Rapid detection of anti-mutant citrullinated vimentin antibody in micro blood sample by microneedle patch technology and preparation method thereof

Abstract

The application provides a rapid detection microneedle patch technology of anti-mutant citrullinated vimentin antibody in a trace blood sample and a preparation method thereof, and belongs to the technical field of medical detection. The needle body of the microneedle patch is coated with mutant citrullinated vimentin and blocked with bovine serum albumin, and the prepared microneedle patch can rapidly and quantitatively detect the anti-mutant citrullinated vimentin antibody in a trace blood sample. The preparation method of the microneedle patch is simple, the cost is controllable, the microneedle patch has the characteristics of high sensitivity and high accuracy of traditional blood tests, the required blood sample amount is greatly reduced compared with traditional blood tests, and the microneedle patch has a good market application prospect.

Description

Technical Field

[0001] This invention belongs to the field of medical testing technology, specifically relating to a microneedle patch technology for rapid detection of anti-mutant citrulline vimentin antibodies in trace blood samples and its preparation method. Background Technology

[0002] Anti-mutated citrullinated vimentin antibody (anti-MCV antibody) is a novel autoantibody biomarker for rheumatoid arthritis (RA) with high specificity and good sensitivity. This antibody recognizes citrullinated mutant vimentin and has a high detection rate in RA patients, with a specificity as high as 90%. Especially in patients who are negative for rheumatoid factor (RF) or anti-CCP antibodies, anti-MCV antibodies may still be positive, which helps improve the early diagnosis rate. Studies have shown that anti-MCV antibody levels are closely related to disease activity and the degree of joint damage, and have certain prognostic assessment and treatment monitoring value.

[0003] Blood tests are a mature diagnostic technology. Patients can have their liver function, hemoglobin, hepatitis B, HIV, and other related indicators tested through blood tests. Patients can also have anti-mutant citrullinated vimentin antibody levels detected through blood tests. Currently, the main method for detecting anti-mutant citrullinated vimentin antibodies is enzyme-linked immunosorbent assay (ELISA). This method requires drawing venous blood to prepare serum for testing. This process requires a large sample volume, is relatively complex, and the sample is susceptible to contamination. Furthermore, traditional blood tests require a large amount of blood, resulting in significant trauma for patients, leading to strong resistance to ELISA. In addition, ELISA testing is time-consuming, requires a high level of expertise from the personnel, and is costly. Therefore, it is necessary to develop a detection device that can detect trace amounts of blood samples, enabling the quantitative detection of anti-mutant citrullinated vimentin antibodies in these samples.

[0004] Microneedles offer the unique advantage of requiring small sample volumes, making them suitable for the quantitative detection of specific biomarkers in minute amounts of blood samples. Currently, known studies primarily utilize hydrogel-swollen microneedles and solid hollow microneedles for detection. Both strategies mainly involve extracting interstitial fluid from the skin and then separating and detecting specific biomarkers in vitro. The disadvantages of this strategy include small sample volumes, challenging biomarker separation, difficulty in meeting detection requirements, poor specificity, and susceptibility to contamination. Furthermore, there are reports of combining microneedles with electrochemistry to monitor electrolytes and glucose in interstitial fluid for monitoring human health. However, the invention of using solid microneedles for the specific detection of antibodies against mutant citrullinated vimentin has not yet been reported. Summary of the Invention

[0005] To address the problems existing in the prior art, the primary objective of this invention is to provide a rapid detection microneedle patch for anti-mutant citrulline vimentin antibodies in trace blood samples and its preparation method, which can rapidly and quantitatively detect anti-mutant citrulline vimentin antibodies in trace blood samples.

[0006] A second objective of this invention is to provide the application of the aforementioned rapid detection microneedle patch in the quantitative detection of anti-mutant citrulline vimentin antibodies in blood samples.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for preparing a microneedle patch for rapid detection of anti-mutant citrulline vimentin antibody in trace blood samples, comprising the following steps: To prepare a microneedle patch, the needles of the microneedle patch were immersed in a 2-100 μg / mL mutant citrulline vimentin solution for protein coating, then washed and dried with 0.05% PBS+T solution. The dried needles were then immersed in a 1-3% bovine serum albumin PBS solution for blocking, then washed and dried with 0.05% PBS+T solution to obtain a rapid detection microneedle patch for anti-mutant citrulline vimentin antibody in trace blood samples. The protein coating conditions were room temperature coating for 2 hours or overnight coating at 4°C; the blocking conditions were room temperature blocking for 1-3 hours.

[0008] Preferably, the material of the microneedle patch includes epoxy resin and polystyrene; the needle shape of the microneedle patch is conical or pyramidal, the needle length is 430~1200μm, and the needle tip distance is 580~1000μm; the bottom diameter of the conical needle is 220μm, and the bottom side length of the pyramidal needle is 320~500μm.

[0009] Preferably, the microneedle patch undergoes surface treatment before protein coating; the surface treatment includes any of the following: (1) Perform plasma surface activation treatment on the needle body surface; (2) The needle body surface is subjected to plasma surface activation treatment and polyethylene glycol grafting treatment in sequence; (3) The needle surface is subjected to plasma surface activation treatment and L-polylysine solution treatment in sequence; (4) The needle body surface is subjected to plasma surface activation treatment, polyethylene glycol grafting treatment and L-polylysine solution treatment in sequence; (5) Treat the surface of the needle with L-polylysine solution.

