A microneedle patch for diagnosing shrimp-allergic patients and a preparation method and application thereof

Abstract

The application discloses a microneedle patch for diagnosing shrimp allergy patients and a preparation method and application thereof. In the application, the microneedle technology is combined with a visual signal probe, type I specific biological ink and type II specific biological ink are prepared first, and then the microneedle patch is assembled through a microneedle mold. The microneedle patch combines the advantages of microneedle microtrauma, painlessness and signal molecule visualization, takes the allergic markers sIgE and total IgE as detection objects, and can realize qualitative diagnosis of shrimp allergy patients through the specific binding discoloration phenomenon of the allergic markers and the signal probe, so that the application has a good application prospect.

Description

Technical Field

[0001] This invention relates to the field of immunoassay diagnostic technology for allergic diseases, and in particular to a microneedle patch for diagnosing shrimp allergy patients, its preparation method, and its application. Background Technology

[0002] With rising living standards and changing dietary habits, shrimp is playing an increasingly important role in people's daily diets. However, this has also brought a host of health problems, among which shrimp allergy is a major health issue with a wide impact, high prevalence, and serious consequences. When shrimp allergy sufferers come into contact with shrimp-containing foods, they may experience allergic reactions. These reactions can include respiratory symptoms (such as asthma), digestive symptoms (such as enteritis), and skin symptoms (such as urticaria). In severe cases, it can be life-threatening. Therefore, developing allergy diagnostic technologies to help allergy sufferers identify their specific allergens and avoid allergenic foods can effectively prevent allergic reactions.

[0003] Existing diagnostic technologies can be broadly categorized into two types: in vivo diagnostic technologies (performed on the human body) and in vitro diagnostic technologies (collecting blood samples, etc.). In vivo diagnostic technologies include food challenge tests, skin prick tests, and patch tests, while in vitro diagnostic technologies primarily refer to ImmunoCAP. The advantages of in vivo diagnostics are simplicity, economy, and accuracy; the disadvantage is that direct contact between the patient and potential allergens may lead to local or systemic adverse reactions. The advantages of in vitro diagnostic methods are safety and accuracy; the disadvantages are time-consuming and expensive testing. Furthermore, existing diagnostic methods generally require specialized personnel.

[0004] To address the issues of low safety, time-consuming nature, and the need for professional operators in existing diagnostic methods, and to fill the gap in rapid home screening for allergens, it is necessary to develop a microneedle patch preparation method for on-site, rapid, and convenient diagnosis of shrimp allergy patients. Summary of the Invention

[0005] The primary objective of this invention is to overcome the shortcomings and deficiencies of the prior art and provide a method for preparing a microneedle patch for diagnosing patients with shrimp allergies.

[0006] Another object of the present invention is to provide a microneedle patch prepared by the method for diagnosing patients with shrimp allergies.

[0007] Another object of the present invention is to provide the application of the microneedle patch for diagnosing patients with shrimp allergies.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] A method for preparing a microneedle patch for diagnosing shrimp allergy patients includes the following steps:

[0010] (1) Preparation of Type I Specific Bio-ink

[0011] 10,12-Ticosadecanoic acid (PCDA) and 1,2-dimyristic-sn-glycerol-3-phosphorylcholine (DMPC) were dissolved in chloroform to obtain PCDA solution and DMPC solution, respectively. The PCDA solution and DMPC solution were then mixed evenly and dried to obtain PCDA / DMPC film. The film was then resuspended in water and mixed evenly with ultrasonic and magnetic stirring at 80±5℃ to obtain PCDA / DMPC suspension. The PCDA / DMPC suspension was then filtered after standing to obtain PCDA / DMPC vesicles. The PCDA / DMPC vesicles were mixed with HRP-labeled anti-human IgE antibody and incubated overnight. Bovine serum albumin (BSA) was then added, and cross-linking was induced by ultraviolet light to obtain type I specific bio-ink.

[0012] (2) Preparation of type II specific bio-ink

[0013] MES buffer was processed using a plasma device to prepare plasma-activated water. Then, 10,12-pentacarboxylic acid (PCDA), carbodiimide (EDC), N-hydroxysuccinimide (NHS), and the epitope corresponding to the shrimp allergen were added sequentially. The mixture was shaken and mixed thoroughly, and impurities were removed by liquid chromatography to obtain epitope-PCDA. Next, epitope-PCDA and 1,2-dimyristoyl-sn-glycerol-3-phosphorylcholine (DMPC) were dissolved in chloroform to obtain epitope-PCDA solution and DMPC solution, respectively. The epitope-PCDA solution and DMPC solution were then mixed thoroughly and dried to obtain epitope-PCDA / DMPC film. The film was resuspended in water and mixed thoroughly at 60±5℃ with ultrasonic and magnetic stirring to obtain epitope-PCDA / DMPC turbidity. After standing and filtration, epitope-PCDA / DMPC vesicles were obtained. Finally, bovine serum albumin (BSA) was added, and cross-linking was induced by ultraviolet light irradiation to obtain type II specific bio-ink.

