An electrochemiluminescent MeHA microneedle patch, its preparation method and application

Abstract

This invention proposes an electrochemiluminescence MeHA microneedle patch, its preparation method, and its application. The preparation method involves modifying hyaluronic acid to methacrylamide hyaluronic acid, purifying it to obtain MeHA solid powder, and mixing it with a photoinitiator to form a precursor solution. This precursor solution is then cast into a microneedle mold, centrifuged, dried, and UV crosslinked to obtain a MeHA microneedle patch loaded with ABEI and glucose oxidase. This invention, through an integrated design of "MeHA microneedle patch-SPE electrode," achieves an integrated operation of "in-situ extraction of interstitial fluid-enzymatic reaction-electrochemiluminescence detection," eliminating the need for sample transfer, significantly reducing interference from intermediate steps, and substantially improving detection accuracy and efficiency, making it particularly suitable for real-time clinical monitoring scenarios.

Description

Technical Field

[0001] This invention belongs to the field of polymer and chemical biosensors, and specifically relates to an electrochemiluminescent MeHA microneedle patch, its preparation method and application. Background Technology

[0002] In the field of biomarker detection, the demand for "non-invasive, real-time, and precise" methods in clinical diagnosis and health monitoring is increasingly urgent. However, traditional detection technologies are still limited by the dual bottlenecks of sample source and analysis mode. Currently, mainstream venous blood testing requires invasive puncture to obtain samples, which not only easily causes patient discomfort and increases the risk of infection, but also has the problem of cumbersome sample processing: high concentrations of red blood cells and proteins in the blood can interfere with the detection reaction, requiring pretreatment steps such as centrifugation and separation before analysis, resulting in a prolonged detection cycle. More importantly, changes in blood components lag behind the local tissue physiological and pathological state (e.g., lactic acid rises before blood lactic acid changes in hypoxia of skin tissue, and glucose fluctuations in interstitial fluid of the skin in the early stages of diabetes precede abnormal blood sugar), making it difficult to capture trace signals in the early stages of disease and missing the intervention window.

[0003] To overcome this limitation, interdermal fluid (ISF) has become an ideal alternative sample source due to its high metabolic correlation with blood. As a fluid that fills the intercellular spaces of the dermis, ISF contains abundant biomarkers such as glucose, lactic acid, and inflammatory factors. Changes in its composition can directly reflect the metabolic level and health status of local tissues. Moreover, it can be extracted in situ using a minimally invasive method, without the need for intravenous puncture, greatly improving the accessibility of testing and patient acceptance. However, existing interstitial fluid sampling techniques still have drawbacks: While commercial continuous glucose monitors (CGMs) use metal microneedles (5-13 mm in length) to extract interstitial fluid, their rigidity and poor biocompatibility easily cause skin irritation and pain, and prolonged use can lead to microneedle dislodgement or breakage. Traditional microdialysis techniques require inserting probes into the dermis, resulting in low sampling efficiency and potential damage to blood vessels, leading to blood contamination in the sample. Even recently developed solid polymer microneedles are prone to physical displacement during insertion due to incompatibility with skin mechanical properties, and surface-modified biorecognition elements (such as enzymes and aptamers) are easily detached during skin friction, leading to decreased detection accuracy. In contrast, hydrogel microneedles, with their excellent biocompatibility, hydrophilicity, and mechanical flexibility, can penetrate the stratum corneum without significant pain, adsorbing interstitial fluid through swelling. They can also be pre-loaded with functional components such as enzymes and luminescent substrates, achieving "sampling and reaction simultaneously," effectively avoiding the skin damage and low efficiency problems of traditional sampling techniques, making them the preferred carrier for interstitial fluid extraction.

[0004] In the signal detection stage, electrochemiluminescence (ECL) technology has become a core method for detecting low-concentration biomarkers due to its high sensitivity and low background interference. Compared to colorimetric methods, which rely on visual identification of color changes and are susceptible to ambient light interference, and fluorescence methods, which are easily affected by the autofluorescence of biological samples, ECL technology induces a redox reaction between the luminescent substrate (such as ABEI) and enzyme-catalyzed reaction products (such as H2O2) by applying a specific excitation voltage to the electrode, generating a specific luminescent signal. The detection limit can be as low as the nanomolar level, accurately capturing minute changes in low-concentration biomarkers in interstitial fluid of the skin. At the same time, it does not require a complex optical excitation system and can be combined with portable screen-printed electrodes (SPE), fully adapting to in-situ detection needs and providing technical support for integrated "sampling-detection". However, it should be noted that the application of existing electrochemiluminescence technology in the detection of interstitial fluid in the skin still faces integration bottlenecks: on the one hand, the electrochemiluminescence reaction is subject to stringent environmental conditions (such as pH and ionic strength), and the swelling process of hydrogel microneedles will change the local reaction environment, resulting in a decrease in luminescence efficiency; on the other hand, the bonding between hydrogel and electrode often relies on physical pressing or high-concentration adhesives, which can easily lead to poor interfacial contact and signal blockage, or the adhesive can penetrate into the hydrogel and interfere with the enzymatic and luminescent reactions, weakening the detection stability.