[0010] Preferably, the plasma surface activation treatment includes the following steps: placing the microneedle patch 0.5-2.5 cm below the spray gun of the plasma treatment machine, and performing plasma treatment for 1-5 minutes.

[0011] Preferably, the polyethylene glycol grafting treatment includes the following steps: The microneedle patch was immersed in the mixed solution and reacted at 60°C for 1 h, followed by ultrasonic cleaning with deionized water; then immersed in MES buffer containing 50 mM EDC and 25 mM NHS, and shaken at room temperature for 30 min, before being transferred to PBS solution containing 1~2 w / v% NH2-PEG-COOH, reacted at room temperature for 4~6 h, and ultrasonically cleaned with deionized water. The mixed solution is obtained by mixing equal volumes of 1 v / v % malonic acid solution and 30 v / v % H2O2 solution, or by mixing equal volumes of 1 v / v % citric acid solution and 30 v / v % H2O2 solution.

[0012] Preferably, the L-polylysine solution treatment includes the following steps: immersing the microneedle patch in the L-polylysine solution; the concentration of the L-polylysine solution is 0.01~0.1 w / v%, the molecular weight is 15~300 kDa, and the solvent is deionized water or PBS; the immersion conditions are immersion at room temperature for 30~120 min.

[0013] This invention also provides a microneedle patch for rapid detection of anti-mutant citrulline vimentin antibodies in trace blood samples prepared by the above preparation method.

[0014] This invention also provides the application of the microneedle patch for rapid detection of anti-mutant citrulline vimentin antibodies in trace blood samples in the quantitative detection of anti-mutant citrulline vimentin antibodies in blood samples.

[0015] Preferably, the quantitative detection method includes the following steps: microneedle patches are immersed in diluted blood samples for 2 hours, washed, and dried; then immersed in horseradish peroxidase-linked rabbit anti-human IgG antibody solution for incubation, followed by washing; then immersed in TMB single-component chromogenic solution for color development for 5-15 minutes, and the content of anti-mutant citrulline vimentin antibody in the blood sample is obtained based on the color.

[0016] Preferably, the quantitative detection method includes the following steps: microneedle patch is immersed in diluted blood sample for 2 hours, washed and dried; then immersed in fluorescein-conjugated rabbit anti-human IgG antibody solution for incubation, washed and dried after incubation, and the content of anti-mutant citrulline vimentin antibody in blood sample is obtained according to fluorescence intensity.

[0017] Compared with the prior art, the beneficial effects of the technical solution of the present invention are as follows: The rapid detection microneedle patch preparation method provided by this invention is simple, cost-controllable, and possesses the high sensitivity and accuracy of traditional blood tests. Compared to traditional blood tests, the rapid detection microneedle patch of this invention significantly reduces the amount of blood sample required, eliminates the need for venous blood draws, and can be used with finger-prick blood, resulting in minimal trauma, high patient compliance, and low requirements for the professional operation of the testing personnel. Compared to existing microneedle detection technologies, the rapid detection microneedle patch of this invention can achieve quantitative detection of anti-mutant citrullinated vimentin antibodies in trace amounts of blood samples, providing more reliable results and showing promising market application prospects. Attached Figure Description

[0018] Figure 1 : A flowchart of the microneedle surface treatment process in this invention; Figure 2 The overall flow chart of microneedle in vivo detection technology and microneedle patch preparation in this invention; Figure 3 : Fabrication process of the microneedle molding method for surface mount technology; Figure 4 : Load-displacement curve of polystyrene microneedles; Figure 5 : Load-displacement curve of epoxy resin microneedles; Figure 6 Comparison of fluorescence intensity among different surface treatment groups; Figure 7 The standard curves for color and concentration in Experiment Example 1; Figure 8 : Detection results of mouse blood samples in Experiment Example 1. Detailed Implementation

[0019] This invention provides a method for preparing a microneedle patch for rapid detection of anti-mutant citrulline vimentin antibody in trace blood samples, comprising the following steps: To prepare a microneedle patch, the needles of the microneedle patch were immersed in a 2-100 μg / mL mutant citrulline vimentin solution for protein coating, then washed and dried with 0.05% PBS+T solution. After drying, the needles were immersed in a 1-3% bovine serum albumin PBS solution for blocking, then washed and dried with 0.05% PBS+T solution to obtain a microneedle patch for rapid detection of anti-mutant citrulline vimentin antibody in trace amounts of blood samples.

[0020] Preferably, the microneedle patch of the present invention is made of epoxy resin or polystyrene. Alternatively, the microneedle patch of the present invention can be prepared using a molding method or a 3D printing method.

[0021] As an optional implementation, the steps of the molding method of the present invention include: Preparation of epoxy resin microneedles: Epoxy resin and curing agent (including toluene dimethylamine) are thoroughly mixed at a mass ratio of epoxy resin to curing agent of 100:(15~20), poured into a silicone microneedle mold, placed in a vacuum machine for vacuum defoaming, and then removed and molded under natural conditions for 24~30 hours to obtain epoxy resin solid microneedles.

[0022] Preparation of polystyrene microneedles: Polystyrene is dissolved in dichloromethane to prepare a 15%~25% (w / v, g / mL) polystyrene dichloromethane solution, preferably 20%. The solution is poured into a silicone mold, placed in a vacuum machine to defoam, and then dried under natural conditions for 6~12 hours to form the microneedles.

