Surface functionalized microneedle for orientable immobilization of modified antibodies and preparation method and application thereof

Abstract

This invention discloses a surface-functionalized microneedle capable of targeted immobilization of modified antibodies, its preparation method, and its applications; belonging to the field of microneedle surface engineering technology. The invention uses a microneedle array as a substrate and sequentially includes: activating the microneedle surface to introduce hydroxyl groups and improve surface reactivity; silanizing to introduce amino functional groups onto the microneedle surface; covalently reacting the amino groups with a biotin-polyethylene glycol coupling agent containing NHS active ester to form a biotinylated layer with hydrophilic spacer arms; and then constructing a stable interface through high-affinity binding of avidin or streptavidin to biotin, thereby achieving site-specific immobilization of biotinylated capture antibodies or their fragments, and supporting the replacement of different capture antibodies according to detection requirements. The surface-functionalized microneedles of this invention can be used to immobilize different modified antibodies to capture target analytes in interstitial fluid, and can be used in conjunction with immunochromatographic or fluorescent methods for subsequent detection.

Description

Technical Field

[0001] This invention belongs to the field of microneedle manufacturing and surface functionalization. Specifically, it relates to a method for constructing a functionalized interface on the surface of a microneedle that can be universally coupled with modified antibodies, and can be used for subsequent biomarker detection. Background Technology

[0002] Microneedle arrays can penetrate or contact the stratum corneum of the skin and interstitial fluid under minimally invasive conditions, providing a low-invasive technical approach for the collection and detection of biomarkers in vivo. To achieve specific recognition of target analytes by microneedles, an immunorecognition interface is typically constructed on the microneedle surface. The immobilization method of the capture antibody on the microneedle surface has a decisive impact on detection performance. In existing technologies, antibody immobilization on microneedles or other polymer substrates mainly includes physical adsorption and direct covalent coupling. However, these methods generally suffer from the following problems in microneedle applications: random orientation of the antibody on the surface leads to the easy obscuring of active sites, resulting in decreased effective binding capacity; insufficient stability of the immobilization layer makes it prone to elution during washing or in complex biological environments; high levels of non-specific adsorption on the surface affect detection repeatability and signal-to-noise ratio; and rapid, interchangeable loading of different capture antibodies is difficult, hindering multi-target detection needs. Therefore, there is an urgent need for an antibody immobilization surface modification method suitable for microneedle substrates, with good stability and versatility, to achieve controllable immobilization and interchangeable loading of modified antibodies on the microneedle surface without significantly increasing process complexity. Summary of the Invention

[0003] This invention aims to address the problems of insufficient stability, random orientation, and high non-specific adsorption in the existing microneedle surface antibody immobilization technology. Instead, it provides a surface-functionalized microneedle for the directional immobilization of modified antibodies, its preparation method, and its application. A universal biotin / avidin antibody linking site is constructed on the surface of the microneedle to achieve controllable immobilization and replaceable loading of modified antibodies.

[0004] To address the aforementioned technical problems, the present invention adopts the following technical solution: This invention uses a microneedle array as a substrate, and sequentially performs surface activation treatment to introduce hydroxyl groups, silanization treatment to introduce amino functional groups, and biotin-polyethylene glycol-NHS coupling agent to covalently react with amino groups to form a biotinylated layer. Then, a stable connection interface is constructed through the high affinity binding of biotin with avidin or streptavidin, and finally, the site-specific immobilization of the biotinylated capture antibody or its fragment on the microneedle surface is achieved, thereby obtaining surface-functionalized microneedles.

[0005] The biotin-polyethylene glycol coupling agent is preferably NHS-PEG4-biotin, which provides a hydrophilic spacer arm, helping to reduce non-specific adsorption and improve antibody accessibility.

[0006] Optionally, conventional sealing treatments in the art can be used to further reduce non-specific adsorption and improve detection repeatability.

[0007] The purpose of this invention is to provide a method for preparing surface-functionalized microneedles capable of directionally immobilizing modified antibodies, comprising the following steps: Step 1: Prepare the microneedle array substrate; Step 2: Perform surface activation treatment on the surface of the microneedle array to form an activation layer rich in hydroxyl groups on the surface of the microneedles, thereby improving surface hydrophilicity and reactivity; Step 3: Perform silanization treatment on the surface-activated microneedle array to introduce amino functional groups on the surface of the microneedles; Step 4: React the silanized microneedle array with a biotin-polyethylene glycol coupling agent containing NHS active ester, so that biotin-polyethylene glycol is fixed on the surface of the microneedles through the covalent reaction of NHS-ester and amino groups, forming a biotinylated layer. Step 5: Combine the biotinylated layer with avidin or streptavidin to form an avidin / streptavidin functional layer; Step 6: Bind the biotinylated capture antibody or its fragment to the avidin / streptavidin functional layer to immobilize the capture antibody on the microneedle surface, thereby obtaining surface-functionalized microneedles.

