Microneedle array electrochemical sensor for monitoring sepsis marker C-reactive protein in skin interstitial fluid

Abstract

The invention discloses a microneedle array electrochemical sensor for monitoring a sepsis marker C-reactive protein in skin interstitial fluid. According to the novel-arrangement cactus-like structure microneedle array electrode, the enrichment efficiency of detection liquid is remarkably improved through the design that the structure sequentially extends from the center to the periphery, and good attachment to skin tissue is achieved in combination with a flexible substrate material. Electrode interface construction of the electrochemical sensor is based on electrochemical deposition, a C-reactive protein aptamer is taken as a recognition object, a highly uniform nucleic acid C-reactive protein aptamer probe single layer is formed by combining a freezing-unfreezing method self-assembly technology, and the nucleic acid C-reactive protein aptamer probe single layer has a good linear relationship with a detection electric signal in a C-reactive protein concentration range of 0.5-100 ng / mL; the kit has the properties of high sensitivity, high specificity, rapid response, real-time monitoring, minimally invasive property and the like, is beneficial to rapid detection of C-reactive protein and dynamic monitoring of sepsis course change, and also provides technical support for development of biosensors for biomarker detection in complex media.

Description

Technical Field

[0001] The present invention relates to the technical fields of microneedles and electrochemical sensors, and particularly relates to a microneedle array electrochemical sensor for monitoring C-reactive protein, a sepsis biomarker, in interstitial fluid of the skin. Background Art

[0002] Sepsis is a clinical syndrome caused by a dysregulated systemic response to infection, leading to life-threatening organ dysfunction, and is a major cause of morbidity and mortality in critically ill patients. The condition of sepsis progresses rapidly and may rapidly develop into septic shock in the short term, triggering abnormal circulation, cells, and metabolism in the body, resulting in a significantly increased fatality rate. Currently, the diagnosis of sepsis mainly relies on the Sequential Organ Failure Assessment (SOFA) and the Quick Sequential Organ Failure Score (qSOFA) of the Sepsis 3.0 diagnostic criteria. However, these diagnostic criteria rely on retrospective studies and need to be continuously verified through clinical practice, resulting in a lag in the diagnosis and treatment of sepsis. In addition, the treatment cycle of sepsis is long and costly, significantly occupying medical resources, accounting for more than 40% of the Intensive Care Unit (ICU), causing a huge economic burden on patients, their families, and society. At the same time, the duration of use of antibacterial drugs is closely related to the mortality rate of septic shock. Patients with septic shock should be immediately medicated within one hour after diagnosis and continuously monitored dynamically. Before introducing expensive intervention measures, basic monitoring standards must be implemented, otherwise it may affect their effectiveness. Therefore, it is urgent to monitor sepsis, detect and intervene in a timely manner to prevent the deterioration of the condition, evaluate the severity of the condition with the help of monitoring results, effectively provide drug treatment, reduce organ damage, improve the survival rate, improve the long-term prognosis of patients, adjust the plan in a timely manner according to the disease progression, and at the same time optimize the utilization of resources and improve the quality of medical care.

[0003] The occurrence of sepsis begins with infection. After infection, components such as pathogens and their toxins enter the body, activating the body's inflammatory response cells, producing and releasing a large number of inflammatory mediators, and causing local or systemic inflammatory responses. These inflammatory mediators form a cascade effect and cytokine storm through a positive feedback mechanism, expanding the body's inflammatory response and ultimately leading to out-of-control systemic inflammatory response. During this pathological process, C-reactive protein, as an acute-phase reaction protein synthesized by the liver, has a sharp increase in plasma levels during infection or tissue injury. It not only has the functions of activating complement and activating monocytes and phagocytes, but also participates in the inflammatory response. As a systemic inflammatory response syndrome, C-reactive protein has high sensitivity in the early diagnosis of sepsis. The increase in C-reactive protein reflects the degree of the body's inflammatory response to infection. In the early stage of the disease and in the intensive care unit, C-reactive protein is a sensitive marker for differentiating sepsis from non-sepsis. At the same time, studies have found that the C-reactive protein level in sepsis patients is positively correlated with the severity of their condition. The C-reactive protein levels in patients with severe sepsis and septic shock are usually higher than those in patients with common sepsis.

[0004] The composition of interstitial fluid is similar to that of blood and is considered a potential substitute for blood for biomarker detection and disease monitoring. Some studies have shown that C-reactive protein exists in blister fluid, and blister fluid is obtained by extracting interstitial fluid from the skin, so CRP also exists in interstitial fluid. The C-reactive protein in the plasma of healthy humans is generally in the range of 1-10 μg / mL. When the C-reactive protein level in human plasma is greater than 10 μg / mL, it indicates a risk of high inflammation or sepsis. However, the protein concentration in interstitial fluid is usually lower than that in blood because interstitial fluid can be regarded as an ultrafiltrate of plasma, and the passage of macromolecules such as proteins is restricted. Recent proteomic studies have shown that the levels of many key analytes in sweat are highly correlated with those in interstitial fluid. C-reactive protein exists in sweat, and the C-reactive protein level in sweat is at the picomolar level. At the same time, the C-reactive protein level in blister fluid is also at the picomolar level. Therefore, interstitial fluid can detect C-reactive protein, and the C-reactive protein level should be at the picomolar level. It is necessary to explore a detection technique that can meet the monitoring of low-concentration C-reactive protein.

[0005] Aptamers are DNA or RNA molecules obtained through in vitro selection that can specifically bind to target molecules (such as proteins). They possess high affinity and specificity, similar to antibodies, but have advantages such as smaller molecular weight, better stability, and ease of modification. The C-reactive protein aptamer can form stable Au-S bonds with the gold electrode surface through thiolation modification. This immobilization method ensures the stability and biological activity of the C-reactive protein aptamer on the electrode surface. When the target protein is present, it will specifically bind to the C-reactive protein aptamer on the electrode surface, and this binding causes a conformational change in the C-reactive protein aptamer. The C-reactive protein aptamer can form diverse secondary and tertiary structures through the folding and structural changes of its sequence, which determines the binding characteristics and affinity of the C-reactive protein aptamer for the target molecule, enabling the C-reactive protein aptamer to precisely capture the target molecule in complex biological samples, thereby improving the sensitivity and specificity of the sensor.

[0006] The key step in the assembly process of the C-reactive protein aptamer sensor is the immobilization of the C-reactive protein aptamer. The C-reactive protein aptamer is vulnerable to desorption over time and changes in storage conditions, which can affect electrochemical measurements. In traditional methods, DNA molecules form a molecular recognition layer at the liquid-solid interface through chemical reactions or adsorption. However, functional DNA is usually in a single-stranded structure, and non-specific adsorption is prone to occur during the modification process, making it difficult to control the DNA conformation at the interface and further affecting the sensing performance. The C-reactive protein aptamer, as a type of DNA, has been verified to be stretchable and aligned through freezing, allowing for faster Au-S ligation using nanogold. Freezing droplets containing DNA on the gold surface can form a uniform DNA monolayer with a perpendicular upright and laterally separated conformation. Through the thawing process, the DNA molecules form a highly uniform monolayer on the gold surface, significantly improving the stability and target recognition ability of the DNA. Compared with the DNA surface obtained by traditional methods, the freezing-induced surface is very stable in electrochemical detection and long-term storage. The uniform conformation increases the binding affinity of the DNA by more than 40 times. In addition, the microneedles have a large specific surface area, which can provide more binding sites for probes, thereby improving the detection sensitivity.

