An integrated microfluidic electrochemical sensing platform based on high-osmotic-pressure microneedles and methods of use thereof

Abstract

This invention belongs to the field of point-of-care testing (POCT) technology, specifically relating to an integrated microfluidic electrochemical sensing platform based on high-osmotic pressure microneedles and its usage method. This platform, using high-osmotic pressure, highly absorbent MeHA / Glu-CaCl2 microneedle patches, can extract approximately 25 μL of interstitial fluid within 3 minutes, achieving a biomarker recovery rate of 96%. Combined with a screen-printed electrode modified with Prussian blue probes and a microfluidic chip, it utilizes capillary force to achieve automatic sample dispensing, enabling simultaneous detection of four indicators—IL-6, CRP, SAA, and PCT—with detection limits as low as 0.48 pg / mL, 1.68 μg / mL, 3.02 μg / mL, and 22.28 pg / mL, respectively. The platform adopts an integrated "extraction-detection-integration" design, requiring only four steps, and is compact (5 cm in diameter, ≤50 g), with detection results highly consistent with the ELISA gold standard. Its modular components can be replaced independently, and the detection range can be easily expanded by changing antibodies. It has the advantages of low cost, easy production, and good biocompatibility, and is suitable for clinical point-of-care diagnosis and home health monitoring.

Description

Technical Field

[0001] This invention belongs to the field of point-of-care testing (POCT) technology, specifically relating to an integrated microfluidic electrochemical sensing platform based on high-osmotic pressure microneedles and its usage method. This platform efficiently extracts interstitial fluid through microneedle patches with the synergistic effect of high osmotic pressure and high water absorption. It integrates screen-printed electrodes and a microfluidic chip to achieve simultaneous detection of four inflammatory markers: interleukin-6 (IL-6), C-reactive protein (CRP), serum amyloid A (SAA), and procalcitonin (PCT). This makes it suitable for rapid point-of-care diagnosis, home health monitoring, and early screening of inflammation-related diseases. Background Technology

[0002] Inflammation is a defensive pathological process in which the body responds to damaging stimuli. Changes in the levels of related biomarkers (such as IL-6, CRP, SAA, and PCT) are key indicators reflecting the degree of inflammation, infection type, and disease progression. Timely and accurate detection of these biomarkers is of great significance for early screening, clinical diagnosis, and treatment monitoring of inflammation-related diseases. Currently, commonly used clinical detection methods mainly involve blood sampling combined with enzyme-linked immunosorbent assay (ELISA) and electrochemiluminescence analysis. However, blood sampling is an invasive procedure, which can easily lead to complications such as hematoma and infection. Furthermore, it relies on professional medical personnel and large-scale instruments, has a long testing cycle, and poor portability, making it difficult to meet the needs of point-of-care testing (POCT) and home health monitoring.

[0003] To address the limitations of traditional detection methods, minimally invasive or non-invasive biomarker detection technologies have become a research hotspot. Interstitial fluid (ISF), as an endogenous body fluid highly correlated with blood components, directly reflects the body's physiological and pathological state through its biomarker content. Furthermore, the sampling process can be performed minimally invasively, significantly reducing patient discomfort. Existing ISF sampling techniques include nylon aspirators, aspiration blister packs, and microdialysis, but these generally suffer from low extraction efficiency, complex operation, and unsuitability for continuous monitoring or home use. Microneedle technology, as a minimally invasive sampling tool at the micrometer scale, has gradually become a core technology for ISF extraction and detection due to its advantage of directly penetrating the epidermis and making direct contact with ISF. It has already been applied to the monitoring of biomarkers such as glucose and metabolites.

[0004] Electrochemical sensing technology has become the preferred detection principle for microneedle sensors due to its high sensitivity, rapid response, and potential for device miniaturization. Currently, microneedle-based electrochemical sensing platforms are mainly divided into two modes: "post-extraction detection" and "in-situ detection." However, both have significant technical drawbacks: the former relies heavily on single water absorption or osmosis, resulting in low ISF extraction efficiency, and requires additional sample transfer to the detection device, making the operation cumbersome; the latter's microneedle sensing interface is easily contaminated by skin secretions, and biomolecules and cellular components in ISF can interfere with the detection signal, leading to decreased specificity and accuracy. Therefore, developing an integrated microneedle electrochemical sensing platform that combines high-efficiency ISF extraction capabilities, anti-contamination and anti-interference performance, and the ability to simultaneously detect multiple biomarkers is crucial to overcoming existing technological bottlenecks and promoting the widespread adoption of immediate detection of inflammatory biomarkers. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing technologies and solve the technical problems of low ISF extraction efficiency, complex operation of inflammatory biomarker detection, and difficulty in simultaneous detection of multiple indicators, so as to achieve minimally invasive, rapid, sensitive, and specific detection of four inflammatory biomarkers in ISF. Here, we have developed an integrated microfluidic electrochemical sensing platform based on high osmotic pressure and high absorbency microneedles. This platform integrates the capabilities of high-efficiency ISF extraction and immunoelectrochemical sensing for the detection of four inflammatory biomarkers: IL-6, CRP, SAA, and PCT. These four biomarkers are specific indicators reflecting the degree of inflammation and the type of infection, and are therefore selected as the core analytes for inflammation detection. A fabricated screen-printed electrode (SPE) is combined with a Prussian blue (PB) signal probe and a specific antibody for the specific identification and detection of the extracted inflammatory biomarkers. After extraction, the high-osmotic-hyperabsorbency MeHA / Glu-CaCl2 microneedle patch is combined with a microfluidic chip integrating SPE. The ISF sample extracted by the microneedle patch enters the microfluidic detection cell under capillary force. The target biomarkers in the sample specifically bind to the antibodies on the electrode surface, causing changes in electrochemical signals through steric hindrance. Using an electrochemical workstation, the current signals corresponding to different biomarkers can be accurately collected, enabling simultaneous quantitative detection of multiple indicators. Simultaneously, the platform ensures high sensitivity and specificity of the detection by optimizing the microneedle formulation and electrode modification conditions. Quality control of the detection results is achieved through comparison and validation with enzyme-linked immunosorbent assay (ELISA). Therefore, this integrated multi-indicator sensing strategy makes the immediate and accurate detection of inflammatory biomarkers possible.