[0008] Further specifying, in step 1, the microneedle array substrate is composed of an integrally formed substrate and an array of multiple microneedles. The microneedle array has a 4×4 structure, and each microneedle is a cone with a height of 800-1200 μm and a bottom diameter of 300-500 μm.

[0009] To further specify, in step 1, the substrate material for the microneedle array is polymethyl methacrylate, which is prepared by injection molding, compression molding, and 3D printing.

[0010] Further specifying, in step 2, oxygen plasma treatment is used to introduce hydroxyl groups, with an oxygen plasma power of 80-120 W.

[0011] Further specifying, in step 3, the activated substrate is immersed in anhydrous ethanol of APTES solution and reacted at room temperature. After the reaction is complete, it is rinsed with anhydrous ethanol and dried. The volume fraction of APTES is 1.5% to 3%.

[0012] Further specified, in step 4, the concentration of NHS-PEG4-biotin is 1.2–3 mg / mL, the pH of PBS is 7.4, and the reaction is carried out at room temperature for 1 h.

[0013] Further specifying, in step 5, the streptavidin concentration is 8-20 μg / mL, and the incubation is carried out at room temperature for 1 h.

[0014] Further specifying, in step 6, the antibody concentration is 8–12 μg / mL, and the incubation is carried out at 37 °C for 1 h.

[0015] In this invention, the fabrication of the microneedle array substrate is illustrated using a polymethyl methacrylate (PMMA) microneedle array as an example. The microneedle array can be fabricated using photopolymerization 3D printing, preferably with a 4×4 structure, a single needle height of approximately 1000 μm, and a bottom diameter of approximately 400 μm. After printing, uncured resin can be removed using isopropanol, followed by UV post-curing to improve structural stability. Polymer microneedle arrays can also be fabricated using injection molding, compression molding, or other methods.

[0016] In this invention, the surface of the microneedle array is subjected to surface activation treatment. Under preferred conditions, oxygen plasma treatment is used to introduce hydroxyl groups and improve hydrophilicity. This is followed immediately by silanization: the activated microneedles are immersed in an amino-containing organosilane solution (preferably a solution of APTES in anhydrous ethanol) to introduce amino functional groups onto the microneedle surface; after the reaction, the microneedles are rinsed with anhydrous ethanol and dried.

[0017] In a preferred embodiment: oxygen plasma power of about 100 W, treatment for about 1 min; APTES volume fraction of about 2% (v / v), reaction at room temperature for about 1 h.

[0018] In this invention, silanized microneedles are reacted with a biotin-polyethylene glycol-NHS coupling agent to achieve biotinylation. After the reaction, the microneedles are washed to remove unreacted reagents. Subsequently, the biotinylated microneedles are incubated with avidin or streptavidin solution to form a stable interface. Finally, a biotinylated capture antibody is incubated with the microneedles, immobilizing them on the microneedle surface through biotin / avidin high affinity; washing is performed after incubation. Optionally, a blocking treatment can be performed after antibody immobilization to reduce non-specific adsorption.

[0019] In a preferred embodiment: NHS-PEG4-biotin concentration of about 2 mg / mL, reacted in PBS (pH about 7.4) at room temperature for about 1 h; streptavidin concentration of about 10 μg / mL, incubated at room temperature for about 1 h; biotinylated capture antibody concentration of about 10 μg / mL, incubated at 37 °C for about 1 h.

[0020] The present invention also provides a surface-functionalized microneedle, which is prepared by any of the above methods, and has an activation layer, an amino-containing silanized layer, a biotin-polyethylene glycol layer, an avidin / streptavidin functional layer, and a biotinylated capture antibody or fragment thereof layer on the surface of the microneedle in sequence.

[0021] The substrate for the microneedle array is preferably a polymer material, and the polymer material includes at least polymethyl methacrylate.

[0022] This invention also provides the use of surface-functionalized microneedles in biomarker detection, wherein the surface-functionalized microneedles are brought into contact with the sample to be tested so that the capture antibody captures the target analyte, and then the signal is read by immunochromatography or fluorescence.

[0023] The sample to be tested is interstitial fluid; preferably, it is interstitial fluid (ISF) obtained percutaneously or by contact.

[0024] A biomarker detection method includes: contacting the surface-functionalized microneedles with the skin surface or superficial tissue for 1 to 10 minutes to adsorb / contact interstitial fluid and capture the target analyte; performing an immunoassay reaction after removing the microneedles and reading the detection signal to obtain quantitative or semi-quantitative results of the target analyte.