[0005] Currently, although the advantages of interstitial fluid sampling, hydrogel microneedles sampling, and electrochemiluminescence detection have been validated, the synergistic integration of these three technologies still faces key technical shortcomings, making it difficult to meet the needs of practical clinical applications. Firstly, existing integrated devices are mostly designed for single targets (e.g., detecting only glucose). If switching to detect other markers such as lactic acid is required, the entire electrode and microneedle system must be replaced, which is cumbersome, costly, and lacks versatility. Secondly, the swelling environment of hydrogel microneedles does not match the optimal conditions for electrochemiluminescence reactions, resulting in low reaction efficiency and failing to fully leverage the high sensitivity of electrochemiluminescence. Thirdly, the interfacial bonding between microneedles and electrodes is unstable, easily displaced by skin movement, leading to signal fluctuations and low reproducibility. Summary of the Invention

[0006] In view of the above, the main objective of this invention is to provide an electrochemiluminescent MeHA microneedle patch, its preparation method, and its application, in order to solve the aforementioned technical problems.

[0007] This invention proposes a method for preparing electrochemiluminescent MeHA microneedle patches, the method comprising the following steps:

[0008] Step 1: Place the phosphate buffer solution in a beaker and slowly add sodium hydroxide solution dropwise while stirring to obtain a stirring solution; add low molecular weight sodium hyaluronate powder to the stirring solution and stir overnight until completely dissolved to form a transparent solution.

[0009] Step 2: Add N,N-dimethylformamide to the transparent solution and stir. After stirring, add methacrylic anhydride dropwise. Immediately after adding, adjust the pH with sodium hydroxide and stir overnight to obtain a standing solution.

[0010] Step 3: Add sodium chloride to the standing liquid and stir until completely dissolved. After dissolution, add ethanol to precipitate and obtain a mixed solution. Centrifuge the mixed solution and discard the supernatant after centrifugation. Wash the solution repeatedly with anhydrous ethanol to obtain a washing solution. Redissolve the washing solution in ultrapure water and transfer it to a dialysis bag for dialysis. After dialysis, pre-freeze the solution in a refrigerator until it forms solid ice blocks and then freeze-dry it in a vacuum freeze dryer to obtain MeHA solid powder.

[0011] Step 4: Add MeHA solid powder and photoinitiator to phosphate buffer solution and stir to dissolve, forming HC-MeHA precursor solution;

[0012] Step 5: Slowly pour the HC-MeHA precursor solution into the cavity of the silicone microneedle mold to obtain a microneedle mold containing the precursor solution; place the microneedle mold containing the precursor solution into a centrifuge tube and fix it in place; then place the centrifuge tube containing the silicone microneedle mold into a centrifuge for centrifugation; after centrifugation, remove the silicone microneedle mold and place it in an oven to dry; after drying, demold to obtain MeHA microneedle patches loaded with ABEI and glucose oxidase.

[0013] This invention also proposes an application of an electrochemiluminescent MeHA microneedle patch. The MeHA microneedle patch loaded with ABEI and glucose oxidase, prepared by the above-mentioned method, is applied to the in-situ real-time electrochemiluminescence detection of biomarkers (such as glucose and lactic acid) in the interstitial fluid of the skin to achieve real-time concentration analysis.

[0014] Beneficial effects:

[0015] 1. Traditional methods for detecting interstitial fluid in the skin require a step-by-step process of "sampling-transfer-detection," which can easily lead to sample loss, contamination, and signal attenuation. However, this invention achieves an integrated operation of "in-situ extraction of interstitial fluid in the skin - enzymatic reaction - electrochemiluminescence detection" through the integrated design of "MeHA (methacrylic anhydride modified hyaluronic acid) microneedle patch - SPE electrode." This eliminates the need for sample transfer, significantly reduces interference from intermediate steps, and greatly improves the accuracy and efficiency of detection, making it particularly suitable for real-time clinical monitoring scenarios.

[0016] 2. Traditional electrochemiluminescence detection devices are mostly designed with fixed targets. When switching the biomarker to be detected (such as from glucose to lactate), the entire electrode and reaction system need to be replaced, which is cumbersome and costly. In contrast, this invention only requires replacing the MeHA microneedle patch loaded with the corresponding enzyme (glucose oxidase / lactic acid oxidase). Combined with the SPE electrode and LC-MeHA adhesion layer, it has strong versatility, greatly reduces the cost of multi-target detection, and adapts to the diverse detection needs of clinical practice.

[0017] 3. Traditional microneedle patch-electrode integration often relies on physical pressing or high-concentration adhesives, which can easily lead to poor contact or signal blocking. This invention adopts the "LC-MeHA semi-dry adhesive with secondary UV crosslinking" process, which not only ensures the stable bonding between the microneedle patch and the SPE electrode (the structure is not easy to fall off after air drying), but also avoids the interference of the adhesive layer on the electrochemiluminescence reaction. Combined with the highly sensitive luminescence signal mediated by ABEI (luminol analogue), it can accurately detect low-concentration skin interstitial fluid biomarkers.

[0018] 4. The core raw materials used in this invention (MeHA, SPE electrode, LAP photoinitiator, ABEI, etc.) are all commercially available reagents and do not require special customization; the MeHA microneedle patch is prepared by centrifugation using a silicone mold, which is simple, controllable, and highly reproducible (microneedle size deviation <5%); the integrated device assembly process does not require large-scale equipment, which facilitates large-scale production and commercialization, and has significant industrialization value.

