneedle patches:

[0110] (1) Preparation of SERS substrate: First, prepare the gold growth solution: Add 17.1 mL H2O, 0.4 mL HAuCl4 and 0.5 mL 100 mM NaOH to a beaker. After adding sodium hydroxide, shake quickly and let stand for two minutes. Immediately after two minutes, add 2 mL 10 mM sodium sulfite and shake evenly. Next, prepare the nanoparticles: First, mix 15.2 mL H2O, 2 mL 100 mM CTAC, 0.2 mL NPs (10 times concentrated), 1.8 mL 10 mM CTAC, and 0.8 mL 10 mM NaOH. -6 M 4-MBA and 0.2 mL 100 mM AA. Inject 20 mL of gold growth solution into the above-prepared solution at a rate of 1 mL / min, while continuously shaking for two hours; centrifuge the shaken solution at 4000 r / min for 10 min; aspirate and discard the supernatant, leaving only the precipitate; add 2 mL 10 mM CTAC; finally, assemble the core-internal standard molecule-shell structured nanoparticles 1 and 80 nm porous alumina 2 by vacuum filtration;

[0111] (2) Preparation of SERS microneedle patches: First, prepare the microneedle preparation mixture: Take 10000 g of polyethylene glycol and 10 mL of 2-methoxyethanol. Add the weighed 10000 g of polyethylene glycol to 2-methoxyethanol and mix. Then, mix at 400 r / min in a 100℃ water bath. Take a clean magnetic rotor and place it in a brown glass bottle. Cut tin foil and wrap it around the side of the glass bottle. Take 2.5 mL of glycidyl methacrylate and start stirring at 400 r / min. Add 1.545 mL of trimethylolpropane trimethacrylate and triethylene glycol dimethyl acrylate. 3.925 mL of ester and 25 mg of 1-hydroxycyclohexylacetone were added; the mixture was heated in a water bath at 65°C for 3 hours; the two solutions were mixed and stirred overnight at 400 rpm in a water bath at 65°C, and stored in a refrigerator at 4°C; next, microneedles were prepared: the mixture was heated to liquid state and stirred at 400 rpm for 5 minutes; 500 μL of the mixture was taken out and injected into a PDMS mold; the mold was placed in a vacuum drying oven at 35°C and evacuated to 0.7 atm for 15 minutes; the mold was removed and air bubbles were removed with a pipette; steps three and four were repeated twice; the SERS substrate was immersed in 10 -4 Remove the SERS microneedle patch from the OPA solution and air dry it. Use a UV lamp to irradiate the mold from the top to solidify the microneedles. After irradiation for 15 seconds, place the air-dried SERS substrate on the mold. Continue to irradiate the mold from the top with a UV lamp for 2 minutes. Remove the SERS microneedle patch and place it in a mixture of 5 mL ultrapure water and 5 mL methanol. Incubate in a water bath at 65°C for 12 hours. After the water bath, remove the SERS microneedle patch and dry it in a sealed container at 50°C for 1 hour.

Claims

1. A method for preparing a SERS microneedle patch, characterized by The SERS microneedle patch comprises a SERS substrate and a porous microneedle patch; The SERS substrate is composed of nanoparticles with a core-inner marker molecule-shell structure and porous alumina, and the porous microneedle patch comprises a plurality of microneedle needles arranged uniformly and a hole formed on each microneedle needle; The preparation steps are as follows: Step (1), construction of the SERS substrate: (1.1), mix the prepared H2O, HAuCl4 and NaOH, then place them in a shaking machine for shaking and standing; Add sodium sulfite solution to the standing mixed solution, continue to shake and mix, and obtain solution C; (1.2), mix the prepared H2O, CTAC, NPs, 4-MBA and AA to obtain solution D, inject the prepared solution C into solution D, and continue to shake; Centrifuge the shaken solution, discard the supernatant, retain the precipitate, and add CTAC to obtain a solution of nanoparticles with a core-inner marker molecule-shell structure; (1.3), assemble the nanoparticles with a core-inner marker molecule-shell structure in the solution of nanoparticles with a core-inner marker molecule-shell structure to the surface of the prepared porous alumina by suction filtration, thereby obtaining the SERS substrate; Step (2), preparation of the SERS microneedle patch: (2.1), mix the prepared polyethylene glycol 10000 and 2-methoxyethanol under water bath condition to obtain solution A; (2.2), take a clean magnetic rotor into a brown glass bottle, wrap a piece of tin paper around the side of the glass bottle, and take glycidyl methacrylate for stirring; Then add the prepared trimethylolpropane trimethylacrylate, triethylene glycol dimethyl acrylate and 1-hydroxycyclohexyl ketone for mixing, and obtain solution B by water bath heating; Mix solution A and solution B, and store them in a refrigerator after water bath stirring overnight; (2.3), heat the above-mentioned frozen mixture to a liquid state, stir uniformly, and obtain a mixed solution; (2.4), take 500 microliters of the mixed solution into a PDMS mold, then place the mold in a vacuum drying box for vacuum drying, take out the mold, and use a pipette gun to suck out the bubbles, and repeat the step twice; Soak the prepared SERS substrate in an o-phthaldehyde solution, and take it out for air drying; Use a UV lamp to irradiate the mixed solution in the mold from the top, so that the mixed solution at the bottom is solidified first, then place the air-dried SERS substrate horizontally on the surface of the mixed solution which is not completely solidified at the top, continue to irradiate the mixed solution from the top using the UV lamp, and completely solidify the mixed solution, thereby obtaining a SERS microneedle patch without removing polyethylene glycol 10000; (2.5), take out the SERS microneedle patch without removing polyethylene glycol 10000, place it in a mixed solution containing ultrapure water and methanol for water bath to remove polyethylene glycol 10000, take it out after water bath, and dry it in a sealed state, finally obtaining the SERS microneedle patch.

