Microneedle patch for in vivo detection of creatinine in interstitial fluid, preparation method and application thereof

Abstract

The application provides a microneedle patch for in vivo detection of creatinine in interstitial fluid, a preparation method and application. The microneedle patch is a three-electrode system, and the three-electrode system comprises a working electrode, a reference electrode and a counter electrode, and the working electrode, the reference electrode and the counter electrode all contain a microneedle array and a conductive polymer coated on the surface of the microneedle array; the working electrode further comprises copper ions fixed on the surface of the conductive polymer through coordination and perfluorosulfonic acid resin coated on the surfaces of the conductive polymer and the copper ions; and the reference electrode further comprises a reference metal and a reference metal halide coated on the surface of the conductive polymer. The application can solve the problem that it is difficult to detect creatinine in vivo in the prior art, and is suitable for the technical field of biosensing and medical detection.

Description

Technical Field

[0001] This invention relates to the field of biosensing and medical detection technology, and more specifically, to a microneedle patch for in vivo detection of creatinine in interstitial fluid, its preparation method, and its application. Background Technology

[0002] Chronic kidney disease (CKD) has become a major global public health problem, and early diagnosis and long-term monitoring are crucial for its prevention and control. Creatinine is a core biomarker of CKD, and its serum concentration is highly correlated with kidney function impairment. Since the concentration of creatinine in interstitial fluid is similar to that in serum, real-time, non-invasive monitoring of creatinine in interstitial fluid has significant clinical implications. Currently, the gold standard for creatinine detection is isotope dilution high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS), which, while highly accurate, is expensive, complex, time-consuming, and requires professional personnel, making it difficult to implement in the general population. In recent years, researchers have developed various alternative methods, including colorimetric methods, fluorescence methods, mass spectrometry, high-performance liquid chromatography, and electrochemical sensing methods. Among these methods, fluorescence and colorimetric methods largely rely on chromogenic reagents and isothermal incubation, while mass spectrometry and high-performance liquid chromatography require large instruments, making it difficult to achieve equipment miniaturization and portability. Although some electrochemical methods have integration potential, they generally rely on a three-enzyme catalytic system to convert creatinine into hydrogen peroxide for detection. However, hydrogen peroxide is easily consumed by reducing substances in body fluids and drugs, leading to signal attenuation and low results. Furthermore, enzyme activity is greatly affected by temperature and microorganisms, resulting in poor storage stability and making it difficult to meet the needs of long-term home use. Enzyme-free electrochemical sensing methods utilize nanomaterials for direct catalytic oxidation, but they generally suffer from problems such as easy shedding of nanomaterials during skin penetration, insufficient sensitivity (detection limits are often higher than 100 μM), poor selectivity, and susceptibility to interference from proteins and anions. Moreover, most methods still require the collection of serum or urine and in vitro pretreatment such as centrifugation and dilution, which is painful during the sampling process and cannot achieve truly painless, pretreatment-free, and real-time in vivo detection.

[0003] Among the aforementioned technologies, existing methods generally suffer from multiple drawbacks, including difficulty in integrating equipment, cumbersome operation procedures, reliance on in vitro sample processing, invasive or painful detection processes, insufficient sensitivity and selectivity, and poor material stability. These shortcomings prevent them from meeting the urgent needs of CKD patients for daily, frequent, non-invasive, and automated home monitoring. In particular, there is a lack of a miniaturized in vivo detection device that can directly contact interstitial fluid, requires no pre-sampling treatment, and can complete highly sensitive, highly selective quantitative detection in a short time with just a simple press, while also being low-cost and capable of long-term stable use. This severely restricts the popularization of early screening and long-term management of CKD. Summary of the Invention

[0004] The main objective of this invention is to provide a microneedle patch, its preparation method, and its application for in vivo detection of creatinine in interstitial fluid, in order to solve the problem of difficulty in in vivo detection of creatinine in the prior art.

[0005] To achieve the above objectives, according to a first aspect of the present invention, a microneedle patch for in vivo detection of creatinine in interstitial fluid is provided. The microneedle patch is a three-electrode system comprising: a working electrode, a reference electrode, and a counter electrode. Each of the working electrode, the reference electrode, and the counter electrode contains a microneedle array and a conductive polymer coated on the surface of the microneedle array. The working electrode further comprises: copper ions immobilized on the surface of the conductive polymer through coordination, and perfluorosulfonic acid resin coated on the conductive polymer and the surface of the copper ions. The reference electrode further comprises: a reference metal and a reference metal halide coated on the surface of the conductive polymer. The microneedle patch has the following function: upon contact with the interstitial fluid, electrochemical scanning is performed to detect Cu in situ generated on the electrode surface. 0 Oxidized to Cu + - Creatine anhydride complexes are formed, thus creating an independent oxidation current peak, which enables in vivo quantitative detection of creatine anhydride without in vitro pretreatment.

[0006] Furthermore, the aforementioned conductive polymer includes poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonic acid).

[0007] Furthermore, the aforementioned perfluorosulfonic acid resin includes Nafion.

[0008] Furthermore, the substrate of the microneedle array comprises polycarbonate; optionally, the height of the microneedles is less than 700 μm; optionally, the tip diameter of the microneedles is less than 10 μm.

[0009] Furthermore, the reference metal mentioned above includes Ag, and the reference metal halide mentioned above includes AgCl.

[0010] To achieve the above objectives, according to a second aspect of the present invention, a method for preparing the microneedle patch for in vivo detection of creatinine in interstitial fluid is provided. The method includes: a) providing the microneedle array; b) coating the conductive polymer on the surface of the microneedle array to form a conductive layer; dividing the conductive layer into a working electrode region, a reference electrode region, and a counter electrode region; c) loading copper ions in the working electrode region and achieving chemical fixation of the copper ions through coordination with the conductive polymer; further covering the surface of the working electrode region with the perfluorosulfonic acid resin; d) depositing and coating a reference metal in the reference electrode region and halogenating it to form a reference metal halide; e) retaining the exposed conductive polymer in the counter electrode region; f) sealing the electrode substrate with a non-water-soluble insulating material and leading out three electrode wires to form a complete three-electrode system, thereby obtaining the microneedle patch.

[0011] Further, the microneedle array is based on polycarbonate; optionally, the conductive polymer includes poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonic acid); optionally, d) includes: coating the reference electrode region with metallic silver and subjecting it to chlorination to form the reference electrode region containing Ag and AgCl.

[0012] Furthermore, the coating of the above-mentioned poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonic acid) includes: immersing the above-mentioned microneedle array in a PEDOT:PSS aqueous solution and then drying it to form a film.

[0013] Further, the loading of copper ions includes: immersing the working electrode region in a copper-containing salt solution for a certain period of time, followed by washing with deionized water and drying; preferably, the salt solution includes one or more of CuSO4, Cu(NO3)2, CuCl2 or Cu(CH3COO)2.

[0014] Furthermore, the coating with the perfluorosulfonic acid resin includes: applying a solution of the perfluorosulfonic acid resin to the working electrode area, allowing it to stand, and then drying it at room temperature; preferably, the solution of the perfluorosulfonic acid resin includes an ethanol solution of Nafion.

[0015] Furthermore, the preparation of the reference electrode region containing Ag and AgCl includes: coating the reference electrode region with silver paste, drying it, and then immersing it in FeCl3 solution for chlorination to form the reference electrode region containing Ag and AgCl.

[0016] To achieve the above objectives, according to a third aspect of the present invention, an application is provided of the above-described microneedle patch, or a microneedle patch prepared by the above-described preparation method, in in vivo detection of creatinine content in interstitial fluid.

[0017] To achieve the above objectives, according to a fourth aspect of the present invention, a method for in vivo detection of creatinine content in interstitial fluid is provided, the method comprising: (1) cleaning the sensing area of ​​the microneedle patch or the microneedle patch prepared by the above preparation method with deionized water or phosphate buffer to remove copper ions physically adsorbed on the surface; (2) connecting the cleaned microneedle patch to an electrochemical detection device and pressing it vertically onto the surface of the hair-removed skin so that the microneedles penetrate the stratum corneum and contact the interstitial fluid, maintaining the attachment time for not less than 10 seconds, so that the electrode contacts the interstitial fluid; (3) scanning using square wave voltammetry, collecting and analyzing the differential current values ​​of characteristic peaks in the range of -0.08 ~ -0.12 V, and calculating the creatinine content based on the differential current values.