[0023] Preferably, the microneedle patch of the present invention has a conical or pyramidal needle body shape; the needle body length is 430~1200μm, more preferably 500μm, 600μm, 700μm, 800μm, 900μm, 1000μm or 1100μm; the needle tip distance is 580~1000μm, more preferably 600μm, 700μm, 800μm or 900μm; the bottom diameter of the conical needle body is 220μm, and the bottom side length of the pyramidal needle body is 320~500μm, more preferably 400μm. As an optional implementation, the array number of the microneedle patch of the present invention includes 10×10, 11×11 or 12×12, and the side length of the entire microneedle patch is 9.8mm×9.8mm, 11.7mm×11.7mm, 11.8mm×11.8mm or 14.5mm×14.5mm.

[0024] Preferably, the concentration of the mutant citrulline vimentin solution of the present invention is 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL or 90 μg / mL; the protein coating conditions are coating at room temperature for 2 hours or coating overnight at 4°C; the washing is performed 3 times, 5 minutes each time; the drying is preferably done by blotting with absorbent paper.

[0025] Preferably, the PBS solution containing 1-3% bovine serum albumin in this invention refers to a PBS solution containing 1-3% (w / v, g / mL) bovine serum albumin, and the concentration of bovine serum albumin is more preferably 2%; the blocking conditions are blocking at room temperature for 1-3 hours, more preferably 2 hours; the washing is performed 3 times, each time for 5 minutes; the drying is preferably done by blotting with absorbent paper.

[0026] In this invention, the 0.05% PBS+T solution refers to Triton X 0.05% (v / v).

[0027] Preferably, the microneedle patch undergoes surface treatment before protein coating (especially when the microneedle patch is made of epoxy resin, surface treatment is beneficial for protein coating). The surface treatment includes introducing active functional groups onto the needle surface, performing surface carboxylation enhancement modification and / or PEG grafting treatment, and L-polylysine grafting treatment; the surface treatment is a hydrophilic treatment. This invention, through the specific surface treatment technology, is more conducive to coating the needle surface with anti-mutagenic citrulline vimentin. The surface treatment process of this invention is illustrated as follows: Figure 1 As shown. Preferably, the surface treatment includes any of the following: (1) Perform plasma surface activation treatment on the needle body surface; (2) The needle body surface is subjected to plasma surface activation treatment and polyethylene glycol grafting treatment in sequence; (3) The needle surface is subjected to plasma surface activation treatment and L-polylysine solution treatment in sequence; (4) The needle body surface is subjected to plasma surface activation treatment, polyethylene glycol grafting treatment and L-polylysine solution treatment in sequence; (5) Treat the surface of the needle with L-polylysine solution.

[0028] Preferably, the plasma surface activation treatment of the present invention includes the following steps: placing the microneedle patch at a distance of 0.5~2.5cm below the spray gun of the plasma treatment machine, and performing plasma treatment for 1~5min; the distance is more preferably 1cm, 1.5cm or 2cm; the treatment time is more preferably 2min, 3min or 4min; the present invention can introduce microneedle patches with active functional groups on the surface of the needle through plasma surface activation treatment, which is beneficial for subsequent protein coating.

[0029] Preferably, the polyethylene glycol grafting treatment of the present invention includes the following steps: Microneedle patches were immersed in a mixed solution and reacted at 60°C for 1 hour to further increase the surface carboxyl group density through chemical oxidation. The patches were then ultrasonically cleaned with deionized water to remove residual reagents. Next, they were immersed in MES buffer (pH 5.5) containing 50 mM EDC and 25 mM NHS and shaken at room temperature for 30 minutes to activate the surface carboxyl groups. The patches were then transferred to PBS solution (pH 7.4) containing 1–2% (w / v, g / mL) NH2-PEG-COOH (5 kDa) and reacted at room temperature for 4–6 hours (or overnight at 4°C). Unreacted PEG was removed by ultrasonic cleaning with deionized water, forming a PEG layer with carboxylic acid groups retained at the ends for subsequent protein coating. The mixed solution was prepared by mixing equal volumes of 1% (v / v) malonic acid solution and 30% (v / v) H2O2 solution, or by mixing equal volumes of 1% (v / v) citric acid solution and 30% (v / v) H2O2 solution. The ultrasonic cleaning with deionized water is preferably performed 3 times, and the ultrasonic conditions can be selected as 40kHz, 30℃, and 10 minutes.

[0030] Preferably, the L-polylysine solution treatment of the present invention includes the following steps: immersing the microneedle patch (needle tip) in an L-polylysine solution; the concentration of the L-polylysine solution is 0.01~0.1% (w / v, g / mL), the molecular weight is 15~300 kDa, and the solvent is deionized water or PBS; the immersion conditions are immersion at room temperature for 30~120 min, more preferably 40 min, 50 min, 60 min, 70 min, 80 min, 90 min, 100 min, or 110 min. After immersion, the patch is allowed to stand for 60 min, then washed and dried; the washing and drying are preferably performed by washing three times with 0.05% PBS+T solution and then blotting with absorbent paper.

[0031] This invention also provides a rapid detection microneedle patch for anti-mutant citrulline vimentin antibody in trace blood samples prepared by the above preparation method. The microneedle patch has high sensitivity and high accuracy; compared with traditional blood tests, the amount of blood sample required is greatly reduced, requiring only 2~5μL of blood sample (whole blood), without the need for venous blood collection, and can be tested by finger prick; the detection time is only 20 minutes.