[0006] This invention is achieved through the following technical solution: An integrated microfluidic electrochemical sensing platform based on high-osmotic-pressure microneedles includes: A MeHA / Glu-CaCl2 composite microneedle patch for efficient extraction of interstitial fluid; A screen-printed electrode modified with a Prussian blue signal probe and a specific antibody for immunoelectrochemical detection; An integrated microfluidic chip for automatic sample dispensing and simultaneous multi-pool detection; The microneedle patch, electrode, and chip work together in an integrated manner of "extraction-detection-integration".

[0007] Furthermore, the microneedle patch is constructed using a composite system of hyaluronic acid methacrylate and glucose-calcium chloride, which has a three-dimensional porous structure and a synergistic effect of high osmotic pressure and high water absorption. The microneedles are arranged in a four-sided pyramidal array with a bottom side length of 380 μm, a height of 850 μm, and a distance of 600 μm between two adjacent needle tips, totaling 221 needles.

[0008] Furthermore, the screen-printed electrode includes four working electrodes, which are respectively immobilized with IL-6, CRP, SAA and PCT antibodies. The electrode surface is sequentially modified with PB, Au and MPA, and the PB self-assembles for 18 cycles.

[0009] Furthermore, the microfluidic chip has a circular structure with a diameter of 5 cm and a thickness of 5 mm. It has a 2 mm diameter inlet in the center and is connected to four detection cells with a diameter of 7 mm and a depth of 2.5 mm through four microchannels with a width of 1 mm and a depth of 0.5 mm. Automatic sample distribution is achieved by using capillary force.

[0010] A detection method based on the platform of claim 1 includes the following steps: Microneedle patches are pressed onto the skin surface to extract interstitial fluid; The extracted microneedle patch was centrifuged to recover the sample. The sample is dropped into the microfluidic chip inlet; The electrochemical workstation was activated to acquire signals and quantitatively analyze the concentrations of inflammatory markers.

[0011] Furthermore, the inflammatory markers include IL-6, CRP, SAA, and PCT, which are detected simultaneously.

[0012] Furthermore, the microneedle patch extraction time is 3 min, the centrifugation recovery conditions are 10000 rpm for 3 min, the sample loading volume is 50 μL, and the settling time is 30 s.

[0013] Furthermore, the electrochemical detection employs square wave voltammetry (SWV) with a potential range of -0.3 V to 0.4 V.

[0014] Furthermore, the microneedle patch has good biocompatibility, a cell survival rate of >80%, and mechanical strength that meets the requirements for skin puncture.

[0015] Furthermore, the platform has an overall diameter of 5 cm, a thickness of 5 mm, and a weight of ≤50 g, making it suitable for bedside diagnosis and home health monitoring.

[0016] The present invention has the following advantages over the prior art: 1. This invention develops an integrated microfluidic electrochemical sensing platform based on high-osmotic pressure microneedles. As a minimally invasive, efficient, and portable analytical tool, it can rapidly achieve simultaneous detection of four inflammatory biomarkers in ISF. Thanks to its carefully designed integrated "extraction-detection-integration" architecture and immunoelectrochemical sensing strategy, the platform combines efficient ISF extraction capabilities with multi-indicator specific detection functions. MeHA / Glu-CaCl2 microneedle patches, with their synergistic effect of high osmotic pressure and high water absorption, can extract sufficient IS (approximately 25 μL) within 3 minutes, achieving a biomarker recovery rate of 96%, significantly superior to the single extraction mechanism of traditional microneedles. SPEs modified with PB signal probes eliminate the cumbersome probe loading steps of traditional immunoassays. Combined with the capillary force automatic sample dispensing design of microfluidic chips, they enable simultaneous detection of four indicators—IL-6, CRP, SAA, and PCT—in a single loading operation, with detection limits as low as 0.48 pg / mL, 1.68 μg / mL, 3.02 μg / mL, and 22.28 pg / mL, respectively, demonstrating significantly improved sensitivity and specificity. Furthermore, the platform's detection results are highly consistent with the ELISA gold standard. By blocking non-specific sites with BSA and optimizing electrode modification processes, interference from biomolecules and small molecule metabolites in ISF is effectively resisted, further ensuring the reliability of the detection.

[0017] 2. The platform possesses significant practicality and promotional value. Its modular design allows for independent fabrication and replacement of microneedle patches, SPE electrodes, and microfluidic chips. By replacing specific antibodies, it can be expanded to detect other biomarkers, offering high flexibility. Core components are fabricated using low-cost materials and simple processes. Microneedle patches are mass-produced using a molding method, the SPE electrode modification process is standardized, and microfluidic chips can be rapidly manufactured via 3D printing, facilitating large-scale production and cost control. The entire detection process requires only four steps: microneedle extraction, centrifugation recovery, sample loading, and electrochemical detection. It requires no specialized medical personnel or large instruments, is compact (5 cm in diameter, 5 mm in thickness), and lightweight (≤50 g), making it suitable for various scenarios such as point-of-care diagnosis, home health monitoring, and early screening for inflammatory diseases. It provides a practical solution for the widespread adoption of point-of-care testing (POCT) technology for inflammatory biomarkers.