[0018] Using the technical solution of this application, after the microneedle patch comes into contact with the interstitial fluid, it can generate Cu originating from in situ through electrochemical scanning. 0 Oxidized to Cu + - An independent oxidation current peak of the creatinine complex. Utilizing this microneedle patch and current peak, in vivo quantitative detection of creatinine can be easily achieved. This current peak exhibits high specificity, directly responding to changes in creatinine concentration in interstitial fluid. Furthermore, based on the coordination immobilization of copper ions with the conductive polymer and the selective permeation of perfluorosulfonic acid resin, the sensing interface ensures stable response in complex biological environments. This microneedle patch possesses the capability for minimally invasive, painless, real-time online detection of creatinine in interstitial fluid, overcoming the dependence on sample pretreatment and invasive sampling in traditional biochemical detection. It provides a high-precision, low-interference, wearable in vivo analytical approach for in vivo creatinine detection, especially for long-term and frequent in vivo creatinine detection. Attached Figure Description

[0019] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0020] Figure 1 The diagram illustrates the preparation method of the microneedle patch according to Embodiment 1 of the present invention and the schematic diagram of the in vivo detection principle of creatinine.

[0021] Figure 2 The SEM results according to Embodiment 2 of the present invention are shown. Figure 2 Image A in the image is the SEM result of PC / PEDOT:PSS. Figure 2 B in the middle is PC / PEDOT:PSS / Cu 2+ SEM results for Nafion.

[0022] Figure 3AThe SEM electron image, elemental distribution map, and energy-dispersive X-ray spectrum of PC / PEDOT:PSS according to Embodiment 2 of the present invention are shown, with a scale bar of 100 μm.

[0023] Figure 3B PC / PEDOT:PSS / Cu according to Embodiment 2 of the present invention is shown. 2+ SEM electron image, elemental distribution map and energy-dispersive X-ray spectrum of / Nafion, scale bar 100 μm.

[0024] Figure 4 A feasibility test of microneedle penetration into the skin according to Embodiment 2 of the present invention is shown. Figure 4 A shows PP / Cu 2+ / Nafion pressure-displacement curve results Figure 4 Image B shows the morphology of the Bama fragrant pig skin surface before microneedles were inserted. Figure 4 C shows the morphology of the Bama fragrant pig skin surface before microneedles were inserted. Figure 4 The image in Figure D shows the morphology of the microneedles after insertion into the pigskin.

[0025] Figure 5 Images related to the microneedle patch according to Embodiment 2 of the present invention are shown, wherein, Figure 5 Figure A shows a schematic diagram of the microneedle patch partitioning modification and preliminary integration. Figure 5 Image B shows a physical image of a preliminarily integrated microneedle patch, with a scale bar of 3 mm. Figure 5 Image C shows the combination of microneedle patch and microelectrochemical detection system.

[0026] Figure 6 The SWV plot and peak current-creatinine concentration linear fitting plot of the microneedle patch according to Embodiment 3 of the present invention are shown. Figure 6 In the diagram, A and B represent the SWV plot and the peak current-creatinine concentration linear fitting plot, respectively, for the first microneedle patch. Figure 6 In the middle, C and D are the SWV diagram and peak current-creatinine concentration linear fitting diagram of the second microneedle patch, respectively. Figure 6 E and F in the middle are the SWV diagram and the peak current-creatinine concentration linear fitting diagram of the third microneedle patch, respectively.

[0027] Figure 7 The working curve of creatinine detection using a microneedle patch according to Embodiment 3 of the present invention is shown.

[0028] Figure 8 The graph shows the detection results of serum creatinine according to Example 3 of the present invention. Wherein, Figure 8 Figures A, B, and C show the results of three parallel experiments. Figure 8In the above-mentioned serum, creatinine was detected by ultra-high performance liquid chromatography-mass spectrometry using the standard addition method. The concentration of deuterated creatinine (d3-Crt) was 50.0 μM (N=3).

[0029] Figure 9 Experimental results of rabbit serum according to Example 3 of the present invention are shown. Among them, Figure 9 Image A in the image shows the patch calibration result. Figure 9 Figure B shows the calibration curve results for creatinine detection using microneedle patches. Figure 9 Figure C shows the in vitro detection results of creatinine in rabbit serum.

[0030] Figure 10 The in vivo detection results of creatinine in rabbit interstitial fluid according to Example 3 of the present invention are shown. Figure 10 Figures A and B show the results of establishing the working curves for a microneedle patch used for in vivo detection of creatinine levels in interstitial fluid. Figure 10 The diagram in C is of a microneedle patch. Figure 10 The image in D shows the test results.

[0031] Figure 11 The image shown is a picture of rabbit ear skin according to Embodiment 3 of the present invention. Figure 11 Image A is a picture of the rabbit's ear skin at the time of the test. Figure 11 Image B is a picture of the rabbit's ear skin 15 minutes after the test was completed.

[0032] Figure 12 The diagram shows the SWV (Symptoms of microneedle patches according to Example 4 of the present invention) in PBS solution containing 75.0 μM creatinine before and after storage at room temperature for 2 months. Detailed Implementation

[0033] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0034] As mentioned in the background section, existing creatinine detection methods generally suffer from problems such as reliance on in vitro sample processing, insufficient sensitivity, poor selectivity, low material stability, and difficulty in achieving non-invasive in vivo real-time monitoring. In particular, there is a lack of a miniaturized sensing device that can directly contact interstitial fluid, require no pretreatment, and achieve highly specific quantitative detection through simple physical contact.

[0035] Therefore, in this application, the inventors attempted to develop a novel microneedle patch and, based on this, proposed a series of protection schemes for this application.

[0036] In a first typical embodiment of this application, a microneedle patch for in vivo detection of creatinine in interstitial fluid is provided. The microneedle patch is a three-electrode system, comprising a working electrode, a reference electrode, and a counter electrode. Each of the working electrode, the reference electrode, and the counter electrode contains a microneedle array and a conductive polymer coated on the surface of the microneedle array. The working electrode further comprises copper ions fixed to the surface of the conductive polymer through coordination, and perfluorosulfonic acid resin coated on the conductive polymer and the surface of the copper ions. The reference electrode further comprises a reference metal and a reference metal halide coated on the surface of the conductive polymer. The microneedle patch has the following function: after contacting the interstitial fluid, electrochemical scanning is performed to detect Cu in situ generated on the electrode surface. 0 Oxidized to Cu + - Creatine anhydride complexes are formed, thus creating an independent oxidation current peak, which enables in vivo quantitative detection of creatine anhydride without in vitro pretreatment.

[0037] The aforementioned microneedle patch for in vivo detection of creatinine in interstitial fluid utilizes a core sensing mechanism based on the high-intensity coordination-redox synergistic effect between copper ions and creatinine molecules, constituting a non-enzymatic sensing system that does not require the participation of biological enzymes. The microneedle patch comprises: an array of microneedles made of biocompatible polymer materials, serving as a mechanical support and puncture structure, capable of minimally invasively penetrating the stratum corneum and contacting the interstitial fluid of the dermis; and a conductive polymer coated on the surface of the microneedles, acting as an electron conduction medium, providing not only a stable electrochemical interface, but also sulfonate (-SO3) groups carried on its molecular chains. - The functional group also serves as a coordination anchor for copper ions, enabling Cu... 2+ The fixation ensures that it does not fall off or leak in the physiological fluid environment.

[0038] Cu fixed on the surface of the conductive layer 2+ During the electrochemical scanning process, it is first reduced to Cu. 0 (Zero-valent copper), while creatine molecules, due to the presence of a highly coordinating secondary amine group in their molecular structure, can significantly stabilize Cu. + And form stable Cu + The complexation with creatinine produces a characteristic oxidation current peak during potential scanning, independent of background interference. The intensity of this current peak is linearly positively correlated with the concentration of creatinine in the interstitial fluid, and its specificity stems from the cation-selective permeability of Nafion and the effect of creatinine on Cu. + / Cu 2+ Its competitive coordination binding is different from other common small molecules in body fluids (such as urea, glucose, ascorbic acid, etc.).