[0032] This invention also provides the application of the microneedle patch for rapid detection of anti-mutant citrulline vimentin antibodies in trace blood samples in the quantitative detection of anti-mutant citrulline vimentin antibodies in blood samples.

[0033] The first method for quantitative detection according to the present invention includes the following steps: a microneedle patch is immersed in a diluted blood sample for 2 hours, washed, and dried; then incubated with a horseradish peroxidase-linked rabbit anti-human IgG antibody solution, followed by washing; then immersed in a TMB single-component chromogenic solution for 5-15 minutes for color development, and the content of anti-mutant citrulline vimentin antibody in the blood sample is obtained based on the colorimetric value. The content of anti-mutant citrulline vimentin antibody in the blood sample is calculated based on a standard curve.

[0034] As an optional implementation, the method for plotting the standard curve is as follows: Anti-mutant citrulline vimentin antibody standards are dissolved in PBS and serially diluted, with at least 5 gradients, more preferably 7 gradients (6.25 ng / ml, 12.5 ng / ml, 25 ng / ml, 50 ng / ml, 100 ng / ml, 200 ng / ml, 400 ng / ml). Microneedle patches are immersed in each gradient of standard samples for 2 hours, washed, and dried; then incubated in horseradish peroxidase-linked rabbit anti-human IgG antibody solution, followed by washing; then immersed in TMB single-component chromogenic solution for 5-15 minutes for color development, and a standard curve of colorimetry and concentration is plotted based on the colorimetry.

[0035] The blood sample described in this invention is diluted 1600-2000 times. The washing is performed three times with 0.05% PBS+T solution, each time for 5 minutes. The drying is done by blotting with absorbent paper. The concentration of the horseradish peroxidase-linked rabbit anti-human IgG antibody solution is 5 μg / mL. The incubation time is 15-90 minutes, more preferably 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 70 minutes, or 80 minutes. The colorimetry is obtained by rapidly placing the microneedle patch under an imaging system and taking a picture, and then analyzing the image.

[0036] The second method for quantitative detection according to the present invention includes the following steps: a microneedle patch is immersed in a diluted blood sample for 2 hours, washed, and dried; then it is incubated in a fluorescein-conjugated rabbit anti-human IgG antibody solution, washed, and dried after incubation, and the content of anti-mutant citrulline vimentin antibody in the blood sample is obtained based on the fluorescence intensity. The content of anti-mutant citrulline vimentin antibody in the blood sample is calculated according to a standard curve.

[0037] As an optional implementation, the standard curve is plotted as follows: Anti-mutant citrulline vimentin antibody standards are dissolved in PBS and serially diluted at least five times, more preferably seven times (6.25 ng / ml, 12.5 ng / ml, 25 ng / ml, 50 ng / ml, 100 ng / ml, 200 ng / ml, 400 ng / ml). Microneedle patches are immersed in each gradient of standard samples for 2 hours, washed, and dried; then incubated in a fluorescein-conjugated rabbit anti-human IgG antibody solution, washed, and dried; a standard curve of fluorescence intensity versus concentration is plotted based on fluorescence intensity.

[0038] The blood samples described in this invention are diluted 1600-2000 times. The washing is performed three times with 0.05% PBS+T solution, each time for 5 minutes. The drying is done by blotting with absorbent paper. The concentration of the fluorescein-conjugated rabbit anti-human IgG antibody solution is 5 μg / mL. The incubation is performed in the dark for 15-90 minutes, more preferably 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 70 minutes, or 80 minutes. The fluorescence intensity is obtained by taking pictures and analyzing the microneedle patch under a fluorescence imaging system.

[0039] The overall flow chart of the microneedle in vivo detection technology and microneedle patch preparation in this invention is as follows: Figure 2 As shown, this invention can rapidly obtain quantitative data on anti-mutant citrulline vimentin antibodies in trace blood samples.

[0040] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0041] Unless otherwise specified, the following embodiments are all conventional methods.

[0042] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0043] Example 1 Fabrication using a microneedle molding method (fabrication process as follows) Figure 3 (as shown) Preparation of epoxy resin microneedles: Epoxy resin and curing agent (toluene dimethylamine) are thoroughly mixed in a ratio (mass ratio of 100:20), poured into a silicone microneedle mold, placed in a vacuum machine for vacuum defoaming, and then removed and allowed to solidify under natural conditions for 30 hours to obtain epoxy resin solid microneedles.

[0044] Preparation of polystyrene microneedles: Polystyrene was dissolved in dichloromethane to prepare a 20% (w / v, g / mL) polystyrene dichloromethane solution. The solution was poured into a silicone mold, placed in a vacuum machine to defoam, and then dried under natural conditions for 12 hours to form the microneedles.

[0045] Four different types of microneedles were prepared for each material, and the dimensional data are shown in the table below: Table 1. Dimensions of different microneedle models Four different types of microneedles made of two different materials were placed in a Shimadzu mechanical testing instrument to test the load-displacement curves of the microneedle materials. The load-displacement curve of the polystyrene microneedle is shown below. Figure 4 As shown, the load-displacement curve of epoxy resin microneedles is as follows: Figure 5 As shown in the figure, the data indicates that all four types of microneedles made of both materials meet the mechanical requirements for skin penetration, demonstrating good mechanical properties of both the materials and the microneedle structure.

