Transdermal diagnosis device for autoimmune disease oral mucosal lesion, manufacturing method and using method

Abstract

The invention provides a transdermal diagnosis device for oral mucosal lesion caused by autoimmune diseases, a manufacturing method and a using method, and belongs to the technical field of immune pathological diagnosis applied to autoimmune skin diseases. Comprising a transdermal assembly, a supporting assembly and a fixing assembly, the transdermal assembly comprises at least one group of microneedles capable of penetrating through mucosa, and each microneedle comprises an inner needle body and an outer coating; the outer coating is arranged on the outer surface of the inner needle body; the supporting assembly is connected with the transdermal assembly and the fixing assembly; the fixing assembly is used for fixing the transdermal assembly and the supporting assembly to the inner surface of the oral mucosa. According to the present invention, the kit can rapidly penetrate through the oral mucosa, can be combined with the protein marker existing in the mucosa tissue, can display the deposition mode of the protein marker in the mucosa by using the fluorescent molecule reduction in vitro, and can accurately distinguish the autoimmune oral mucosa damage from the non-autoimmune oral mucosa damage according to different fluorescence intensities or different fluorescence modes;

Description

Technical Field

[0001] The present invention belongs to the technical field of immunopathological diagnosis applied to autoimmune skin diseases, and relates to a transdermal diagnosis device for oral mucosal lesions of autoimmune diseases, a manufacturing method and a use method. Background Art

[0002] Autoimmune diseases are a type of disease that causes destruction of tissues and organs due to immune system disorders, including oral pemphigus, mucosal oral pemphigoid, oral lupus erythematosus, etc. The core feature is that the human body produces autoreactive immune cells and autoantibodies, which recognize and attack skin tissues, leading to chronic inflammation and tissue damage, and can affect the skin, mucosa and other organs. Oral mucosal lesions can be an early manifestation or even the only manifestation of such diseases. At present, pathological biopsy and direct immunofluorescence can be used to distinguish autoimmune and non-autoimmune oral lesions, with both specificity, sensitivity and intuitiveness. However, oral mucosal biopsy is difficult to obtain, and it is easy to aggravate the patient's pain and original mucosal damage. In addition, during the long course of the disease, patients have very poor compliance with multiple biopsies. Although indirect immunofluorescence and ELISA tests overcome the shortcomings of biopsy, the former requires a sensitive substrate, otherwise the positive rate of the test results is low, and the latter is less intuitive. Therefore, developing a minimally invasive, simple, and intuitive method for diagnosing and identifying autoimmune oral mucosal lesions to replace traditional pathological biopsy and immunopathology methods is an important issue for further optimizing the diagnosis of autoimmune diseases. Summary of the invention

[0003] In order to solve the shortcomings of existing autoimmune skin disease diagnosis methods, the purpose of the present invention is to provide a transdermal diagnostic device to achieve minimally invasive, simple and intuitive diagnosis of autoimmune skin diseases, which can be used to replace indirect immunofluorescence and has similar functions to direct immunofluorescence.

[0004] The present invention provides a transdermal diagnostic device for oral mucosal damage of autoimmune diseases, comprising a transdermal component, a supporting component and a fixing component; The transdermal component includes at least one group of microneedles capable of penetrating the mucosa, each microneedle including an inner needle body and an outer coating, the outer surface of the inner needle body is provided with a capture fork, the capture fork is used to capture protein markers present in the oral mucosa of autoimmune diseases; the outer coating is arranged on the outer surface of the inner needle body, is used to protect the inner needle body and is dissolved in the mucosa; One end of the support component is attached to the transdermal component, and the other end is connected to the fixing component; The fixing component is used to fix the transdermal component and the supporting component on the inner surface of the oral mucosa.

[0005] Optionally, the material of the inner needle body is set to at least one of polymethyl vinyl ether / maleic anhydride, hyaluronic acid, methacrylate hyaluronic acid, polyvinyl pyrrolidone, polyvinyl alcohol or polylactic acid.

[0006] Optionally, the total height H1 of the inner needle body is 400 μm-600 μm, the base width D1 is 200 μm-300 μm, and the needle distance X2 between the centers of adjacent microneedles is 600 μm.

[0007] Optionally, the inner needle body includes a needle tip and a needle body, the shape of the needle tip is set to be conical, pyramidal, bullet-shaped, hemispherical, truncated cone or prism-shaped, and the shape of the needle body is set to be truncated cone, cylindrical or prism-shaped.

[0008] Optionally, the height H3 of the needle tip is 100 μm-200 μm, and the base width D2 is 50 μm-300 μm; The height H4 of the needle body is 300 μm-400 μm, the width D2 of the upper base is 50 μm-300 μm, and the width of the lower base is 200 μm-300 μm; The material of the outer coating is set to be at least one of polysaccharides, cellulose, polypeptides, proteins or glues; The material of the support component is set to at least one of polymethyl vinyl ether / maleic acid, hyaluronic acid, methacrylate hyaluronic acid, polyvinyl pyrrolidone, polyvinyl alcohol or polylactic acid.

[0009] Optionally, the material of the outer coating layer contains a quick dissolving agent, and the quick dissolving agent is set to be a water-soluble divalent inorganic salt or sodium alginate; The outer coating covers the needle tip and the surface of the inner needle body, and the thickness H2 of the outer coating is 50-60 μm; The shape of the support component is set to be an elongated rectangle, a square or a circle, and the thickness of the support component is 200 μm-1000 μm.

[0010] Optionally, when the shape of the support component of the transdermal diagnostic device is set to be an elongated rectangle, the side length combination includes 20mm×15mm, 40mm×15mm or 60mm×15mm; When the shape of the support component of the transdermal diagnostic device is set to be a square, the side length X1 thereof is set to be 7 mm, 14 mm or 28 mm; When the shape of the support component of the transdermal diagnostic device is set to be circular, its diameter is set to 7 mm, 14 mm or 28 mm.