[0039] It should be noted that, in the practical application of the microneedle patch of this application, the inventors discovered that the microneedle patch of this application can generate a Cu peak with a very good shape during electrochemical scanning. 0 Oxidized to Cu + The creatine complex exhibits an oxidation peak, which is virtually unaffected by any common interfering substances in the body fluid. This principle and mechanism distinguish it from other existing technologies that utilize copper ions for electrochemical detection. This application utilizes this novel mechanism to achieve accurate in vivo detection of creatine.

[0040] To enhance the anti-interference capability of the sensing interface, a layer of perfluorosulfonic acid resin is further coated on the surface of the microneedles. This polymer has a network of sulfonic acid groups with a high negative charge density, which can effectively repel negatively charged biomolecules (such as albumin and globulin) and anionic interfering substances (such as Cl-). - HPO4 2- This prevents it from approaching the electrode surface, thus ensuring that the detection signal is transmitted solely by creatine-Cu. + The electrochemical response of the complex is dominant.

[0041] This microneedle patch, without requiring any sample collection, centrifugation, dilution, or pretreatment, only needs to be in direct contact with interstitial fluid and a specific potential scanning program applied to achieve in-situ, real-time, and quantitative detection of creatinine through its independent oxidation peak. Its sensing principle is entirely based on physicochemical processes and does not rely on biomolecular recognition elements. It possesses high stability, repeatability, and structural integrability, making it suitable for constructing non-invasive, portable, and long-term wearable monitoring devices. It is the first to achieve enzyme-free in vivo detection of creatinine in interstitial fluid. Furthermore, the microneedle patch has a simple structure, low manufacturing cost, and is easy to industrialize, market, and apply.

[0042] In a preferred embodiment, the conductive polymer described above comprises poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonic acid) (PEDOT:PSS).

[0043] The conductive polymer in the microneedle patch of this application includes PEDOT:PSS, wherein poly(3,4-ethylenedioxythiophene) (PEDOT) provides electronic conductivity, while poly(styrene sulfonic acid) (PSS), as a macromolecular anionic dopant, not only achieves oxidative doping of PEDOT to improve conductivity, but also features densely distributed sulfonate groups (–SO3) on its molecular chain. - It is also Cu² +It provides high-density coordination sites to achieve stable chemical immobilization of copper ions. At the same time, PEDOT:PSS has good water dispersibility, film-forming properties and electrochemical stability. It can form a uniform, dense and strongly adherent conductive coating on the microneedle surface. In addition, its resistance to biofouling in physiological environments is better than that of metal or carbon-based materials, making it suitable as a functional conductive interface for microneedle electrodes.

[0044] In a preferred embodiment, the perfluorosulfonic acid resin described above includes Nafion.

[0045] The perfluorosulfonic acid resin in the microneedle patch of this application includes Nafion, a cation exchange polymer containing perfluorosulfonic acid groups. Its molecular structure comprises a highly hydrophobic polytetrafluoroethylene backbone and hydrophilic sulfonic acid end groups, forming a nanoscale phase-separated ion channel network. This structure endows Nafion with excellent anion repulsion properties, forming a selective molecular sieve layer on the electrode surface, which can effectively block negatively charged biomolecules (such as serum albumin, globulin, heparin, etc.) and small molecule anions (such as Cl-). - HPO4² - The urate group (creatine ion) is located close to the sensing interface, thereby significantly reducing signal drift and baseline interference caused by nonspecific adsorption. As an ultrathin coating, it can self-assemble into a continuous film through solvent evaporation without damaging the physical structure of the microneedle tip or blocking the diffusion and penetration of the target analyte, creatine, thus achieving synergistic optimization of selective shielding and high-throughput detection.

[0046] In a preferred embodiment, the substrate of the microneedle array comprises polycarbonate (PC); optionally, the height of the microneedles is 500–700 μm; optionally, the tip diameter of the microneedles is less than 3 μm.

[0047] The substrate of the aforementioned microneedle array includes PC, a thermoplastic engineering plastic with high mechanical strength, good thermal stability, and bioinertness. It is suitable for precision compression molding processes, enabling high-precision mass replication of the microneedle structure. This ensures that the microneedles have sufficient sharpness to reduce puncture resistance, improve insertion success rate, and provide a structurally reliable foundation for stable contact and monitoring of interstitial fluid.

[0048] In a preferred embodiment, the microneedle patch is a three-electrode system, comprising: a working electrode, a reference electrode, and a counter electrode; wherein the working electrode comprises: a composite layer formed by the copper ions, PEDOT:PSS, and perfluorosulfonic acid resin; the reference electrode comprises: a reference metal and a reference metal halide; the counter electrode comprises: the conductive polymer; optionally, the reference metal comprises Ag, and the reference metal halide comprises AgCl.

[0049] In a second typical embodiment of this application, a method for preparing the microneedle patch for in vivo detection of creatinine in interstitial fluid is provided. The method includes: a) providing the microneedle array; b) coating the conductive polymer on the surface of the microneedle array to form a conductive layer; dividing the conductive layer into a working electrode region, a reference electrode region, and a counter electrode region; c) loading copper ions in the working electrode region and achieving chemical fixation of the copper ions through coordination with the conductive polymer; further covering the surface of the working electrode region with the perfluorosulfonic acid resin; d) depositing and coating a reference metal in the reference electrode region and halogenating it to form a reference metal halide; e) retaining the exposed conductive polymer in the counter electrode region; f) sealing the electrode substrate with a non-water-soluble insulating material and leading out three electrode wires to form a complete three-electrode system.

[0050] In the above preparation method, the miniaturized integration of the three-electrode system is achieved through a structured stepwise construction. The core of this method lies in the chemical modification and spatial isolation of the functional regions on the microneedle surface. First, a conductive polymer is coated onto the surface of the microneedle array to form a continuous conductive layer with high charge transport capability. This layer not only serves as an electron conduction medium but also provides coordination sites for the subsequent chemical anchoring of metal ions. Subsequently, by distinguishing the physical structure of this conductive layer, the working electrode region, reference electrode region, and counter electrode region are spatially electrically independent, ensuring no crosstalk between the three electrodes. This fulfills the necessary foundation for stable potential control and current response in electrochemical detection. In the working electrode region, copper ions react with sulfonate groups (-SO3) in the conductive polymer. - The coordination of Cu achieves fixation, and this coordination has a strong binding force, making Cu... 2+The microneedle remains stable in a physiological environment while retaining its ability to function as a redox active site. The subsequent coating of perfluorosulfonic acid resin, through its dense hydrophobic ion exchange network, forms a selective barrier on the microneedle surface, effectively blocking negatively charged proteins, amino acids, and anionic interfering substances in the interstitial fluid from approaching the electrode interface, thereby enhancing the specificity and anti-contamination capability of the detection signal. In the reference electrode region, a reference metal / reference metal halide redox couple is generated in situ through coating with a reference metal and the action of a halogenating agent. This system provides a stable and repeatable reference potential under physiological pH and ionic strength, and its electrochemical response is unaffected by fluctuations in the biological medium, which is crucial for accurate quantitative detection. The counter electrode region retains an unmodified conductive polymer layer as a charge balance pathway, ensuring the integrity of the current loop during detection. Finally, the microneedle substrate is completely sealed with a non-water-soluble insulating material (such as nail polish), leaving only the tips of each microneedle exposed to prevent short circuits between electrodes and electrochemical responses in non-target areas. Then, a complete three-electrode system is formed by leading out the electrodes with wires, thereby achieving seamless integration from micron-level structures to macroscopically connectable electrode systems, providing a structurally stable and functionally complete sensing platform for subsequent in vivo electrochemical detection.

[0051] In a preferred embodiment, the microneedle array is based on PC; optionally, the conductive polymer includes PEDOT:PSS; optionally, d) includes: depositing metallic silver in the reference electrode region and chlorinating it to form the reference electrode region containing Ag and AgCl.

[0052] In the above preparation method, PC can be used as the microneedle substrate, which combines excellent mechanical strength and biocompatibility, supporting high-precision microfabrication and skin penetration. PEDOT:PSS, as a preferred conductive polymer, has excellent conductivity, electrochemical window stability, and film-forming adaptability. The reference electrode region can optionally be formed into an Ag / AgCl structure through silver deposition and chlorination, which is the preferred configuration for achieving a stable reference potential. Its preparation process is simple, has high response repeatability, and is suitable for low-cost batch preparation.

[0053] In a preferred embodiment, the coating of the above-mentioned PEDOT:PSS includes: immersing the microneedle array in a 0.5–5.0 wt% PEDOT:PSS aqueous solution for 1–5 minutes, followed by drying at 110–130°C for 10–60 minutes to form a film.