[0019] 5. Traditional skin detection devices often suffer from skin irritation (such as metal microneedles) or poor biocompatibility. This invention uses MeHA (hyaluronic acid derivative) as the substrate for microneedles and adhesive layers, which has excellent biocompatibility. It only penetrates the stratum corneum of the skin (without damage to the dermis), and there is no obvious pain or irritation when it is applied. Patients accept it highly, and it is more suitable for long-term dynamic monitoring (such as daily blood glucose monitoring for diabetic patients).

[0020] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by means of embodiments of the invention. Attached Figure Description

[0021] Figure 1 This is a fabrication diagram of the "MeHA microneedle patch-SPE electrode" integrated device;

[0022] Figure 2 This is a schematic diagram of the preparation and detection process of glucose in interstitial fluid of the skin;

[0023] Figure 3 Comparison of 1H NMR spectra of methacrylamide hyaluronic acid and hyaluronic acid with ECL signals from different modified electrodes;

[0024] Figure 4Optical photographs of the microneedle patch and topographic images of the integrated device;

[0025] Figure 5 Force-displacement curves for HC-MeHA microneedles, HC+LC-MeHA microneedles, and force-displacement curves for different volumes of LC-MeHA;

[0026] Figure 6 The graph shows the effect of pH and GODx (glucose oxidase) concentration on ECL signal.

[0027] Figure 7 HE-stained sections and appearance of mouse skin;

[0028] Figure 8 The standard curve for glucose and the ECL spectrum are shown. Detailed Implementation

[0029] Embodiments of the present invention are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0030] These and other aspects of the embodiments of the present invention will become clear from the following description and accompanying drawings. In these descriptions and drawings, some specific embodiments of the present invention are specifically disclosed to illustrate some ways of implementing the principles of the embodiments of the present invention; however, it should be understood that the scope of the embodiments of the present invention is not limited thereto.

[0031] Example 1:

[0032] This embodiment provides a method for preparing an electrochemiluminescent MeHA microneedle patch, the method comprising the following steps:

[0033] Step 1: Place 200 mL of phosphate buffer solution in a 500 mL beaker, and slowly add 5 M sodium hydroxide solution dropwise to the beaker to adjust the pH of the solution in the beaker to 8. Stir for 10 min to obtain a stirring solution. Add 4 g of low molecular weight sodium hyaluronate powder to the stirring solution at 4 °C and stir overnight for 12 h until completely dissolved to form a transparent solution.

[0034] Step 2: Add 130 mL of N,N-dimethylformamide to the clear solution and stir for 30 min. After stirring, add 4.7 mL of methacrylic anhydride dropwise. Immediately after adding, adjust the pH to between 8 and 9 with 0.5 M sodium hydroxide and stir overnight at 4 °C for 12 h to obtain the standing solution.

[0035] Step 3: Add 0.5M sodium chloride to the standing liquid and stir at 4°C until completely dissolved. After dissolution, add ethanol at a volume three times the total volume of the solution to precipitate and obtain a mixed solution. Transfer the mixed solution to a centrifuge tube and centrifuge at 4000 rpm for 5 min. After centrifugation, discard the supernatant of the mixed solution and wash it three times with anhydrous ethanol to obtain a washing solution. Redissolve the washing solution in 200 mL of ultrapure water and transfer it to a dialysis bag for dialysis for 3 days, changing the ultrapure water every 8 hours. After dialysis, pre-freeze the solution in a -80°C freezer until it forms solid ice blocks, and then freeze-dry it in a vacuum freeze dryer for 3 days to obtain MeHA solid powder.

[0036] Step 4: Take 0.08g of MeHA solid powder and 0.0039g of photoinitiator (2,4,6-trimethylbenzoyl) and add them to a phosphate buffer solution with a pH of 8.5 and a volume of 2mL. Stir and dissolve at 4℃ for 4h to form HC-MeHA precursor solution; wherein, the phosphate buffer solution contains 40μM ABEI and 10U / mL glucose oxidase;

[0037] Step 5: Slowly pour the HC-MeHA precursor solution into a silicone microneedle mold cavity with a microneedle height of 900μm, a bottom diameter of 410*410μm, a needle spacing of 750μm, a number array of 12*12, a microneedle area of ​​8.7*8.7mm, and a patch size of 12*12mm to obtain a microneedle mold filled with the precursor solution. Place a 5mm thick piece of wood at the bottom of a centrifuge tube, and then place the microneedle mold filled with the precursor solution into the centrifuge tube. At the same time, use foam blocks to fill the gap between the mold and the tube wall in the centrifuge tube to ensure that the mold does not shift or rotate in the centrifuge tube. Then, centrifuge the centrifuge tube containing the silicone microneedle mold at 4000rpm for 5min. After centrifugation, remove the silicone microneedle mold and dry it in a 37℃ oven for 10h. After drying, peel off the edge of the mold with tweezers to obtain the MeHA microneedle patch loaded with ABEI and glucose oxidase, denoted as A1.

[0038] Among them, after obtaining the MeHA microneedle patch loaded with ABEI and glucose oxidase, it was further UV crosslinked for 5 min under a 365nm UV crosslinker to enhance the stability of the microneedle structure.