[0007] Therefore, the present invention provides a microneedle array electrochemical sensor for monitoring C-reactive protein, a sepsis marker, in interstitial fluid of the skin. A microneedle array female mold that is designed to be cactus-like arranged to effectively enrich the detection liquid is used. The conductive nanocomposite is solidified and formed through the microneedle mold and further wire-connected and encapsulated to obtain a microneedle array electrode. Methylene blue is used as the intrinsic electrochemical signal. The C-reactive protein aptamer is fixed on the surface of the electrode modified with gold nanoparticles by the freeze-thaw method to construct a microneedle C-reactive protein aptamer electrochemical sensor for monitoring C-reactive protein, a sepsis marker, in interstitial fluid of the skin, which has potential application value in the rapid diagnosis of sepsis and the real-time monitoring of the disease condition. Summary of the Invention

[0008] 1. The object of the present invention is to provide a microneedle array electrochemical sensor for monitoring C-reactive protein, a sepsis marker, in interstitial fluid of the skin.

[0009] 2. The microneedle array electrochemical sensor for monitoring C-reactive protein, a sepsis marker, in interstitial fluid of the skin according to the present invention uses a resin composite material as the electrode substrate, which has good mechanical properties and biocompatibility, avoiding problems such as fracture and infection that occur after the microneedles are inserted into the skin; and the operation is simple, batch production can be achieved, and the cost can be effectively reduced.

[0010] 3. The microneedle array electrochemical sensor for monitoring C-reactive protein, a sepsis marker, in interstitial fluid of the skin according to the present invention improves the array arrangement and substrate of the microneedles, making it have better skin puncture ability and flexibility to fit the skin. The microneedles are arranged in a cactus-like manner, growing orderly from the center to the periphery, effectively enriching the detection liquid, and thus obtaining higher sensing performance.

[0011] 4. The microneedle array electrochemical sensor for monitoring C-reactive protein, a sepsis marker, in interstitial fluid of the skin according to the present invention uses methylene blue as the intrinsic electrochemical signal. The C-reactive protein aptamer is fixed on the surface of the electrode modified with gold nanoparticles by the freeze-thaw method to form a highly uniform DNA monolayer, significantly improving the stability and target recognition ability of DNA, and enabling the direct monitoring of C-reactive protein in interstitial fluid of the skin.

[0012] 5. The manufacturing process of the microneedle array electrochemical sensor for monitoring C-reactive protein, a sepsis marker, in interstitial fluid of the skin according to the present invention sequentially includes the following steps:

[0013] (1) Using epoxy resin as the matrix, dichloromethane as the solvent, compounding multi-walled carbon nanotubes, adding an epoxy resin curing agent after uniform dispersion to obtain a conductive material mixture;

[0014] (2) Fill the conductive material mixture into the female mold of the microneedle array electrode made of polydimethylsiloxane, and cure it to obtain the microneedle array. A total of 3 microneedle arrays are made;

[0015] (3) The bases of the 3 microneedle arrays are energized through wires respectively. The bases for receiving the 3 microneedle arrays are obtained by curing the filled resin, and the wire arrangement is encapsulated and demolded to obtain the microneedle array electrode;

[0016] (4) Coat the photocurable resin on the area of the microneedle array electrode except for the tips of the microneedles. The exposed parts of the wires are insulated by the photocurable resin through the polyvinyl chloride hose, and insulation treatment is carried out;

[0017] (5) Pretreat the microneedle array electrode, including steps of washing with dilute sulfuric acid, washing with water, and drying with nitrogen, to obtain the pretreated microneedle array electrode (MAE);

[0018] (6) Deposit nano-gold on the surface of the pretreated microneedle array electrode MAE obtained in step (5) by electro-deposition method to prepare the nano-gold / microneedle array electrode AuNPs / MAE;

[0019] (7) First, reduce the C-reactive protein aptamer with tris(2-carboxyethyl)phosphine, and then assemble the C-reactive protein aptamer with MB signal onto the surface of the nano-gold / microneedle array electrode AuNPs / MAE prepared in step (6) through gold-sulfur bonds to obtain the C-reactive protein aptamer / nano-gold / microneedle array electrode CRP-Apt / AuNPs / MAE;

[0020] (8) Drop 6-mercaptohexanol on the surface of the C-reactive protein aptamer / nano-gold / microneedle array electrode CRP-Apt / AuNPs / MAE prepared in step (7) to obtain the 6-mercaptohexanol / C-reactive protein aptamer / nano-gold / microneedle array electrode MCH / CRP-Apt / AuNPs / MAE;

[0021] (9) Place this electrode MCH / CRP-Apt / AuNPs / MAE in the interstitial fluid containing C-reactive protein, and perform electrochemical detection by square wave pulse voltammetry. Calculate the concentration of C-reactive protein in the sample through the change amount of the electrical signal.

[0022] Specifically, a microneedle array electrochemical sensor for monitoring C-reactive protein, a sepsis marker in skin interstitial fluid, according to the present invention is characterized by including the following specific steps:

[0023] (1) Design of the microneedle female mold: The design drawing of the microneedle female mold includes a multi-array microneedle module, an array microneedle base, and a total substrate module. Specifically, a cylindrical substrate with a diameter of 20 mm and a thickness of 2 mm is used as the total substrate module; three array microneedle bases (with a diameter of 5 mm and a thickness of 1.5 mm), with a spacing of 3.66 mm between the bases; a conical microneedle array with a needle length of 800 μm, a needle base diameter of 320 μm, and a tip-to-tip distance of 800 μm, which is composed of a cactus-like arrangement with 8 microneedles in each of the inner and outer circles centered on 1 microneedle, is used as the multi-array microneedle module;

[0024] (2) Preparation of the conductive material mixture: Using epoxy resin E51 as the base, multi-walled carbon nanotubes (≥95%, inner diameter: 5 - 12 nm, outer diameter: 30 - 50 nm, length: 10 - 20 μm) are compounded. The mass concentration of the multi-walled carbon nanotubes is 15 wt%. Dichloromethane is used as the solvent, and ultrasonic oscillation is carried out at a power of more than 95% for 30 min while keeping the temperature below 37 °C to ensure that all the multi-walled carbon nanotubes in the material are dissolved and dispersed evenly. Based on this, an epoxy resin curing agent W93 is added, with a mass ratio to the epoxy resin of 1:2, and vortex mixing is carried out thoroughly to obtain the conductive material mixture;

[0025] (3) Preparation of the conductive microneedles: The conductive material mixture is added to the 3 arrays of the female mold of the microneedles made of polydimethylsiloxane in 3 portions with a volume of 30 μL each to ensure that there are no bubbles in the liquid in the mold. Then the mold is transferred to a 50 mL centrifuge tube (with a wooden column at the bottom), the mold cover is covered, and they are balanced in pairs and centrifuged at a speed of 4000 rpm for 10 min. The mold is taken out, and the conductive material mixture is added again until it is flush with the top of the grooves of the microneedle female mold, and it is cured at room temperature for 2 h and then at 40 °C for 1 h to obtain the array conductive microneedles;

[0026] (4) Encapsulation of the microneedle array electrode: The photocurable resin is coated with a watercolor pen on the 3 bases with conductive microneedles. The silver wire is bent into an arc shape to increase the contact surface and is connected to the base with the conductive microneedles through the photocurable resin, and it is cured under a 405 nm ultraviolet lamp for 30 s, which is carried out in three times to ensure that the silver wire is fixed to the base. Finally, a waterborne polyurethane with a solid content of 60 wt% is poured onto the top layer of the mold and cured overnight at 40 °C to obtain a flexible substrate connecting the 3 array electrode bases. The flexible substrate is the total substrate module, and after demolding, the microneedle array electrode (MAE) is obtained;

[0027] (5) Electrode insulation treatment; The area of the microneedle array electrode except for the tips of the microneedles is coated with the photocurable resin and cured under a 405 nm ultraviolet lamp for 60 s to form an insulating layer. The exposed part of the silver wire connected to the bottom of the microneedles is covered with a polyvinyl chloride hose and cured under a 405 nm ultraviolet lamp with the photocurable resin for 30 s for insulation treatment;

[0028] (6) Pretreatment of the micro-needle array electrode (MAE): The micro-needle array electrode was placed in a 0.5 mol / L H 2 SO 4 solution and scanned under cyclic voltammetry. The scanning range was 0 - 1.5 V, the scanning speed was 1 V / s, and the number of scanning cycles was 30. Then the electrode was washed with ultrapure water and dried with nitrogen to obtain the pretreated micro-needle array electrode MAE for standby;