[0014] (3) Preparation of microneedle patches

[0015] The cut absorbent paper was immersed in the type I specific bio-ink obtained in step (1) and the type II specific bio-ink obtained in step (2), respectively. Then it was taken out and air-dried to obtain air-dried absorbent paper I and II. Gelatin methacrylate (GelMA) was filled into the bottom of the microneedle mold, vacuum degassed, plasma-treated for catalytic cross-linking, and air-dried. Then the air-dried absorbent paper I and II were filled into the microneedle mold at the same time. This operation (filling with GelMA, vacuum degassed, plasma-treated for catalytic cross-linking and air-drying) was repeated until the mold groove was completely covered. After air-drying, it was demolded to obtain a microneedle patch for diagnosing patients with shrimp allergies.

[0016] The molar ratio of 10,12-pentadecanodiyne (PCDA) and 1,2-dimyristicoyl-sn-glycerol-3-phosphorylcholine (DMPC) in step (1) is 3 to 4:1; preferably 4:1.

[0017] The concentration of the PCDA solution mentioned in step (1) is preferably 10 mmol / L.

[0018] The concentration of the DMPC solution mentioned in step (1) is preferably 10 mmol / L.

[0019] The drying described in steps (1) and (2) is vacuum drying.

[0020] The water mentioned in steps (1) and (2) is ultrapure water.

[0021] The concentration of PCDA in the PCDA / DMPC turbid solution in step (1) is 3-4 mmol / L; preferably 4 mmol / L.

[0022] The conditions for ultrasound in steps (1) and (2) are as follows: the ultrasound power is 130-150W (preferably 140W), intermittent processing is adopted, and one ultrasound cycle is 10s ultrasound on and 10s ultrasound off.

[0023] The stirring speed in steps (1) and (2) is 100-140 r / min; preferably 120 r / min.

[0024] In steps (1) and (2), the total processing time for ultrasonic and magnetic stirring to achieve uniform mixing is 40 to 50 minutes; preferably 50 minutes.

[0025] The settling time mentioned in steps (1) and (2) is 8 to 12 hours; preferably 12 hours.

[0026] The filtration described in steps (1) and (2) is performed using a 0.22 μm filter membrane.

[0027] The amount of HRP (horseradish peroxidase) labeled anti-human IgE antibody used in step (1) is calculated based on its final concentration in the reaction system being 1 to 1.5 μg / mL; preferably, it is calculated based on its final concentration in the reaction system being 1 μg / mL.

[0028] The amount of bovine serum albumin (BSA) used in steps (1) and (2) is calculated based on its final concentration in the reaction system being 0.1% by mass.

[0029] The conditions for UV irradiation in steps (1) and (2) are: UV intensity of 500 mW / cm². 2 UV treatment time is 3-5 minutes.

[0030] The MES buffer mentioned in step (2) is a pH 6.7 MES buffer.

[0031] The plasma equipment described in step (2) can be a conventional plasma discharge experimental device in the field, such as an atmospheric low-temperature plasma discharge experimental device (Nanjing Suman Plasma Technology Co., Ltd., model DBD-50), etc.

[0032] The conditions for preparing plasma-activated water in step (2) are: treatment for 1 min under the conditions of voltage 50V, current 1mA and electrode spacing 13mm.

[0033] The amount of 10,12-pentadecanoic acid (PCDA) mentioned in step (2) is calculated based on its final concentration in the reaction system being 8-10 mmol / L; preferably, it is calculated based on its final concentration in the reaction system being 10 mmol / L.

[0034] The amount of carbodiimide (EDC) used in step (2) is calculated based on a final concentration of 45-50 mmol / L in the reaction system; preferably, it is calculated based on a final concentration of 50 mmol / L in the reaction system.

[0035] The amount of N-hydroxysuccinimide (NHS) used in step (2) is calculated based on a final concentration of 45-50 mmol / L in the reaction system; preferably, it is calculated based on a final concentration of 50 mmol / L in the reaction system.

[0036] The amount of shrimp allergen epitope used in step (2) is calculated based on a final concentration of 3-5 mg / mL in the reaction system; preferably, it is calculated based on a final concentration of 4 mg / mL in the reaction system.