[0011] The present invention also provides a method for manufacturing a transdermal diagnostic device for oral mucosal damage of autoimmune diseases, comprising the following steps: Step 1, preparation of inner layer microneedles and basal layer solution; S1.1. Weigh the materials for preparing the needle tip, needle body or base layer of the inner layer microneedle and the photocrosslinking agent and directly dissolve them in distilled water at a suitable temperature, or dissolve them separately in distilled water and then mix them, so that the mass volume concentration of the materials after mixing in distilled water is 10% to 24%, and the mass volume concentration of the photocrosslinking agent in distilled water is 0.1%, so that it becomes a uniform solution and bubbles are arranged side by side, and the inner layer microneedle needle tip, needle body or base layer solution is obtained; S1.2, the inner layer microneedle materials are polymethyl vinyl ether / maleic anhydride and methacrylate hyaluronic acid, and the photocrosslinker is lithium phenyl-2,4,6-trimethylbenzoylphosphonate; at room temperature, the two materials are dissolved in distilled water respectively, and then the two solutions are fully mixed and the bubbles are removed. The mass volume concentrations of the mixed polymethyl vinyl ether / maleic anhydride and methacrylate hyaluronic acid are 20% to 24% and 10% to 12% respectively, and the mass volume concentration of the photocrosslinker LAP is 0.1%; S1.3. Methacrylate hyaluronic acid is selected as the material of the microneedle base layer, and LAP is selected as the photocrosslinker. At room temperature, methacrylate hyaluronic acid is fully dissolved in distilled water and bubbles are removed, the mass volume concentration is 20-24%, and the mass volume concentration of the photocrosslinker is 0.1%; Step 2: Preparation of capture antibody solution of autoantibodies; Weigh the capture antibody solution of autoantibody IgG and mix it evenly, then add morpholineethanesulfonic acid buffer containing N-hydroxysuccinimide and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide as a carbodiimide crosslinker to ensure that the capture antibody concentration in the final solution is set to 0.5μg / ml-1.5μg / ml and the ratio is set to 1:1; the concentrations of NHS and EDC are set to 5-10mg / ml and the ratio is set to 1:1; Step 3: Preparation of coating solution; Take appropriate amounts of materials for preparing the coating layer, photocrosslinking agent, and quick dissolving agent and directly dissolve them in distilled water at a suitable temperature, so that the mass volume concentration of the coating layer materials after mixing in distilled water is 20% to 24%, the mass volume concentration of the photocrosslinking agent in distilled water is 0.1%, and the mass volume concentration ratio of the quick dissolving agent in distilled water is 0.1% to 10%; Step 4: preparing inner microneedles and a basal layer; The inner microneedles and the base layer were obtained by using the mold method, droplet spraying method, stereolithography method and 3D printing method; Step 5, coating the inner microneedles with the capture antibody of the autoantibody IgG and the coating layer of the coated microneedles; The capture antibody coated with autoantibody IgG and the coated microneedle coating layer are obtained by using deposition method, dip coating method and spray coating method; Step 6: Manufacturing fixed components; The fixing components are fixed by a pasting method, and the fixing components are manufactured by a mold method, an electrospinning method, an electrospraying method and a 3D printing method; Alternatively, the fixing component is fixed by an adsorption method, and the fixing component is manufactured by a mold method, a hot pressing method, and a 3D printing method; Alternatively, the fixing components are fixed by a clamping method, and the fixing components are manufactured by a mold method, a hot pressing molding method, and a 3D printing method.

[0012] The present invention also provides a method for using a transdermal diagnostic device for oral mucosal damage caused by autoimmune diseases, comprising a sampling step and a diagnostic step; wherein: 1. Sampling procedures; The transdermal diagnostic device is introduced into the junction of the patient's skin lesions and normal skin and stays there for 20-40 minutes; the microneedle enters the deep epidermis, the coating layer decomposes, the inner needle body absorbs interstitial fluid and swells, and the capture antibody in its capture fork binds to the autoantibody; (ii) diagnostic steps; The diagnostic steps include incubation with biotinylated detection antibodies, incubation with streptavidin-containing fluorophores, and observation with a fluorescence microscopy imager; ①, Incubation of biotinylated detection antibody; Incubate the transdermal diagnostic device with 5 ml of a standard protein solution having a biotinylated detection antibody concentration of 2 μg / ml-4 μg / ml at room temperature for 1-2 h, and wash with a phosphate buffer solution; ②, Incubation of streptavidin fluorophore; Incubate the transdermal diagnostic device after step ① with 5 ml of a standard protein solution containing 3 μg / ml to 5 μg / ml streptavidin fluorophore for 30 min to 45 min, and wash with a phosphate buffer solution; ③ Observation by fluorescence microscope; Observe the transdermal diagnostic device that has completed step ② using a fluorescence microscope, and obtain a corresponding diagnostic result based on whether fluorescence is observed; If strong fluorescence is observed, it indicates that the patient has an autoimmune skin disease; If weak / no fluorescence is observed, it suggests that the patient has a non-autoimmune skin disease.

[0013] Optionally, a preliminary identification of the specific type of autoimmune skin disease is performed by: ⅰ. Strong fluorescence was observed, indicating that the patient's oral mucosal damage was caused by an autoimmune disease; ii. The fluorescence was observed to be distributed in a continuous linear pattern near the needle tip of the inner needle body, indicating that the protein marker was deposited in a continuous linear pattern between the mucosal epithelium and the mucosal lamina propria, suggesting that the oral mucosal damage was caused by mucosal oral pemphigoid; ⅲ. Fluorescence was observed to be distributed in the inner needle body near the needle tip in a discontinuous linear or granular manner, indicating that the protein marker was deposited in a discontinuous linear or granular manner between the mucosal epithelium and the mucosal lamina propria, suggesting that the oral mucosal damage was caused by oral lupus erythematosus; ⅳ. The fluorescence was observed to be irregularly distributed near the needle tip of the inner needle body, indicating that the protein marker was irregularly deposited between the mucosal epithelium and the mucosal lamina propria, suggesting that the oral mucosal damage was caused by lichen planus; ⅴ. Fluorescence was observed to be distributed in a grid-like pattern at the needle tip and the needle body, indicating that the protein marker was deposited between the mucosal epithelial cells, suggesting that the oral mucosal damage was caused by oral pemphigus; ⅵ. Fluorescence was observed, but it was different from the forms of ⅰ-ⅴ and was deposited in the inner needle body in a non-specific manner, indicating that protein markers were deposited, suggesting that the oral mucosal damage was caused by other autoimmune diseases; ⅶ. Weak / no fluorescence was observed, suggesting that the patient's oral mucosal damage was caused by non-autoimmune diseases.

[0014] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention provides a rapid and minimally invasive method for detecting oral lesions of autoimmune diseases, which can improve the early detection rate of autoimmune diseases involving the oral mucosa.

[0015] (2) The transdermal component of the diagnostic system is mainly made of polymer materials and has high biocompatibility.

[0016] (3) The outer coating of the microneedle in the transdermal component of the diagnostic system is hard, which can protect the inner needle body and capture fork and reduce the damage of the needle body during insertion into the mucosa; it can be quickly decomposed in the mucosa and does not prevent the capture fork from binding to the biomarker.

[0017] (4) Since the entire surface of the inner needle is covered with capture forks, the color pattern (fluorescence intensity, fluorescence position, etc.) after capturing the protein marker reflects its deposition pattern, quantity and depth in the skin, which is highly intuitive.

[0018] (5) The transdermal diagnostic system proposed in the present invention can realize minimally invasive, simple and intuitive diagnosis of oral mucosal lesions of autoimmune diseases. Compared with indirect immunofluorescence, it has a higher positive detection rate and breaks through the limitation of direct immunofluorescence that requires the sampling of pathological tissue.