[0029] (7) Deposition of gold nanoparticles (AuNPs): The micro-needle array electrode MAE was placed in a 2.8 mmol / L chloroauric acid solution, and the electrodeposition of gold nanoparticles AuNPs was carried out by the chronoamperometry method. The deposition potential was -0.4 V, and the time was 500 s. After washing with ultrapure water and N 2 drying, the gold nanoparticle / micro-needle array electrode AuNPs / MAEE was prepared for standby;

[0030] (8) Assembly of C-reactive protein aptamer: 10 μL of a 1 μmol / L C-reactive protein aptamer solution pre-reacted for 1 h was dropped onto the surface of the gold nanoparticle / micro-needle array electrode AuNPs / MAE, and incubated at -20 °C for 15 min. After thawing for 2 min, it was washed with 20.0 mmol / L Tris-HCl buffer and dried with nitrogen to obtain the C-reactive protein aptamer / gold nanoparticle / micro-needle array electrode CRP-Apt / AuNPs / MAE for standby; The above C-reactive protein aptamer solution was diluted with 20.0 mmol / L Tris-HCl buffer after being reduced by a 1:1 volume ratio of 100 μmol / L C-reactive protein aptamer stock solution and 100 mmol / L tris(2-carboxyethyl)phosphine;

[0031] (9) Blocking with 6-mercaptohexanol: 10 μL of a 1 mmol / L 6-mercaptohexanol solution was dropped onto the surface of the C-reactive protein aptamer / gold nanoparticle / micro-needle array electrode CRP-Apt / AuNPs / MAE, incubated at room temperature for 30 min, washed with 20.0 mmol / L Tris-HCl buffer, and dried with nitrogen to obtain the 6-mercaptohexanol / C-reactive protein aptamer / gold nanoparticle / micro-needle array electrode MCH / CRP-Apt / AuNPs / MAE;

[0032] (10) The above 6-mercaptohexanol / C-reactive protein aptamer / gold nanoparticle / micro-needle array electrode MCH / CRP-Apt / AuNPs / MAE was placed in 1 mL of a test solution containing C-reactive protein at 10 ng / mL, incubated at room temperature for 30 min, and directly subjected to square wave pulse voltammetry electrochemical detection to record the change in the electrical signal. The scanning voltage was -0.4 V to 0 V.

[0033] Furthermore, a microneedle array electrochemical sensor for monitoring C-reactive protein, a sepsis marker in interstitial fluid, is prepared by the above preparation method of the present invention.

[0034] Specifically, a microneedle array electrochemical sensor for monitoring C-reactive protein, a sepsis marker in interstitial fluid, of the present invention; includes a microneedle array electrode, and the microneedle array electrode is prepared by the following method, which is characterized in that it includes the following steps: (1) Design of the female mold of the microneedles: Design a multi-array microneedle module, an array microneedle base and a total substrate module. There are three array microneedle bases on the total substrate module, and each array microneedle base has a multi-array microneedle module. The multi-array microneedle module is composed of a plurality of microneedles arranged in an array form; (2) To meet the electrochemical performance of the microneedles, prepare a conductive material mixture of epoxy resin as the base and multi-walled carbon nanotubes; (3) Use the centrifugation method to transfer the conductive material mixture into the female mold of the microneedles and cure and form; (4) Connect each array microneedle base with a silver wire to conduct electricity, and finally pour water-based polyurethane on the top layer of the female mold of the microneedles to obtain a microneedle three-electrode connected by a flexible backing. The microneedle three-electrode is the working electrode, the reference electrode, and the counter electrode. Coating the area other than the microneedle tip of the microneedle three-electrode with a photocurable resin for the final insulation operation to obtain a microneedle array electrode.

[0035] The method for detecting the concentration of C-reactive protein of a microneedle array electrochemical sensor for monitoring C-reactive protein, a sepsis marker in interstitial fluid, of the present invention is characterized in that it includes the following steps: (1) Use the electrochemical deposition method to prepare gold nanoparticles on the surface of the microneedle array electrode. The C-reactive protein aptamer with the intrinsic signal electrochemical signal methylene blue and mercapto forms a gold-sulfur bond with the gold nanoparticles through the reduction of tris(2-carboxyethyl)phosphine; (2) Use the freeze-thaw method to form a highly uniform nucleic acid monolayer on the surface of the gold nanoparticles, significantly improving the stability and target recognition ability of the nucleic acid; (3) Modify 6-mercaptohexanol on the surface of the C-reactive protein aptamer of the microneedle array electrode to form an anti-fouling coating to resist non-specific adsorption caused by serum proteins, cells, and plasma; (4) Use square wave pulse voltammetry to detect the current change before and after incubation with the interstitial fluid sample for continuous dynamic monitoring of C-reactive protein in a skin simulation model and monitoring of C-reactive protein levels in a Franz diffusion cell transdermal experiment.

[0036] The preparation method of a microneedle array electrochemical sensor for monitoring C-reactive protein, a sepsis marker in interstitial fluid, of the present invention is characterized in that it includes the following steps:

[0037] (1) Using epoxy resin as the matrix, dichloromethane as the solvent, compounding multi-walled carbon nanotubes, and adding an epoxy resin curing agent after uniform dispersion to obtain a conductive material mixture;

[0038] (2) Fill the conductive material mixture into the female mold of the microneedles made of polydimethylsiloxane, and after curing, obtain a microneedle array. A total of 3 microneedle arrays are made;

[0039] (3) The bases of the 3 microneedle arrays are energized respectively through wires. The bases that hold the 3 microneedle arrays are obtained by filling and curing resin, and the wire arrangement is encapsulated and demolded to obtain microneedle array electrodes;

[0040] (4) Coat the photocurable resin on the area of the microneedle array electrode except for the microneedle tips. The exposed parts of the wires are insulated by polyvinyl chloride hoses through the photocurable resin for insulation treatment;

[0041] (5) Pretreat the microneedle array electrode, including steps of cleaning with dilute sulfuric acid, washing with water, and drying with nitrogen, to obtain a pretreated microneedle array electrode (MAE);

[0042] (6) Deposit nano-gold (AuNPs) on the surface of the pretreated microneedle array electrode MAE obtained in step (5) by electrodeposition method to prepare a nano-gold / microneedle array electrode AuNPs / MAE;

[0043] (7) First, reduce the C-reactive protein aptamer with tris(2-carboxyethyl)phosphine, and then assemble the C-reactive protein aptamer (CRP-Apt) with MB signal onto the surface of the nano-gold / microneedle array electrode AuNPs / MAE prepared in step (6) through gold-sulfur bonds to obtain a C-reactive protein aptamer / nano-gold / microneedle array electrode CRP-Apt / AuNPs / MAE;

[0044] (8) Drop 6-mercaptohexanol (MCH) onto the surface of the C-reactive protein aptamer / nano-gold / microneedle array electrode CRP-Apt / AuNPs / MAE prepared in step (7) to obtain a 6-mercaptohexanol / C-reactive protein aptamer / nano-gold / microneedle array electrode MCH / CRP-Apt / AuNPs / MAE;

[0045] (9) Place this electrode MCH / CRP-Apt / AuNPs / MAE in the interstitial fluid containing C-reactive protein, and perform electrochemical detection by square wave pulse voltammetry. Calculate the concentration of C-reactive protein in the sample through the change in the electrical signal.