[0037] The amino acid sequences of the shrimp allergen epitopes mentioned in step (2) are: LENRSLSDEERMDALENQ, DRLEDELVNEKEKYKSITDE, LAEEADRKYDEVARK, and AMKLEKDNAMDRA, with a molar ratio of 1:1:1:1.

[0038] The concentration of the epitope-PCDA solution in step (2) is preferably 10 mmol / L.

[0039] The concentration of the DMPC solution mentioned in step (2) is preferably 10 mmol / L.

[0040] The molar ratio of epitope-PCDA to 1,2-dimyristic-sn-glycerol-3-phosphocholine (DMPC) in step (2) is 3 to 4:1; preferably 3:1.

[0041] The concentration of epitope-PCDA in the epitope-PCDA / DMPC turbidity in step (2) is 5 to 6 mmol / L; preferably 6 mmol / L.

[0042] The absorbent paper mentioned in step (3) is an ultra-thin absorbent paper, and its size can be cut according to actual needs. Preferably, it is 2.5mm long, 2.5mm wide, and 0.2mm thick.

[0043] The immersion time in step (3) is 5 to 10 minutes; preferably 10 minutes.

[0044] The microneedle mold mentioned in step (3) is a PDMS mold, and its size and shape can be set according to actual needs. The PDMS mold is preferably a mold with a cubic base and a cylindrical tip. The dimensions of the base are: 10mm long, 10mm wide, and 1.5mm high. The dimensions of the microneedle tip are: 0.8-1mm long, 0.01-0.02mm tip diameter, 0.25-0.35mm bottom diameter, 0.5-0.7mm tip distance, and (9*9)-(11*11) array number. The preferred dimensions of the microneedle tip are: 0.8mm long, 0.015mm tip diameter, 0.3mm bottom diameter, 0.6mm tip distance, and 11*11 array number.

[0045] The concentration of the gelatin (GelMA) in step (3) is 18-22% by mass; preferably 20% by mass.

[0046] The number of repetitions in step (3) is 5 times, that is, the amount of gelatin methacrylate (GelMA) filled each time is 100 μL, and a total of 600 μL is filled.

[0047] The vacuum degassing conditions described in step (3) are: 0.1 MPa, treatment for 3 to 5 minutes (preferably 5 minutes).

[0048] The plasma treatment conditions described in step (3) are: voltage 160-169kV, current 150-200mA, electrode distance 2.5-3cm, and treatment time 5-10min; preferably: voltage 169kV, current 200mA, electrode distance 2.5cm, and treatment time 5min.

[0049] A microneedle patch for diagnosing patients with shrimp allergies is prepared by any of the methods described above.

[0050] The application of the microneedle patch for diagnosing shrimp allergy in the preparation of a reagent kit for diagnosing shrimp allergy.

[0051] The method of using the kit is as follows:

[0052] (a) Sample extraction:

[0053] The microneedle patch is applied directly to the skin surface of the test subject, or the serum of the test subject is first extracted and prepared into GelMA gel, and then the microneedle patch is inserted into the surface of the GelMA gel and waited for 10 to 15 minutes (preferably 10 minutes).

[0054] (b) Result Interpretation:

[0055] If neither the total IgE nor the sIgE region changes color (blue), the individual is considered to be at low risk of allergies. If both the total IgE and sIgE regions change from blue to red, the individual is considered to have a high risk of shrimp allergy. If the total IgE region changes from blue to red, but the sIgE region does not change color (blue), the individual is considered to have other allergy risks. If the total IgE region does not change color (blue), but the sIgE region changes from blue to red, this is an invalid result and retesting is required.

[0056] The GelMA gel described in step (a) is prepared by the following method: gelatin methacrylate (GelMA) containing a photoinitiator is filled into a 24-well plate and subjected to ultraviolet cross-linking treatment. After being placed at room temperature for 10-14 hours (preferably 12 hours), it is frozen at -80±5℃ for 2 hours and then freeze-dried at -65±5℃ to obtain lyophilized GelMA. Serum is then added to the lyophilized GelMA, mixed evenly, placed at room temperature for 1-3 hours (preferably 2 hours), and then placed in a 4℃ refrigerator to obtain the final product.

[0057] The photoinitiator content in the methacrylic gelatin containing the photoinitiator is 0.5% by mass.

[0058] The concentration of the gelatin (GelMA) is 18-22% by mass; preferably 20% by mass.

[0059] The photoinitiator is photoinitiator 2959.