[0039] Example 2

[0040] This embodiment provides a method for preparing an electrochemiluminescent MeHA microneedle patch, the method comprising the following steps:

[0041] Step 1: Place 200 mL of phosphate buffer solution in a 500 mL beaker, and slowly add 5 M sodium hydroxide solution dropwise to the beaker to adjust the pH of the solution in the beaker to 8.5. Stir for 10 min to obtain a stirring solution. Add 4 g of low molecular weight sodium hyaluronate powder to the stirring solution at 4 °C and stir overnight for 12 h until completely dissolved to form a transparent solution.

[0042] Step 2: Add 132 mL of N,N-dimethylformamide to the transparent solution and stir for 30 min. After stirring, add 4.75 mL of methacrylic anhydride dropwise. Immediately after adding, adjust the pH to 8.5 with 0.5 M sodium hydroxide and stir overnight at 4 °C for 12 h to obtain the standing solution.

[0043] Step 3: Add 0.5M sodium chloride to the standing liquid and stir at 4°C until completely dissolved. After dissolution, add ethanol at a volume three times the total volume of the solution to precipitate and obtain a mixed solution. Transfer the mixed solution to a centrifuge tube and centrifuge at 4000 rpm for 7.5 min. After centrifugation, discard the supernatant of the mixed solution and wash it three times with anhydrous ethanol to obtain a washing solution. Redissolve the washing solution in 200 mL of ultrapure water and transfer it to a dialysis bag for dialysis for 5 days, changing the ultrapure water every 8 hours. After dialysis, pre-freeze the solution in a -80°C freezer until it forms solid ice blocks, and then freeze-dry it in a vacuum freeze dryer for 4 days to obtain MeHA solid powder.

[0044] Step 4: Take 0.1g of MeHA solid powder and 0.0040g of photoinitiator (2,4,6-trimethylbenzoyl) and add them to a phosphate buffer solution with a pH of 8.5 and a volume of 2mL. Stir and dissolve at 4℃ for 6h to form HC-MeHA precursor solution; wherein, the phosphate buffer solution contains 40μM ABEI and 10U / mL glucose oxidase;

[0045] Step 5: Slowly pour the HC-MeHA precursor solution into a silicone microneedle mold cavity with a microneedle height of 900μm, a bottom diameter of 410*410μm, a needle spacing of 750μm, a number array of 12*12, a microneedle area of ​​8.7*8.7mm, and a patch size of 12*12mm to obtain a microneedle mold filled with the precursor solution. Place a 7.5mm thick piece of wood at the bottom of a centrifuge tube, and then place the microneedle mold filled with the precursor solution into the centrifuge tube. At the same time, use foam blocks to fill the gap between the mold and the tube wall in the centrifuge tube to ensure that the mold does not shift or rotate in the centrifuge tube. Then, centrifuge the centrifuge tube containing the silicone microneedle mold at 4000rpm for 5min. After centrifugation, remove the silicone microneedle mold and dry it in a 37℃ oven for 10h. After drying, peel off the edge of the mold with tweezers to obtain the MeHA microneedle patch loaded with ABEI and glucose oxidase, denoted as A2.

[0046] Among them, after obtaining the MeHA microneedle patch loaded with ABEI and glucose oxidase, it was further UV crosslinked for 5 min under a 365nm UV crosslinker to enhance the stability of the microneedle structure.

[0047] Example 3

[0048] This embodiment provides a method for preparing an electrochemiluminescent MeHA microneedle patch, the method comprising the following steps:

[0049] Step 1: Place 200 mL of phosphate buffer solution in a 500 mL beaker, and slowly add 5 M sodium hydroxide solution dropwise to the beaker to adjust the pH of the solution in the beaker to 9. Stir for 10 min to obtain a stirring solution. Add 4 g of low molecular weight sodium hyaluronate powder to the stirring solution at 4 °C and stir overnight for 12 h until completely dissolved to form a transparent solution.

[0050] Step 2: Add 134 mL of N,N-dimethylformamide to the transparent solution and stir for 30 min. After stirring, add 4.8 mL of methacrylic anhydride dropwise. Immediately after adding, adjust the pH to 9 with 0.5 M sodium hydroxide and stir overnight at 4 °C for 12 h to obtain the standing solution.

[0051] Step 3: Add 0.5M sodium chloride to the standing liquid and stir at 4°C until completely dissolved. After dissolution, add ethanol at a volume three times the total volume of the solution to precipitate and obtain a mixed solution. Transfer the mixed solution to a centrifuge tube and centrifuge at 4000 rpm for 10 min. After centrifugation, discard the supernatant of the mixed solution and wash it three times with anhydrous ethanol to obtain a washing solution. Redissolve the washing solution in 200 mL of ultrapure water and transfer it to a dialysis bag for dialysis for 7 days, changing the ultrapure water every 8 hours. After dialysis, pre-freeze the solution in a -80°C freezer until it forms solid ice blocks, and then freeze-dry it in a vacuum freeze dryer for 5 days to obtain MeHA solid powder.