[0046] Preferably, the preparation method of a microneedle array electrochemical sensor for monitoring C-reactive protein, a sepsis marker in skin interstitial fluid, is characterized by including the following specific steps:

[0047] (1) Design of the female mold of microneedles: The design drawing of the female mold of microneedles includes a multi-array microneedle module, an array microneedle base, and a total substrate module; specifically, a cylindrical substrate with a diameter of 20 mm and a thickness of 2 mm is used as the total substrate module; the size of each array microneedle base is a diameter of 5 mm and a thickness of 1.5 mm, and the interval between 3 array microneedle bases is 3.66 mm; the multi-array microneedle module is a cactus-like arrangement with 8 microneedles in each of the inner and outer circles centered on 1 microneedle, forming a conical microneedle array multiple holes with a needle length of 800 μm, a needle bottom diameter of 320 μm, and a tip distance of 800 μm for the array microneedles;

[0048] (2) Preparation of the conductive material mixture: Using epoxy resin E51 as the base, multi-walled carbon nanotubes are compounded, and the mass concentration of multi-walled carbon nanotubes is 15 wt%. Using dichloromethane as the solvent, ultrasonic oscillation is carried out at a power of more than 95% for 30 min, and the temperature is kept below 37 °C to ensure that all the multi-walled carbon nanotubes in the conductive material are dissolved and dispersed evenly; then epoxy resin curing agent W93 is added, and the mass ratio of epoxy resin curing agent W93 to epoxy resin is 1:2, and vortex mixing is carried out thoroughly to obtain the conductive material mixture;

[0049] (3) Preparation of conductive microneedles: The conductive material mixture is added to the 3 microneedle arrays of the female mold made of polydimethylsiloxane in 3 portions with a volume of 30 μL each to ensure that there are no bubbles in the liquid in the female mold. Then the female mold is transferred to a 50 mL centrifuge tube. There is a wooden column at the bottom of the centrifuge tube. The mold cover is put on, and they are balanced in pairs and centrifuged at a speed of 4000 rpm for 10 min. The female mold is taken out, and the conductive material mixture is added again until it is flush with the top of the groove of the female mold of the microneedles. After curing at room temperature for 2 h and then curing at 40 °C for 1 h, the array of conductive microneedles is obtained;

[0050] (4) Encapsulation of the microneedle array electrode: The photocurable resin is coated with a watercolor pen on the 3 bases with conductive microneedles. The silver wire is bent into an arc shape to increase the contact surface and is connected to the base with conductive microneedles through the photocurable resin. It is cured under a 405 nm ultraviolet lamp for 30 s, and this is carried out in three times to ensure that the silver wire is fixed to the base with conductive microneedles; finally, the aqueous polyurethane with a solid content of 60 wt% is poured onto the top layer of the mold and cured overnight at 40 °C to obtain a flexible substrate connecting the 3 array electrode bases. The flexible substrate is the total substrate module, and after demolding, the microneedle array electrode (MAE) is obtained;

[0051] (5) Electrode insulation treatment: The area of the microneedle array electrode except for the tips of the microneedles is coated with photocurable resin and cured under a 405 nm ultraviolet lamp for 60 s to form an insulating layer. The exposed part of the silver wire connected to the bottom of the microneedles is covered by a polyvinyl chloride hose and cured under a 405 nm ultraviolet lamp with photocurable resin for 30 s for insulation treatment;

[0052] (6) Pretreatment of the microneedle array electrode (MAE): The microneedle array electrode was placed in a 0.5 mol / L H 2 SO 4 solution and scanned under cyclic voltammetry. The scanning range was 0 - 1.5 V, the scanning speed was 1 V / s, and the number of scanning cycles was 30. Then the electrode was washed with ultrapure water and dried with nitrogen to obtain the pretreated microneedle array electrode MAE for standby;

[0053] (7) Deposition of gold nanoparticles (AuNPs): The microneedle array electrode MAE was placed in a 2.8 mmol / L chloroauric acid solution, and the electrodeposition of gold nanoparticles AuNPs was carried out by chronoamperometry. The deposition potential was -0.4 V, and the time was 500 s. After washing with ultrapure water and 2 drying with N, the gold nanoparticle / microneedle array electrode AuNPs / MAE was prepared for standby;

[0054] (8) Assembly of C-reactive protein aptamer: 10 μL of the 1 μmol / L C-reactive protein aptamer solution pre-reacted for 1 h was dropped onto the surface of the gold nanoparticle / microneedle array electrode AuNPs / MAE, and incubated at -20 °C for 15 min. After thawing for 2 min, it was washed with 20.0 mmol / L Tris-HCl buffer and dried with nitrogen to obtain the C-reactive protein aptamer / gold nanoparticle / microneedle array electrode CRP-Apt / AuNPs / MAE for standby. The above C-reactive protein aptamer solution was diluted with 20.0 mmol / L Tris-HCl buffer after being reduced by mixing 100 μmol / L C-reactive protein aptamer stock solution and 100 mmol / L tris(2-carboxyethyl)phosphine in a volume ratio of 1:1;

[0055] (9) Blocking with 6-mercaptohexanol: 10 μL of 1 mmol / L 6-mercaptohexanol solution was dropped onto the surface of the C-reactive protein aptamer / gold nanoparticle / microneedle array electrode CRP-Apt / AuNPs / MAE, incubated at room temperature for 30 min, washed with 20.0 mmol / L Tris-HCl buffer, and dried with nitrogen to obtain the 6-mercaptohexanol / C-reactive protein aptamer / gold nanoparticle / microneedle array electrode MCH / CRP-Apt / AuNPs / MAE;

[0056] (10) Place the above 6-mercaptohexanol / C-reactive protein aptamer / gold nanoparticle / microneedle array electrode

[0057] MCH / CRP-Apt / AuNPs / MAE in 1 mL of the test solution containing C-reactive protein at a concentration of 10 ng / mL, incubate at room temperature for 30 min, and directly perform square wave pulse voltammetry electrochemical detection to record the change in the electrical signal. The scanning voltage was -0.4 V to 0 V.

[0058] Advantages of the present invention:

[0059] In the present invention, epoxy resin is selected as the matrix to compound carbon nanotubes, and microneedles are prepared and directly energized to be encapsulated into an array electrode. Microneedles with required properties and safety are prepared from cheap and conventional materials under mild conditions, and their applicability as a penetrable skin biosensor is determined, which is cost-effective and can be mass-produced; the array arrangement and substrate of the microneedles are improved to make them have better skin puncture ability and flexibility to fit the skin. The microneedles are arranged in a cactus-like manner and grow orderly from the center to the periphery, effectively enriching the detection liquid, thereby obtaining higher sensing performance; methylene blue is used as the intrinsic electrochemical signal, and the C-reactive protein aptamer is fixed on the surface of the electrode modified with gold nanoparticles by the freeze-thaw method to form a highly uniform DNA monolayer, significantly improving the stability of DNA and the target recognition ability, and enabling the direct in-situ monitoring of C-reactive protein in interstitial fluid of the skin. This method is expected to be used for the instant, efficient, and continuous bedside detection of C-reactive protein to assist in the detection and treatment process evaluation of sepsis. Description of the Drawings

[0060] Figure 1 Schematic diagram of a microneedle array electrochemical sensor for monitoring C-reactive protein, a sepsis biomarker, in interstitial fluid according to the present invention;

[0061] Figure 2 Physical diagram of the microneedle array electrode of a microneedle array electrochemical sensor for monitoring C-reactive protein, a sepsis biomarker, in interstitial fluid according to the present invention;

[0062] Figure 3 Morphology characterization diagram of the microneedle array of a microneedle array electrochemical sensor for monitoring C-reactive protein, a sepsis biomarker, in interstitial fluid according to the present invention;

[0063] Figure 4 Mechanical property characterization of the microneedle array of a microneedle array electrochemical sensor for monitoring C-reactive protein, a sepsis biomarker, in interstitial fluid according to the present invention;

[0064] Figure 5 、 6 Skin puncture performance characterization of the microneedle array of a microneedle array electrochemical sensor for monitoring C-reactive protein, a sepsis biomarker, in interstitial fluid according to the present invention;

[0065] Figure 7 Flexible backing characterization of a microneedle array electrochemical sensor for monitoring C-reactive protein, a sepsis biomarker, in interstitial fluid according to the present invention;

[0066] Figure 8 SEM result diagram of the electrode modification of a microneedle array electrochemical sensor for monitoring C-reactive protein, a sepsis biomarker, in interstitial fluid according to the present invention;