[0060] The conditions for UV crosslinking are: UV intensity of 500 mW / cm². 2 The UV treatment time is 3 to 5 minutes (preferably 5 minutes).

[0061] The freeze-drying time is preferably 48 hours or more.

[0062] Other allergies mentioned in step (a) include milk allergy, etc.

[0063] The diagnostic principle of the diagnostic microneedle patch in this invention is as follows:

[0064] (i) Sample extraction: The microneedle patch is applied to the skin surface of the test subject. When the microneedle is inserted into the skin, the subcutaneous tissue fluid is enriched under the swelling and adsorption effect of the microneedle.

[0065] (ii) Specific marker identification: After enrichment, the tissue fluid comes into contact with absorbent paper containing visual probes fixed in microneedles. Allergy markers (total IgE, sIgE) contained in the tissue fluid bind specifically to the visual probes, causing changes in the probe structure and triggering a color change reaction.

[0066] (iii) Result interpretation: By observing the color changes of signal molecules at different locations, it is possible to determine whether there is a risk of food allergy.

[0067] The present invention has the following advantages and effects compared with the prior art:

[0068] (1) The method of the present invention combines microneedle technology with visual signal probes, combining the advantages of microneedle minimally invasive and painless and the visualization of signal molecules. It uses allergy markers sIgE and total IgE as detection objects, and realizes the qualitative diagnosis of food allergy by observing the phenomenon of color change of sIgE and total IgE specifically binding with the signal probe.

[0069] (2) The present invention uses plasma-activated water as a buffer system for EDC-NHS coupling, which provides abundant electron transfer pathways for activation and improves activation efficiency.

[0070] (3) This invention uses plasma to replace traditional photoinitiators and ultraviolet light to promote cross-linking of GelMA, which is green, environmentally friendly and efficient.

[0071] (4) This invention can utilize interstitial fluid in the skin to achieve on-site, rapid and convenient diagnosis of patients with shrimp allergies. Furthermore, by changing the type of epitope in the preparation process of type II specific bio-ink, it can also diagnose other types of food allergies besides shrimp allergies. It has the potential to be extended to the diagnosis of other allergic diseases and has a wide range of application prospects. Attached Figure Description

[0072] Figure 1 This is a schematic diagram of the microneedle patch preparation process in Embodiment 1 of the present invention.

[0073] Figure 2 This is a schematic diagram illustrating the diagnostic process and result determination of the diagnostic microneedle patch prepared in this invention.

[0074] Figure 3 This is a comparison chart of the swelling performance of Example 3 of the present invention.

[0075] Figure 4 This is a graph showing the comparison results of protein adsorption performance in Example 4 of the present invention.

[0076] Figure 5 The images show the preparation and diagnostic results of the diagnostic microneedle patch in Embodiment 5 of the present invention; wherein, (a) is a physical image of the diagnostic microneedle patch; and (b) is a diagnostic result image. Detailed Implementation

[0077] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field. Test methods in the following embodiments that do not specify specific experimental conditions are generally performed according to conventional experimental conditions or experimental conditions recommended by the manufacturer. Unless otherwise specified, the reagents and raw materials used in the present invention are commercially available.

[0078] Example 1

[0079] (1) Preparation of Type I specific bio-ink:

[0080] 10,12-Ticosadecanodiyne (PCDA; CAS: 66990-32-7) and 1,2-dimyristoyl-sn-glycerol-3-phosphocholine (DMPC; CAS: 18194-24-6) were dissolved separately in chloroform to prepare 10 mM PCDA and 10 mM DMPC solutions. The 10 mM PCDA and 10 mM DMPC solutions were mixed at a PCDA:DMPC molar ratio of 4:1. The PCDA / DMP was obtained by vacuum drying. C membrane; resuspended in ultrapure water, and then subjected to treatment, heating, and magnetic stirring (ultrasonic power of 140W, intermittent treatment, one ultrasonic cycle is 10s on and 10s off; heating temperature is 80℃; magnetic stirring speed is 120r / min, total treatment time is 50min) to obtain PCDA / DMPC turbidity (the concentration of PCDA in the resuspended turbidity is 4mM); let stand for 12h, filter with a 0.22μm filter membrane to obtain PCDA / DMPC vesicles. The prepared 4mM PCDA / DMPC vesicles were mixed with 1 mg / mL goat anti-human IgE H&L (HRP) (abcam73901) (the final concentration of PCDA / DMPC vesicles was close to 4mM, and the final antibody concentration was 1 μg / mL), and the reaction was incubated overnight. A protective protein, bovine serum albumin (BSA), was added, with a final BSA concentration of 0.1% (w / w). The mixture was then irradiated with ultraviolet light (UV treatment time 5 min, intensity 500 mW / cm²). 2 Cross-linking was induced to obtain type I specific bio-ink.