[0018] 3. The microneedle patch of this invention uses MeHA material with excellent biocompatibility. It has been verified to be non-toxic by cytotoxicity tests and has good mechanical properties. It can effectively puncture the epidermis, and the minimally invasive detection method reduces the pain of the subject. The sensor has high detection specificity and stability, and the detection results are in good agreement with the clinical ELISA method. It provides a reliable and practical technical means for the early diagnosis of inflammatory diseases and daily health monitoring. Attached Figure Description

[0019] Figure 1 The principle of MeHA synthesis; Figure 2 Optimization of microneedle patch fabrication parameters; Figure 3 This is a schematic diagram of a microneedle patch; Figure 4 Characterization of the morphology and structure of MeHA / Glu-CaCl2 microneedle patch; Figure 5 Mechanical performance testing of MeHA / Glu-CaCl2 microneedle patches; Figure 6 The survival rate of L929 cells after culturing in MeHA / Glu-CaCl2 for 24 h, 48 h and 72 h; Figure 7 For in vitro recovery of target substances using microneedle patches; Figure 8 This is a schematic diagram illustrating the modification process and principle of the SPE electrode. Figure 9 SEM images of the electrode surface when the number of PB self-assembly turns is 5, 10, 20 and 30 turns; Figure 10 Optimization of the number of self-assembly loops for PB; Figure 11 For surface morphology and structure characterization and elemental analysis; Figure 12 The changes in electrochemical performance at each step of SPE modification; Figure 13 Characterization of the electrochemical performance of the SPE electrode; Figure 14 A schematic diagram of a microneedle electrochemical sensor for the immediate detection of inflammatory markers in interstitial fluid; Figure 15 Schematic diagram and physical image of a microfluidic chip; Figure 16 Optimize IL-6 detection conditions; Figure 17 Optimize CRP detection conditions; Figure 18 Optimize SAA testing conditions; Figure 19 Optimize PCT detection conditions; Figure 20 Analysis and detection methods and sensing performance for IL-6; Figure 21 Analysis and detection methods and sensing performance of CRP; Figure 22 Analysis and detection methods and sensing performance of SAA; Figure 23 This describes the analysis and detection methods and sensing performance of PCT. Detailed Implementation

[0020] To further explain the present invention, the following specific embodiments are described.

[0021] The experimental materials of this invention mainly include hyaluronic acid (HA), methacrylic anhydride (MA), photoinitiator (Irgacure 2959), glucose (Glu), calcium chloride (CaCl2), Prussian blue (PB), tetrachloroauric acid (HAuCl4), 3-mercaptopropionic acid (MPA), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), N-hydroxysuccinimide (NHS), IL-6 antibody / antigen, CRP antibody / antigen, SAA antibody / antigen, PCT antibody / antigen, bovine serum albumin (BSA), rhodamine B (RhoB), agarose, PBS buffer, etc., and all reagents are of analytical grade or corresponding special grade.

[0022] Experimental instruments included an electrochemical workstation (CHI 660E / CHI 1040C, Autolab PGSTAT M204) and a scanning electron microscope (HITACHI SU8010, Phenom). TM Pro G6, Atomic Force Microscope (Dimension® Icon) TM Equipment includes: electronic universal testing machine (UTM4103), microplate reader (SPARK 10 M), centrifuge (L600-A), 3D printer, ultraviolet crosslinker, etc.

[0023] Example 1: Preparation of MeHA / Glu-CaCl2 microneedle patch 1.1 Synthesis of hyaluronic acid methacrylate (MeHA) like Figure 1As shown in the figure, the synthesis process of MeHA is described in detail. 1.0 g of hyaluronic acid (HA) was weighed and dissolved in 50 mL of deionized water. The solution was stirred overnight at 4°C until completely dissolved. 33 mL of N,N-dimethylformamide (DMF) was added to make the water-to-DMF volume ratio 3:2. 1.2 mL of methacrylic anhydride (MA) was slowly added dropwise under vigorous stirring. The pH was adjusted to 8-9 with 1 mol / L NaOH solution. The grafting reaction was completed by stirring overnight at 4°C. 2.47 g of NaCl was added to a concentration of 0.5 mol / L. After stirring to dissolve, anhydrous ethanol was poured in to precipitate the precipitate. The precipitate was collected by centrifugation and dissolved in 200 mL of deionized water. The precipitate was purified for 5 days using a dialysis bag with a molecular weight cutoff of 7 kDa. After freeze-drying, the precipitate was stored at -20°C.

[0024] 1.2 Optimization of Microneedle Patch Parameters The extraction performance of microneedle patches is closely related to the preparation parameters. Key parameters were determined through systematic optimization, such as... Figure 2 As shown, this figure contains four sub-figures, corresponding to the optimization results of the type of penetrant, the ratio of MeHA to Glu-CaCl2, the ratio of Glu to CaCl2, and the UV crosslinking time. (a) The figure compares the effects of Glu, Suc, NaCl, CaCl2, and various mixed penetrants on SBF extraction yield, showing that the Glu-CaCl2 mixed penetrant has the best extraction effect; (b) The figure explores the effect of different mass ratios of MeHA to Glu-CaCl2 (4:1, 3:2, 1:1, 2:3, 1:4), determining that the SBF extraction yield reaches its peak at a ratio of 1:1; (c) The figure optimizes the mass ratio of Glu to CaCl2 (4:1, 3:2, 1:1, 2:3, 1:4), concluding that the extraction effect is best at a ratio of 2:3; (d) The figure analyzes the balancing effect of different UV crosslinking times (1, 5, 10, 15, 20 min) on swelling performance and mechanical properties, finally selecting 15 min as the crosslinking time.