2. The method of claim 1, wherein the SERS microneedle patch is prepared by the steps of: In step (1.1), the addition amount of H2O is 15-20 mL, the addition amount of HAuCl4 is 0.4-0.5 mL, the concentration of NaOH is 100 mM, and the addition amount is 0.4-0.6 mL; The standing time is 2-3 min; The concentration of the sodium sulfite solution is 10 mM, and the added amount is 2-3 mL.

3. The method for preparing a SERS microneedle patch according to claim 1, characterized in that, In step (1.2), the added amounts of reagents are as follows: H2O 15-20 mL, 10-6 M 4-MBA 1-2 mL, 100 mM AA 0.2-0.5 mL, and 10-fold concentrated NPs 0.2-0.5 mL. In the solution D, the added CTAC includes 100 mM CTAC and 10 mM CTAC. The added amounts of CTAC are as follows: 100 mM CTAC 1-2 mL and 10 mM CTAC 1.5-2 mL. The injection speed is 1 mL / min, and the oscillation time is 2-3 h. The centrifugation condition is 4000 r / min for 10-20 min. The concentration of the added CTAC in the solution of the core-inner marker molecule-shell structure nanoparticles is 10 mM, and the added amount is 2-5 mL.

4. The method for preparing a SERS microneedle patch according to claim 1, characterized in that, In step (1.3), the prepared core-inner marker molecule-shell structure nanoparticles have a diameter of 100 nm, an Ag shell, an Au core, and 4-MBA as the inner marker molecule. The pore size of the porous alumina is 80 nm.

5. The preparation method of the SERS microneedle patch according to claim 1, characterized in that, In step (2.1), the mixing is performed at a speed of 400 r / min under the condition of a water bath at 100℃. The added amount of polyethylene glycol 10000 is 2-3 g, and the added amount of 2-methoxyethanol is 10-12 mL.

6. The preparation method of the SERS microneedle patch according to claim 1, characterized in that, In step (2.2), the added amount of glycidyl methacrylate is 2.5-5 mL, and the stirring condition is 400 r / min. The added amounts of the added reagents are as follows: trimethylolpropane trimethacrylate 1.5-2 mL, triethylene glycol dimethacrylate 4-5 mL, and 1-hydroxycyclohexyl ketone 25-30 mg. The solution configuration condition is heating at 65℃ for 2-3 h to obtain solution B. The solution configuration condition for mixing solution A and B is stirring at 400 r / min under the condition of a water bath at 65℃, and the mixed solution is stored in a refrigerator at 4℃.

7. The method for preparing a SERS microneedle patch according to claim 1, characterized in that, In step (2.3), the stirring condition is 400 r / min for 5-10 min. The porous microneedle patch has a side length of 1 cm and a main body thickness of 2 mm, one side of the patch has a microneedle array composed of 144 microneedles, the microneedle length is 500 μm, and the microneedle bottom diameter is 240 μm. The micropore diameter of the porous microneedle is 2 μm.

8. The method for preparing a SERS microneedle patch according to claim 1, characterized in that, In step (2.4), the vacuum drying box environment is 35℃, the vacuum degree is 0.6-0.8 atm, and the vacuum time is 15-20 min. The concentration of the phthaldehyde solution is 10 -4 M; The ultraviolet lamp is first irradiated for 15-20 s, and then irradiated for 2-5 min after placing the SERS substrate.

9. The method for preparing a SERS microneedle patch according to claim 1, characterized in that, In step (2.5), the mixed solution configuration condition is a water bath at 65℃ for 12-24 h, the added amount of ultrapure water is 5-7 mL, and the added amount of methanol is 5-6 mL. The closed dry condition is closed dry for 1-2 hours at 50℃.

10. Use of a SERS microneedle patch prepared by the method of any one of claims 1-9 in stroke risk monitoring.

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