[0081] (2) Preparation of type II specific bio-ink:

[0082] 10 mL of MES buffer (pH 6.7) (Aladdin M301885) was placed in a small media-barrier plasma treatment chamber (Nanjing Suman Plasma Technology Co., Ltd., model DBD-50) and treated for 1 min at 50 V, 1 mA, and 13 mm electrode spacing to obtain plasma-activated water. 10,12-carbodiyne acid (PCDA), carbodiimide (EDC), N-hydroxysuccinimide (NHS), and epitopes corresponding to shrimp allergens (LENRSLSDEERMDALENQ, DRLEDELVNEKEKYKSITDE, LAEEADRKYDEVARK, AMKLEKDNAMDRA) were added sequentially to the plasma-activated water, followed by thorough shaking. The final concentrations were: PCDA 10 mM, EDC and NHS 50 mM each, and epitopes 4 mg / mL, with a molar ratio of 1:1:1:1 for the four epitope sequences. The solution was then analyzed by preparative liquid chromatography (reference: Roeder M, Wiacek). C,LankampF,et al.Improved Sensitivity of Allergen Detection by Immunoaffinity LC-MS / MSUsing Ovalbumin as a Case Study[J].Foods,2021.DOI:10.3390 / foods10122932.) Separate and purify to obtain epitope-PCDA; dissolve epitope-PCDA and DMPC separately in chloroform to prepare 10 mM epitope-PCDA solution and 10 mM DMPC solution, respectively. Mix the 10 mM epitope-PCDA solution and 10 mM DMPC solution at a molar ratio of 3:1; obtain epitope-PCDA / DMPC film by vacuum drying; add ultrapure water for resuspension, and treat simultaneously with ultrasound, heating and magnetic stirring (ultrasound power is 140 W, intermittent treatment, one ultrasound cycle is 10 s ultrasound on and 10 s ultrasound off; heating to 60 °C; magnetic stirring speed is 120 r / min, total treatment time is 50 min) to obtain 6 mM epitope-PCDA / DMPC turbidity. After standing for 12 hours, the vesicles were filtered through a 0.22 μm filter to obtain epitope-PCDA / DMPC vesicles. Protective protein BSA was added to 6 mM epitope-PCDA / DMPC vesicles (final BSA concentration: 0.1% (w / w)). The vesicles were then irradiated with ultraviolet light (UV treatment time: 3 min, intensity: 500 mW / cm²). 2 Cross-linking was induced to obtain type II specific bio-ink.

[0083] (3) Preparation of diagnostic microneedle patches:

[0084] Cut several pieces of ultra-thin absorbent paper (absorbent cotton paper, made of cotton) of appropriate size (2.5mm long, 2.5mm wide, and 0.2mm thick), immerse them in type I specific bio-ink and type II specific bio-ink respectively for 10 minutes, then remove them and let them air dry naturally to obtain air-dried absorbent paper I and II.

[0085] Prepare a microneedle mold (PDMS mold). The base of the microneedle mold groove is a cube with dimensions of 10mm in length, 10mm in width, and 1.5mm in height. The tip of the microneedle mold groove is a cylinder with dimensions of 0.8mm in length, 0.015mm in tip diameter, 0.3mm in bottom diameter, and 0.6mm in tip distance. The array size is 11*11.

[0086] 100 μL of gelatin methacrylate (GelMA, 20% (W / V)) was filled into the bottom of the prepared PDMS mold. Then, vacuum degassing (0.1 MPa, 5 min), plasma treatment for catalytic crosslinking (169 kV, 200 mA, electrode distance 2.5 cm, 5 min), and natural air drying were performed. After crosslinking, air-dried absorbent paper I and II were filled into the mold (both types of absorbent paper can be filled simultaneously, the order is not critical). The process of filling GelMA, vacuum degassing, plasma treatment for catalytic crosslinking (169 kV, 200 mA, electrode distance 2.5 cm, 5 min), and natural air drying was repeated 5 times until the mold groove was completely covered. The amount of GelMA filled each time was 100 μL, for a total of 600 μL. After natural air drying (24 h), the mold was demolded to obtain the GelMA microneedle patch, i.e., the diagnostic microneedle patch. The results are as follows: Figure 1 As shown.