[0054] In the above preparation method, the microneedle array is immersed in a 0.5–5.0 wt% PEDOT:PSS aqueous solution for 1–5 minutes and dried at 110–130℃ for 10–60 minutes to form a film. This allows PEDOT:PSS to be fully adsorbed onto the PC microneedle surface under limited concentration and time conditions. Subsequently, precise temperature-controlled drying enables slow solvent evaporation and polymer chain rearrangement, significantly enhancing the interfacial bonding between the conductive layer and the microneedle substrate. Simultaneously, it promotes the uniform construction of the conductive network and effectively avoids mechanical detachment and electrochemical signal drift caused by excessively thin, discontinuous, or poorly adhered coatings. This film-forming process improves the density and stability of the conductive layer structure while ensuring the subsequent Cu... 2+ By leveraging the coordination fixation of sulfonic acid groups and the selective permeability and charge transport properties of the perfluorosulfonic acid resin membrane, the electrochemical response consistency of the three-electrode system is maintained during minimally invasive skin penetration and contact with interstitial fluid, providing a reliable signal basis for high-precision in vivo quantitative detection of creatinine.

[0055] In a preferred embodiment, the loading of copper ions includes: immersing the working electrode region in a salt solution containing 1–100 mM copper ions for 1–30 minutes, followed by rinsing with deionized water and drying; preferably, the salt solution includes one or more of CuSO4, Cu(NO3)2, CuCl2 or Cu(CH3COO)2.

[0056] In the above preparation method, the working electrode region of the microneedle array is immersed in 1–100 mM Cu. 2+ Incubation in salt solution for 1–30 minutes, followed by washing with deionized water and drying, yielded Cu 2+ Controllable and stable chemical immobilization based on the coordination of sulfonic acid groups on the PEDOT:PSS conductive layer; the range of process parameters ensures the stability of Cu. 2+ The thorough binding with sulfonic acid groups effectively removes uncoordinated ions during the cleaning process, leaving only firmly bound Cu. 2+ This maintains the stability of active sites on the electrode surface within the interstitial fluid environment; any one of CuSO4, Cu(NO3)2, CuCl2, or Cu(CH3COO)2 can effectively provide coordinateable Cu. 2+ The ions, and their anions do not interfere with the PEDOT:PSS structure, nor do they affect conductivity and biocompatibility; combined with the barrier effect of the perfluorosulfonic acid resin membrane on macromolecular interferences and the stable potential output of the Ag / AgCl reference electrode, this Cu 2+The fixation method significantly improves the sensitivity, repeatability, and anti-interference ability of the working electrode to the electrochemical response of creatinine. It eliminates the need for external fixatives or complex chemical modifications, enabling truly non-invasive, painless in vivo quantitative detection without in vitro pretreatment. This provides a highly reliable and mass-producible microneedle sensing platform for real-time monitoring of physiological indicators.

[0057] In a preferred embodiment, the coating of the perfluorosulfonic acid resin includes: applying a solution of the perfluorosulfonic acid resin of 0.1–2.0 wt% to the working electrode area, allowing it to stand, and then drying it at room temperature; preferably, the solution of the perfluorosulfonic acid resin includes an ethanol solution of Nafion.

[0058] In the above preparation method, a perfluorosulfonic acid resin membrane is formed by drop-coating a 0.1–2.0 wt% Nafion ethanol solution onto the working electrode area and allowing it to stand before drying at room temperature. This membrane, due to its highly selective cation exchange properties, can effectively block interfering substances such as anions, proteins, and negatively charged amino acids in the interstitial fluid from approaching the Cu atoms loaded on the PEDOT:PSS sulfonic acid groups. 2+ The active site allows the target molecule creatine to diffuse freely to the electrode surface, thus significantly improving the selectivity and anti-interference capability of the detection process. Nafion solutions in this concentration range can form a dense and uniform film, ensuring efficient shielding of biomolecules, while avoiding signal reduction due to excessive film thickness inhibiting electron and ion transport. Combined with Cu... 2+ The stable coordination structure with PEDOT:PSS enables highly specific and stable electrochemical detection of creatinine without in vitro pretreatment, providing reliable technical support for minimally invasive and painless in vivo quantitative analysis.

[0059] In a preferred embodiment, the preparation of the reference electrode region containing Ag and AgCl includes: coating the reference electrode region with silver paste, drying it, and then immersing it in a 0.01–0.5 M FeCl3 solution for chlorination for 1–20 minutes to form the reference electrode region containing Ag and AgCl.

[0060] In the above preparation method, after coating the reference electrode region with silver paste and drying it, it is immersed in a 0.01–0.5 M FeCl3 solution for chlorination for 1–20 minutes. This allows the deposited metallic silver to undergo a controllable and uniform interfacial reaction with chloride ions in the solution, forming a dense and stable AgCl layer in situ. This process is suitable for the microscale characteristics of microneedle structures and effectively avoids the problem of reference electrode potential drift and failure caused by uneven reaction or excessive corrosion in small areas. The generated Ag / AgCl layer and the Cu² loaded in the working electrode region... +The PEDOT:PSS composite system and the exposed PEDOT:PSS layer in the counter electrode region work together to form a stable, low-noise three-electrode electrochemical circuit, ensuring the accuracy and long-term stability of the potential reference under conditions of no in vitro pretreatment and minimally invasive insertion into the interstitial fluid, thereby achieving highly sensitive and repeatable in vivo quantitative detection of creatinine.

[0061] In a third typical embodiment of this application, an application is provided for the microneedle patch described above, or a microneedle patch prepared using the above preparation method, in the in vivo detection of creatinine content in interstitial fluid.

[0062] In the aforementioned applications, this patch is based on an integrated three-electrode structure and PEDOT:PSS / Cu 2+ The Nafion sensing interface allows direct contact with interstitial fluid via skin micro-puncture without any in vitro pretreatment or sample separation, utilizing creatinine's effect on Cu. + The complexation stabilizing effect generates a characteristic oxidation current signal in square wave voltammetry (SWV) scanning, enabling a quantitative response to creatinine. This application utilizes the minimally invasive structure of the microneedle (600 μm in length) for painless sampling, while avoiding the drawbacks of traditional methods such as susceptibility to enzymatic interference, nanomaterial detachment, and cumbersome sample pretreatment. The detection process is completed within 90 seconds, with a linear range covering 5.00–300 μM and a detection limit of 1.00 μM, accurately reflecting changes in creatinine concentration in the interstitial fluid of healthy individuals and patients with early-stage chronic kidney disease. The patch cost is less than 0.5 yuan per patch, exhibits good stability, and maintains a 97% response retention rate after two months of storage at room temperature. It can also be seamlessly integrated with portable electrochemical systems, allowing results to be read directly via a mobile phone, enabling users without professional training to perform long-term home monitoring and meeting the clinical needs for large-scale screening and continuous management of chronic kidney disease.

[0063] In a preferred embodiment, SWV is used for detection, with specific parameters including but not limited to the following: scan potential range of –0.3 V to +0.1 V, potential increment of 10–15 mV, amplitude of 20–30 mV, frequency of 0.5–2 Hz, and rest time of 20–40 s; wherein, the copper species immobilized on PEDOT:PSS and coated with Nafion are first reduced to CuO, and then, in the presence of creatine (Crt), the following electrochemical reaction occurs during the electrochemical scan:

[0064] Cu 0 + Crt – e - → Cu + –Crt.

[0065] In the absence of creatine, only Cu-induced oxidation occurs on the electrode surface. 2+– A PSS / Nafion-dominated redox process; the electrochemical potential of this process differs from that in the presence of creatinine, and Cu 2+ The binding strength with PSS and Nafion is also weaker than its binding strength with creatinine. When creatinine is present in the interstitial fluid, it forms a stable Cu with copper ions. + –Crt complex, therefore during detection, the initial potential of the scan is set at Cu 2+ Reduced to Cu 0 The potential is then scanned from low to high, and a highly selective oxidation current peak will appear in the range of -0.08 V to -0.12 V. This peak only appears in the presence of creatinine, and its peak current intensity is linearly positively correlated with the concentration of creatinine in the interstitial fluid, R²≥0.99, thus enabling in vivo quantitative detection without blood collection or pretreatment.