[0046] Four different types of microneedles made of two materials were inserted into fresh pigskin. The morphology of the needle tip and body was observed during and after insertion. The results showed that the morphology of the needle tip and body remained intact during and after insertion, without any breakage.

[0047] Example 2 Preparation of microneedle patches for rapid detection of anti-mutant citrullinated vimentin antibodies in trace blood samples: 1. The material of the microneedle patch is epoxy resin. It is prepared by the molding method described in Example 1. The shape is designed as a cone. The dimensions are: the length of each needle is 430μm, the diameter of the bottom cone is 220μm, the distance between the needle tips is 580μm, the number of arrays is 11×11, and the side length of the whole microneedle patch is 9.8mm×9.8mm.

[0048] 2. Surface hydrophilic treatment of microneedle patches: Place the microneedle patches obtained in step 1 at a distance of 1 cm below the spray gun of the plasma treatment machine, and perform plasma treatment for 3 min to obtain microneedle patches with active functional groups introduced on the surface of the needle body.

[0049] 3. Coat the surface-treated microneedle patch obtained in step 2 with protein: Immerse the microneedle patch needles in a 5 μg / mL mutant citrulline vimentin solution at 4°C overnight. After removal, wash three times with 0.05% PBS+T solution for 5 min each time, and blot dry with absorbent paper. Immerse the needles in a 2% bovine serum albumin PBS solution and block at room temperature for 2 h. Wash three times with 0.05% PBS+T solution for 5 min each time, and blot dry with absorbent paper to obtain a microneedle patch with a surface coated with capture protein.

[0050] Example 3 Preparation of microneedle patches for rapid detection of anti-mutant citrullinated vimentin antibodies in trace blood samples: 1. The material of the microneedle patch is epoxy resin. It is prepared by the molding method described in Example 1. The shape is designed as a four-sided pyramid with the following dimensions: the length of each needle is 720 μm, the side length of the bottom of the four-sided pyramid is 320 μm, the distance between the needle tips is 680 μm, the number of arrays is 12×12, and the side length of the entire microneedle patch is 11.8 mm × 11.8 mm.

[0051] 2. The microneedle patch undergoes surface hydrophilic treatment: (1) Place the microneedle patch obtained in step 1 at a distance of 1 cm below the spray gun of the plasma treatment machine and perform plasma treatment for 3 min to obtain a microneedle patch with active functional groups introduced on the surface of the needle body.

[0052] (2) The microneedle patch obtained in (1) was modified by surface carboxylation enhancement: the microneedle patch was immersed in a 1:1 mixed solution of 1% malonic acid (or citric acid) and 30% H2O2, reacted at 60℃ for 1h, and then cleaned three times with deionized water by ultrasonication (40kHz, temperature 30℃, time 10min) to remove residual reagents. (3) Grafting PEG onto the microneedle patch obtained in (2): First, activate the surface carboxyl groups of the microneedle patch by shaking at room temperature for 30 min with 50 mM EDC and 25 mM NHS in pH 5.5 MES buffer. Then, transfer the microneedle patch to 2% NH2-PEG-COOH (5 kDa) in pH 7.4 PBS solution and incubate overnight at 4°C. Finally, remove unreacted PEG by ultrasonic washing three times with deionized water to form a PEG layer with carboxylic acid groups retained at the ends for subsequent protein coating.

[0053] 3. Coat the surface-treated microneedle patch obtained in step 2 with protein: The microneedle patch was immersed in a 10 μg / mL mutant citrulline vimentin solution and coated at room temperature for 2 h. After removal, it was washed three times with 0.05% PBS+T solution for 5 min each time, and then dried with absorbent paper. The patch was then immersed in a 2% bovine serum albumin PBS solution and blocked at room temperature for 1 h. After washing three times with 0.05% PBS+T solution for 5 min each time, it was dried with absorbent paper to obtain a microneedle patch coated with capture protein.

[0054] Example 4 Preparation of microneedle patches for rapid detection of anti-mutant citrullinated vimentin antibodies in trace blood samples: 1. The material of the microneedle patch is epoxy resin. It is prepared by the molding method described in Example 1. The shape is designed as a four-sided pyramid with the following dimensions: each needle body is 1000 μm long, the bottom side length of the four-sided pyramid is 390 μm, the needle tip distance is 700 μm, the array number is 10×10, and the side length of the entire microneedle patch is 11.7 mm × 11.7 mm.

[0055] 2. The microneedle patch undergoes surface hydrophilic treatment: (1) Place the microneedle patch obtained in step 1 at a distance of 0.5-2.5 cm below the spray gun of the plasma treatment machine and treat it with plasma for 1-5 min to obtain a microneedle patch with active functional groups introduced on the surface of the needle.

[0056] (2) Immerse the microneedle patch tip obtained in (1) in L-polylysine solution (L-polylysine solution concentration is 0.05% (g / mL), molecular weight is 200 kDa, dissolved in deionized water). The tip immersion conditions are 50 min at room temperature, 60 min standing, 3 washes with 0.05% PBS+T solution for 5 min each time, and then absorbent paper to dry.

[0057] 3. Coat the surface-treated microneedle patch obtained in step 2 with protein: The microneedle patch was immersed in a 50 μg / mL mutant citrulline vimentin solution and coated at room temperature for 2 h. After removal, it was washed three times with 0.05% PBS+T solution for 5 min each time, and then dried with absorbent paper. The patch was then immersed in a 2% bovine serum albumin PBS solution and blocked at room temperature for 3 h. After washing three times with 0.05% PBS+T solution for 5 min each time, it was dried with absorbent paper to obtain a microneedle patch coated with capture protein.