[0019] In addition to the above-described purposes, features and advantages, the present invention has other purposes, features and advantages. The present invention will be further described in detail with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The drawings constituting a part of this application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings: Figure 1 This is a schematic diagram of the overall structure of a transdermal diagnostic device for oral mucosal damage caused by autoimmune diseases in Example 1 of the present invention; Figure 2 yes Figure 1 Schematic diagram of the connection between two adjacent microneedles; Figure 3 yes Figure 1 Schematic diagram of the structure of a single microneedle; Figure 4 yes Figure 1 Schematic diagram of the inner needle body, capture fork and outer coating; Figure 5 is a schematic diagram of a transdermal diagnostic device fixed by adsorption method in Embodiment 1 of the present invention; Figure 6 is a schematic diagram of a transdermal diagnostic device fixed by a clamping method in Embodiment 1 of the present invention; Figure 7 This is a flow chart of manufacturing the inner needle body and the support assembly using a mold method in the second embodiment of the present invention; Figure 8 This is a flow chart of manufacturing a support component using a mold method in Embodiment 2 of the present invention; Fig. 9 This is a flow chart of manufacturing an inner needle body by using a droplet spraying method in Embodiment 2 of the present invention; Fig.10 This is a flow chart of manufacturing an inner needle body by using a droplet spraying method in Embodiment 2 of the present invention; Fig.11 This is a flow chart of coating the capture fork using a deposition method in Example 2 of the present invention; Fig.12 This is a flow chart of coating a capture fork using a dip coating method in Example 2 of the present invention; Fig.13 This is a flow chart of coating a capture fork using a spray coating method in Embodiment 2 of the present invention; Fig.14 This is a flow chart of using a deposition method to coat an outer coating in Example 2 of the present invention; Fig.15 This is a flow chart of applying an outer coating using a dip coating method in Example 2 of the present invention; Fig.16This is a flow chart of using a spray coating method to coat an outer coating in Example 2 of the present invention; Fig.17 This is a flow chart of using a mold method to manufacture a fixing component in which the fixing method is an adhesive method in Embodiment 2 of the present invention; Fig.18 This is a flow chart of using a mold method to manufacture a fixing component using an adsorption method in Embodiment 2 of the present invention.

[0021] in: 1. Transdermal component, 2. Support component, 3. Fixing component, 31. Clamp, 32. Clamp handle, 33, 4. Microneedle, 41. Inner needle body, 42. Outer coating, 411. Needle tip, 412. Needle body. DETAILED DESCRIPTION

[0022] In order to make the above-mentioned purposes, features and advantages of the present invention more clear and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the accompanying drawings. It should be noted that the drawings of the present invention are all simplified and use non-precise proportions, which are only used to conveniently and clearly assist in explaining the implementation of the present invention; the "number" mentioned in the present invention is not limited to the specific number in the examples in the accompanying drawings; the directions or positional relationships indicated by "front", "middle", "back", "left", "right", "up", "down", "top", "bottom", "middle", etc. mentioned in the present invention are based on the directions or positional relationships shown in the drawings of the present invention, and do not indicate or imply that the devices or components referred to must have a specific direction, nor can they be understood as limitations on the present invention.

[0023] See also Figures 1 to 6 As shown, a transdermal diagnostic device for oral mucosal damage caused by autoimmune diseases provided by the present invention includes a transdermal component 1, a supporting component 2, and a fixing component 3.

[0024] The transdermal component 1 includes a group of conical microneedles 4 , each of which is composed of an inner needle body 41 and an outer coating 42 .

[0025] The inner needle body 41 consists of a needle tip 411 and a needle body 412 , with a total height H1 of 400 μm-600 μm (preferably set to 600 μm), a base width D1 of 200 μm-300 μm (preferably set to 300 μm), and a needle distance X2 between adjacent microneedle centers of 600 μm.

[0026] The surface of the inner needle body 41 is provided with a capture fork.

[0027] The shape of the needle tip 411 is set to be conical, pyramidal, bullet-shaped, hemispherical, truncated cone or prism-shaped, and the pyramidal shape can include triangular pyramid and quadrangular pyramid. Preferably, the shape of the needle tip 411 is preferably set to be conical, and its height H3 is 100μm-200μm (preferably set to 200μm), and the base width D2 is 50μm-300μm (preferably set to 100μm).

[0028] The needle body 412 is truncated cone-shaped, with a height H4 of 300-400 μm (preferably 400 μm), an upper base width D2 of 50-300 μm (preferably 100 μm), and a lower base width of 200-300 μm (preferably 300 μm).

[0029] The outer coating 42 covers the needle tip 411 of the inner needle body 41 and the surface of the needle body 412 , and the thickness H2 is 50-60 μm.

[0030] The shape of the support component 2 is set to be rectangular, circular or diamond. Preferably, the shape of the support component 2 is preferably set to be square and elongated rectangle (strip) to adapt to the sampling requirements of connective tissue diseases and autoimmune bullous skin diseases. Specifically, for the diagnosis of autoimmune bullous diseases, the shape of the support component of the transdermal diagnostic device should preferably be an elongated rectangle (strip); for other autoimmune diseases such as oral lupus erythematosus, scleroderma, dermatomyositis and other connective tissue diseases, the shape of the support component of the transdermal diagnostic device should preferably be square. Further preferably, when the shape of the support component 2 is a square, its side length X1 is set to 7mm, 14mm, and 28mm, respectively matching the number of microneedles 25 (5×5), 100 (10×10), and 400 (20×20); when the shape of the support component 2 is an elongated rectangle, its side length combination may include but is not limited to 20mm×15mm, 40mm×15mm, or 60mm×15mm, to adapt to microneedle arrangements including 150 (15×10), 250 (25×10), and 400 (40×10). The thickness Z1 of the support component 2 is 200μm.

[0031] Furthermore, the material of the inner needle body 41 is set to one or more of polymethyl vinyl ether / maleic anhydride, hyaluronic acid, methacrylic hyaluronic acid, polyvinyl pyrrolidone, polyvinyl alcohol or polylactic acid. Further preferably, considering that the inner needle body 41 should have the characteristics of high hardness, strong swelling and strong binding ability with the capture fork, the material of the inner needle body 41 is preferably set to polymethyl vinyl ether / maleic acid (anhydride) and methacrylic hyaluronic acid.

[0032] Further, the material of the outer coating 42 is set to one or more of polysaccharides, celluloses, polypeptides, proteins, and gums. Further preferably, the polysaccharides include hyaluronic acid, sucrose, maltose, starch, chitosan, dextran, chondroitin sulfate, alginic acid and agarose; the celluloses include one or more of carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, hydroxyethyl cellulose, and cellulose ether; the polypeptides include one or more of polyglutamic acid, polylysine, polyleucine, and polyaspartic acid; the proteins include one or more of silk fibroin and collagen; the gums include one or more of methacryloyl gelatin, gelatin, guar gum, xanthan gum, carrageenan, and donkey-hide gelatin. Further preferably, considering the accessibility, dissolution rate, hardness and other characteristics of the material, the material of the outer coating 42 is preferably set to one or more of hyaluronic acid, methacryloyl gelatin, silk fibroin, and polyglutamic acid, and is specifically preferably set to hyaluronic acid.