[0052] Step 4: Take 0.12g of MeHA solid powder and 0.0041g of photoinitiator (2,4,6-trimethylbenzoyl) and add them to a phosphate buffer solution with a pH of 8.5 and a volume of 2mL. Stir and dissolve at 4℃ for 8h to form HC-MeHA precursor solution; wherein, the phosphate buffer solution contains 40μM ABEI and 10U / mL glucose oxidase;

[0053] Step 5: Slowly pour the HC-MeHA precursor solution into a silicone microneedle mold cavity with a microneedle height of 900μm, a bottom diameter of 410*410μm, a needle spacing of 750μm, a number array of 12*12, a microneedle area of ​​8.7*8.7mm, and a patch size of 12*12mm to obtain a microneedle mold filled with the precursor solution. Place a 10mm thick piece of wood at the bottom of a centrifuge tube, and then place the microneedle mold filled with the precursor solution into the centrifuge tube. At the same time, use foam blocks to fill the gap between the mold and the tube wall in the centrifuge tube to ensure that the mold does not shift or rotate in the centrifuge tube. Then, centrifuge the centrifuge tube containing the silicone microneedle mold at 4000rpm for 5min. After centrifugation, remove the silicone microneedle mold and dry it in a 37℃ oven for 10h. After drying, peel off the edge of the mold with tweezers to obtain the MeHA microneedle patch loaded with ABEI and glucose oxidase, denoted as A3.

[0054] Among them, after obtaining the MeHA microneedle patch loaded with ABEI and glucose oxidase, it was further UV crosslinked for 5 min under a 365nm UV crosslinker to enhance the stability of the microneedle structure.

[0055] Application Example 1

[0056] Application Example 1 provides an application of an electrochemiluminescent MeHA microneedle patch. The MeHA microneedle patch loaded with ABEI and glucose oxidase, prepared using the above-mentioned method, is applied to the in-situ real-time electrochemiluminescence detection of biomarkers (such as glucose and lactic acid) in the interstitial fluid of the skin, realizing its real-time concentration analysis.

[0057] The method specifically includes the following steps:

[0058] Take 0.065±0.05g of MeHA solid powder and 0.001±0.0005g of photoinitiator (phenylbis(2,4,6-trimethylbenzoyl)phosphine oxide) and place them in a 5mL centrifuge tube. Add 2mL of ultrapure water and stir at 4℃ for 4-8h to form LC-MeHA precursor solution.

[0059] Use scissors to trim the MeHA microneedle patch loaded with ABEI and glucose oxidase in A2 to the preset size (1±0.1cm×1±0.1cm) to obtain the trimmed MeHA microneedle patch.

[0060] Take a commercial SPE electrode (working electrode: carbon electrode, reference electrode: Ag / AgCl, counter electrode: carbon electrode), mark the surface of the SPE electrode with the size of the trimmed MeHA microneedle patch as a reference to form the marked area of ​​the SPE electrode, and use a pipette to measure 100±10μL of LC-MeHA precursor solution and evenly drop it onto the marked area of ​​the SPE electrode to completely cover the marked area (it can slightly exceed the edge of the marked area, but the excess range should be controlled within 1-2mm to ensure full adhesion with the microneedle patch later). After the LC-MeHA precursor solution is evenly spread in the marked area of ​​the SPE electrode, put the SPE electrode into a 37℃ oven to dry for 30-40min, so that the LC-MeHA precursor solution on the SPE electrode is in a semi-dry adhesive state to form an LC-MeHA layer on the SPE electrode.

[0061] The trimmed MeHA microneedle patch is smoothly attached to the LC-MeHA layer of the SPE electrode and gently pressed for 5-10 seconds to ensure a tight bond between the microneedle patch and the electrode, thus obtaining an integrated device. The integrated device is then air-dried in a 37°C oven for 1-2 hours to completely cure the LC-MeHA layer of the SPE electrode and form a stable adhesive layer, thus obtaining an integrated device with an adhesive layer. The integrated device with the adhesive layer is then UV crosslinked for 5 minutes to obtain an SPE with an integrated microneedle patch.

[0062] Mice weighing 20-25g were selected as experimental targets. They were fasted for 12 hours before the experiment but allowed free access to water to eliminate the interference of food metabolism on the glucose concentration in the interstitial fluid of the skin. During the experiment, mice were anesthetized by intraperitoneal injection of chloral hydrate (400mg / kg). After the corneal reflex disappeared and the limbs were no longer moving spontaneously, the skin of the shaved area was wiped with a cotton ball soaked in 75% ethanol. Within a preset time, the SPE with integrated microneedle patch was pressed onto the skin surface of the shaved area of ​​the mouse. After removing the SPE with integrated microneedle patch, the hydrogel microneedles, due to their hydrophilicity and swelling properties, slowly adsorbed the interstitial fluid of the dermal layer of the mouse skin into the microneedles through the osmotic pressure gradient. The SPE electrode with integrated microneedle patch that absorbed the interstitial fluid was inserted into the adapter, placed in the electrochemiluminescence detector, and the detection parameters were set.

[0063] The specific detection parameters are as follows: excitation voltage of 0.2-1.2V, scan rate of 0.1V / s, detection time of 25s, amplification stage of 4, and photomultiplier tube voltage of 800V, for detection.

[0064] To verify the effectiveness of the present invention, a data comparison and characterization experiment was conducted. The sensor (denoted as M-ECL) prepared by the MeHA microneedle patch using the process described in Application Example 1 consists of a methacrylamide hyaluronic acid hydrogel microneedle array (MeHA MN), a luminol analog, a specific oxidase (GOX / LOX), and a highly expandable hydrogel integrated electrode. Figure 1The schematic diagram illustrates the sensor's fabrication process and detection principle.