[0066] In a fourth typical embodiment of this application, a method for in vivo detection of creatinine content in interstitial fluid is provided. The method includes: (1) cleaning the sensing area of ​​the microneedle patch with deionized water or phosphate buffer solution with pH 7.0–7.4 to remove copper ions physically adsorbed on the surface; (2) connecting the cleaned microneedle patch to a portable electrochemical detection device and pressing it vertically onto the surface of the hair-removed skin so that the microneedles penetrate the stratum corneum and contact the interstitial fluid, maintaining the attachment time for not less than 10 seconds to ensure that the electrode is in full contact with the interstitial fluid; (3) scanning with square wave voltammetry, collecting and analyzing the differential current values ​​of characteristic peaks in the range of -0.08 ~ -0.12 V, and calculating the creatinine content based on the differential current values. Optionally, when using SWV for scanning, the scanning potential range is –0.3 V to +0.1 V, the potential increment is 4–15 mV, the amplitude is 15–50 mV, the frequency is 0.5–15 Hz, and the rest time is 0–100 s.

[0067] This application also provides a method for in vivo detection of creatinine in interstitial fluid. The method involves briefly cleaning the microneedle sensing area with deionized water or physiological buffer solution to effectively activate the electrode interface and remove physically adsorbed copper ions, ensuring the stability of the subsequent detection signal. The microneedle patch is vertically pressed onto the surface of the hair-removed skin and maintained for at least 10 seconds, allowing the microneedle to fully penetrate the stratum corneum and stably contact the interstitial fluid, achieving non-invasive sampling and real-time bio-environmental response. Scanning is performed using optimized SWV parameter ranges (potential increment 10–15 mV, amplitude 20–30 mV, frequency 0.5–2 Hz, rest time 20–40 s), which can effectively improve the detection of Cu. 0 Oxidized to Cu +The system leverages the resolution of the electrochemical process involving creatine complexes to reduce background noise interference. Combined with a pre-set linear calibration curve (k = 0.010–0.012 μA / μM, b = 1.0–1.5 μA), it enables rapid concentration calculation. The entire process requires no blood collection, centrifugation, or incubation, and quantitative detection can be completed in approximately 90 seconds. Furthermore, the detection results show good consistency with serum creatine concentrations, with a relative error of less than 5%, meeting the accuracy and convenience requirements for long-term home monitoring.

[0068] It should be noted that the detection of creatinine concentration or content in this application is only used for quantitative analysis of the electrochemical signal of creatinine molecules in interstitial fluid. Its purpose is to obtain objective physicochemical data reflecting the concentration level of this metabolite in biological fluids. It does not involve medical assessment of the subject's renal function, disease staging, clinical diagnostic decision-making, or treatment plan formulation. The sensing system and detection method constructed in this application do not introduce any medical diagnostic standards, clinical threshold determination rules, or disease-related algorithms, nor do they provide any conclusive opinions related to disease status. Its output results are only quantifiable current response values ​​and corresponding creatinine concentration values, which fall within the scope of physicochemical detection and do not constitute a "diagnosis or treatment method for disease".

[0069] The beneficial effects of this application will be explained in more detail below with reference to specific embodiments.

[0070] Example 1: Preparation of microneedle patches

[0071] Step 1: Fabrication of PC microneedle array

[0072] Place the Micropoint microneedle template in a horizontal rotor centrifuge (centrifuge speed limit 6000 rpm), and add 120-180 μL (preferably 150 μL) of 10-20 w / v% (preferably 15 w / v%) PC dichloromethane solution. Centrifuge at -10-10℃ (preferably -10℃) at 2500-6000 rpm (preferably 5900 rpm) for 5-45 minutes (preferably 25 minutes). Then place the template in a refrigerator at 4℃ or -20℃ (preferably -20℃) to air dry naturally before demolding to obtain the PC microneedle array.

[0073] Step 2: Fabrication of PC / PEDOT:PSS coated microneedle array

[0074] Preparation steps are as follows Figure 1The process includes: adding 20 μL of a 0.5-5 g / L (preferably 2 g / L) decafluoropentane solution of perfluoropolyether to a PC microneedle array, and then irradiating it under a UV / ozone lamp with a power of 5-30 W (preferably 15 W) for 6-24 hours (preferably 12 hours) to improve the hydrophilicity of PC. The PC microneedle array is then immersed in a 0.1-1.1 wt% (preferably 1.1 wt%) PEDOT:PSS solution (purchased from Sigma-Aldrich, high conductivity grade) for 0.5-5 minutes (preferably 2 minutes), and then dried at room temperature to -140 ℃ (preferably 120 ℃) ​​to obtain a PC / PEDOT:PSS coated microneedle array.

[0075] The fabrication principle in the assembly and testing process of the microneedle patch in step 2 is as follows:

[0076] A PC dichloromethane solution was dropped onto a solid microneedle template and centrifuged at -10 °C to prepare a PC solid microneedle array (MN) approximately 600 μm in length. The high rotation speed ensured the solution filled the tip of the template, giving the microneedles sufficient sharpness, while the low temperature prevented voids formed in the microneedles due to rapid dichloromethane evaporation, ensuring the strength of the microneedles. A perfluoropolyether decafluoropentane solution was then dropped onto the microneedle array. Under UV light and ozone, hydroxyl groups were introduced onto the PC surface to improve its hydrophilicity, facilitating subsequent modification of PEDOT:PSS. Subsequently, using this microneedle array as a substrate, [further details needed]. Figure 1 The method shown completes the construction of the sensing interface.

[0077] The PC microneedle array was immersed in a PEDOT:PSS solution, allowing the solution to be naturally adsorbed onto the surface of the microneedle array. After drying at 120°C to form a film, methanol was used to improve its conductivity, thus obtaining PC / PEDOT:PSS.

[0078] Step 3: PC / PEDOT:PSS Segmentation and Partitioning

[0079] The PC / PEDOT:PSS was immersed in methanol for 3-60 minutes (preferably 15 minutes), then removed and dried at room temperature to -140°C (preferably 120°C). The microneedle array was attached to the PC membrane, and a portion of the PEDOT:PSS on the PC surface was scraped off with a knife to divide it into three unconnected sections. Then, 20 μL of non-water-soluble nail polish was dropped onto the patch surface, and the mixture was centrifuged at 500-5000 rpm (preferably 2000 rpm) for 0.5-10 minutes (preferably 2 minutes) to seal the electrode substrate. This process was repeated 0-3 times (preferably once), and the mixture was dried at room temperature. The number of microneedles in the working electrode area was determined to be 1-30 (preferably 6 for in vitro testing, and 14 for in vivo testing), and the number of microneedles in both the counter electrode and reference electrode areas was 14. Next, the three areas were treated as follows:

[0080] Part 1 PC / PEDOT:PSS is used directly as the counter electrode portion of the microneedle patch.

[0081] Coat the second part with 20 μL of silver paste, centrifuge at 500-3000 rpm (preferably 2000 rpm) for 0.5-5 minutes (preferably 2 minutes), and then dry at 80-140 °C (preferably 120 °C). Add 20 μL of 0.01-0.5 M (preferably 0.1 M) FeCl3 to its surface, and chlorinate for 1-20 minutes (preferably 3 minutes) to obtain PC / PEDOT:PSS / Ag / AgCl, which is the reference electrode part of the microneedle patch.

[0082] In the third part, 20 μL of 1-1000 mM (preferably 25 mM) CuSO4 solution was added dropwise, incubated for 1-30 minutes (preferably 5 minutes), and then washed with deionized water to obtain PC / PEDOT:PSS / Cu 2+ Add 5 μL of a 0.1-2.0 wt% (preferably 0.5 wt%) Nafion ethanol solution, and dry at room temperature to obtain PC / PEDOT:PSS / Cu. 2+ / Nafion coated microneedle array, this part is the working electrode part of the microneedle patch.

[0083] Finally, silver conductive adhesive is used to lead out each part to connect to an electrochemical workstation or portable electrochemical detection system.

[0084] The specific preparation procedures in this application are shown in Experimental Examples 1 and 2 below. The patch prepared in Experimental Example 1 was used for the in vitro creatinine detection experiment described below; the patch prepared in Experimental Example 2 was used for the in vivo detection experiment described below. The characterization and in vitro detection data described below were obtained from the microneedle patch prepared in Experimental Example 1, and the in vivo detection data described below were obtained from the microneedle patch prepared in Experimental Example 2.