[0033] As a further embodiment of the present invention, a certain proportion of a quick dissolving agent needs to be added to the material of the outer coating 42, and the quick dissolving agent is set to one or more of the water-soluble divalent inorganic salts, or the quick dissolving agent is set to sodium alginate. Specifically, the water-soluble divalent inorganic salt includes one or more of magnesium chloride, zinc chloride, calcium chloride, magnesium sulfate, zinc sulfate, calcium sulfate, magnesium nitrate, zinc nitrate, calcium nitrate, magnesium acetate, zinc acetate, and calcium acetate. Preferably, considering the accessibility and commonness of the quick dissolving agent, the further preferred quick dissolving agent is preferably set to sodium alginate.

[0034] Furthermore, the material of the support component 2 is set to one or more of polymethyl vinyl ether / maleic acid (anhydride), hyaluronic acid, methacrylic hyaluronic acid, polyvinyl pyrrolidone, polyvinyl alcohol, and polylactic acid. Further preferably, considering that the support component 2 needs to have the characteristics of flexibility, moderate hardness, and easy adhesion to mucous membranes, in one embodiment, the material of the support component 2 is preferably set to acrylic hyaluronic acid.

[0035] Optionally, the method for fixing the transdermal diagnostic device to the oral mucosa by the fixing component 3 may be one or more of the methods such as pasting, adsorption, extrusion fixation, magnetic attraction, clamping, etc., or any other method that can fix the transdermal diagnostic device to the oral mucosa, and can firmly fix the transdermal diagnostic device to the oral mucosa.

[0036] Considering the accessibility of the manufacturing method and the compatibility with the oral structure, the preferred fixing methods are bonding, adsorption and clamping.

[0037] In one embodiment, the material and structure of the fixing component 3 using the adhesive method are as follows: The material of the fixing component 3 can be one or more of any material with strong adhesion and good biocompatibility, such as natural adhesive material, semi-synthetic polymer adhesive material, synthetic polymer adhesive material, etc. The natural adhesive material includes hyaluronic acid, gelatin, starch, chitosan, deacetylated chitosan, lectin, etc. The semi-synthetic polymer adhesive material includes various celluloses, chitosan, chitosan, etc. The synthetic polymer adhesive material includes carbopol (carbomer), monoglyceride, polyvinyl alcohol, polyacrylic acid and polyethylene glycol, etc. Preferably, the material of the fixing component 3 is carbopol.

[0038] The fixing component 3 is a thin film in the shape of an elongated rectangle or square, which matches the shapes of the supporting component 2 and the transdermal component 1 .

[0039] Optionally, when the fixing component 3 of the transdermal diagnostic device is in the shape of a square, the side length X3 thereof is set to 14 mm, 28 mm or 56 mm.

[0040] Optionally, when the fixing component 3 of the transdermal diagnostic device is in the shape of an elongated rectangle, the side length combinations thereof include 20 mm×30 mm, 40 mm×30 mm or 60 mm×30 mm.

[0041] Specifically, the thickness Z2 of the fixing component 3 is 1 mm-2 mm, preferably 1 mm.

[0042] In another embodiment, the fixing component 3 whose fixing method is adsorption method has the following materials and structures: The material of the fixing component 3 can be any material with strong biocompatibility and elasticity, such as silicone, nitrile rubber, polyisoprene, polytetrafluoroethylene, polydimethylsiloxane, thermoplastic polyurethane, polycarbonate, polyacrylic resin, etc. Preferably, the material of the fixing component 3 is silicone.

[0043] The fixing component 3 is a truncated cone suction cup, which matches the shapes of the supporting component 2 and the transdermal component 1 . The supporting component 2 is connected to the bottom of the truncated cone suction cup, and the transdermal component 1 is connected to the supporting component 2 .

[0044] Optionally, when the fixing component 3 of the transdermal diagnostic device is shaped as a truncated cone suction cup, before deformation, the inner diameter D3 of the top circle is 7 mm, 14 mm or 28 mm, the inner diameter D4 of the bottom circle is 14 mm, 28 mm or 56 mm, the inner height H5 is 5 mm-10 mm, and the thickness H6 is 1 mm-2 mm.

[0045] In another embodiment, the material and structure of the fixing component 3 in which the fixing method is the clamping method are as follows: The fixing component 3 includes a splint 31, a clamp handle 32 and 33. The splint 31 is divided into an intraoral splint and an extraoral splint. The intraoral splint has the supporting component 2 and the transdermal component 1. By pressing or releasing the clamp handle 32, the spring connection part 33 is opened or closed, thereby fixing the transdermal diagnostic device to the oral mucosa.

[0046] The material of the splint 31 and the clamp handle 32 is any relatively hard, biocompatible and easily available polymer material such as acetal diethanolamine, polypropylene, polycarbonate, acrylonitrile-butadiene-styrene, polystyrene, polyvinyl chloride, polyetheretherketone, polytetrafluoroethylene, etc. The preferred material of the splint 31 and the clamp handle 32 is acetal diethanolamine.

[0047] The splint 31 is in the shape of an elongated rectangle or a square, matching the shapes of the supporting component 2 and the transdermal component 1 .

[0048] Optionally, when the shape of the clamping plate 31 is an elongated rectangle, the side length combinations include 20 mm×30 mm, 40 mm×30 mm or 60 mm×30 mm.

[0049] Optionally, when the shape of the clamping plate 31 is a square, the side length X4 thereof is set to 14 mm, 28 mm or 56 mm.

[0050] Optionally, the thickness Z3 of the clamping plate 31 is 1 mm-2 mm, preferably 1 mm.

[0051] The clamp handle 32 has a total length of 6cm-9cm, a rectangular side, a width X5 of 7mm, 14mm, 28mm, preferably 7mm, and a thickness Z4 of 1mm-2mm, preferably 1mm. There is a 120°-150° angle at 2 / 3 of the length of the clamp handle 32, which divides the clamp handle into an intraoral section 321 (front 2 / 3) and an extraoral section 322 (rear 1 / 3), with lengths of X6 (4-6cm) and X7 (2-3cm) respectively.

[0052] The spring connection part 33 is a section of spring, both ends of which are connected to the clamp handle 32, and the material is stainless steel.

[0053] See also Figures 7 to 18 As shown, the present invention also provides a method for manufacturing a transdermal diagnostic device for oral mucosal damage caused by autoimmune diseases, comprising the following steps: Step 1: Preparation of inner needle body and support component solution; Weigh appropriate proportions of materials for preparing the needle tip, needle body or support component of the inner needle body and a photocrosslinker, and directly dissolve them in distilled water at a suitable temperature, or dissolve them separately in distilled water and then mix them, so that the mass volume concentration (w / v) of the materials after mixing in distilled water is 10% to 24%, and the mass volume concentration (w / v) of the photocrosslinker in distilled water is 0.1%, so that it becomes a uniform solution with side-by-side bubbles, and obtains a solution of the inner needle tip, needle body or support component.