[0067] Figure 9 EIS characterization of the assembly process of a microneedle array electrochemical sensor for monitoring C-reactive protein, a sepsis biomarker, in interstitial fluid according to the present invention;

[0068] Figure 10 Detection feasibility characterization of a microneedle array electrochemical sensor for monitoring C-reactive protein, a sepsis biomarker, in interstitial fluid according to the present invention;

[0069] Figures 11 - 13 Linear investigation of a microneedle array electrochemical sensor for monitoring C-reactive protein, a sepsis biomarker, in interstitial fluid according to the present invention in PBS solution, interstitial fluid, and a skin simulation model;

[0070] Figure 14 Specificity investigation of a microneedle array electrochemical sensor for monitoring C-reactive protein, a sepsis biomarker, in interstitial fluid according to the present invention;

[0071] Figures 15 - 17 Stability investigation of a microneedle array electrochemical sensor for monitoring C-reactive protein, a sepsis biomarker, in interstitial fluid according to the present invention during detection in solution;

[0072] Figure 18 Long-term stability investigation of a microneedle array electrochemical sensor for monitoring C-reactive protein, a sepsis biomarker, in interstitial fluid according to the present invention during detection in a skin simulation model;

[0073] Figure 19 Continuous monitoring investigation of a microneedle array electrochemical sensor for monitoring C-reactive protein, a sepsis biomarker, in interstitial fluid according to the present invention in a skin simulation model;

[0074] Figure 20 Detection comparison graph of C-reactive protein achieved by a microneedle array electrochemical sensor for monitoring C-reactive protein, a sepsis biomarker, in interstitial fluid according to the present invention through a Franz diffusion cell transdermal test. Detailed implementation manners

[0075] In order to make the technical problems, technical solutions, and effects to be solved by the present invention clearer, the present invention will be further described in detail below with reference to the embodiments and the drawings.

[0076] A microneedle array electrochemical sensor for monitoring C-reactive protein, a sepsis biomarker, in skin interstitial fluid according to the present invention is used for monitoring C-reactive protein in skin interstitial fluid, and the specific operation steps are as follows:

[0077] (1) Preparation of instruments, reagents, and solutions for detection

[0078] The instruments and equipment used in this invention were all purchased from the following companies:

[0079]

[0080]

[0081]

[0082] (2) Design of the micro-needle female mold

[0083] The design drawing of the micro-needle female mold includes a multi-array micro-needle module, an array micro-needle base, and a total substrate module. There are three array micro-needle bases on the total substrate module, and each array micro-needle base has a multi-array micro-needle module. The multi-array micro-needle module consists of multiple micro-needles arranged in an array. Specifically, 1 cylindrical substrate with a diameter of 20 mm and a thickness of 2 mm serves as the total substrate module; 3 array micro-needle bases (each with a diameter of 5 mm and a thickness of 1.5 mm), with a spacing of 3.66 mm between the array micro-needle bases; a conical micro-needle array with a needle length of 800 μm, a needle base diameter of 320 μm, and a tip-to-tip distance of 800 μm, composed of a cactus-like arrangement with 8 micro-needles in each of the inner and outer circles centered on 1 micro-needle, is used as the multi-array micro-needle module.

[0084] (3) Preparation of the conductive material mixture

[0085] Using epoxy resin E51 as the base, multi-walled carbon nanotubes (≥95%, inner diameter: 5 - 12 nm, outer diameter: 30 - 50 nm, length: 10 - 20 μm) are compounded. The mass concentration of the multi-walled carbon nanotubes is 15 wt%. Using dichloromethane as the solvent, ultrasonic oscillation is carried out at a power of more than 95% for 30 min, while keeping the temperature below 37 °C to ensure that all the multi-walled carbon nanotubes in the conductive material mixture are dissolved and evenly dispersed. Based on this, epoxy resin curing agent W93 is added. The mass ratio of the epoxy resin curing agent W93 to the epoxy resin is 1:2, and thorough vortex mixing is carried out to obtain the conductive material mixture.

[0086] (4) Preparation of the conductive micro-needles

[0087] The conductive material mixture is added to the 3 arrays of the micro-needle female mold made of polydimethylsiloxane in 3 portions with a volume of 30 μL each, ensuring that there are no bubbles in the liquid in the female mold. Then, the female mold is transferred to a 50 mL centrifuge tube (with a wooden post at the bottom), the mold cover is put on, and they are balanced in pairs and centrifuged at a speed of 4000 rpm for 10 min. The female mold is taken out, and the conductive material mixture is added again until it is flush with the top of the groove of the micro-needle female mold. After curing at room temperature for 2 h and then at 40 °C for 1 h, the array of conductive micro-needles is obtained.

[0088] (5) Encapsulation of the micro-needle array electrode

[0089] The photocurable resin was coated onto 3 array electrode bases (each array electrode base has an array of conductive micro - needles, and the array of conductive micro - needles is the multi - array micro - needle module) with a watercolor pen. The silver wire was bent into an arc shape to increase the contact area and connected to the base with conductive micro - needles through the photocurable resin. It was cured under a 405 nm ultraviolet lamp for 30 s, and this was done in three times to ensure that the silver wire was fixed to the base with conductive micro - needles. Finally, a water - borne polyurethane with a solid content of 60 wt% was poured onto the top layer of the female mold and cured overnight at 40 °C to obtain a flexible substrate connecting 3 array electrode bases. The flexible substrate is the total substrate module. Each array electrode base is provided with a multi - array micro - needle module. The multi - array micro - needle module is a conical micro - needle array with multiple holes arranged in a cactus - like pattern, with a needle length of 800 μm, a needle - base diameter of 320 μm, and a tip - to - tip distance of 800 μm. Specifically, with 1 micro - needle as the center, there are 8 micro - needles in both the inner and outer circles, arranged in a circular pattern as shown in Figure 3 . After demolding, a micro - needle array electrode (MAE) was obtained.

[0090] (6) Electrode insulation treatment

[0091] The area of the micro - needle array electrode except for the tips of the micro - needles was coated with photocurable resin and cured under a 405 nm ultraviolet lamp for 60 s to form an insulating layer. The exposed part of the silver wire connected to the bottom of the micro - needles was covered with a polyvinyl chloride hose and cured under a 405 nm ultraviolet lamp for 30 s with photocurable resin for insulation treatment.

[0092] (7) Pretreatment of micro - needle array electrode (MAE)

[0093] The micro - needle array electrode was placed in a 0.5 mol / L H 2 SO 4 solution and scanned by cyclic voltammetry. The scanning range was 0 - 1.5 V, the scanning speed was 1 V / s, and the number of scanning cycles was 30. Then the electrode was washed with ultrapure water and dried with nitrogen to obtain a pretreated micro - needle array electrode MAE for standby.

[0094] (8) Deposition of nano - gold (AuNPs)

[0095] The micro - needle array electrode MAE was placed in a 2.8 mmol / L chloroauric acid solution, and time - current method was used for the electrodeposition of nano - gold AuNPs. The deposition potential was: - 0.4 V, the time was: 500 s. It was washed with ultrapure water and dried with N 2 to obtain a nano - gold / micro - needle array electrode AuNPs / MAEE for standby.

[0096] (9) Assembly of C - reactive protein aptamer

[0097] 10 μL of 1 μmol / L C-reactive protein aptamer solution pre-reacted for 1 h was dropped onto the surface of the nano-gold / micro-needle array electrode AuNPs / MAE, and incubated at -20 °C for 15 min. After thawing for 2 min, it was washed with 20.0 mmol / L Tris-HCl buffer and dried with nitrogen to obtain the C-reactive protein aptamer / nano-gold / micro-needle array electrode CRP-Apt / AuNPs / MAE for standby; the above C-reactive protein aptamer solution was prepared by reducing 100 μmol / L C-reactive protein aptamer stock solution and 100 mmol / L tris(2-carboxyethyl)phosphine in a volume ratio of 1:1 and then diluting with 20.0 mmol / L Tris-HCl buffer.