[0025] 1.3 Preparation of microneedle patches Weigh 5 mg MeHA and dissolve it in 100 μL of deionized water. Stir to dissolve and degas under vacuum for 10 min. Add 100 μL of 0.5 mg / mL photoinitiator (Irgacure 2959) and an appropriate amount of 50 mg / mL Glu-CaCl2 mixed solution (Glu to CaCl2 mass ratio 2:3) to make the MeHA to Glu-CaCl2 mass ratio 1:1. Mix well and degas under vacuum again for 10 min. Take 300 μL of the mixed precursor solution and pour it into a PDMS microneedle mold. Centrifuge at 4000 rpm for 5 min to fill the mold gaps. Air dry in a fume hood overnight. Crosslink with 360 nm ultraviolet light for 15 min. Peel it from the mold and trim the edges. Seal and store for later use.

[0026] The MeHA / Glu-CaCl2 microneedle patch is circular in shape, with a concentrated microneedle area diameter of 9 mm, approximately the size of a thumb, facilitating thumb pressure. It consists of an array of 221 pyramidal microneedles, each with a base side length of 380 μm, a height of 850 μm, and a distance of 600 μm between adjacent needle tips. After drying, it possesses a three-dimensional porous structure, exhibiting both high water absorption and high osmotic pressure. This structural design ensures effective microneedle penetration into the skin and efficient extraction of ISF. Its manufacturing process, external dimensions, and individual microneedle structure are described below. Figure 3 As shown.

[0027] Figure 3 (a) Schematic diagram of the manufacturing process of the cross-linked MeHA-MN patch; (b) Schematic diagram of the external dimensions of the microneedle patch; (c) Schematic diagram of the dimensions of a single microneedle (cross section).

[0028] Example 2: Performance Verification of Microneedle Patches 2.1 Morphological and structural characterization The preparation of MeHA / Glu-CaCl2 microneedle patches requires the synthesis of MeHA first, followed by molding, cross-linking and other steps. Its morphology and structure directly affect the extraction performance.

[0029] SEM characterization clearly revealed the macroscopic morphology and microstructure of the microneedles. The dried microneedles exhibited a complete quadrangular pyramid shape, with a neatly arranged array and a three-dimensional porous surface. This structure increases the contact area with ISF and provides capillary force. Although the microneedles deformed slightly after swelling, they maintained structural integrity without collapse, ensuring stable recovery of biomarkers during the extraction process. Specific morphological characteristics are as follows: Figure 4 As shown.

[0030] Figure 4 (a) Actual image of the microneedle patch; (b) SEM image of the dried MeHA / Glu-CaCl2; (c) Microneedle array before swelling; (d) Microneedle array after swelling.

[0031] 2.2 Mechanical Performance Testing The mechanical properties of microneedle patches are key to their ability to effectively pierce the skin, and need to be verified through force-displacement curve testing and puncture experiments.

[0032] Force-displacement curves show that the microneedles undergo plastic deformation at approximately 22.4 N, with a critical buckling failure strength of about 0.1 N / needle, meeting the requirements for skin puncture. Puncture experiments with 10 layers of paraffin film (1.29 mm thick, simulating the epidermis) and agarose gel (simulating the dermis) demonstrate that the microneedles can successfully penetrate the paraffin film and leave clear puncture marks on the gel surface, proving that they possess sufficient mechanical strength to cope with practical application scenarios. Relevant test results are as follows: Figure 5 As shown.

[0033] Figure 5 (a) Force-displacement curve of microneedle patch (inset is a schematic diagram of applying compressive force to microneedle); (b) Comparison of microneedle patch before and after penetrating paraffin film; (c) Comparison of microneedle patch before and after piercing agarose gel.

[0034] 2.3 Biocompatibility Testing Biocompatibility is a core requirement for the in vivo application of microneedle patches. The cytotoxicity of the MeHA / Glu-CaCl2 material was tested using the CCK-8 assay. L929 cells were co-cultured with the material for 24 h, 48 h, and 72 h, and cell viability was measured. The results showed that the survival rate in the experimental group was consistently above 80%, with no significant difference compared to the control group that had not been exposed to the material. This demonstrates that the material is non-cytotoxic, meets biocompatibility requirements, and is safe for human testing. Cell viability data are shown below. Figure 6 As shown.

[0035] 2.4 ISF extraction and target recovery capabilities of microneedle patches The ISF extraction and target recovery capabilities of microneedle patches need to be verified through in vitro experiments. Using RhoB as a biomarker can visually demonstrate the extraction and recovery effects. The experimental procedure involves inserting microneedle patches into an agarose gel containing RhoB, extracting the sample, transferring it to a centrifuge tube, adding deionized water, and centrifuging at different speeds and times for recovery. The absorbance at 552 nm is measured using a microplate reader, and the recovery rate is calculated using a standard curve. The microneedle patches after RhoB extraction show a uniform pink color, indicating that RhoB is uniformly integrated into the microneedle structure. Optimized results for different centrifugation parameters and extraction times show that a recovery rate of over 96% is achieved when centrifuging at 10,000 rpm for 3 min and inserting the patch into the gel for 3 min, meeting the requirements for sample volume and purity for subsequent detection. The experimental procedure and results are as follows. Figure 7 As shown.

[0036] Figure 7 (a) Schematic diagram of the experiment; (b) Actual image of the microneedle patch after RhoB extraction; (c) Magnified image of the microneedles after RhoB extraction; (d) Absorbance of different concentrations of RhoB at 552 nm; (e) Effect of different rotation speeds and centrifugation times on RhoB recovery rate; (f) Recovery rate of RhoB extracted from the microneedle patch at gel insertion times of 1, 3, 5, 10 and 15 min.