[0054] S1.1. The inner needle body material is polyvinyl methyl ether / maleic acid (anhydride) and methacrylic hyaluronic acid, and the photocrosslinker is lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP). At room temperature, the two materials are dissolved in distilled water respectively, and then the two solutions are fully mixed and the bubbles are removed. The mass volume concentrations of the mixed polyvinyl methyl ether / maleic acid (anhydride) and methacrylic hyaluronic acid are 20-24% and 10-12% respectively, and the mass volume concentration of the photocrosslinker LAP is 0.1%.

[0055] S1.2, the material of the support component is methacrylate hyaluronic acid, and the photocrosslinker is LAP. At room temperature, methacrylate hyaluronic acid is fully dissolved in distilled water and bubbles are removed, the mass volume concentration is 20-24%, and the mass volume concentration of the photocrosslinker is 0.1%.

[0056] Step 2: Preparation of capture fork solution; Weigh the capture fork solution of the Fc segment of autoantibody IgG or complement C3, C4 and mix them evenly. The capture fork can be an antibody molecule or nucleic acid in a protein. Then add morpholineethanesulfonic acid (MES) buffer containing N-hydroxysuccinimide (NHS) and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) as a carbodiimide cross-linking agent to ensure that the capture fork concentration in the final solution is 0.5μg / ml-1.5μg / ml, with a ratio of 1:1; the concentrations of NHS and EDC are 5-10mg / ml, with a ratio of 1:1.

[0057] Step 3: Preparation of outer coating solution; Take appropriate amounts of materials for preparing the outer coating, photocrosslinking agent, and quick dissolving agent and directly dissolve them in distilled water at a suitable temperature, so that the mass volume concentration (w / v) of the outer coating materials after mixing in distilled water is 20% to 24%, the mass volume concentration (w / v) of the photocrosslinking agent in distilled water is 0.1%, and the mass volume concentration ratio (w / v) of the quick dissolving agent in distilled water is 0.1%-10%.

[0058] Preferably, the material of the outer coating is hyaluronic acid, the photocrosslinking agent is LAP, and the quick dissolving agent is sodium alginate.

[0059] Table 1 Materials and ratios of transdermal diagnostic devices

[0060] Note: The above materials and material ratios were used to make microneedles using the mold method. The mechanical strength of the microneedles was measured when the displacement of the microneedles on the pressure sensor reached 0.3 mm; the swelling rate of the microneedles was calculated by the weight ratio of the microneedles after being pressed into the agarose gel for 10 minutes to the weight before being pressed.

[0061] Step 4: Prepare the inner needle body and support assembly; The inner needle body and the supporting components were obtained by using a mold method, a droplet spraying method, a stereolithography method and a 3D printing method.

[0062] Preferably, the process of manufacturing the inner needle body and the support assembly by the mold method is specifically as follows: A transdermal diagnostic device is manufactured using a PDMS microneedle mold. The mold has an inner needle body negative mold and a support component negative mold. The support component negative mold is 200 μm high and has a square bottom with a side length of 14 mm. There are 100 (10×10) inner needle body negative molds on it. The inner needle body negative molds are all conical in shape, with a height of 600 μm, a base width of 300 μm, and a needle distance of 600 μm between the centers of adjacent microneedles.

[0063] Take the inner needle tip, needle body or support component solution obtained in step 1 and add it once or multiple times to the microneedle mold containing the inner needle tip, needle body and support component structure negative mold, and vacuumize once or multiple times for 10 minutes at a vacuum degree of 0.07MPa, so that the inner needle body solution is evenly injected into the inner needle body structure of the microneedle mold, remove the excess inner needle body solution, and ventilate and dry; if the microneedle tip and needle body materials are different, first inject the inner needle body tip solution evenly into the inner needle body tip structure of the microneedle mold, ventilate and dry, then inject the inner needle body solution evenly into the inner needle body structure of the microneedle mold, ventilate and dry again; then apply the support component solution to the support component structure of the microneedle mold. Place the microneedle tip, needle body and support component together with the microneedle mold under blue light for curing and crosslinking for 10-15 minutes, then place it in a room temperature dryer for drying for 15-24 hours, and demold to obtain an integrated inner needle tip and needle body.

[0064] Table 2 The number of support components and inner needles in the PDMS mold

[0065] Note: According to the optimal materials and material ratios in the examples, the inner needle body was manufactured by the mold method, and each outer layer was coated by the deposition method to obtain the microneedle. The water absorption of the manufactured microneedle was uniformly calculated by the weight ratio of the microneedle after being pressed into the agarose gel for 10 minutes to that before.

[0066] Table 3 Negative mold of inner needle body in PDMS mold

[0067] Note: According to the optimal materials and material ratios in the examples, the inner needle body was manufactured using the mold method, and each outer layer was coated using the deposition method to obtain a microneedle. The microneedle was pressed into the ex vivo pig skin using a pressure of 0.6N, and then pulled out after 1 minute. The average depth of the pinholes on the pig skin was measured as the actual penetration depth.

[0068] Preferably, the method of manufacturing the support assembly by the mold method is: Take the support component solution obtained in step 1 and add it once or multiple times to the mold containing the support component structure negative mold, and evacuate once or multiple times at a vacuum degree of 0.07 MPa for 10 minutes to evenly inject the support component solution into the support component negative mold. Place it under blue light for curing and cross-linking for 10-15 minutes, then place the entire mold in a room temperature dryer and dry it for 15-24 hours, and demold it to obtain the support component.

[0069] Preferably, the droplet spraying method is used to manufacture the inner needle body with the same needle tip and needle body material: The support assembly made by the mold method is used as the upper and lower substrates. A solution dispenser and an automatic X, Y, and Z robot are used to discharge for 0.05 s at 0.6 kg·f / cm, and 40 μl of the inner needle solution obtained in step (1) is dropped on the lower substrate, with each droplet having a diameter of 300 μm and a distance of 600 μm.

[0070] Move the upper group of substrates downward to contact the droplets, separate the two groups of substrates at a speed of 5 mm / min and move to a distance of 2000 μm, blow air from the four sides of the two substrates toward the center in a symmetrical direction at room temperature for 5 minutes, and finally separate the two substrates at a speed of 30 mm / min to obtain two groups of support components and an inner needle body.

[0071] Preferably, the droplet spraying method is used to manufacture the inner needle body with different needle tip and needle body materials: The support assembly made by the mold method is used as the upper and lower substrates. A solution dispenser and an automatic X, Y, and Z robot are used to discharge for 0.05 s at 0.6 kg·f / cm. First, 10 μl of the inner needle body solution obtained in step (1) is dripped on the upper and lower substrates, and then 20 μl of the inner needle tip solution obtained in step (1) is dripped on the lower substrate.