[0025] Compared with the prior art, the present invention has the following beneficial effects: This invention achieves a strong binding between the antibody linking site and the microneedle surface through stepwise modification of surface activation, silanization, and NHS-ester covalent coupling, resulting in good washability and reproducibility. This invention introduces PEG spacer arms and optional blocking treatment to reduce non-specific adsorption and improve the signal-to-noise ratio in complex samples. This invention utilizes the high affinity binding of biotin / avidin to achieve modular replacement of capture antibodies, making it easy to expand to different antibody and multiple biomarker detection scenarios; The functionalized interface of the present invention is preferably applicable to polymer microneedle substrates (especially PMMA) and processing technologies such as 3D printing, injection molding or compression molding, and has strong process versatility.

[0026] For a deeper understanding of the features and technical content of this invention, please refer to the accompanying detailed description and drawings. It should be noted that the drawings are provided for illustrative purposes only and are not intended to limit the scope of the invention. Attached Figure Description

[0027] Figure 1 This is a schematic diagram illustrating the use of surface-functionalized microneedles to construct an immune recognition interface for subsequent detection. Figure 2 This is a schematic diagram of the microneedle array structure and its mechanical compression curve. Figure 2 a is a diagram of the microneedle array structure. Figure 2 b is a schematic diagram of the mechanical compression curve; Figure 3 This is a schematic diagram illustrating the preparation process of surface-functionalized microneedles and antibody immobilization according to the present invention.

[0028] Figure 4 The linear response diagram for the detection of surface-functionalized microneedles is shown. Detailed Implementation

[0029] The present invention will be described in detail below with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but should not be considered as limiting the present invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention.

[0030] Example 1: Step 1: In this embodiment, a polymethyl methacrylate (PMMA) microneedle array substrate is used. The microneedle array substrate is prepared by photopolymerization 3D printing, and the structure is as follows. Figure 2 As shown in Figure a, the array has a 4×4 structure with a height of 800–1200 μm, preferably 1000 μm, and a bottom diameter of 300–500 μm, preferably 400 μm. After printing, uncured resin is removed by cleaning with isopropanol, and then UV cured for 10 minutes (wavelength 365 nm) to improve structural stability.

[0031] Step 2: Surface activation treatment is performed on the surface of the microneedle array. Under preferred conditions, oxygen plasma treatment is used to introduce hydroxyl groups and improve hydrophilicity. The oxygen plasma power is 80–100 W, preferably 100 W, and the treatment time is 1 min. The APTES volume fraction is approximately 2% (v / v), and the reaction is carried out at room temperature for approximately 1 h. Step 3: Immediately afterwards, silanization was performed: the activated microneedles were immersed in an anhydrous ethanol solution of APTES with a volume fraction of 2% (v / v) (the solution obtained by dissolving APTES in anhydrous ethanol was reacted at room temperature for 1 h to introduce amino functional groups on the surface of the microneedles; after the reaction, the microneedles were rinsed with anhydrous ethanol and dried at 25 °C (room temperature). Step 4: React the silanized microneedle array with a biotin-polyethylene glycol coupling agent containing NHS active ester, so that biotin-polyethylene glycol is fixed on the surface of the microneedles through the covalent reaction of NHS-ester and amino groups, forming a biotinylated layer. The reaction was carried out at room temperature for 1 h in NHS-PEG4-biotin at a concentration of 2 mg / mL and PBS at pH 7.4. Step 5: Combine the biotinylated layer with streptavidin to form a streptavidin / avidin functional layer; Streptavidin concentration was 10 μg / mL, and incubation was carried out at room temperature for 1 h. Step 6: Bind the biotinylated capture antibody or its fragment to the avidin / streptavidin functional layer to immobilize the capture antibody on the microneedle surface. The concentration of the biotinylated capture antibody is about 10 μg / mL. Incubate at 37 °C for about 1 h to obtain surface-functionalized microneedles.

[0032] Step 7: First, place the functionalized microneedle array in TNF-α standard solutions with concentrations of 0, 7.81, 31.25, 62.5, 250, and 500 pg / mL, respectively, and incubate with gentle shaking at 37°C for 1 hour to complete antigen capture.

[0033] Step 8: After incubation, wash the microneedles three times with 200 μL of phosphate buffer.

[0034] Step 9: The microneedles are then immersed in horseradish peroxidase-labeled detection antibody at twice the standard concentration prepared in phosphate buffer and incubated at 37°C for 30 minutes to allow them to specifically bind to the captured antigen.

[0035] Step 10: After washing three times with phosphate buffer, add an appropriate concentration of streptavidin-horseradish peroxidase solution, incubate at 37°C for 30 minutes, and wash three more times with phosphate buffer.

[0036] Step 11: Under light-protected conditions at room temperature, incubate the microneedles in tyramine working solution for 15 minutes. After the tyramine reaction is complete, thoroughly wash the microneedles to remove unreacted reagents.