[0065] Figure 2 This invention demonstrates a complete technical chain connecting "device preparation - signal generation - in vivo application," which not only intuitively presents the functional division of each component but also clarifies the core principle of electrochemiluminescence detection, providing visual support for understanding the technical solution of this invention.

[0066] The ECL (Extended Cl-Layer) is composed of three functional components: ① a core reaction layer (MeHA hydrogel microneedle array loaded with ABEI and specific oxidase, MeHA-ABEI-Enzyme MNAs), responsible for interstitial fluid extraction and enzymatic luminescence reaction; ② an interface bonding layer (highly expandable hydrogel "bio-glue", LC-MeHA), achieving stable integration of microneedles and electrodes; and ③ a signal detection layer (SPE electrodes: carbon working electrode, Ag / AgCl reference electrode, and carbon counter electrode), responsible for the acquisition and conversion of luminescence signals. The fabrication process of each component has been optimized to ensure functional synergy.

[0067] The synthesized MeHA was characterized by ¹H-NMR spectroscopy, and the differences in characteristic peaks between the HA raw material and MeHA were compared. The degree of methacrylation (the ratio of the peak area of ​​the olefin hydrogen introduced by methacrylic anhydride to the peak area of ​​the hydroxyl hydrogen in HA) was calculated by integrating the characteristic peaks. Figure 3 As shown in the ¹H-NMR comparison of MeHA and HA, MeHA exhibits a distinct olefinic hydrogen characteristic peak at 5.5-6.2 ppm (corresponding to the C=C bond of methacrylic anhydride), while HA does not, proving that methacrylic anhydride was successfully grafted onto the HA molecular chain. Through integral calculation, the degree of methacrylation is 40-65%, which is within the optimal range for hydrogel formation (below 40% results in insufficient cross-linking and easy breakage of microneedles; above 65% results in poor hydrogel flexibility and increased skin irritation).

[0068] exist Figure 3 Figure b (Comparison of ECL signals with different modified electrodes) shows that only the "SPE / ABEI-GODx-MeHA microneedles" system (the core system of this invention) produces a strong ECL signal (intensity of approximately 4000), while the ECL intensities of blank SPE, SPE / MeHA microneedles, and SPE / ABEI-MeHA microneedles are all below 500. This result indicates that the ECL signal originates from the electrochemiluminescence reaction between ABEI and GODx catalyzing the formation of H2O2 from glucose, completely eliminating non-specific luminescence interference from the MeHA substrate, ABEI itself, or the electrode material. This directly verifies the scientific validity and specificity of the electrochemiluminescence detection system, laying the foundation for subsequent quantitative detection.

[0069] It should be noted that, Figure 3In the figure, 'a' is a comparison of the 1H NMR spectra of MeHA and HA; Figure 3 In the figure, b is a comparison chart of the ECL feasibility verification of the ABEI / GODx-HC LC-MeHA microneedle sensor.

[0070] Combined with appendix Figure 4 (Morphological diagrams of microneedles and integrated devices) and mechanical test data, the effectiveness of the microneedle patch fabrication process can be verified from three aspects: Figure 4 Image a (optical photograph) shows that the microneedle array has no missing needles or deformation. Figure 4 In image b (SEM image), the microneedles are visible as square pyramids with sharp tips (tip diameter <50μm) and uniform height (900±20μm), consistent with the pre-set mold dimensions. Figure 4 Image c (integrated device diagram) shows that the microneedle patch adheres tightly to the SPE electrode without any obvious gaps. This demonstrates that the 4000 rpm centrifugation process for 5 minutes ensures the precursor solution completely fills the mold cavity without any air bubbles, and that the drying and UV crosslinking steps do not damage the microneedle structure. Universal testing machine tests show that the MeHA microneedle's single-needle puncture force reaches 0.35 N, exceeding the stratum corneum tensile strength (approximately 0.2 MPa), allowing it to penetrate human skin without significant needle breakage. Multiple batches of microneedles prepared showed a dimensional deviation of <5%, proving that the centrifugation, drying, and crosslinking process parameters (37℃ drying for 10 hours, UV crosslinking for 5 minutes) are stable and meet the requirements for large-scale production.

[0071] It should be noted that, Figure 4 In the image, 'a' represents an optical photograph of the HC-MeHA microneedle patch. Figure 4 In the image, b represents the SEM image of the microneedle array. Figure 4 In the image, 'c' represents the actual SPE electrode with integrated HC-MeHA microneedle patch.

[0072] exist Figure 5 The tensile-displacement curves in section b of the figure jointly verify the rationality of the integration process: tests on different LC-MeHA volumes show that the applied tensile force corresponding to 100μL LC-MeHA reaches 3.7N, significantly higher than that of 50μL (1.9N), and the tensile force difference between 120μL and 100μL is only 0.03N. Figure 5 In the case of LC-MeHA (a), excessive LC-MeHA is prone to overflowing from the electrode area, so 100±10μL is determined to be the optimal volume. The tension under this parameter can resist the lateral force (about 0.2N) generated by daily skin activities, thus preventing microneedles from falling off.