[0087] Example 2

[0088] According to the method of Example 1, a plasma-catalyzed GelMA microneedle patch (B) was prepared. Simultaneously, referring to the method of Example 1, a photoinitiator and UV-catalyzed GelMA microneedle patch (A) was prepared under the conditions shown in Table 1, with the specific differences being: gelatin methacrylate (GelMA, concentration 20% (W / V)) was replaced with GelMA (concentration 20% (W / V)) doped with 0.5% (W / V) photoinitiator 2959 (CAS: 106797-53-9); and plasma-treated catalytic crosslinking (voltage 169kV, current 200mA, electrode distance 2.5cm, treatment 5min) was replaced with UV-treated crosslinking (500mW / cm). 2 (Process for 5 minutes).

[0089] Table 1. GelMA preparation conditions

[0090] Group Photoinitiator Vacuum defoaming Plasma crosslinking UV crosslinking A √ √ √ B √ √

[0091] Example 3

[0092] The swelling characteristics of two types of microneedle patches, A and B, obtained in Example 2 were tested. To calculate the swelling rate of the GelMA microneedle patch, both microneedle patch samples were incubated in simulated skin (2% (w / v) agarose solution) at 37°C for 0–30 min, with data collected every 5 min. After the specified duration, residual liquid on the patch surface was removed and the wet weight (Ww) was recorded. The dry weight (Wd) was measured after lyophilization. The swelling rate was calculated using the formula [(Ww-Wd) / Wd]×100%. The experiment was conducted in triplicate.

[0093] The results are as follows Figure 3 As shown, the plasma-catalyzed GelMA microneedle patch exhibits better overall swelling performance, reaching 77.26% at 30 min, which is slightly higher than the 72.03% of the photoinitiator and UV-catalyzed GelMA microneedle patch.

[0094] Example 4

[0095] The in vitro protein extraction efficiency of microneedle patches A and B obtained in Example 2 was tested. Using 2% (w / w) low-melting-point agarose (Shanghai Sangon Biotech A600015) solution as solvent, BSA protein solutions with concentration gradients of 0.25, 0.5, 0.75, 1, and 1.5 mg / mL were prepared. After solidification, agarose gels were obtained to simulate interstitial fluid (ISF) extraction in vitro. Microneedle patches A and B were respectively applied to the surface of the gel containing protein solutions. After 10 min, they were removed, and the protein concentration in the microneedles was detected using a Bradford protein concentration assay kit. The experiment was repeated three times.

[0096] The results are as follows Figure 4 As shown, the protein concentration measurements of both microneedles showed a linear relationship in the range of 0.25–1.5 mg / mL, while the results measured using plasma-catalyzed GelMA microneedle patches were closer to the actual results.

[0097] Example 5

[0098] Blood was collected from 14 volunteers (including shrimp allergy patients, other allergy patients, and healthy volunteers). Serum was separated and the specific IgE content was measured (according to the ImmunoCAP method in reference: Johnston EB, Kamath SD, Iyer SP, et al. Defining specific allergens for improved component-resolved diagnosis of shrimp allergy in adults[J]. Molecular immunology, 2019, 112:330. https: / / doi.org / 10.1016 / j.molimm.2019.05.006). The serum information obtained is shown in Table 1, where P1-P8 are shrimp allergy samples, P9-P11 are milk allergy samples, and P12-P14 are healthy samples.

[0099] Table 1 Serum information of allergic patients

[0100] serial number age gender <![CDATA[Specific IgE (kU / L) a > allergens P1 33 male 64.1 shrimp P2 38 female 24.5 shrimp P3 52 male 60.1 shrimp P4 61 male 34.983 shrimp P5 31 male 26.7 shrimp P6 21 female 22.1 shrimp P7 27 female 17.5 shrimp P8 29 female 20.1 shrimp P9 19 female milk P10 25 male milk P11 24 male milk P12 43 male - - P13 20 female - - P14 38 male - -

[0101] Note: a A specific IgE level >0.35 kU / L indicates a positive result.

[0102] The diagnostic microneedle patches prepared in Example 1 were inserted into the surfaces of GelMA gels prepared with serum from shrimp allergy patients (shrimp allergy patient group), GelMA gels prepared with serum from milk allergy patients (other allergy patient groups), and GelMA gels prepared with serum from healthy individuals (healthy individual group), respectively, and the results were observed after 10 minutes.

[0103] The specific preparation method of serum-containing GelMA gel is as follows: 1 mL of GelMA doped with 0.5% (w / v) photoinitiator 2959 (concentration 20% (w / v)) is filled into a 24-well plate; UV treatment for crosslinking (500 mW / cm²) is performed. 2 Process for 5 min); after standing at room temperature for 12 h, freeze at -80℃ for 2 h; then freeze-dry (48 h, cold trap temperature -65℃); mix 1 mL of serum into the lyophilized GelMA, stand at room temperature for 2 h, and then store at 4℃ for later use. The method for determining the microneedle patch is described in [link to relevant documentation]. Figure 2 .