[0037] Example 3: Modification and Optimization of SPE Electrode 3.1 Modification and Optimization of SPE Electrodes The modification process of the SPE electrode directly affects the detection performance, such as... Figure 8As shown in the figure, the complete modification process of the SPE electrode is clearly illustrated, sequentially including PB self-assembly, Au electrodeposition, MPA linkage, EDC / NHS activation, antibody immobilization, and BSA blocking. The detection principle is also visually presented: the specific binding of the antigen to the antibody on the electrode surface creates a steric hindrance effect, leading to a decrease in the peak current of the PB probe. The concentration of the biomarker is quantitatively detected by measuring this current change. The number of PB self-assembly cycles is a key parameter affecting the electrocatalytic performance of the electrode. Figure 9 As shown in the figure, this diagram illustrates the electrode surface morphology after 5, 10, 20, and 30 self-assembly cycles. At 5 cycles, the PB deposition is insufficient; at 10 cycles, the deposition is uniform; and at 20 and 30 cycles, the film is too thick, resulting in cracks. This provides a clear morphological basis for subsequent optimization. Figure 10 As shown, (a) Figure presents the SWV curves of the PB / SPE electrode corresponding to different self-assembly cycles (5, 10, 15, 18, 20, 30 cycles), and the peak current shows a trend of first increasing and then decreasing with the number of cycles; (b) Figure shows that the peak current is the largest at 18 cycles through peak current value statistics, which verifies from the perspective of electrochemical performance that 18 cycles is the optimal number of self-assembly cycles.

[0038] The specific modification process of the electrode involved preparing two PB self-assembly solutions: Solution 1 was a mixed solution of 10 mmol / L K₄[Fe(CN)₆], 0.1 mol / L KCl, and 0.1 mol / L HCl; Solution 2 was a mixed solution of 10 mmol / L FeCl₃, 0.1 mol / L KCl, and 0.1 mol / L HCl. The SPE electrode was sequentially immersed in Solution 1 (1 min), deionized water (30 s), Solution 2 (1 min), and deionized water (30 s), repeating this cycle 18 times to obtain the PB / SPE electrode. This electrode was then immersed in a 0.1 mg / mL HAuCl₄ solution, and a constant potential of -200 mV was applied for 30 s. After rinsing with deionized water and drying with nitrogen, an Au / PB / SPE electrode was obtained. 10 μL of 10 mmol / L MPA solution was drop-coated onto the electrode surface, and the electrode was left at room temperature for 2 h. Then, 10 μL of 0.1 mg / mL HAuCl₄ solution was drop-coated onto the electrode surface. Activate with a mol / L EDC / NHS mixed solution (1:1) at room temperature for 1 h. After rinsing with PBS, drop 10 μL of the corresponding antibody (IL-6: 60 μg / mL, CRP: 80 μg / mL, SAA: 60 μg / mL, PCT: 80 μg / mL) onto each of the four working electrodes and incubate overnight at 4 °C. Finally, drop 10 μL of 1 mg / mL BSA solution onto the electrodes, block at room temperature for 1 h, rinse with PBS, and dry with nitrogen.

[0039] 3.2 Electrode Characterization The effects of electrode modification need to be verified through multi-dimensional characterization, such as... Figure 11As shown, (a) is the SEM image of PB / SPE and the distribution images of Fe and Cl elements. The uniform distribution of Fe elements proves that PB was successfully deposited; (b) is the EDS energy spectrum of PB / SPE, which shows that it contains elements such as C, N, O, Cl, and Fe, consistent with the composition of PB; (c) is the Au element distribution image of Au / PB / SPE. The uniform coverage of Au elements indicates that electrodeposition was successful; (d) is the EDS energy spectrum of Au / PB / SPE. The addition of Au element peaks verifies that the Au layer modification was completed.

[0040] like Figure 12 As shown, (a) Figure 1 shows the EIS images of four electrodes with different modification stages, namely SPE, PB / SPE, Au / PB / SPE, and mAb / Au / PB / SPE, in a 5 mmol / L K3[Fe(CN)6] solution. The diameter of the semicircle in the Nyquist plot changes sequentially, reflecting the change in interfacial resistance, which proves that the antibody was successfully immobilized. (b) Figure 2 shows the SWV images of the above electrodes in a PBS solution containing 0.1 mol / L KCl. The peak current shows a regular decrease with each modification step, further verifying the effectiveness of each modification step.

[0041] like Figure 13 As shown, (a) Figure shows the CV curves at different scan rates (10, 20, 40, 80, 120, 160 mV / s). The oxidation peak and reduction peak are symmetrical and the peak current increases with the increase of scan rate. (b) Figure shows the linear relationship between the redox peak current and the square root of the scan rate. The fitting coefficient is close to 1, which proves that the electron transport at the electrode interface is mainly controlled by diffusion.

[0042] The SPE is a small-sized model requiring a small sample volume. It features an integrated structure including a working electrode, a counter electrode, and a reference electrode. The four working electrodes (WEs) are arranged in an array, corresponding to the immobilization of IL-6, CRP, SAA, and PCT antibodies, respectively, forming a specific detection array. The electrode surfaces are sequentially modified with PB, a gold layer, and 3-mercaptopropionic acid (MPA) to provide a stable foundation for antibody immobilization and signal transduction. The overall sensor structure and detection principle are as follows: Figure 14 As shown.

[0043] Example 4: Fabrication of Microfluidic Chips The chip model (5 cm in diameter and 5 mm in thickness, including one 2 mm diameter injection port, four 1 mm wide and 0.5 mm deep microchannels, and four 7 mm diameter and 2.5 mm deep detection cells) was designed using SolidWorks software. The chip mold was printed using a 3D printer (photosensitive resin material), poured with PDMS and cured. After demolding, the edges were trimmed, cleaned with ethanol and dried for later use.

[0044] The microfluidic chip is circular, 5 cm in diameter and 5 mm thick, with a compact structure for easy portability. It features a 2 mm diameter sample inlet at its center, connected to four detection cells via four 1 mm wide and 0.5 mm deep microchannels. The detection cells are 7 mm in diameter and 2.5 mm deep, perfectly matching the diameter of the SPE sensing area. This ensures complete electrode contact with the electrolyte while minimizing sample volume, enabling rapid sample dispensing and simultaneous detection. The chip structure and sample flow characteristics are as follows: Figure 15 As shown.