[0098] (10) Blocking with 6-mercaptohexanol

[0099] 10 μL of 1 mmol / L 6-mercaptohexanol solution was dropped onto the surface of the C-reactive protein aptamer / nano-gold / micro-needle array electrode CRP-Apt / AuNPs / MAE, incubated at room temperature for 30 min, washed with 20.0 mmol / L Tris-HCl buffer and dried with nitrogen to obtain the 6-mercaptohexanol / C-reactive protein aptamer / nano-gold / micro-needle array electrode MCH / CRP-Apt / AuNPs / MAE.

[0100] (10) Detection of C-reactive protein

[0101] The above 6-mercaptohexanol / C-reactive protein aptamer / nano-gold / micro-needle array electrode MCH / CRP-Apt / AuNPs / MAE was placed in 1 mL of the test solution containing C-reactive protein at 10 ng / mL, incubated at room temperature for 30 min, and directly subjected to square wave pulse voltammetry electrochemical detection, recording the change in the electrical signal, and the scanning voltage was -0.4 V to 0 V.

[0102] In the embodiment of the present invention:

[0103] Figure 1 It is a schematic diagram of a micro-needle array electrochemical sensor for monitoring C-reactive protein, a sepsis biomarker in interstitial fluid. The electrochemical biosensor prepared through the above steps was subjected to relevant experiments.

[0104] Figure 2This is a physical diagram of the microneedle array electrode of a microneedle array electrochemical sensor for monitoring the sepsis biomarker C-reactive protein in interstitial fluid. The microneedle array electrode consists of a total substrate supporting 3 array microneedle bases. Both the total substrate and the array microneedle bases are cylinders. The microneedle array electrode on each array microneedle base is centered on 1 microneedle, with 8 microneedles in both the inner and outer circles, arranged in a circular pattern. For the prepared microneedle array electrochemical sensor, the total substrate is a cylindrical substrate with a bottom diameter of 20 mm and a thickness of 2 mm. There are 3 microneedle array electrodes on the total substrate, which are the working electrode, reference electrode, and counter electrode respectively. The array microneedle bases of each microneedle array electrode are cylinders with a bottom diameter of 5 mm and a thickness of 1.5 mm. Each microneedle array electrode is connected to a silver wire for power supply through each array microneedle base. The exposed part of the wire is insulated by a polyvinyl chloride hose through photocuring resin. The distance between each microneedle array electrode is 3.66 mm.

[0105] Figure 3 This is a morphological characterization diagram of the microneedle array of a microneedle array electrochemical sensor for monitoring the sepsis biomarker C-reactive protein in interstitial fluid. Each microneedle array electrode is arranged in a circular pattern, orderly arranged from 1 central microneedle to the surrounding. There are 8 arrays in both the inner and outer circles. The bottom diameter of each microneedle is 320 μm, the distance between the tips of the inner and outer circle microneedles is 800 μm. The microneedles are conical in shape, with a needle height of 800 μm. The microneedle array is complete, and no fracture or bending occurs at the needle tips. The microneedle array imitates the cactus structure. The cactus has the ability to continuously collect dew and grows orderly from the center to the surrounding. The periodic array structure can provide a large contact area and effectively enrich the detection liquid, thereby obtaining higher sensing performance. At the same time, there are literature reports that the microneedle array arranged in a circular pattern has better skin puncture performance and mechanical properties than the microneedle array arranged in a square pattern.

[0106] Figure 4 This is the mechanical property characterization of the microneedle array of a microneedle array electrochemical sensor for monitoring the sepsis biomarker C-reactive protein in interstitial fluid. According to the stress-strain curve ( Figure 4 in A), the compression modulus is 14.28 MPa, and the compression strength is 37.49 MPa. The volume change of the microneedle is small when compressed, with good stability and anti-deformation ability. And the microneedle material can withstand a large pressure without damage when compressed. The force-displacement curve ( Figure 4 in B) shows that a single needle can withstand a force greater than 0.7 N / needle without breaking, which is greater than the usually reported breaking force of 0.3 - 0.4 N / needle, and far higher than the force required for skin penetration (0.1 N / MN).

[0107] Figure 5 、 6Characterization of the skin puncture performance of the microneedle array of a microneedle array electrochemical sensor for monitoring C-reactive protein, a sepsis biomarker, in interstitial fluid. The skin puncture performance of the microneedle array was characterized using Parafilm and mouse skin, and the results are as Figure 5 and 6 shown. Figure 5 For the first four layers of the Parafilm, three holes of the 17 microneedle array appeared, with each layer of the sealing film being about 126 μm, and the puncture depth of the microneedle array being about 400 μm, meeting the conditions for puncturing the stratum corneum, and the piercing rate reaching 100%. Figure 6 The mouse skin puncture results showed three complete 17-array micropores, and these micropores generated by the microneedles were almost re-closed within 20 min, as Figures 2 - 10 shown, returning to the original state with little damage to the skin.

[0108] Figure 7 Characterization of the flexible backing of a microneedle array electrochemical sensor for monitoring C-reactive protein, a sepsis biomarker, in interstitial fluid. Figure 7 In [reference], A is a schematic diagram of the three-point bending test of the backing (total substrate) of the microneedle array electrode in a bent state, Figure 7 and the force-displacement curve obtained in the B test of [reference] shows that the backing of the microneedle array has good bending performance, with a bending strength of 0.90 MPa and a bending modulus of 1.67 MPa. When the maximum force of 15 N, which is the force verified to meet the microneedle array puncture and not break as described above, was applied, no breakage occurred during the test process. At the same time, due to the relatively soft polyurethane base plate, the prepared microneedle array electrochemical sensor has good flexibility ( Figure 7 C in [reference]). Therefore, the backing (total substrate) prepared with polyurethane has flexible characteristics, can fit the skin more closely, and is convenient for force application.

[0109] Figure 8 SEM result diagrams of the electrodes of a microneedle array electrochemical sensor for monitoring C-reactive protein, a sepsis biomarker, in interstitial fluid. Figure 8 In [reference], A is the SEM image of the microneedle array electrode MAE, with a rough surface and the tubular structure of multi-walled carbon nanotubes clearly visible; Figure 8 B in [reference] is the SEM image of the nano-gold / microneedle array electrode AuNPs / MAE, with flower-shaped gold nanoparticles growing on the surface of the microneedle electrode; Figure 8 C in [reference] is the C-reactive protein aptamer / nano-gold / microneedle array electrode CRP-APt / AuNPs / MAE, with fibrous structures connected to the electrode surface; Figure 8 D in [reference] is the C-reactive protein / 6-mercaptohexanol / C-reactive protein aptamer / nano-gold / microneedle array electrode

[0110] For CRP / MCH / CAP-APt / AuNPs / MAE, the fine structure on the surface of the fibrous structure is passivated, and a film-like structure is formed on the surface, indicating the successful assembly of CRP / MCH / CAP-APt / AuNPs / MAE.

[0111] Figure 9 This is the EIS characterization of the assembly process of a microneedle array electrochemical sensor for monitoring the sepsis marker C-reactive protein in interstitial fluid according to the present invention. The impedance of the bare microneedle array electrode is about 6.5 kΩ. After modification with nano-gold, the impedance is reduced to 1 kΩ. After the assembly of the C-reactive protein aptamer, the impedance increases to 2.5 kΩ. After blocking with 6-mercaptohexanol, the impedance increases again to 5 kΩ. After the assembly of C-reactive protein, the impedance further increases to 7.5 kΩ. This is because nano-gold has good conductivity, while the non-conductive C-reactive protein aptamer hinders electron transfer. 6-Mercaptohexanol blocks non-specific adsorption on the interface, further hindering electron transfer. Finally, the binding of C-reactive protein further hinders electron transmission, and the resistance value reaches the maximum. This result indicates the successful assembly of the CRP / MCH / CRP-APt / AuNPs / MAE sensor.