[0085] Example 2: Characterization, Testing, and Integration of Microneedle Patches

[0086] I. Characterization of the microneedle patch assembly process

[0087] The assembly of the subsequently coated microneedles was characterized using SEM and EDS. First, SEM showed that... Figure 2 PC / PEDOT:PSS with a height of 600 μm, a substrate width of 200 μm, and a pin spacing of 300 μm are neatly arranged, as shown in the EDS elemental distribution diagram (e.g.). Figure 3A and Figure 3BAs shown in the diagram, the signal distribution of the S element indicates that PEDOT:PSS is uniformly distributed in areas other than the microneedle tip. This gives the microneedle both high conductivity and sharpness, ensuring it can effectively penetrate the stratum corneum and contact the ISF in the dermis without causing pain. The weaker F element signal originates from the residue of perfluoropolyether. After incubation with CuSO4, the signal distribution of Cu element in the EDS elemental distribution diagram is consistent with that of S element, confirming that Cu... 2+ The microneedles were successfully immobilized on PEDOT:PSS. Finally, they were sealed with Nafion. The signal distribution of the F element matched the secondary electron image of the microneedles, indicating that Nafion was uniformly distributed on the microneedle surface. This effectively improves the anti-contamination performance and detection selectivity of the sensing interface. Furthermore, the microneedle array maintained high sharpness, with a tip diameter of less than 3 μm.

[0088] II. Feasibility Analysis of Microneedle Patches Penetrating the Skin

[0089] The feasibility of this microneedle array piercing the skin can be confirmed by the pressure-displacement curve test of the microneedles and the trypan blue staining experiment of Bama pig skin before and after microneedle insertion. Figure 4 ).

[0090] Studies have shown that the force required for microneedles to penetrate human skin is 0.08 N / needle, while the pressure-displacement curve of the microneedles indicates that the experimentally prepared microneedles can withstand a pressure greater than 0.16 N / needle without breaking. Figure 4 (A) This indicates that the microneedles prepared in the experiment possess sufficient mechanical strength to pierce human skin. Since Bama miniature pig skin has the closest physiological structure and biochemical properties to human skin, it was used to further investigate the feasibility of microneedles piercing human skin. Figure 4 As shown in Figure B, the initial Bama miniature pig skin structure is intact. After inserting and withdrawing the microneedle array into the skin and staining with trypan blue, it can be seen that each microneedle can form a closed insertion wound without damaging the surrounding skin. Figure 4 (C) This confirms that the microneedle array can effectively penetrate the skin. The microneedles, after removal, did not break but only bent slightly. Figure 4 (D), confirming its feasibility for use in electrochemical in vivo detection.

[0091] III. Integration of Microneedle Patches

[0092] To achieve the integration of the three-electrode system, according to... Figure 5 The process is segmented and partitioned as shown in Figure A, where the working electrode (WE) is composed of PC / PEDOT and PSS / Cu. 2+ / Nafion; Reference electrode (RE): PC / PEDOT:PSS / Ag / AgCl; Counter electrode (CE): PC / PEDOT:PSS. The microneedle patch was adhered to a PC film, and the various parts were brought out using Ag conductive adhesive to connect to the electrochemical workstation. A physical image of the microneedle patch is shown below. Figure 5 As shown in Figure B, its size is as small as 0.9 cm × 1.6 cm, and its weight is as light as 80 (±6) mg, making it easy to carry and use. Furthermore, the patch also exhibits good flexibility. This patch structure is used for the in vitro detection of creatinine.

[0093] When using patches for in vivo detection of creatinine in interstitial fluid, according to... Figure 5 The C-type connection is used to connect to the miniaturized electrochemical detection system HY-FlexiSens (Shenzhen, Haoyang Technology).

[0094] Example 3: Method for detecting creatinine

[0095] Step 1: Drawing the working curve

[0096] The prepared coated microneedle array was placed in PBS buffer, and its blank SWV curve was recorded. Then, standard creatine solutions of different concentrations were added to the solution sequentially, and after mixing, their SWV curves were recorded sequentially. The peak current value of the reoxidation peak near -0.1 V increased with increasing creatine concentration.

[0097] When the concentration of creatinine is 5.00-300 μM, the peak current value I of the modified electrode is... p There is a linear relationship between I and the concentration C of creatinine: p / μA = 0.0110 C / μM + 1.25, R 2 =0.998, which is the basis for quantitative detection of creatinine.

[0098] To evaluate the precision of microneedle patches in detecting creatinine, a working curve was established using three parallel microneedle patches (named the first microneedle patch, the second microneedle patch, and the third microneedle patch). Figure 6 (A in AF). Although there were some differences in the SWV peak current among the different patches, their linear range for detecting creatinine was 5.00 ~ 300 μM, and the lowest detection concentration was 1.00 μM. Based on the average response current of the three patches, the linear regression equation was calculated as: Ip / μA = 0.0110 C / μM + 1.25, R 2 =0.998 ( Figure 7Since the reference concentrations of creatinine in the serum of healthy adult men and women are 53-106 μM and 44-97 μM, respectively, the microneedle patches prepared in this experiment can well meet the needs of actual detection.

[0099] Step 2: Testing of actual samples

[0100] Step 2-1: In vitro detection of creatinine in human serum or rabbit serum

[0101] Before the experiment, a working curve for detecting creatinine using microneedle patches was established using three standard creatinine solutions. The sensing area of ​​the microneedle patch was rinsed in deionized water for 5-30 minutes (preferably 15 minutes). Then, the microneedle patch was placed in serum, and its SWV curve was immediately recorded. The peak current was then substituted into the established working curve to calculate the creatinine content in the sample.

[0102] If the sample is whole blood, it should be allowed to settle naturally first, and then centrifuged at 2500 rpm for 15 minutes. The supernatant is the serum.

[0103] The experimental results of human serum are as follows Figure 8 As shown in Table 1.

[0104] Table 1. In vitro detection results of creatinine in human serum (N=3)

[0105]

[0106] Three parallel microneedle patches were prepared to detect the same human serum sample, and the spiked recoveries were determined. The spiked concentrations were 30.0 μM and 90.0 μM, respectively. The measured values ​​of the three patches were similar, with an average of 65.7 μM, and the spiked recoveries ranged from 95.3% to 98.1%. Figure 8 (See Table 1). The concentration of creatinine in this serum was determined using the national standard isotope-labeled UPLC-MS method. Before the test, 800 μL of methanol was added to 200 μL of the sample to remove protein. To further eliminate interference from the blank matrix, the standard addition method was selected for quantification. Figure 8 (D). The concentration of creatinine was plotted against the ratio of the peak areas of creatinine and deuterated creatinine, and the data were linearly fitted to obtain the linear equation A. Crt / A d3-Crt = 0.0156 C / μM + 0.994, R 2 = 0.999, so the concentration of creatinine is 63.7 μM, which is very close to the measurement result of the patch, with a relative error of 3.1%. The above results indicate that the microneedle patch has good anti-interference ability and accuracy.

[0107] The experimental results of rabbit serum are as follows Figure 9 As shown

[0108] New Zealand rabbits (male, 3.3 kg) were used as an animal model to verify the ability of microneedle patches to detect creatinine in vivo. To compare with in vivo results, in vitro detection of creatinine in rabbit serum was first performed using the microneedle patches provided by this technology. To improve the accuracy of the detection, the patch was calibrated with three creatinine standard solutions before testing. Figure 9 (A) The working curve for detecting creatinine using this patch was obtained: Ip / μA = 0.0109 C / μM + 1.19, R 2 = 0.996 ( Figure 9 (B), the calculated detection result was 59.2 μM ( Figure 9 (C)

[0109] Step 2-2: In vivo detection of creatinine in rabbit interstitial fluid

[0110] The patch was connected to the portable electrochemical detection system HY-FlexiSens (Shenzhen, Haoyang Technology). A working curve was established using serum (with creatinine concentration determined by isotopic dilution UPLC-MS) and two serum-spiked samples. The sensing area of ​​the microneedle patch was rinsed in deionized water for 5-30 minutes (preferably 15 minutes). The sensing portion of the patch was pressed into the hair-removed rabbit ear skin by hand, and the test was started using a mobile phone. The SWV curve obtained after 90 seconds could be read on the phone. The peak current was then substituted into the working curve to calculate the creatinine content in the sample.