[0045] Figure 15 (a) Schematic diagram of the microfluidic chip device; (b) State of the sample test solution at 0 s, 1 s, 3 s and 30 s after pumping in (Note: RhoB in pink is used as the sample test solution model).

[0046] Example 5: Assembly and Use of the Sensing Platform The assembly of portable sensing devices requires the organic integration of antibody-modified SPEs with microfluidic chips and microneedle patches. The smoothness of the detection process directly affects the user experience. After the recovered ISF sample is added to the microfluidic chip inlet, the detection liquid fills the microchannel within 3 seconds under capillary force, and completely wets the four detection electrodes after 30 seconds. No additional power device is required, achieving rapid sample distribution. Combined with the SWV detection method, simultaneous detection of four biomarkers can be completed in a short time. The operation is convenient and efficient, meeting the application requirements of point-of-care testing. The sample flow state is as follows: Figure 15 As shown in (b).

[0047] The specific usage method is as follows: First, assemble the components. Take out the 3D-printed microfluidic chip and precisely embed the SPE electrodes modified with specific antibodies (IL-6: 60 μg / mL, CRP: 80 μg / mL, SAA: 60 μg / mL, PCT: 80 μg / mL) into the four detection cells of the chip. Ensure that the electrode sensing area is completely attached to the detection cell without any offset or gaps. Check whether the chip inlet and microchannels are unobstructed. Wipe the surface impurities with a lint-free paper. Then, connect the positive and negative electrodes and the reference electrode interface of the electrochemical workstation to the working electrode, counter electrode, and reference electrode of the SPE, respectively, ensuring that the wiring is firm and not loose. Place the microfluidic chip stably to avoid displacement during detection. Next, extract the sample. Wipe the skin on the inside of the subject's forearm with a 75% alcohol swab and let it air dry. Then, apply the MeHA / Glu-CaCl2 microneedle patch to the clean area and press vertically with your thumb for 3 seconds. After ensuring the microneedles effectively penetrate the epidermal layer to extract ISF, gently peel off the patch. Then, sample recovery is performed by placing the extracted microneedle patch into a centrifuge tube, adding 200 μL of deionized water, and centrifuging at 10,000 rpm for 3 minutes. The supernatant is collected as the ISF sample to be tested. Next, sample loading is performed by pipetting 50 μL of ISF sample and slowly adding it to the central inlet of the microfluidic chip. After standing for 30 seconds, the sample is automatically distributed to the four detection cells through the microchannels under capillary force, completely wetting the electrode surfaces. Electrochemical detection is then performed by turning on the electrochemical workstation, setting the SWV detection mode to a potential range of -0.3V to 0.4V, starting the detection program, acquiring the peak current signals corresponding to each electrode, and recording the detection data. Finally, results analysis is performed by calculating the concentrations of IL-6, CRP, SAA, and PCT based on pre-plotted standard curves for each biomarker and the obtained peak current changes, completing simultaneous quantitative analysis of multiple indicators.

[0048] Example 6: Optimization of Detection Conditions The detection conditions for each inflammatory marker need to be optimized specifically, such as Figure 16 As shown in the figure, this figure is an optimization diagram of IL-6 detection conditions. (a) Figure explores the effect of different IL-6 antibody concentrations (10, 20, 40, 60, 80, 100 μg / mL) on the current difference. The current difference is the largest at 60 μg / mL, which is determined to be the optimal antibody concentration for IL-6 detection. (b) Figure analyzes the effect of different IL-6 antibody fixation times (2, 4, 6, 8, 10, 12 h) on the current difference. The current difference reaches its peak at 10 h, which is determined to be the optimal fixation time for IL-6 detection. (c) Figure optimizes different IL-6 antigen incubation times (10, 20, 30, 40, 50, 60 min). The current difference stabilizes at its maximum value at 30 min, which is determined to be the optimal incubation time for IL-6 detection.

[0049] Figure 16 In the middle: (a) antibody concentration; (b) antibody fixation time; (c) antigen incubation time.

[0050] Note: I is the peak current value after incubation with IL-6 at a concentration of 1000 pg / mL; I0 is the peak current value before incubation with the antigen.

[0051] like Figure 17 As shown in the figure, this figure is an optimization diagram of CRP detection conditions. (a) Figure compares the effects of different CRP antibody concentrations (20, 40, 60, 80, 100, 120 μg / mL) on the current difference. The current difference is the largest at 80 μg / mL, which is determined to be the optimal antibody concentration for CRP detection. (b) Figure analyzes the effects of different CRP antibody fixation times (4, 6, 8, 10, 12, 14 h) on the current difference. The current difference is the best at 8 h, which is determined to be the optimal fixation time for CRP detection. (c) Figure optimizes different CRP antigen incubation times (10, 20, 30, 40, 50, 60 min). The current difference reaches its peak at 20 min, which is determined to be the optimal incubation time for CRP detection.

[0052] Figure 17 In the middle: (a) antibody concentration; (b) antibody fixation time; (c) antigen incubation time.

[0053] Note: I is the peak current value after incubation with CRP at a concentration of 100 μg / mL; I0 is the peak current value before incubation with antigen.

[0054] like Figure 18 As shown in the figure, this figure is an optimization diagram of SAA detection conditions. (a) Figure compares the effects of different SAA antibody concentrations (20, 40, 60, 80, 100 μg / mL) on the current difference. The current difference is the largest at 60 μg / mL, which is determined to be the optimal antibody concentration for SAA detection. (b) Figure analyzes the effects of different SAA antibody fixation times (6, 8, 10, 12, 14 h) on the current difference. The current difference is the best at 10 h, which is determined to be the optimal fixation time for SAA detection. (c) Figure optimizes different SAA antigen incubation times (15, 20, 30, 40, 50 min). The current difference reaches its peak at 30 min, which is determined to be the optimal incubation time for SAA detection.