[0104] The total IgE region and shrimp sIgE region of the diagnostic microneedle patch prepared in Example 1 were blue ( Figure 5 (a)); The prepared diagnostic microneedle patch was inserted into the gel prepared with serum in each group for 10 minutes, and the results were as follows. Figure 5As shown in (b): In the healthy population, both the total IgE and shrimp sIgE regions remain blue; in the shrimp allergy patient group, both the total IgE and shrimp sIgE regions, which were originally blue, turn red; in other allergy patient groups, the total IgE region turns red, while the sIgE region remains blue; in the healthy population, both the total IgE and shrimp sIgE regions remain blue, indicating that the diagnostic microneedle patch can specifically respond to shrimp allergy patients. If both the total IgE and shrimp sIgE regions are red, the individual can be identified as being at risk of shrimp allergy; if the total IgE region is red and the shrimp sIgE region is blue, the individual can be identified as being at risk of other allergies; if both the total IgE and shrimp sIgE regions remain blue, the individual can be identified as being at low risk of allergies.

[0105] Table 2 shows the test results for the serum samples in Table 1. The results show that the detection accuracy was 100% for 8 shrimp allergy patients, 3 other allergy patients, and 3 healthy individuals.

[0106] Table 2. Test results of serum samples

[0107] serial number allergens IgE region sIgE-Shrimp Area Diagnostic results Do the results match? P1 shrimp red red Shrimp allergy risk yes P2 shrimp red red Shrimp allergy risk yes P3 shrimp red red Shrimp allergy risk yes P4 shrimp red red Shrimp allergy risk yes P5 shrimp red red Shrimp allergy risk yes P6 shrimp red red Shrimp allergy risk yes P7 shrimp red red Shrimp allergy risk yes P8 shrimp red red Shrimp allergy risk yes P9 milk blue red Other allergy risks yes P10 milk blue red Other allergy risks yes P11 milk blue red Other allergy risks yes P12 - blue blue Low risk of allergies yes P13 - blue blue Low risk of allergies yes P14 - blue blue Low risk of allergies yes

[0108] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing a microneedle patch for diagnosing shrimp allergic patients, characterized by, The method comprises the following steps: (1) Preparation of type I specific bio-ink Dissolve 10, 12-pentacosadiynoic acid and 1, 2-dimyristoyl-sn-glycerol-3-phosphocholine into chloroform respectively to obtain PCDA solution and DMPC solution; then mix the PCDA solution and DMPC solution uniformly, dry to obtain PCDA / DMPC film; then resuspend with water, mix uniformly under the condition of 80±5 ℃ with the aid of ultrasonic and magnetic stirring, obtain PCDA / DMPC turbidity; then let the PCDA / DMPC turbidity stand, filter to obtain PCDA / DMPC vesicles; mix the PCDA / DMPC vesicles with HRP-labeled anti-human IgE antibody, incubate overnight, then add bovine serum albumin, induce cross-linking by ultraviolet light irradiation to obtain type I specific bio-ink; (2) Preparation of type II specific bio-ink Use plasma equipment to process MES buffer into plasma active water, then add 10, 12-pentacosadiynoic acid, carbodiimide, N-hydroxysuccinimide and the corresponding epitopes of shrimp allergen in sequence, shake to mix uniformly, remove impurities by liquid chromatography to obtain epitope-PCDA; then dissolve the epitope-PCDA and 1, 2-dimyristoyl-sn-glycerol-3-phosphocholine into chloroform respectively to obtain epitope-PCDA solution and DMPC solution; then mix the epitope-PCDA solution and DMPC solution uniformly, dry to obtain epitope-PCDA / DMPC film; resuspend with water, mix uniformly under the condition of 60±5 ℃ with the aid of ultrasonic and magnetic stirring to obtain epitope-PCDA / DMPC turbidity; let it stand, filter to obtain epitope-PCDA / DMPC vesicles; finally add bovine serum albumin, induce cross-linking by ultraviolet light irradiation to obtain type II specific bio-ink; (3) Preparation of microneedle patch Submerge the cut water-absorbing paper in the type I specific bio-ink obtained in step (1) and the type II specific bio-ink obtained in step (2) respectively, then take out and air dry to obtain air-dried water-absorbing paper I and II; fill the methacrylic acid gelatin in the bottom of the microneedle mold, vacuum to remove bubbles, catalyze cross-linking by plasma treatment, air dry, then fill the air-dried water-absorbing paper I and II into the microneedle mold at the same time, repeat the operation of filling methacrylic acid gelatin, vacuum to remove bubbles, catalyze cross-linking by plasma treatment, air dry until the concave grooves of the mold are completely covered; after air drying, demold to obtain the microneedle patch for diagnosing shrimp allergy patients.