[0112] Figure 10 This is the detection feasibility characterization of a microneedle array electrochemical sensor for monitoring the sepsis marker C-reactive protein in interstitial fluid according to the present invention. When the concentration of C-reactive protein is 10 ng / mL, the difference △Ⅰ between the experimental group and the control group is 0.75 μA, indicating that the CRP / MCH / CRP-APt / AuNPs / MAE of the present invention, that is, the electrochemical sensor of the present invention, is feasible for detecting C-reactive protein.

[0113] Figures 11 - 13 This is the linearity investigation of a microneedle array electrochemical sensor for monitoring the sepsis marker C-reactive protein in interstitial fluid according to the present invention in PBS solution, interstitial fluid and skin simulation model. The results show that in the range of 0.5 ng / mL to 100 ng / mL, there is a good linear relationship between the current difference △I and lg C CRP and the detection limit is 0.3391 ng / mL. The linear range and detection limit in interstitial fluid and skin simulation model are basically the same as those in PBS solution.

[0114] Figure 14Specificity investigation of a microneedle array electrochemical sensor for monitoring C-reactive protein, a sepsis biomarker, in interstitial fluid. Considering the differences in the content of interfering substances in skin interstitial fluid, 10.0 ng / mL SAA, 5.0 ng / mL PCT, 0.5 ng / mL IL-6, 0.5 ng / mL D-D, 10 ng / mL FN, and 20.0 mg / mL BSA were added to explore the interference with the signal of 10.0 ng / mL C-reactive protein. The results showed that the current signals of SAA, PCT, IL-6, D-D, FN, and BSA were all similar to the background value, with small differences, and were significantly different from the current signal of C-reactive protein, indicating that the microneedle array electrochemical sensor used in this study for C-reactive protein monitoring has good specificity and anti-interference ability.

[0115] Figures 15 - 17 Stability investigation of a microneedle array electrochemical sensor for monitoring C-reactive protein, a sepsis biomarker, in interstitial fluid when detected in solution. To study the repeatability and stability of the microneedle array sensor, the same microneedle array sensor was used to detect five times, and C-reactive protein samples with a concentration of 10 ng / mL were detected using five microneedle array sensors of the same batch, five different microneedle array sensors, and five microneedle array sensors of different batches. The results are as Figure 15 shown, the RSD of a microneedle array sensor used for 5 cycles was 3.41%; Figure 16 shown, the RSD of different microneedle array sensors within the group was 4.47%; Figure 17 shown, the RSD of different microneedle array sensors between groups was 4.97%. This indicates that the change in current response of the microneedle array sensor is small during the usage cycle and between different batches of microneedle array sensors, and the current response can meet the requirements of actual detection.

[0116] Figure 18 Long-term stability investigation of a microneedle array electrochemical sensor for monitoring C-reactive protein, a sepsis biomarker, in interstitial fluid when detected in a skin simulation model. The microneedle array electrochemical sensor was repeatedly inserted into a skin simulation model filled with 10 ng / mL C-reactive protein at different time points of 0, 1, 2, 4, 8, 12, 24, 48, and 72 h. Figure 18 shown, the response of the sensor to C-reactive protein remained relatively constant (<10% change), indicating that the microneedle array electrochemical sensor used for C-reactive protein monitoring has good long-term stability.

[0117] Figure 19Continuous monitoring investigation of a microneedle array electrochemical sensor for monitoring C-reactive protein, a sepsis marker in interstitial fluid, on a skin simulation model. The microneedle array electrochemical sensor can meet the detection of C-reactive protein from low concentration to high concentration (0, 0.5, 1, 5, 10, 50, 100 ng / mL), and the signal gradually increases; it can also meet the detection of C-reactive protein from high concentration to low concentration (100, 50, 10, 5, 1, 0.5, 0 ng / mL), and the signal gradually decreases; that is, the change of the response current signal is consistent with the change of the C-reactive protein concentration. Due to the different degrees of wear of the sensing interface caused by the insertion of the microneedles, as the microneedles are inserted into the skin model with different concentrations of C-reactive protein, the current signal response decreases, but in the rising stage and the falling stage, the current signals at the same C-reactive protein concentration are basically the same, with a small difference, indicating that the electrochemical sensor of this microneedle array has the ability to continuously monitor C-reactive protein in the skin simulation model, which helps to continuously and real-time respond to the C-reactive protein signal in the skin.

[0118] Figure 20 Comparison diagram of C-reactive protein monitoring achieved by a microneedle array electrochemical sensor for monitoring C-reactive protein, a sepsis marker in interstitial fluid, in a Franz diffusion cell transdermal experiment of the present invention. Figure 20 A in it shows the change of SWV signal caused by the increase of C-reactive protein concentration in the receptor chamber. In practical applications, the level of C-reactive protein affects the disease severity of sepsis patients. When sepsis patients are given drug treatment, the C-reactive protein level changes from high concentration to low concentration due to the remission of the body's inflammatory reaction degree. Therefore, it is necessary to quickly detect the inflammatory level of C-reactive protein by monitoring the clearance rate of C-reactive protein in the human body, that is, to monitor the C-reactive protein level in ISF from high concentration to low concentration. Figure 20 B in it shows the change of SWV signal caused by the decrease of C-reactive protein concentration in the receptor chamber under the Franz diffusion cell. The decrease and reduction of the concentration are accompanied by the decrease and reduction of the signal. At the same time, in the range of 0.5 - 100 ng / mL C-reactive protein, the rising trend and the falling trend tend to be consistent, respectively proving the analytical ability of the microneedle array electrochemical sensor to monitor the C-reactive protein receptor chamber level through the skin.

Claims

1. A microneedle array electrochemical sensor for monitoring C-reactive protein, a sepsis marker, in skin interstitial fluid; comprising a microneedle array electrode, wherein the microneedle array electrode is prepared by the following method, characterized in that: The method comprises the following steps: (1) designing a negative mold of a microneedle: designing a multi-array microneedle module, an array microneedle base and a total base module, wherein the total base module has three array microneedle bases, each array microneedle base has a multi-array microneedle module, and the multi-array microneedle module is composed of a plurality of microneedles arranged in an array; (2) preparing a conductive material mixture liquid with epoxy resin as a base and composite multi-walled carbon nanotubes to meet the electrochemical performance of the microneedles; (3) transferring the conductive material mixture liquid to the negative mold of the microneedle by a centrifugal method and curing it into a shape; (4) connecting each array microneedle base through silver wire and electrifying it, and finally pouring waterborne polyurethane into the top layer of the negative mold of the microneedle to obtain a microneedle three-electrode connected by a flexible backing, wherein the microneedle three-electrode is a working electrode, a reference electrode and a counter electrode, and coating the area of ​​the microneedle three-electrode except the microneedle needle tip with a photocurable resin and performing a final insulation operation to obtain a microneedle array electrode.

2. The method for detecting the concentration of C-reactive protein using a microneedle array electrochemical sensor for monitoring the sepsis marker C-reactive protein in skin interstitial fluid according to claim 1, characterized in that: The method comprises the following steps: (1) preparing gold nanoparticles on the surface of a microneedle array electrode by electrochemical deposition, and reducing a C-reactive protein aptamer having an intrinsic electrochemical signal of methylene blue and a thiol group with the nanogold to form a gold-sulfur bond through tri(2-carboxyethyl)phosphine reduction; (2) forming a highly uniform nucleic acid monolayer on the surface of the nanogold by a freeze-thaw method, thereby significantly improving the stability of the nucleic acid and the target recognition ability; (3) modifying the surface of the C-reactive protein aptamer of the microneedle array electrode by 6-mercaptohexanol to form an anti-fouling coating to resist the non-specific adsorption caused by serum proteins, cells, and plasma; and (4) detecting the current change before and after incubation with an interstitial fluid sample by square wave pulse voltammetry, which is used for continuous dynamic monitoring of C-reactive protein in a skin simulation model and monitoring of C-reactive protein levels in a Franz diffusion cell transdermal test.