[0111] SWV parameters: potential increment of 12 mV, amplitude of 25 mV, frequency of 1 Hz, rest time of 30 s, and the signal used for quantification is differential current (Difference).

[0112] In vivo detection results of creatinine in rabbit interstitial fluid are as follows: Figure 10 and Figure 11 As shown.

[0113] To facilitate better adhesion of the microneedle patch to the rabbit ear, the number of microneedles used for sensing was increased to 14. To further simulate the physicochemical environment of the interstitial fluid, before in vivo detection, the microneedle patch was connected to an integrated electrochemical system. Then, a working curve for detecting serum creatinine using the microneedle patch was established using human serum with a known creatinine concentration: Ip / μA = 0.0540 C / μM + 5.99, R 2 = 0.994 ( Figure 10 (A and B in the middle). Then, press the microneedle patch onto the rabbit ear by hand (see diagram of microneedle patch as shown). Figure 10 (As shown in Figure C), an electrochemical scan was then performed immediately. The resulting electrochemical curve is shown in Figure C. Figure 10 As shown in Figure D, the peak shape is similar to but slightly different from that of the in vitro detection, which may be due to the bending deformation that occurred when the microneedles pierced the skin. Substituting the peak current value of the SWV peak into the calibration curve, the concentration of creatinine in the rabbit interstitial fluid was calculated to be 60.9 μM. This result is close to the in vitro detection result of serum mentioned above, and the detection time is only about 90 seconds, confirming that the microneedle patch can realize rapid in vivo detection of creatinine in interstitial fluid and has high application potential.

[0114] After the test was completed, the patch was removed, and micropores left on the surface of the rabbit ear due to the insertion of the microneedles could be observed, confirming the effective penetration of the microneedles. Figure 11 (A). Furthermore, nearly colorless interstitial fluid was observed flowing from the micropores, and no bleeding was observed, confirming that the microneedle patch did not puncture blood vessels, which is the basis for painless and minimally invasive testing. After 15 minutes, all the micropores closed. Figure 11 (B) This rapid closure confirms the minimally invasive nature of the microneedle patch, which can effectively reduce the risk of infection caused by microbial invasion.

[0115] Example 4 Stability Assessment

[0116] After the prepared microneedle patch was stored at room temperature for 2 months (at an ambient humidity of approximately 30%), 97% of its response value to 75.0 μM creatinine was retained. Figure 12 This indicates that the microneedle patch provided by this technology has excellent stability, which is very beneficial for its practical application.

[0117] Experimental Example 1

[0118] Step 1: Fabrication of PC microneedle array

[0119] A solid microneedle template (Micropoint, Singapore) was placed in a horizontal rotor centrifuge, and 150 μL of 15w / v% PC dichloromethane solution was added. After centrifugation at 5900 rpm for 25 minutes at -5 ℃, the template was placed in a -20 ℃ freezer to air dry naturally before demolding to obtain the PC microneedle array.

[0120] Step 2: Fabrication of PC / PEDOT:PSS coated microneedle array

[0121] 20 μL of a 2 g / L perfluoropolyether solution in decafluoropentane was dropped onto a PC microneedle array, and then the array was irradiated under a 15W UV / ozone lamp for 12 hours to improve the hydrophilicity of the PC. The PC microneedle array was then immersed in a 1.1 wt% PEDOT:PSS solution for 2 minutes, removed, and dried at 120 °C to obtain PC / PEDOT:PSS. The electrode was then immersed in MeOH for 15 minutes and dried at 120 °C to improve the conductivity of the PEDOT:PSS.

[0122] Step 3: Integration of the three-electrode system

[0123] Using a knife, scrape off a portion of the PEDOT:PSS from the PC surface to divide it into three unconnected parts. Add approximately 20 μL of non-water-soluble nail polish to the electrode surface, centrifuge at 2000 rpm for 2 minutes to seal the electrode substrate, repeat once, and dry at room temperature. Determine the number of microneedles for the working electrode area to be 6, and the number of microneedles for the counter and reference electrode areas to be 14 each. Insulate the remaining microneedles with nail polish and dry at room temperature. The first part is the working electrode. Add 20 μL of 25 mM CuSO4 solution to the electrode surface, incubate for 5 minutes, and then wash with deionized water to obtain PC / PEDOT:PSS / Cu 2+ Add 5 μL of 0.5 wt% Nafion dropwise, and dry at room temperature to obtain PC / PEDOT:PSS / Cu. 2+ / Nafion coated microneedle array. A suitable amount of silver paste was coated onto the second part, centrifuged at 2000 rpm for 2 minutes, and then dried at 120 °C for 10 minutes. 20 μL of 0.1 M FeCl3 was added to its surface, and after chlorination for 3 minutes, a PC / PEDOT:PSS / Ag / AgCl reference electrode was obtained. The third part was PC / PEDOT:PSS, which was used directly as the counter electrode. The microneedle array was attached to a PC film, and each part was led out using silver conductive adhesive to connect to an electrochemical workstation.

[0124] Step 4: Electrochemical detection of creatinine

[0125] The prepared coated microneedle array was placed in PBS buffer, and its blank SWV curve was recorded using an electrochemical workstation CHI660E (Shanghai, Chenhua). Then, standard creatinine solutions of different concentrations were added sequentially, mixed, and their SWV curves were recorded. The peak current value of the oxidation peak near -0.1 V increased with increasing creatinine concentration. The peak current value I... pA linear fit was performed on the creatinine concentration C to obtain the working curve. The parameters of the SWV are as follows: potential increment of 12 mV, amplitude of 25 mV, frequency of 1 Hz, rest time of 30 s, and the signal used for quantification is differential current (Difference).

[0126] Step 5: In vitro detection of creatinine

[0127] The sensing area of ​​the microneedle patch was placed in human serum, and the SWV curve was immediately recorded and the peak current value was read. Substituting the peak current value into the working curve, the creatinine content in the serum sample was obtained.

[0128] Experiment Example 2

[0129] Step 1: Fabrication of PC microneedle array

[0130] A solid microneedle template (Micropoint, Singapore) was placed in a horizontal rotor centrifuge, and 150 μL of 15w / v% PC dichloromethane solution was added. After centrifugation at 5900 rpm for 25 minutes at -5 ℃, the template was placed in a -20 ℃ freezer to air dry naturally before demolding to obtain the PC microneedle array.

[0131] Step 2: Fabrication of PC / PEDOT:PSS coated microneedle array

[0132] 20 μL of a 2 g / L perfluoropolyether solution in decafluoropentane was dropped onto a PC microneedle array, and then the array was irradiated under a 15W UV / ozone lamp for 12 hours to improve the hydrophilicity of the PC. The PC microneedle array was then immersed in a 1.1 wt% PEDOT:PSS solution for 2 minutes, removed, and dried at 120 °C to obtain PC / PEDOT:PSS. The electrode was then immersed in MeOH for 15 minutes and dried at 120 °C to improve the conductivity of the PEDOT:PSS.

[0133] Step 3: Integration of the three-electrode system

[0134] Using a knife, scrape off a portion of the PEDOT:PSS from the PC surface to divide it into three unconnected parts. Add approximately 20 μL of non-water-soluble nail polish to the electrode surface, centrifuge at 2000 rpm for 2 minutes to seal the electrode substrate, repeat once, and dry at room temperature. Determine the number of microneedles used for sensing to be 14; the remaining microneedles are insulated with nail polish and dried at room temperature. The first part is the working electrode. Add 20 μL of 25 mM CuSO4 solution to the electrode surface, incubate for 5 minutes, and then wash with deionized water to obtain PC / PEDOT:PSS / Cu 2+Add 5 μL of 0.5 wt% Nafion dropwise, and dry at room temperature to obtain PC / PEDOT:PSS / Cu. 2+ / Nafion coated microneedle array. A suitable amount of silver paste was coated onto the second part, centrifuged at 2000 rpm for 2 minutes, and then dried at 120 °C for 10 minutes. 20 μL of 0.1 M FeCl3 was added to its surface, and after chlorination for 3 minutes, a PC / PEDOT:PSS / Ag / AgCl reference electrode was obtained. The third part was PC / PEDOT:PSS, which was used directly as the counter electrode. The microneedle array was attached to a PC film, and each part was led out using silver conductive adhesive to connect to a miniaturized electrochemical detection system.