[0073] It should be noted that, Figure 5 In the figure, 'a' represents a comparison of the force-displacement curves of different MeHA microneedles. Figure 5In the figure, b is a comparison of the tension-displacement curves when different volumes of LC-MeHA (100 μL and 50 μL) are added.

[0074] Appendix Figure 6 (The effects of pH and GODx concentration on ECL signal) clarified the optimal parameters of the detection system, and the specific analysis is as follows: Figure 6 In example a, when the buffer pH=8.5, the ECL intensity reached its maximum (approximately 4500), with a signal-to-noise ratio (S / N) of 35, significantly higher than other pH values. This is because as the pH increases further, GODx activity decreases at higher pH levels, and the increase in ECL intensity is attributed to the rapid generation of ROS and the deprotonation of luminol. At pH=8.5, the glucose-glucose oxidase-ABEI system exhibited the highest luminescence efficiency (avoiding ABEI protonation under acidic conditions or enzyme inactivation under alkaline conditions), validating the rationality of the pH=8.5 parameter in Example 2. When the GODx concentration was 10 U / mL, the ECL intensity reached a stable value (approximately 4500) (at...). Figure 6 In step b), further increasing the concentration (15-25 U / mL) resulted in an ECL intensity increase of <5%, with no significant improvement. This result demonstrates that 10 U / mL is the optimal concentration, ensuring sufficient enzymatic reaction while avoiding cost waste and substrate competition caused by excessive enzyme, perfectly matching the final GODx concentration parameter in Example 2.

[0075] It should be noted that, Figure 6 In the figure, 'a' represents the effect of different pH values ​​on the ECL signal. Figure 6 Figure b in the figure shows the effect of different concentrations of glucose oxidase (GODx) on ECL signaling.

[0076] Appendix Figure 7 (H&E stained sections and appearance of mouse skin (in)) Figure 7 (b) This visually demonstrates the biosafety of the device: HE-stained sections (in...) Figure 7 In Figure a (scale bar 300μm), the epidermis of the experimental group was intact, the dermis was free of edema, and no inflammatory cell infiltration such as neutrophils and lymphocytes was observed. This proves that the MeHA microneedles only penetrate the stratum corneum of the skin (without damage to the dermis, avoiding the risk of blood vessel rupture and bleeding). Furthermore, the MeHA substrate (hyaluronic acid derivative) has excellent biocompatibility with human tissues and no immune rejection reaction, making it suitable for scenarios such as long-term dynamic monitoring of diabetic patients.

[0077] It should be noted that, Figure 7 In the image, 'a' is a section of mouse skin stained with hematoxylin and eosin (HE) after microneedle patch application. Figure 7 In the image, b represents the appearance of the microneedle patch applied to the skin of a mouse.

[0078] Appendix Figure 8 (The glucose standard curve and ECL spectrum) were obtained by inserting the sensor into agarose to simulate interstitial fluid detection data, comprehensively verifying the detection performance: within the glucose concentration range of 0.1-15 mM, the ECL intensity showed a good linear relationship with the concentration. Figure 8 In the regression equation y = 369.18x + 434.42, the coefficient of determination R² = 0.9955 (R² is close to 1, indicating excellent linear correlation). Low-concentration detection showed a method limit of detection (LOD) of 0.028 mM (calculated at a signal-to-noise ratio of 3), far lower than the basal glucose concentration in the interstitial fluid of healthy individuals (0.5-5 mM), capable of capturing minute fluctuations in glucose in the interstitial fluid under pathological conditions such as prediabetes (e.g., decreasing from 0.5 mM to 0.1 mM). Figure 8 The ECL spectrum (b) shows that even at a low concentration of 0.1 mM, there is still a clear emission peak with no baseline drift, further demonstrating the synergistic effect of the ABEI-mediated luminescence system and the hydrogel microneedles, which can meet the needs of accurate detection of low-concentration biomarkers. Combining the extraction time experiment and the detection process timing, the entire process of "dermal interstitial fluid extraction-electrochemiluminescence detection" takes only 14 minutes: 10 minutes of microneedle pressing on the skin for extraction (after 10 minutes, the ECL intensity reaches a stable value; extending to 15 or 20 minutes results in an intensity increase of <3%, requiring no additional time), and approximately 4 minutes for electrode insertion into the adapter and ECL detection. This fully meets the core performance indicator of "detection completed within 15 minutes," making it suitable for scenarios with high detection efficiency requirements, such as clinical emergency and real-time monitoring in sports medicine.

[0079] It should be noted that, Figure 8 In the figure, 'a' represents the standard curve of glucose concentration versus electrochemiluminescence intensity. Figure 8 In the diagram, b represents the electrochemiluminescence spectra at different concentrations.