3. A method for preparing a microneedle array electrochemical sensor for monitoring C-reactive protein, a sepsis marker, in skin interstitial fluid, characterized in that: The steps include: (1) Using epoxy resin as a matrix and dichloromethane as a solvent, compounding multi-walled carbon nanotubes, and adding epoxy resin curing agent after uniform dispersion to obtain a conductive material mixed liquid; (2) Filling the conductive material mixture into the negative mold of the microneedle made of polydimethylsiloxane, and curing to obtain a microneedle array, and a total of 3 microneedle arrays were prepared; (3) The bases of the three microneedle arrays are energized through wires, and the base supporting the three microneedle arrays is obtained by curing the filled resin, and the microneedle array electrodes are obtained by wiring, packaging and demolding; (4) coating the microneedle array electrode with a photocurable resin in areas other than the microneedle tips, and insulating the exposed wires with a polyvinyl chloride hose insulated with the photocurable resin; (5) pre-treating the microneedle array electrode, including washing with dilute sulfuric acid, washing with water, and drying with nitrogen, to obtain a pre-treated microneedle array electrode (MAE); (6) depositing gold nanoparticles (AuNPs) on the surface of the pretreated microneedle array electrode MAE obtained in step (5) by an electrodeposition method to prepare gold nanoparticles / microneedle array electrode AuNPs / MAE; (7) first reducing the C-reactive protein aptamer with tri(2-carboxyethyl)phosphine, and then assembling the C-reactive protein aptamer (CRP-Apt) with MB signal onto the surface of the nano-gold / microneedle array electrode AuNPs / MAE prepared in step (6) through a gold-sulfur bond to obtain a C-reactive protein aptamer / nano-gold / microneedle array electrode CRP-Apt / AuNPs / MAE; (8) adding 6-mercaptohexanol (MCH) dropwise onto the surface of the C-reactive protein aptamer / nano-gold / microneedle array electrode CRP-Apt / AuNPs / MAE prepared in step (7) to obtain 6-mercaptohexanol / C-reactive protein aptamer / nano-gold / microneedle array electrode MCH / CRP-Apt / AuNPs / MAE; (9) The MCH / CRP-Apt / AuNPs / MAE electrode was placed in interstitial fluid containing C-reactive protein, and electrochemical detection was performed using square wave pulse voltammetry. The C-reactive protein concentration in the sample was calculated based on the change in the electrical signal.

4. The method for preparing a microneedle array electrochemical sensor for monitoring the sepsis marker C-reactive protein in skin interstitial fluid according to claim 3, characterized in that: The specific steps include: (1) Design of the negative mold of microneedles: The negative mold design of the microneedle includes a multi-array microneedle module, an array microneedle base and a total base module; specifically, a cylindrical base with a diameter of 20 mm and a thickness of 2 mm is used as the total base module; the size of each array microneedle base is 5 mm in diameter and 1.5 mm thick, and the interval between the three array microneedle bases is 3.66 mm; the multi-array microneedle module is a cactus-like arrangement with 8 microneedles in each inner and outer circles with one microneedle as the center. The needle length of the array microneedle is 800 μm, the needle base diameter is 320 μm, and the needle tip distance is 800 μm, which is a conical microneedle array with multiple holes; (2) Preparation of conductive material mixture: Using epoxy resin E51 as the substrate, compounding multi-walled carbon nanotubes, the mass concentration of multi-walled carbon nanotubes is 15wt%, using dichloromethane as the solvent, using ultrasonic oscillation at a power of more than 95% for 30 min, keeping the temperature below 37°C, to ensure that the multi-walled carbon nanotubes in the conductive material are completely dissolved and evenly dispersed; then adding epoxy resin curing agent W93, the mass ratio of epoxy resin curing agent W93 to epoxy resin is 1:2, and vortex mixing is thoroughly performed to obtain a conductive material mixture; (3) Preparation of conductive microneedles: Add the conductive material mixture in a volume of 30 μL three times to the three microneedle arrays of the negative mold of the microneedles made of polydimethylsiloxane, ensuring that there are no bubbles in the liquid in the negative mold. Then transfer the negative mold to a 50 mL centrifuge tube with a wooden column at the bottom of the centrifuge tube, cover the mold cover, balance the two and centrifuge at 4000 rpm for 10 min, remove the negative mold, add the conductive material mixture again until it is flush with the top of the groove of the negative mold of the microneedle, cure at room temperature for 2 h and then cure at 40°C for 1 h to obtain the conductive microneedle array; (4) Encapsulation of microneedle array electrodes: Use a watercolor pen to apply photocurable resin to three bases with conductive microneedles. Bend the silver wire into an arc shape to increase the contact surface. Connect it to the base with conductive microneedles through the photocurable resin and cure it under a 405 nm ultraviolet lamp for 30 s. This is done three times to ensure that the silver wire is fixed to the base with conductive microneedles. Finally, pour water-based polyurethane with a solid content of 60 wt% on the top layer of the mold and cure it at 40 °C overnight to obtain a flexible substrate connected to the three array electrode bases. The flexible substrate is the total substrate module. After demolding, a microneedle array electrode (MAE) is obtained. (5) Electrode insulation treatment: The area of ​​the microneedle array electrode except the microneedle tip is coated with a photocurable resin and cured under a 405 nm ultraviolet lamp for 60 s to form an insulating layer. The exposed part of the silver wire connected to the bottom of the microneedle is covered with a polyvinyl chloride hose and cured with a photocurable resin under a 405 nm ultraviolet lamp for 30 s for insulation treatment. (6) Pretreatment of microneedle array electrode (MAE): The microneedle array electrode was placed in a 0.5 mol / L H2SO4 solution and scanned under cyclic voltammetry with a scanning range of 0-1.5 V, a scanning speed of 1 V / s, and a scanning cycle of 30. The electrode was then washed with ultrapure water and dried with nitrogen to obtain the pretreated microneedle array electrode MAE for use. (7) Deposition of gold nanoparticles (AuNPs): The microneedle array electrode (MAE) was placed in a 2.8 mmol / L chloroauric acid solution and the electrodeposition of gold nanoparticles (AuNPs) was performed using the time-current method. The deposition potential was −0.4 V and the time was 500 s. The electrode was then washed with ultrapure water and dried with N2 to obtain gold nanoparticles / microneedle array electrode (AuNPs / MAE) for later use. (8) Assembly of C-reactive protein aptamer: 10 μL of 1 μmol / L C-reactive protein aptamer solution pre-reacted for 1 h was added dropwise onto the surface of the nanogold / microneedle array electrode AuNPs / MAE, frozen and incubated at -20°C for 15 min, thawed for 2 min, washed with 20.0 mmol / L Tris-HCl buffer, and dried with nitrogen gas to obtain C-reactive protein aptamer / nanogold / microneedle array electrode CRP-Apt / AuNPs / MAE for later use; the above C-reactive protein aptamer solution was prepared by reducing 100 μmol / L C-reactive protein aptamer stock solution and 100 mmol / L tris(2-carboxyethyl)phosphine in a volume ratio of 1:1, and then diluted with 20.0 mmol / L Tris-HCl buffer; (9) Blocking with 6-mercaptohexanol: 10 μL of 1 mmol / L 6-mercaptohexanol solution was dripped onto the surface of C-reactive protein aptamer / nanogold / microneedle array electrode CRP-Apt / AuNPs / MAE, incubated at room temperature for 30 min, washed with 20.0 mmol / L Tris-HCl buffer, and dried with nitrogen gas to obtain 6-mercaptohexanol / C-reactive protein aptamer / nanogold / microneedle array electrode MCH / CRP-Apt / AuNPs / MAE; (10) The 6-mercaptohexanol / C-reactive protein aptamer / nanogold / microneedle array electrode MCH / CRP-Apt / AuNPs / MAE was placed in 1 mL of a test solution containing 10 ng / mL C-reactive protein and incubated at room temperature for 30 min. Square wave pulse voltammetry electrochemical detection was performed directly to record the changes in electrical signals with a scanning voltage of −0.4 V to 0 V.

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