[0072] Move the upper group of substrates downward to contact the droplets, separate the two groups of substrates at a speed of 5 mm / min and move to a predetermined distance (the predetermined distance is set to the sum of the lengths of the two microneedles), blow air from the four sides of the two substrates toward the center from a symmetrical direction at room temperature for 5 minutes, and finally separate the two substrates at a speed of 30 mm / min. The two supporting components obtained both contain inner needles with different needle tip and needle body materials. Place them under blue light for curing and cross-linking for 10-15 minutes, and then dry them at room temperature for 15-24 hours.

[0073] Table 4 Parameters of inner needle body made by droplet spraying method

[0074] Note: According to the optimal materials and material ratios in the examples, the inner needle body with the same needle tip and needle seat material was manufactured by the droplet spray method, and each outer layer was coated by the deposition method to obtain a microneedle. The microneedle was pressed into the ex vivo pig skin with a pressure of 0.6N, and then pulled out after 1 minute. The average depth of the pinholes on the pig skin was measured as the actual penetration depth.

[0075] Step 5: Capture fork coated with autoantibody IgG; The capture forks coated with autoantibody IgG were obtained by deposition, dip-coating and spray-coating methods.

[0076] Preferably, the method of coating the capture fork of the autoantibody IgG by the deposition method is: Take the capture fork solution of autoantibody IgG obtained in step 2 and slowly drip it on the inner needle body. At 4°C, use the carbodiimide method to cross-link the antibody and the microneedle. After incubation for 0.5h-2h, remove the solution, wash with MES, and dry the microneedle at room temperature for 0.5h-1h to obtain the inner needle body covered with the capture fork.

[0077] Preferably, the method of coating the capture fork of the autoantibody IgG by dip coating is: Immerse the needle tip or the entire inner needle body into the capture fork solution of autoantibody IgG obtained in step 2, incubate at 4°C for 0.5h-2h, then take it out, wash it with MES, and dry the microneedle at room temperature for 0.5h-1h. Repeat the above steps to obtain an inner needle body covered with a capture fork.

[0078] Preferably, the method of spray coating the capture fork of the autoantibody IgG is: Use an ultrasonic sprayer to spray the capture fork solution of autoantibody IgG obtained in step 2 on the inner needle body at a speed of 30 μL / min for 15-30 minutes, react at 4°C for 0.5h-2h, wash with MES, and dry the microneedle at room temperature for 0.5h-1h. Repeat the above steps to obtain an inner needle body covered with a capture fork.

[0079] Step 6: coating the microneedles with an outer coating; The outer coating of the coated microneedles was obtained by deposition, dip coating and spray coating.

[0080] Preferably, the method of coating the microneedle outer coating by the deposition method is: Take the outer coating solution obtained in step 3 and slowly drip it on the inner needle body. After incubation at room temperature for 0.5h-2h, remove the solution, cure and cross-link under blue light for 10-15min, wash with distilled water, and then place in a room temperature dryer to dry for 15-24h. Repeat the above steps to obtain the inner needle body covered with the microneedle outer coating.

[0081] Preferably, the method of coating the outer coating of the microneedles by dip coating is: Immerse the tip or the entire inner needle body in the outer coating solution obtained in step three, incubate at room temperature for 0.5h-2h, then take it out, cure and cross-link under blue light for 10-15min, wash with distilled water, and then dry in a room temperature dryer for 15-24h. Repeat the above steps to obtain an inner needle body coated with a microneedle outer coating.

[0082] Preferably, the method of spray coating the microneedle outer coating is: Use an ultrasonic sprayer to spray the outer coating solution obtained in step three on the inner needle body at a speed of 30 μL / min for 15-30 minutes, cure and cross-link under blue light for 10-15 minutes, wash with distilled water, and then place in a room temperature dryer to dry for 15 to 24 hours. Repeat the above steps to obtain an inner needle body covered with a microneedle outer coating.

[0083] Step 7: manufacturing fixed components; The process of manufacturing a fixed component using the mold method with the fixing method being the adhesive method is as follows: The fixed component is manufactured using a PDMS mold, wherein the mold has a female mold for the fixed component, the female mold for the fixed component has a height of 1 mm, and the bottom surface is a square with a side length of 28 mm.

[0084] Prepare a fixed component solution: weigh appropriate proportions of carboxyvinyl polyol and a photocrosslinker and directly dissolve them in distilled water at a suitable temperature, or dissolve them separately in distilled water and then mix them, so that the mass volume concentration (w / v) of the materials after mixing in distilled water is 10% to 24%, and the mass volume concentration (w / v) of the photocrosslinker in distilled water is 0.1%, so that it becomes a uniform solution with side-by-side bubbles, and a fixed component solution is obtained.

[0085] Add the fixed component solution into the mold at one time, evacuate once or multiple times at a vacuum degree of 0.07 MPa for 10 minutes to ensure uniform injection of the solution, remove excess solution, and ventilate and dry; cure and cross-link under blue light for 10-15 minutes, then place in a room temperature dryer and dry for 15-24 hours, and demold to obtain the fixed component.

[0086] The process of using the mold method to manufacture a fixed component whose fixing method is the adsorption method is as follows: A fixed component was manufactured using a PDMS mold. The mold had a female mold for the fixed component, the height of the female mold for the fixed component was 8 mm, and the bottom and top were circles with diameters of 9 mm and 16 mm, respectively. Another PDMS mold had a male mold for the fixed component, the height of the male mold for the fixed component was 7 mm, and the bottom and top were circles with diameters of 14 mm and 7 mm, respectively.

[0087] Prepare the raw materials for the fixed components: Weigh the base polymer and cross-linking agent (curing agent) of silicone in appropriate proportions, mix them thoroughly in a clean container until the color is uniform, and make the ratio of silicone to cross-linking agent in the mixture be 10:1. Vacuum it once or multiple times at a vacuum degree of 0.07 MPa for 10 minutes to remove bubbles.

[0088] Inject the degassed silicone into the female mold, ensure uniform filling, then embed the male mold into the female mold, remove excess silicone, ventilate and dry, allow the silicone to cure in the mold for 8-24 hours, and demold to obtain a fixed component.

[0089] The present invention also provides a method for using a transdermal diagnosis device for oral mucosal damage caused by autoimmune diseases, comprising a sampling step and a diagnosis step.

[0090] 1. Sampling procedures; Introduce the transdermal diagnostic device into the patient's oral mucosal lesions, the junction of oral mucosal lesions and normal mucosa, or other locations specified by the physician, and leave it there for 20-40 minutes; For a transdermal diagnostic device whose fixing method is an adhesive method or an adsorption method, the method of introduction is to place the transdermal diagnostic device at the lesion of the oral mucosa.