[0058] Example 5 Preparation of microneedle patches for rapid detection of anti-mutant citrullinated vimentin antibodies in trace blood samples: 1. The material of the microneedle patch is epoxy resin. It is prepared by the molding method described in Example 1. The shape is designed as a four-sided pyramid with the following dimensions: each needle body is 1000 μm long, the bottom side length of the four-sided pyramid is 390 μm, the needle tip distance is 700 μm, the array number is 10×10, and the side length of the entire microneedle patch is 11.7 mm × 11.7 mm.

[0059] 2. The microneedle patch undergoes surface hydrophilic treatment: (1) Place the microneedle patch obtained in step 1 at a distance of 2 cm below the spray gun of the plasma treatment machine and perform plasma treatment for 3 min to obtain a microneedle patch with active functional groups introduced on the surface of the needle body.

[0060] (2) The microneedle patch obtained in (1) was modified by surface carboxylation enhancement: the microneedle patch was immersed in a 1:1 mixed solution of 1% malonic acid (or citric acid) and 30% H2O2, reacted at 60℃ for 1h, and then cleaned three times with deionized water by ultrasonication (40kHz, temperature 30℃, time 10min) to remove residual reagents. (3) Grafting PEG onto the microneedle patch obtained in (2): First, activate the surface carboxyl groups of the microneedle patch by shaking at room temperature for 30 min with 50 mM EDC and 25 mM NHS in pH 5.5 MES buffer. Then, transfer the microneedle patch to 1-2% NH2-PEG-COOH (5k Da) in pH 7.4 PBS solution and react at room temperature for 5 hours. Finally, remove unreacted PEG by ultrasonic washing with deionized water 3 times to form a PEG layer with carboxylic acid groups retained at the end for subsequent protein coating.

[0061] (4) Immerse the microneedle patch tip obtained in (3) in L-polylysine solution (L-polylysine solution concentration is 0.05% (g / mL), molecular weight is 100 kDa, dissolved in deionized water). The tip immersion conditions are 60 min at room temperature, 60 min standing, 3 washes with 0.05% PBS+T solution for 5 min each time, and then absorbent paper to dry.

[0062] 3. Coat the surface-treated microneedle patch obtained in step 2 with protein: The microneedle patch needles were immersed in a 20 μg / mL mutant citrulline vimentin solution and incubated overnight at 4°C. After removal, the needles were washed three times with 0.05% PBS+T solution for 5 min each time, and then blotted dry with absorbent paper. The needles were then immersed in a 2% bovine serum albumin PBS solution and blocked at room temperature for 2 h. After washing three times with 0.05% PBS+T solution for 5 min each time, the needles were blotted dry with absorbent paper to obtain a microneedle patch with the capture protein coated on the surface.

[0063] Example 6 Preparation of microneedle patches for rapid detection of anti-mutant citrullinated vimentin antibodies in trace blood samples: 1. The material of the microneedle patch is polystyrene. It is prepared by the molding method described in Example 1. The shape is designed as a four-sided pyramid with the following dimensions: each needle is 1200 μm long, the bottom side length of the pyramid is 500 μm, the distance between the needle tips is 1000 μm, the number of arrays is 10×10, and the side length of the entire microneedle patch is 14.5 mm × 14.5 mm.

[0064] 2. The microneedle patch undergoes surface hydrophilic treatment: (1) Immerse the microneedle patch tip obtained in step 1 in L-polylysine solution (L-polylysine solution concentration is 0.05% (g / mL), molecular weight 100 kDa, dissolved in deionized water). The tip immersion conditions are 30 min at room temperature, 60 min standing, 3 washes with 0.05% PBS+T solution for 5 min each time, and then absorbent paper to dry.

[0065] 3. Coat the surface-treated microneedle patch obtained in step 2 with protein: The microneedle patch was immersed in a 60 μg / mL mutant citrulline vimentin solution and coated at room temperature for 2 h. After removal, it was washed three times with 0.05% PBS+T solution for 5 min each time, and then dried with absorbent paper. The patch was then immersed in a 2% bovine serum albumin PBS solution and blocked at room temperature for 2 h. After washing three times with 0.05% PBS+T solution for 5 min each time, it was dried with absorbent paper to obtain a microneedle patch coated with capture protein.

[0066] Example 7 Preparation of microneedle patches for rapid detection of anti-mutant citrullinated vimentin antibodies in trace blood samples: The material of the microneedle patch is polystyrene, and it is prepared by the molding method described in Example 1. The shape is designed as a cone, with the following dimensions: each needle body is 430 μm long, the bottom cone diameter is 220 μm, the needle tip distance is 580 μm, the array number is 11×11, and the side length of the entire microneedle patch is 9.8 mm × 9.8 mm.

[0067] The microneedle patch was immersed in a 100 μg / mL mutant citrulline vimentin solution and coated at room temperature for 2 h. After removal, it was washed three times with 0.05% PBS+T solution for 5 min each time, and then dried with absorbent paper. The patch was then immersed in a 3% bovine serum albumin PBS solution and blocked at room temperature for 3 h. After washing three times with 0.05% PBS+T solution and drying with absorbent paper, the microneedle patch coated with the capture protein was obtained.