[0135] Step 4: Establishing the working curve

[0136] The microneedle patch was connected to the portable miniaturized electrochemical detection system HY-FlexiSens (Shenzhen, Haoyang Technology). 40 μL of serum sample with a known creatinine concentration was dropped onto the patch surface. Then, the "Start" button was clicked in the mobile operating software to record the SWV curve. The same method was then used to record the SWV curves of the microneedle patch in two serum-standardized samples (30.0 μM and 60.0 μM creatinine, respectively). The peak current value of the oxidation peak near -0.1 V increased with increasing creatinine concentration. The peak current value I... p A linear fit was performed on the creatinine concentration C to obtain the working curve.

[0137] Step 5: In vivo detection of creatinine in rabbit interstitial fluid

[0138] Press the sensing area of ​​the patch into the rabbit's ear after hair removal, then tap "Start" in the mobile app to record the SWV curve. Read the peak current value from the obtained SWV curve and substitute it into the working curve to calculate the concentration of creatinine in the rabbit's interstitial fluid.

[0139] The parameters of the SWV are as follows: potential increment is 12 mV, amplitude is 25 mV, frequency is 1 Hz, rest time is 30 s, and the signal used for quantification is differential current (Difference).

[0140] As can be seen from the above description, the embodiments of the present invention achieve the following technical effects:

[0141] 1. Excellent in vivo detection capability. The integrated microneedle patch enables in vivo detection of creatinine in the interstitial fluid of laboratory rabbits, eliminating the need for complex in vitro procedures and taking only about 90 seconds. The relative error between the measured value and the in vitro measured value of creatinine in serum is less than 5%, meeting the requirements for in vivo detection;

[0142] 2. High sensitivity and wide linear range. The linear range for quantitative detection of creatinine is 5.00-300 μM, with a minimum detectable concentration of 1.00 μM. Since the creatinine content in the interstitial fluid of healthy individuals is 44-106 μM, and the creatinine content in early CKD patients can be increased by about 30%, this patch can well meet the monitoring needs of CKD.

[0143] 3. High anti-interference ability and accuracy. In addition to in vivo detection of creatinine in the interstitial fluid of rabbits, this microneedle patch can also realize the in vitro detection of creatinine in human serum, with a relative error of 3.1% and a spiked recovery rate between 95.3% and 98.1% (N=3).

[0144] 4. Extremely low cost. The construction cost of a microneedle patch is less than 0.5 yuan per patch. Furthermore, due to the use of electrochemical detection methods, the cost of a fully integrated patch can also be effectively reduced.

[0145] 5. Minimally invasive and painless. The 600 μm long microneedles can contact interstitial fluid without damaging blood vessels and nerves, enabling painless detection. Furthermore, the needle punctures formed during the test can completely heal within 15 minutes (results from experimental rabbits), effectively preventing infection.

[0146] 6. High stability. After the microneedle patch was stored at room temperature for 2 months, 97% of the response values ​​were retained.

[0147] In summary, in this application, the conductive layer coated on the surface of the microneedle array is made of copper ions (Cu). 2+ This provides stable coordination binding sites for Cu. 2+ Achieving efficient and robust surface loading without relying on exogenous fixatives or chemical modifications; when the microneedle patch contacts the interstitial fluid, Cu 2+ It is reduced in situ to Cu during the electrochemical scanning process. 0 In subsequent testing, Cu 0 It can produce independent and identifiable oxidation to Cu + The current peak of the creatine complex enables a direct electrochemical response to creatine. Simultaneously, the perfluorosulfonic acid resin membrane covering the microneedle surface selectively blocks large molecular interferences (such as proteins, uric acid, etc.) and anions, allowing only cations and neutral small molecules such as creatine to permeate freely. This ensures the specificity and stability of the detection signal without relying on blood sampling, enzymatic reactions, or in vitro pretreatment. This synergistic effect of the structure enables the microneedle patch to detect creatine in interstitial fluid in a minimally invasive, painless, real-time, and highly selective manner. It breaks through the dependence of traditional biochemical detection on sample pretreatment and invasive sampling, providing a new, high-precision, low-interference, wearable in vivo analysis approach for continuous monitoring of renal function.

[0148] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. 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. A microneedle patch for in vivo detection of creatinine in interstitial fluid, characterized in that, The microneedle patch is a three-electrode system, which includes a working electrode, a reference electrode, and a counter electrode. The working electrode, the reference electrode, and the counter electrode each contain a microneedle array and a conductive polymer coated on the surface of the microneedle array. The working electrode further includes: copper ions fixed on the surface of the conductive polymer through coordination, and perfluorosulfonic acid resin coated on the surface of the conductive polymer and the copper ions; The reference electrode further includes: a reference metal and a reference metal halide coated on the surface of the conductive polymer; The microneedle patch has the following function: after contacting the interstitial fluid, through electrochemical scanning, Cu generated in situ on the electrode surface... 0 Oxidized to Cu + - Creatine anhydride complexes form independent oxidation current peaks, enabling in vivo quantitative detection of creatine anhydride without in vitro pretreatment.

2. The microneedle patch according to claim 1, characterized in that, The conductive polymer includes poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonic acid).

3. The microneedle patch according to claim 1, characterized in that, The perfluorosulfonic acid resin includes Nafion.

4. The microneedle patch according to claim 1, characterized in that, The substrate of the microneedle array comprises polycarbonate.

5. The microneedle patch according to any one of claims 1-4, characterized in that, The reference metal includes Ag, and the reference metal halide includes AgCl.

6. A method for preparing a microneedle patch for in vivo detection of creatinine in interstitial fluid as described in any one of claims 1-5, characterized in that, The preparation method includes: a) Provide the microneedle array; b) Coating the conductive polymer onto the surface of the microneedle array to form a conductive layer; dividing the conductive layer into a working electrode region, a reference electrode region, and a counter electrode region; c) Loading the copper ions in the working electrode region and achieving chemical fixation of the copper ions through coordination with the conductive polymer; further, covering the surface of the working electrode region with the perfluorosulfonic acid resin; d) A reference metal is deposited and coated in the reference electrode region, and then subjected to a halogenation treatment to form a reference metal halide; e) The exposed conductive polymer is retained in the counter electrode region; f) The electrode substrate is sealed with a non-water-soluble insulating material, and three electrode wires are led out to form a complete three-electrode system, thereby obtaining the microneedle patch.

7. The preparation method according to claim 6, characterized in that, The microneedle array is based on polycarbonate. The conductive polymer includes poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonic acid). The d) includes: coating the reference electrode region with metallic silver and subjecting it to chlorination to form the reference electrode region containing Ag and AgCl.

8. The preparation method according to claim 7, characterized in that, The coating of poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonic acid) comprises: immersing the microneedle array in a PEDOT:PSS aqueous solution and then drying it to form a film.

9. The preparation method according to claim 6, characterized in that, The loading of copper ions includes: immersing the working electrode region in a salt solution containing copper ions for incubation, followed by washing with deionized water and drying.

10. The preparation method according to claim 6, characterized in that, The perfluorosulfonic acid resin coating includes: applying a solution of the perfluorosulfonic acid resin to the working electrode area, allowing it to stand, and then drying it.

11. The preparation method according to claim 7, characterized in that, The preparation of the reference electrode region containing Ag and AgCl includes: coating the reference electrode region with silver paste, drying it, and then immersing it in FeCl3 solution for chlorination to form the reference electrode region containing Ag and AgCl.

12. The use of the microneedle patch according to any one of claims 1-5, or the microneedle patch prepared by the preparation method according to any one of claims 6-10, in in vivo detection of creatinine content in interstitial fluid.

13. A method for in vivo detection of creatinine content in interstitial fluid, characterized in that, The method includes: (1) The sensing area of ​​the microneedle patch according to any one of claims 1-5, or the microneedle patch prepared by any one of claims 6-10, is cleaned with deionized water or phosphate buffer to remove copper ions physically adsorbed on the surface. (2) After the cleaned microneedle patch is connected to the electrochemical detection equipment, it is pressed vertically onto the surface of the skin to be de-haired, so that the microneedle penetrates the stratum corneum and comes into contact with the interstitial fluid. The patch is maintained for a certain period of time so that the electrode comes into contact with the interstitial fluid. (3) The square wave voltammetry method is used to scan, collect and analyze the current values ​​of characteristic peaks in the range of -0.08 ~ -0.12 V, and calculate the creatinine content based on the differential current values.

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