[0080] In the description of this specification, references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0081] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A method for preparing an electrochemiluminescent MeHA microneedle patch, characterized in that, The method includes the following steps: Step 1: Place the phosphate buffer solution in a beaker and slowly add sodium hydroxide solution dropwise while stirring to obtain a stirring solution; add low molecular weight sodium hyaluronate powder to the stirring solution and stir overnight until completely dissolved to form a transparent solution. Step 2: Add N,N-dimethylformamide to the transparent solution and stir. After stirring, add methacrylic anhydride dropwise. Immediately after adding, adjust the pH with sodium hydroxide and stir overnight to obtain a standing solution. Step 3: Add sodium chloride to the standing liquid and stir until completely dissolved. After dissolution, add ethanol to precipitate and obtain a mixed solution. Centrifuge the mixed solution and discard the supernatant after centrifugation. Wash the solution repeatedly with anhydrous ethanol to obtain a washing solution. Redissolve the washing solution in ultrapure water and transfer it to a dialysis bag for dialysis. After dialysis, pre-freeze the solution in a refrigerator until it forms solid ice blocks, and then freeze-dry it in a vacuum freeze dryer to obtain MeHA solid powder. Step 4: Add MeHA solid powder and photoinitiator to phosphate buffer solution and stir to dissolve, forming HC-MeHA precursor solution; wherein, the phosphate buffer solution contains ABEI and glucose oxidase, and the solute concentration of ABEI is 40 μM, and the solute concentration of glucose oxidase is 10 U / mL. Step 5: Slowly pour the HC-MeHA precursor solution into the cavity of the silicone microneedle mold to obtain a microneedle mold containing the precursor solution; place the microneedle mold containing the precursor solution into a centrifuge tube and fix it, then place the centrifuge tube containing the silicone microneedle mold into a centrifuge for centrifugation. After centrifugation, remove the silicone microneedle mold and place it in an oven to dry. After drying, demold to obtain MeHA microneedle patches loaded with ABEI and glucose oxidase. Among them, the MeHA microneedle patch has a single needle puncture force of 0.35N, and the microneedle size deviation of multiple batches is less than 5%; when the MeHA microneedle patch is used for glucose detection, the ECL intensity reaches a maximum of 4500 and the signal-to-noise ratio is 35 when the buffer pH is 8.

5. In step 4, during the formation of the HC-MeHA precursor solution, the mass of MeHA solid powder weighed is 0.08-0.12g, the mass of photoinitiator is 0.0039-0.0041g, the volume of phosphate buffer solution is 2mL, the pH of phosphate buffer solution is 8.5, the stirring and dissolving temperature is 4℃, and the stirring and dissolving time is 4-8h. Specifically, the photoinitiator is lithium phenyl (2,4,6-trimethylbenzoyl) phosphate. In step 5, during the process of obtaining the MeHA microneedle patch loaded with ABEI and glucose oxidase, the silicone microneedle mold has a microneedle height of 900 μm and a bottom diameter of 410 μm. 410μm, pin spacing 750μm, number array 12 12. The microneedle area is 8.

7. 8.7mm, patch size is 12 12mm; centrifuge speed of 4000rpm, centrifugation time of 5min, drying temperature of 37℃ in oven, drying time of 10h; after obtaining MeHA microneedle patches loaded with ABEI and glucose oxidase, UV crosslinking was performed for 5min under a 365nm UV crosslinker to enhance the stability of the microneedle structure.

2. The method for preparing the electrochemiluminescent MeHA microneedle patch according to claim 1, characterized in that, In step 1, during the formation of the transparent solution, the volume of the phosphate buffer solution is 200 mL, the beaker capacity is 500 mL, the sodium hydroxide concentration is 5 M, the sodium hydroxide solution is added slowly, and the pH value of the solution in the beaker is adjusted to 8-9, and the stirring time is 10 min. The mass of low molecular weight sodium hyaluronate powder is 4g. The temperature at which the low molecular weight sodium hyaluronate is added to the stirring solution is 4℃. The stirring time in the stirring solution is 12h.

3. The method for preparing the electrochemiluminescent MeHA microneedle patch according to claim 1, characterized in that, In step 2, during the process of obtaining the standing solution, the volume of N,N-dimethylformamide added is 130-134 mL, the stirring time is 30 min, the volume of methacrylic anhydride added is 4.7-4.8 mL, the concentration of sodium hydroxide is 0.5 M, the pH value adjusted by sodium hydroxide is between 8 and 9, and the stirring overnight temperature is 4℃ and the time is 12 h.

4. The method for preparing the electrochemiluminescent MeHA microneedle patch according to claim 1, characterized in that, In step 3, during the process of obtaining MeHA solid powder, sodium chloride was added to the standing solution at a concentration of 0.5M, and the temperature at which it was stirred until completely dissolved was 4℃. The volume of ethanol added to precipitate was 3 times the total volume of the solution. The centrifugation speed was 4000 rpm, and the centrifugation time was 5-10 min. The dialysis bag was washed three times with anhydrous ethanol. The volume of ultrapure water was 200 mL. The dialysis bag was pretreated by rinsing it three times with ultrapure water. The molecular weight cutoff of the dialysis bag was 14000 Da. The dialysis time was 3-7 days, and the ultrapure water was replaced every 8 hours during the dialysis time. The temperature conditions in the refrigerator were -80℃. The freeze-drying time in a vacuum freeze dryer was 3-5 days. After obtaining the MeHA solid powder, it was placed in a sealed bag with the desiccant and stored at 4℃.

5. An electrochemiluminescent MeHA microneedle patch, characterized in that, The electrochemiluminescent MeHA microneedle patch is specifically a MeHA microneedle patch loaded with ABEI and glucose oxidase, prepared using the preparation method of the electrochemiluminescent MeHA microneedle patch as described in any one of claims 1-4.

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