[0068] Example 8 The difference between this embodiment and Embodiment 2 is that the material of the microneedle patch is replaced with polystyrene instead of epoxy resin.

[0069] Example 9 The difference between this embodiment and Embodiment 3 is that the material of the microneedle patch is replaced with polystyrene instead of epoxy resin.

[0070] Example 10 The difference between this embodiment and embodiment 4 is that the material of the microneedle patch is replaced with polystyrene instead of epoxy resin.

[0071] Example 11 The difference between this embodiment and Embodiment 5 is that the material of the microneedle patch is replaced with polystyrene instead of epoxy resin.

[0072] Example 12 Verify the protein coating efficiency after surface treatment: The epoxy resin microneedle patch prepared in Example 1 was placed 1 cm below the spray gun of a plasma treatment machine and plasma treated for 3 min to obtain a microneedle patch with active functional groups introduced on the needle surface. The surface-treated microneedles and the untreated microneedles were immersed in 5 μg / ml fluorescent antibody solution (goat anti-rabbit IgG antibody - Alexa Fluor® 555) for coating in the dark, incubated overnight at 4°C, washed three times with 0.05% PBS+T washing buffer for 5 min each time, dried, and then imaged under an inverted fluorescence microscope to measure the fluorescence intensity. The results are as follows: Figure 6 As shown in the figure, the y-axis represents the fluorescence intensity.

[0073] The results showed that the protein coating efficiency was significantly improved after surface treatment.

[0074] Example 13 The microneedle patch prepared in Example 2 was used to test blood samples.

[0075] The anti-mutant citrulline vimentin antibody standard (purchased from the Human Anti-mutant Citrulline Vimentin Antibody (MCV) ELISA Kit) was dissolved in PBS and serially diluted in seven gradients (6.25 ng / ml, 12.5 ng / ml, 25 ng / ml, 50 ng / ml, 100 ng / ml, 200 ng / ml, and 400 ng / ml). Microneedle patches were immersed in each concentration of the standard solution for 2 hours, then washed three times with 0.05% PBS+T solution for 5 minutes each time, and dried. The microneedle patches were then incubated in 5 μg / mL horseradish peroxidase-linked rabbit anti-human IgG antibody solution (purchased from Beijing Solarbio Science & Technology Co., Ltd.) for 30 minutes, followed by washing three times with 0.05% PBS+T solution for 5 minutes each time. The obtained microneedle patch was immersed in TMB single-component colorimetric solution (purchased from Beijing Solarbio Technology Co., Ltd.) for 5 minutes. After the time was up, it was quickly placed under the imaging system to take pictures and analyze the color of the images to obtain the grayscale standard curve of the microneedle in vitro detection image.

[0076] Two μL of blood sample was diluted 2000-fold with PBS. A microneedle patch was immersed in the diluted blood sample for 2 hours. After immersion, the patch was washed three times with 0.05% PBS+T solution for 5 minutes each time, and then dried. The microneedle patch was then incubated in 5 μg / mL horseradish peroxidase-linked rabbit anti-human IgG antibody solution (purchased from Beijing Solarbio Science & Technology Co., Ltd.) for 30 minutes. After incubation, the patch was washed three times with 0.05% PBS+T solution for 5 minutes each time. The resulting microneedle patch was then immersed in TMB single-component chromogenic solution (purchased from Beijing Solarbio Science & Technology Co., Ltd.) for 5 minutes. After the 5 minutes, the patch was quickly placed under an imaging system to take a picture. The colorimetric properties of the image were analyzed, and the concentration of anti-mutant citrullinated vimentin antibody in the blood sample was calculated based on the standard curve.

[0077] Example 14 The microneedle patch prepared in Example 2 was used to test blood samples.

[0078] Anti-mutant citrulline vimentin antibody standard (purchased from the Human Anti-mutant Citrulline Vimentin Antibody (MCV) ELISA Kit) was dissolved in PBS and serially diluted seven times (6.25 ng / ml, 12.5 ng / ml, 25 ng / ml, 50 ng / ml, 100 ng / ml, 200 ng / ml, 400 ng / ml). Microneedle patches were immersed in each concentration of the standard solution for 2 hours, then washed three times with 0.05% PBS+T solution for 5 minutes each time, and dried. The microneedle patches were then incubated in 5 μg / mL fluorescein-conjugated rabbit anti-human IgG antibody solution (purchased from Beijing Solarbio Science & Technology Co., Ltd.) for 30 minutes, followed by washing three times with 0.05% PBS+T solution for 5 minutes each time, and dried. The resulting microneedle patches were then photographed and analyzed using a fluorescence imaging system to obtain a standard curve.

[0079] Two μL of blood sample was diluted 2000-fold with PBS. A microneedle patch was immersed in the diluted blood sample for 2 hours. After immersion, the patch was washed three times with 0.05% PBS+T solution for 5 minutes each time, and then dried. The microneedle patch was then immersed in 5 μg / mL fluorescein-conjugated rabbit anti-human IgG antibody solution (purchased from Beijing Solarbio Science & Technology Co., Ltd.) for 30 minutes. After incubation, the patch was washed three times with 0.05% PBS+T solution for 5 minutes each time, and then dried. The resulting microneedle patch was photographed and its fluorescence intensity analyzed using a fluorescence imaging system. The concentration of anti-mutant citrullinated vimentin antibody in the blood sample was calculated based on the standard curve.