[0055] Figure 18 In the middle: (a) antibody concentration; (b) antibody fixation time; (c) antigen incubation time.

[0056] Note: I is the peak current value after incubation with 100 μg / mL SAA; I0 is the peak current value before incubation with antigen.

[0057] like Figure 19 As shown in the figure, this figure is an optimization diagram of PCT detection conditions. (a) Figure compares the effects of different PCT antibody concentrations (40, 60, 80, 100, 120 μg / mL) on the current difference. The current difference is the largest at 80 μg / mL, which is determined to be the optimal antibody concentration for PCT detection. (b) Figure analyzes the effects of different PCT antibody fixation times (6, 8, 10, 12 h) on the current difference. The current difference is the best at 8 h, which is determined to be the optimal fixation time for PCT detection. (c) Figure optimizes different PCT antigen incubation times (20, 30, 40, 50, 60 min). The current difference reaches its peak at 40 min, which is determined to be the optimal incubation time for PCT detection.

[0058] Figure 19 In the middle: (a) antibody concentration; (b) antibody fixation time; (c) antigen incubation time.

[0059] Note: I is the peak current value after incubation with PCT at a concentration of 500 pg / mL; I0 is the peak current value before incubation with the antigen.

[0060] Example 7: Validation of Inflammatory Marker Sensing Performance After the SPE electrode is modified, the sensing performance of each inflammatory marker needs to be fully verified to ensure the accuracy and reliability of the detection.

[0061] 7.1 IL-6 detection performance For IL-6 detection, the SWV curves of a series of IL-6 concentrations showed that the peak current gradually decreased with increasing concentration, exhibiting a two-segment linear relationship in the ranges of 1-10 pg / mL and 50-1000 pg / mL. The detection limit was 0.48 pg / mL, and the sensitivity was 2.70 μA / (pg / mL). The RSD of 10 consecutive detections on the same electrode was 0.23%, and the RSD after 7 days of storage at 4℃ was 4.59%, demonstrating good stability. In the presence of interfering substances such as CRP, SAA, PCT, IgG, BSA, Glu, and LA, the response current change rate was <5%, indicating excellent selectivity. The correlation curve between the actual IL-6 concentration in the hydrogel and the extracted and recovered concentration had a slope of 0.96, showing good correlation. Specific sensing performance is as follows: Figure 20 As shown.

[0062] Figure 20In the middle section: (a) SWV curves of the electrode at a series of IL-6 concentrations; (b) Linear fitting curves between peak current changes and IL-6 concentrations (n=3); (c) SWV curves of IL-6 at the same concentration scanned 10 times in PBS (the inset shows the peak current value of the SWV curve for each scan); (d) Detection results of IL-6 at the same concentration in PBS for 7 consecutive days (Note: ΔI' and ΔI represent the difference in current reduction obtained on a given date and the first day, respectively, and the same applies below); (e) Response current value of the sensing electrode to IL-6 in the presence of interfering substances (Note: ΔI and ΔI0 represent the peak current changes in solutions with and without interfering substances, respectively, and the same applies below); (f) Correlation between the actual IL-6 concentration in the hydrogel and the calculated IL-6 concentration extracted and recovered based on MeHA / Glu-CaCl2 microneedle patches.

[0063] 7.2 CRP testing performance The sensing performance of CRP detection also showed excellent results. SWV curves for a series of CRP concentrations showed that the peak current decreased with increasing concentration, exhibiting good linearity. The detection limit was 1.68 μg / mL, and the sensitivity was 1.31 μA / (μg / mL). The RSD for 10 consecutive measurements with the same electrode was 0.52%, and the RSD after 7 days of storage at 4℃ was 1.47%, demonstrating good long-term stability. The response current showed minimal change in the presence of interfering substances, indicating excellent selectivity. The correlation curve between the actual CRP concentration in the hydrogel and the extracted and recovered concentration had a slope of 0.95, indicating good correlation. Specific sensing performance details are as follows: Figure 21 As shown.

[0064] Figure 21 In the middle: (a) SWV curves of the electrode at a series of CRP concentrations; (b) linear fitting curves between peak current changes and CRP concentrations (n=3); (c) SWV curves of CRP at the same concentration scanned 10 times in PBS (the inset shows the peak current value of the SWV curve for each scan); (d) Detection results of CRP at the same concentration in PBS for 7 consecutive days; (e) Response current value of the sensing electrode to CRP in the presence of interfering substances; (f) Correlation between the actual CRP concentration in the hydrogel and the calculated CRP concentration extracted and recovered based on MeHA / Glu-CaCl2 microneedle patches.

[0065] 7.3 SAA Detection Performance The sensor performance verification results for SAA detection showed that the peak current of the SWV curves for a series of SAA concentrations gradually decreased with increasing concentration, exhibiting a good linear relationship. The detection limit was 3.02 μg / mL, and the sensitivity was 0.74 μA / (μg / mL). The RSD for 10 consecutive detections using the same electrode was 0.67%, and the RSD after 7 days of storage at 4℃ was 3.55%, demonstrating stability that meets application requirements. Interfering substances had minimal impact on the detection results, indicating excellent selectivity. The correlation curve between the actual SAA concentration and the extracted and recovered concentration showed a slope of 0.96, indicating a good correlation. Specific sensor performance details are as follows: Figure 22 As shown.

[0066] Figure 22 In the middle: (a) SWV curves of the electrode at a series of SAA concentrations; (b) linear fitting curves between peak current changes and SAA concentrations (n=3); (c) SWV curves of SAA at the same concentration scanned 10 times in PBS (the inset shows the peak current value of the SWV curve for each scan); (d) Detection results of SAA at the same concentration in PBS for 7 consecutive days; (e) Response current value of the sensing electrode to SAA in the presence of interfering substances; (f) Correlation between the actual SAA concentration in the hydrogel and the calculated SAA concentration extracted and recovered based on MeHA / Glu-CaCl2 microneedle patches.