[0091] For transdermal diagnostic devices whose fixing method is the clamping method, the introduction method is: by pressing the clamp handle, first open the spring connection part of the transdermal diagnostic device, so that the intraoral section splint transfers the transdermal component of the transdermal diagnostic device to the damaged part of the oral mucosa, and then release the clamp handle to close the spring connection part, thereby fixing the transdermal diagnostic device to the oral mucosa through the extraoral section splint and the intraoral section splint.

[0092] After the transdermal component enters the mucosa, the outer coating decomposes, the inner needle absorbs interstitial fluid and swells, and the capture fork on the surface binds to the protein markers in the mucosa.

[0093] 1. Diagnostic steps: The diagnostic steps include incubation with biotinylated detection antibodies, incubation with fluorophores containing streptavidin, and observation with a fluorescence microscope.

[0094] ①, Incubation of biotinylated detection antibody; The transdermal diagnostic device is incubated in 5 ml of PBS solution (i.e., buffer solution) with a biotinylated detection antibody concentration of 2 μg / ml-4 μg / ml at room temperature for 1-2 hours, and then washed with PBS solution.

[0095] ②, Incubation of streptavidin fluorophore; The transdermal diagnostic device was incubated in 5 ml of a PBS solution containing 3 μg / ml to 5 μg / ml streptavidin fluorophore for 30 min to 45 min, and then washed with PBS.

[0096] ③ Observation by fluorescence microscope; The transdermal diagnostic device is observed using a fluorescence microscopy imager, and a corresponding diagnostic result can be obtained based on whether fluorescence is observed. If strong fluorescence is observed, it indicates that the patient may suffer from oral lesions caused by autoimmune diseases, and if weak / no fluorescence is observed, it indicates that the patient may suffer from oral lesions caused by non-autoimmune diseases, or oral lesions caused by other factors. In addition, the specific type of autoimmune disease can be preliminarily identified based on the observed fluorescence pattern, depth, and location.

[0097] Further, the method for preliminary identification of the specific type of autoimmune disease is: Strong fluorescence was observed, suggesting that the patient's oral mucosal damage was caused by an autoimmune disease; The fluorescence was observed to be distributed in a continuous linear pattern near the needle tip of the inner needle body, indicating that the protein marker was deposited in a continuous linear pattern between the mucosal epithelium and the mucosal lamina propria, suggesting that oral mucosal damage may be caused by mucosal oral pemphigoid; The fluorescence was observed to be distributed in the inner needle body near the needle tip in a discontinuous linear or granular manner, indicating that the protein marker was deposited in a discontinuous linear or granular manner between the mucosal epithelium and the mucosal lamina propria, suggesting that oral mucosal damage may be caused by oral lupus erythematosus; It was observed that the fluorescence was irregularly distributed near the needle tip of the inner needle body, indicating that the protein marker was irregularly deposited between the mucosal epithelium and the mucosal lamina propria, suggesting that oral mucosal damage may be caused by lichen planus; Fluorescence was observed to be distributed in a grid-like pattern at the needle tip and the needle body, indicating that the protein marker was deposited between the mucosal epithelial cells, suggesting that oral mucosal damage may be caused by oral pemphigus; Fluorescence was observed, but it was different from the above forms and was deposited in the inner needle body in a non-specific manner, indicating that protein markers were deposited, suggesting that oral mucosal damage may be caused by other autoimmune diseases; Weak / no fluorescence was observed, suggesting that the patient's oral mucosal damage may be caused by non-autoimmune diseases or other factors (such as trauma).

[0098] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A transdermal diagnostic device for oral mucosal damage in autoimmune diseases, characterized in that: It includes a transdermal component, a support component and a fixing component; The transdermal component includes at least one group of microneedles capable of penetrating the mucosa, each microneedle including an inner needle body and an outer coating, the outer surface of the inner needle body is provided with a capture fork, the capture fork is used to capture protein markers present in the oral mucosa of autoimmune diseases; the outer coating is arranged on the outer surface of the inner needle body, is used to protect the inner needle body and is dissolved in the mucosa; One end of the support component is attached to the transdermal component, and the other end is connected to the fixing component; The fixing component is used to fix the transdermal component and the supporting component on the inner surface of the oral mucosa.

2. The transdermal diagnostic device for oral mucosal lesions of autoimmune diseases according to claim 1, characterized in that: The material of the inner needle body is set to at least one of polymethyl vinyl ether / maleic anhydride, hyaluronic acid, methacrylate hyaluronic acid, polyvinyl pyrrolidone, polyvinyl alcohol or polylactic acid.

3. The transdermal diagnostic device for oral mucosal lesions of autoimmune diseases according to claim 2, characterized in that: The total height H1 of the inner needle body is 400 μm-600 μm, the base width D1 of the inner needle body is 200 μm-300 μm, and the needle distance X2 between the centers of adjacent microneedles is 600 μm.

4. The transdermal diagnostic device for oral mucosal lesions of autoimmune diseases according to claim 3, characterized in that: The inner needle body comprises a needle tip and a needle body, wherein the shape of the needle tip is set to be conical, pyramidal, bullet-shaped, hemispherical, truncated cone or prism-shaped, and the shape of the needle body is set to be truncated cone, cylindrical or prism-shaped.

5. The transdermal diagnostic device for oral mucosal lesions of autoimmune diseases according to claim 4, characterized in that: The height H3 of the needle tip is 100 μm-200 μm, and the base width D2 is 50 μm-300 μm; The height H4 of the needle body is 300 μm-400 μm, the width D2 of the upper base is 50 μm-300 μm, and the width of the lower base is 200 μm-300 μm; The material of the outer coating is set to be at least one of polysaccharides, cellulose, polypeptides, proteins or glues; The material of the support component is set to at least one of polymethyl vinyl ether / maleic acid, hyaluronic acid, methacrylate hyaluronic acid, polyvinyl pyrrolidone, polyvinyl alcohol or polylactic acid.

6. The transdermal diagnostic device for oral mucosal lesions of autoimmune diseases according to any one of claims 1 to 5, characterized in that: The material of the outer coating layer includes a quick dissolving agent, and the quick dissolving agent is set to be a water-soluble divalent inorganic salt or sodium alginate; The outer coating covers the needle tip and the surface of the inner needle body, and the thickness H2 of the outer coating is 50-60 μm; The shape of the support component is set to be an elongated rectangle, a square or a circle, and the thickness of the support component is 200 μm-1000 μm.

7. The transdermal diagnostic device for oral mucosal lesions of autoimmune diseases according to claim 6, characterized in that: When the shape of the support component of the transdermal diagnostic device is set to be an elongated rectangle, the side length combinations include 20mm×15mm, 40mm×15mm or 60mm×15mm; When the shape of the support component of the transdermal diagnostic device is set to be a square, the side length X1 thereof is set to be 7 mm, 14 mm or 28 mm; When the shape of the support component of the transdermal diagnostic device is set to be circular, its diameter is set to 7 mm, 14 mm or 28 mm.