[0080] Experimental Example 1 The tail vein blood of mice was tested using the method described in Example 13.

[0081] The ELISA method was used to detect the anti-mutant citrulline vimentin antibody (coating: antigen fixation; blocking: to prevent non-specific binding; sample loading: primary antibody incubation; sample loading: enzyme-labeled secondary antibody incubation; color development: addition of enzyme substrate to generate a detectable signal). The OD values ​​of the whole blood concentration gradient were obtained, and the OD values ​​of the whole blood were compared with the grayscale values ​​of simultaneous microneedle detection; the data trends were consistent. A standard curve was plotted based on the ELISA detection results, as shown in the figure. Figure 7 As shown.

[0082] Blood was collected from the tail vein of model mice and diluted with Tris buffer at 100, 200, 400, 800, 1600, and 3200 times, respectively. The results of the whole blood dilution tests were obtained. Figure 8 As shown. Figure 8 Grayscale data and Figure 7 The quantitative results of diluted mouse whole blood solutions were obtained through comparison. The results show that the microneedle in vitro micro-volume whole blood detection technology presented in this invention can accurately detect anti-mutant citrullinated vimentin antibodies in trace blood samples with high sensitivity.

[0083] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a microneedle patch for rapid detection of anti-mutant citrullinated vimentin antibody in trace blood samples, characterized in that, Includes the following steps: To prepare a microneedle patch, the needles of the microneedle patch were immersed in a 2-100 μg / mL mutant citrulline vimentin solution for protein coating, then washed and dried with 0.05% PBS+T solution. The dried needles were then immersed in a 1-3% bovine serum albumin PBS solution for blocking, then washed and dried with 0.05% PBS+T solution to obtain a rapid detection microneedle patch for anti-mutant citrulline vimentin antibody in trace amounts of blood samples. The protein coating conditions were room temperature coating for 2 hours or overnight coating at 4°C; the blocking conditions were room temperature blocking for 1-3 hours.

2. The preparation method according to claim 1, characterized in that, The microneedle patch is made of epoxy resin and polystyrene; the needle body of the microneedle patch is conical or pyramidal, with a needle body length of 430~1200μm and a needle tip distance of 580~1000μm; the bottom diameter of the conical needle body is 220μm, and the bottom side length of the pyramidal needle body is 320~500μm.

3. The preparation method according to claim 2, characterized in that, The microneedle patch undergoes a surface treatment before protein coating; the surface treatment includes any of the following: (1) Perform plasma surface activation treatment on the needle body surface; (2) The needle body surface is subjected to plasma surface activation treatment and polyethylene glycol grafting treatment in sequence; (3) The needle surface is subjected to plasma surface activation treatment and L-polylysine solution treatment in sequence; (4) The needle body surface is subjected to plasma surface activation treatment, polyethylene glycol grafting treatment and L-polylysine solution treatment in sequence; (5) Treat the surface of the needle with L-polylysine solution.

4. The preparation method according to claim 3, characterized in that, The plasma surface activation treatment includes the following steps: placing the microneedle patch 0.5-2.5 cm below the plasma treatment machine's spray gun and treating with plasma for 1-5 minutes.

5. The preparation method according to claim 3, characterized in that, The polyethylene glycol grafting treatment includes the following steps: The microneedle patch was immersed in the mixed solution and reacted at 60°C for 1 h, followed by ultrasonic cleaning with deionized water; then immersed in MES buffer containing 50 mM MEDC and 25 mM NHS, and shaken at room temperature for 30 min, before being transferred to PBS solution containing 1~2 w / v% NH2-PEG-COOH, reacted at room temperature for 4~6 h, and then ultrasonically cleaned with deionized water. The mixed solution is obtained by mixing equal volumes of 1 v / v % malonic acid solution and 30 v / v % H2O2 solution, or by mixing equal volumes of 1 v / v % citric acid solution and 30 v / v % H2O2 solution.

6. The preparation method according to claim 3, characterized in that, The L-polylysine solution treatment includes the following steps: the microneedle patch is immersed in the L-polylysine solution; the concentration of the L-polylysine solution is 0.01~0.1 w / v%, the molecular weight is 15~300 kDa, and the solvent is deionized water or PBS; the immersion conditions are immersion at room temperature for 30~120 min.

7. A microneedle patch for rapid detection of anti-mutant citrulline vimentin antibody in trace blood samples prepared by the preparation method according to any one of claims 1 to 6.

8. The application of the microneedle patch for rapid detection of anti-mutant citrulline vimentin antibodies in trace blood samples as described in claim 7 in the quantitative detection of anti-mutant citrulline vimentin antibodies in blood samples.

9. The application according to claim 8, characterized in that, The quantitative detection method includes the following steps: Microneedle patches were immersed in diluted blood samples for 2 hours, washed, and dried. They were then incubated in horseradish peroxidase-linked rabbit anti-human IgG antibody solution and washed. Finally, they were immersed in TMB single-component chromogenic solution for 5-15 minutes. The content of anti-mutant citrulline vimentin antibody in the blood sample was obtained based on the color.

10. The application according to claim 8, characterized in that, The quantitative detection method includes the following steps: Microneedle patches were immersed in diluted blood samples for 2 hours, washed, and dried. They were then incubated in a fluorescein-conjugated rabbit anti-human IgG antibody solution, washed, and dried after incubation. The content of anti-mutant citrulline vimentin antibody in the blood sample was obtained based on the fluorescence intensity.

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