2. The method according to claim 1, wherein the amino acid sequence of the corresponding epitope of shrimp allergen in step (2) is: LENRSLSDEERMDALENQ, DRLEDELVNEKEKYKSITDE, LAEEADRKYDEVARK, AMKLEKDNAMDRA, and the molar ratio of the four epitope sequences is 1:1:1:

1.

3. The method according to claim 1, wherein ​ The molar ratio of 10,12-pentacosadiynoic acid to 1,2-dimyristoyl-sn-glycero-3-phosphocholine in step (1) is 3-4:1; The concentration of PCDA in the PCDA / DMPC turbidity liquid in step (1) is 3-4 mmol / L; The amount of HRP-labeled anti-human IgE antibody in step (1) is calculated according to its final concentration in the reaction system of 1-1.5 μg / mL; The amount of bovine serum albumin in steps (1) and (2) is calculated according to its final concentration in the reaction system of 0.1% by mass; The amount of 10,12-pentacosadiynoic acid in step (2) is calculated according to its final concentration in the reaction system of 8-10 mmol / L; The amount of epitope corresponding to shrimp allergen in step (2) is calculated according to its final concentration in the reaction system of 3-5 mg / mL; The molar ratio of epitope-PCDA to 1,2-dimyristoyl-sn-glycero-3-phosphocholine in step (2) is 3-4:1; The concentration of epitope-PCDA in the epitope-PCDA / DMPC turbidity liquid in step (2) is 5-6 mmol / L; The concentration of methacrylic acid gelatin in step (3) is 18-22% by mass.

4. The method according to claim 3, characterized in that: The molar ratio of 10,12-pentacosadiynoic acid to 1,2-dimyristoyl-sn-glycero-3-phosphocholine in step (1) is 4:1; The concentration of PCDA in the PCDA / DMPC turbidity liquid in step (1) is 4 mmol / L; The amount of HRP-labeled anti-human IgE antibody in step (1) is calculated according to its final concentration in the reaction system of 1 μg / mL; The amount of 10,12-pentacosadiynoic acid in step (2) is calculated according to its final concentration in the reaction system of 10 mmol / L; The amount of epitope corresponding to shrimp allergen in step (2) is calculated according to its final concentration in the reaction system of 4 mg / mL; The molar ratio of epitope-PCDA to 1,2-dimyristoyl-sn-glycero-3-phosphocholine in step (2) is 4:1; The concentration of epitope-PCDA in the epitope-PCDA / DMPC turbidity liquid in step (2) is 6 mmol / L; The concentration of methacrylic acid gelatin in step (3) is 20% by mass.

5. The method according to claim 1, characterized in that: The conditions for the irradiation of the UV lamp described in steps (1) and (2) are: UV intensity of 500 mW / cm 2 , UV treatment time of 3-5 min. The conditions for preparing the plasma active water in step (2) are: treating for 1 min under the conditions of a voltage of 50 V, a current of 1 mA, and a distance between the electrodes of 13 mm; The conditions for the plasma treatment in step (3) are: a voltage of 160-169 kV, a current of 150-200 mA, a distance between the electrodes of 2.5-3 cm, and treating for 5-10 min.

6. The method according to claim 1, characterized in that: The ultrasonic power in steps (1) and (2) is 130-150 W, and the intermittent treatment is used, with 10 s of ultrasonic on and 10 s of ultrasonic off for one ultrasonic cycle; The stirring speed in steps (1) and (2) is 100-140 r / min; The standing time in steps (1) and (2) is 8-12 h; The filtration in steps (1) and (2) is performed by using a 0.22 μm filter membrane; The vacuum bubble removal conditions in step (3) are as follows: 0.1 MPa, for 3-5 min; The immersion time in step (3) is 5-10 min; The number of repetitions in step (3) is 5.

7. A microneedle patch for diagnosing a shrimp-allergic patient, characterized by: The micro-needle patch for diagnosing shrimp-allergic patients is prepared by the method according to any one of claims 1-6.

8. Use of the micro-needle patch for diagnosing shrimp-allergic patients according to claim 7 in the preparation of a kit for diagnosing shrimp-allergic patients.

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