[0067] 7.4 PCT Detection Performance The sensor performance verification results for PCT detection showed that the peak current of the SWV curves for a series of PCT concentrations decreased with increasing concentration, exhibiting a good linear relationship. The detection limit was 22.28 pg / mL, and the sensitivity was 0.09 μA / (μg / mL). The RSD for 10 consecutive detections using the same electrode was 0.64%, and the RSD after 7 days of storage at 4℃ was 3.65%, demonstrating good long-term stability. The response current showed minimal change in the presence of interfering substances, indicating excellent selectivity. The correlation curve between the actual PCT concentration and the extracted and recovered concentration had a slope of 0.97, indicating a good correlation. Specific sensor performance details are as follows: Figure 23 As shown.

[0068] Figure 23 In the middle: (a) SWV curves of the electrode at a series of PCT concentrations; (b) Linear fitting curves between peak current changes and PC concentrations (n=3); (c) SWV curves of PCT at the same concentration scanned 10 times in PBS (the inset shows the peak current value of the SWV curve for each scan); (d) Detection results of PCT at the same concentration in PBS for 7 consecutive days; (e) Response current value of the sensing electrode to PCT in the presence of interfering substances; (f) Correlation between the actual PCT concentration in the hydrogel and the calculated PCT concentration extracted and recovered based on MeHA / Glu-CaCl2 microneedle patches.

[0069] Example 8: Accuracy Verification The accuracy of the sensor needs to be verified by comparison with the clinical gold standard ELISA method. In the IL-6 detection sample recovery experiment, 0, 10, 500, and 1000 pg / mL of IL-6 were added respectively. The recovery rate of this sensor was between 92.28% and 105.16%, with a relative standard deviation (RSD) of 0.81% to 6.05%, directly verifying the accuracy of IL-6 detection. In the CRP detection sample recovery experiment, 0, 1, 10, and 50 μg / mL of CRP were added respectively. The recovery rate of the sensor was between 103.03% and 128.62%, with an RSD of 1.05% to 6.05%, verifying the accuracy of CRP detection. In the SAA detection recovery experiments, with the addition of 0, 10, 50, and 100 μg / mL of SAA, the sensor recovery rates ranged from 86.20% to 104.34%, with RSDs ranging from 1.54% to 6.03%, validating the accuracy of SAA detection. In the PCT detection recovery experiments, with the addition of 0, 10, 250, and 500 pg / mL of PCT, the sensor recovery rates ranged from 89.95% to 107.74%, with RSDs ranging from 1.67% to 9.80%, validating the accuracy of PCT detection. The detection results of this platform are highly consistent with the results of the ELISA gold standard method, validating the accuracy and reliability of this platform. The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An integrated microfluidic electrochemical sensing platform based on high-osmotic-pressure microneedles, characterized in that, include: A MeHA / Glu-CaCl2 composite microneedle patch for efficient extraction of interstitial fluid; A screen-printed electrode modified with a Prussian blue signal probe and a specific antibody for immunoelectrochemical detection; An integrated microfluidic chip for automatic sample dispensing and simultaneous multi-pool detection; The microneedle patch, electrode, and chip work together in an integrated manner of "extraction-detection-integration".

2. The platform according to claim 1, characterized in that, The microneedle patch is constructed using a composite system of hyaluronic acid methacrylate and glucose-calcium chloride, which has a three-dimensional porous structure and a synergistic effect of high osmotic pressure and high water absorption. The microneedles are arranged in a four-sided pyramidal array with a bottom side length of 380 μm, a height of 850 μm, and a distance of 600 μm between two adjacent needle tips, totaling 221 needles.

3. The platform according to claim 1, characterized in that, The screen-printed electrode contains four working electrodes, which are respectively immobilized with IL-6, CRP, SAA and PCT antibodies. The electrode surface is sequentially modified with PB, Au and MPA, and the PB self-assembles in 18 cycles.

4. The platform according to claim 1, characterized in that, The microfluidic chip has a circular structure with a diameter of 5 cm and a thickness of 5 mm. It has a 2 mm diameter inlet in the center and is connected to four detection cells with a diameter of 7 mm and a depth of 2.5 mm through four microchannels with a width of 1 mm and a depth of 0.5 mm. Automatic sample distribution is achieved by using capillary force.

5. A detection method based on the platform described in claim 1, characterized in that, Includes the following steps: Microneedle patches are pressed onto the skin surface to extract interstitial fluid; The extracted microneedle patch was centrifuged to recover the sample. The sample is dropped into the microfluidic chip inlet; The electrochemical workstation was activated to acquire signals and quantitatively analyze the concentrations of inflammatory markers.

6. The method according to claim 5, characterized in that, The inflammatory markers include IL-6, CRP, SAA, and PCT, which are detected simultaneously.

7. The method according to claim 5, characterized in that, The microneedle patch extraction time was 3 min, the centrifugation recovery conditions were 10000 rpm for 3 min, the sample loading volume was 50 μL, and the settling time was 30 s.

8. The method according to claim 5, characterized in that, The electrochemical detection was performed using square wave voltammetry (SWV), with a potential range of -0.3 V to 0.4 V.

9. The platform according to claim 1, characterized in that, The microneedle patch has good biocompatibility, a cell survival rate of >80%, and mechanical strength that meets the requirements for skin puncture.

10. The platform according to claim 1, characterized in that, The platform has an overall diameter of 5 cm, a thickness of 5 mm, and a weight of ≤50 g, making it suitable for bedside diagnosis and home health monitoring.

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