[0037] Step 12: Add tetramethylbenzidine substrate solution and hydrogen peroxide solution to the container holding the microneedles. Incubate at room temperature in the dark for 15 minutes for colorimetric detection. Stop the reaction by adding 50 μL of 2 M sulfuric acid. Transfer the reaction solution to a 96-well plate and measure the absorbance at 450 nm using a microplate reader. The resulting surface-functionalized microneedles were then used to detect samples. The tyramine-amplified immunoassay based on the microneedles showed a considerable linear correlation (R² = 0.8593) in the range of 0–500 pg / mL. Figure 4 As shown.

[0038] Surface-functionalized microneedles are briefly brought into contact with the surface or superficial tissue of the skin to allow the microneedles to adsorb / contact interstitial fluid and capture the target analyte. After removal, conventional immunoassay procedures can be used, such as adding detection antibodies and markers and performing colorimetric or fluorescent readings, to achieve quantitative or semi-quantitative detection of the target analyte. This application is only to illustrate that functionalized microneedles can be used for subsequent detection; the innovation of this invention lies in the preparation of surface-functionalized microneedles and the construction of universal antibody-linking interfaces.

[0039] The specific embodiments of the present invention have been described in detail above. It should be noted that the present invention is not limited to the specific embodiments described above. Various modifications or alterations can be made by those skilled in the art without departing from the scope of protection defined by the claims, and all such modifications or alterations fall within the scope of the present invention.

Claims

1. A method for preparing surface-functionalized microneedles capable of directionally immobilizing modified antibodies, characterized in that, Includes the following steps: Step 1: Prepare the microneedle array substrate; Step 2: Perform surface activation treatment on the substrate surface to generate hydroxyl groups on the surface; Step 3: Then perform silanization treatment; Step 4: The silanized substrate is reacted with a biotin-polyethylene glycol coupling agent containing NHS active ester. Biotin-polyethylene glycol is fixed on the surface of the microneedles through the covalent reaction between NHS-ester and amino groups, forming a biotinylated layer on the surface of the microneedles. Step 5: Then, the biotinylated layer is combined with avidin or streptavidin; Step 6: Then, bind it with the biotinylated capture antibody or its fragment to immobilize the capture antibody on the surface of the microneedle, thus obtaining the surface-functionalized microneedle.

2. The method according to claim 1, characterized in that, The microneedle array substrate consists of an integrally molded substrate and an array of multiple microneedles. The microneedle array has a 4×4 structure, with each microneedle being a cone with a height of 800–1200 μm and a bottom diameter of 300–500 μm. The microneedle array substrate is made of polymethyl methacrylate and is prepared by injection molding, compression molding, and 3D printing.

3. The method according to claim 1, characterized in that, In step 2, oxygen plasma treatment is used to introduce hydroxyl groups, with an oxygen plasma power of 80–120 W.

4. The method according to claim 1, characterized in that, In step 3, the activated substrate is immersed in anhydrous ethanol of APTES solution and reacted at room temperature. After the reaction is complete, it is rinsed with anhydrous ethanol and dried. The volume fraction of APTES is approximately 1.5% to 3% (v / v).

5. The method according to claim 1, characterized in that, In step 4, the concentration of NHS-PEG4-biotin was 1.2–3 mg / mL, the pH of PBS was 7.4, and the reaction was carried out at room temperature for 1 h.

6. The method according to claim 1, characterized in that, In step 5, the streptavidin concentration is 8-20 μg / mL, and the mixture is incubated at room temperature for 1 h.

7. The method according to claim 1, characterized in that, In step 6, the antibody concentration is 8-12 μg / mL, and the mixture is incubated at 37 °C for 1 h.

8. A surface-functionalized microneedle prepared by the method according to any one of claims 1-7, characterized in that, The surface of the microneedle sequentially comprises an activation layer, an amino-containing silanized layer, a biotin-polyethylene glycol layer, an avidin / streptavidin functional layer, and a biotinylated capture antibody or fragment thereof layer.

9. The use of surface-functionalized microneedles prepared by the method according to any one of claims 1-7 in the detection of biomarkers, characterized in that: The surface-functionalized microneedles are brought into contact with the sample to be tested, so that the capture antibody captures the target analyte, and then the signal is read by immunochromatography or fluorescence. The sample to be tested is interstitial fluid; preferably, it is interstitial fluid (ISF) obtained or in contact with the skin.

10. A method for detecting a biomarker, characterized in that, Includes the following steps: The surface-functionalized microneedles prepared by the method according to any one of claims 1-7 are brought into contact with the skin surface or superficial tissue for 8-12 minutes to adsorb / contact interstitial fluid and capture target analytes; After removing the microneedles, an immunoassay reaction is performed, and the detection signal is read to obtain quantitative or semi-quantitative results for the target analyte.

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