8. A method for manufacturing a transdermal diagnostic device for oral mucosal damage in autoimmune diseases, characterized in that: The following steps are involved: Step 1, preparation of inner layer microneedles and basal layer solution; S1.

1. Weigh the materials for preparing the needle tip, needle body or base layer of the inner layer microneedle and the photocrosslinking agent and directly dissolve them in distilled water at a suitable temperature, or dissolve them separately in distilled water and then mix them, so that the mass volume concentration of the materials after mixing in distilled water is 10% to 24%, and the mass volume concentration of the photocrosslinking agent in distilled water is 0.1%, so that it becomes a uniform solution and bubbles are arranged side by side, and the inner layer microneedle needle tip, needle body or base layer solution is obtained; S1.2, the inner layer microneedle materials are polymethyl vinyl ether / maleic anhydride and methacrylate hyaluronic acid, and the photocrosslinker is lithium phenyl-2,4,6-trimethylbenzoylphosphonate; at room temperature, the two materials are dissolved in distilled water respectively, and then the two solutions are fully mixed and the bubbles are removed. The mass volume concentrations of the mixed polymethyl vinyl ether / maleic anhydride and methacrylate hyaluronic acid are 20% to 24% and 10% to 12% respectively, and the mass volume concentration of the photocrosslinker LAP is 0.1%; S1.

3. Methacrylate hyaluronic acid is selected as the material of the microneedle base layer, and LAP is selected as the photocrosslinker. At room temperature, methacrylate hyaluronic acid is fully dissolved in distilled water and bubbles are removed, the mass volume concentration is 20-24%, and the mass volume concentration of the photocrosslinker is 0.1%; Step 2: Preparation of capture antibody solution of autoantibodies; Weigh the capture antibody solution of autoantibody IgG and mix it evenly, then add morpholineethanesulfonic acid buffer containing N-hydroxysuccinimide and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide as a carbodiimide crosslinker to ensure that the capture antibody concentration in the final solution is set to 0.5μg / ml-1.5μg / ml and the ratio is set to 1:1; the concentrations of NHS and EDC are set to 5-10mg / ml and the ratio is set to 1:1; Step 3: Preparation of coating solution; Take appropriate amounts of materials for preparing the coating layer, photocrosslinking agent, and quick dissolving agent and directly dissolve them in distilled water at a suitable temperature, so that the mass volume concentration of the coating layer materials after mixing in distilled water is 20% to 24%, the mass volume concentration of the photocrosslinking agent in distilled water is 0.1%, and the mass volume concentration ratio of the quick dissolving agent in distilled water is 0.1% to 10%; Step 4: preparing inner microneedles and a basal layer; The inner microneedles and the base layer were obtained by using the mold method, droplet spraying method, stereolithography method and 3D printing method; Step 5, coating the inner microneedles with the capture antibody of the autoantibody IgG and the coating layer of the coated microneedles; The capture antibody coated with autoantibody IgG and the coated microneedle coating layer are obtained by using deposition method, dip coating method and spray coating method; Step 6: Manufacturing fixed components; The fixing components are fixed by a pasting method, and the fixing components are manufactured by a mold method, an electrospinning method, an electrospraying method and a 3D printing method; Alternatively, the fixing component is fixed by an adsorption method, and the fixing component is manufactured by a mold method, a hot pressing method, and a 3D printing method; Alternatively, the fixing components are fixed by a clamping method, and the fixing components are manufactured by a mold method, a hot pressing molding method, and a 3D printing method.

9. A method for using a transdermal diagnostic device for oral mucosal damage in autoimmune diseases, characterized in that: It includes a sampling step and a diagnostic step; wherein:

1. Sampling procedures; The transdermal diagnostic device is introduced into the junction of the patient's skin lesions and normal skin and stays there for 20-40 minutes; the microneedle enters the deep epidermis, the coating layer decomposes, the inner needle body absorbs interstitial fluid to swell, and the capture antibody in its capture fork binds to the autoantibody; (ii) diagnostic steps; The diagnostic steps include incubation with biotinylated detection antibodies, incubation with streptavidin-containing fluorophores, and observation with a fluorescence microscopy imager; ①, Incubation of biotinylated detection antibody; Incubate the transdermal diagnostic device with 5 ml of a standard protein solution having a biotinylated detection antibody concentration of 2 μg / ml-4 μg / ml at room temperature for 1-2 h, and wash with a phosphate buffer solution; ②, Incubation of streptavidin fluorophore; Incubate the transdermal diagnostic device after step ① with 5 ml of a standard protein solution containing 3 μg / ml to 5 μg / ml streptavidin fluorophore for 30 min to 45 min, and wash with a phosphate buffer solution; ③. Observation by fluorescence microscope; Observe the transdermal diagnostic device that has completed step ② using a fluorescence microscope, and obtain a corresponding diagnostic result based on whether fluorescence is observed; If strong fluorescence is observed, it indicates that the patient has an autoimmune skin disease; If weak / no fluorescence is observed, it suggests that the patient has a non-autoimmune skin disease.

10. The method for using the transdermal diagnostic device for oral mucosal lesions of autoimmune diseases according to claim 9, characterized in that: The method for initially identifying the specific type of autoimmune skin disease is: ⅰ. Strong fluorescence was observed, indicating that the patient's oral mucosal damage was caused by an autoimmune disease; ii. The fluorescence was observed to be distributed in a continuous linear pattern near the needle tip of the inner needle body, indicating that the protein marker was deposited in a continuous linear pattern between the mucosal epithelium and the mucosal lamina propria, suggesting that the oral mucosal damage was caused by mucosal oral pemphigoid; ⅲ. Fluorescence was observed to be distributed in the inner needle body near the needle tip in a discontinuous linear or granular manner, indicating that the protein marker was deposited in a discontinuous linear or granular manner between the mucosal epithelium and the mucosal lamina propria, suggesting that the oral mucosal damage was caused by oral lupus erythematosus; ⅳ. The fluorescence was observed to be irregularly distributed near the needle tip of the inner needle body, indicating that the protein marker was irregularly deposited between the mucosal epithelium and the mucosal lamina propria, suggesting that the oral mucosal damage was caused by lichen planus; ⅴ. Fluorescence was observed to be distributed in a grid-like pattern at the needle tip and the needle body, indicating that the protein marker was deposited between the mucosal epithelial cells, suggesting that the oral mucosal damage was caused by oral pemphigus; ⅵ. Fluorescence was observed, but it was different from the forms of ⅰ-ⅴ and was deposited in the inner needle body in a non-specific manner, indicating that protein markers were deposited, suggesting that the oral mucosal damage was caused by other autoimmune diseases; ⅶ. Weak / no fluorescence was observed, suggesting that the patient's oral mucosal damage was caused by non-autoimmune diseases.

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