Microneedle array electrochemical sensor for in-situ detection of sepsis marker lactic acid

Abstract

The invention discloses a microneedle array electrochemical sensor for in-situ detection of sepsis marker lactic acid, which comprises a working electrode, a reference electrode and a counter electrode which are arranged on a light-cured resin base, and the three electrodes all take epoxy resin as a matrix and dichloromethane as a dispersing solvent, and the working electrode, the reference electrode and the counter electrode are arranged on the light-cured resin base. A microneedle conductive layer is prepared from a conductive material prepared from composite multi-walled carbon nanotubes, light-cured resin is used as an insulating layer, a working electrode is provided with a lactic acid enzyme electrode modification layer, polyvinylpyrrolidone is used as a protective layer, in-situ detection of lactic acid in interstitial fluid is realized, and the microneedle array electrochemical sensor for in-situ detection of sepsis marker lactic acid is constructed. The kit has the advantages of being easy to process, short in processing period, low in cost, easy to realize large-scale production, high in sensitivity, strong in specificity and the like, can be used for clinical accurate, efficient and rapid sepsis diagnosis and real-time disease course monitoring, and also provides a new thought for biomarker detection of other diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of microneedles and electrochemical sensors, and in particular to a microneedle array electrochemical sensor for in-situ detection of sepsis marker lactate. Background Art

[0002] Sepsis is a clinical syndrome that causes life-threatening organ dysfunction due to a dysregulated systemic response to infection. It is the main cause of morbidity and mortality in critically ill patients. At present, the diagnosis of sepsis mainly relies on the sequential organ failure assessment (SOFA) and 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, which may lead to delays in diagnosis and treatment. Due to the rapid progression of sepsis, it may rapidly develop into septic shock, causing circulatory, cellular and metabolic abnormalities and increasing mortality. At the same time, the use time of antimicrobial drugs is closely related to the mortality rate of septic shock. Patients with septic shock should be treated immediately within one hour and continue to be closely monitored. Therefore, a rapid, instant and simple detection method is needed to monitor the changes in the course of the disease in real time so as to provide patients with medication and other intervention methods in a timely manner.

[0003] At present, the diagnostic criteria for septic shock are mean arterial pressure ≥ 65 mmHg and blood lactate > 2 mmol / L, which are used to identify septic patients with a high risk of death. In clinical practice, capillary lactate (CL) and serum lactate (SL) are also used as prognostic indicators of mortality within 48 hours and 7 days, respectively. Lactate has been shown to be an independent prognostic predictor of mortality in patients with sepsis, and its level is a key biomarker for assessing the progression or recovery of organ function in patients with sepsis. Therefore, testing the lactate content in patients with sepsis can dynamically monitor the development of the disease, quickly and accurately identify sepsis patients with a higher risk of death, conduct appropriate prognostic assessments, and help manage and diagnose sepsis.

[0004] The most commonly used method for measuring lactate is venous blood sampling, but it is invasive, causing pain and secondary physical trauma to patients, increasing the risk of infection, and requiring professional operation. Laboratory analysis after sampling is also time-consuming. In fact, every analyte present in the blood can be considered to be present in the interstitial fluid, including proteins, nucleic acids and small molecules. Blood lactate concentrations above 2mmol / L are used as a reference indicator for septic shock, while the normal level of lactate in the blood is about 0.38-1.38mmol / L, and the interstitial fluid is about 1.14-3.14mmol / L. Given the concentration correlation between the two, interstitial fluid lactate concentrations above 4-5mmol / L can be used as an early warning sign of septic shock. Because interstitial fluid exists near the surface of the skin, biomarkers are easier to obtain and do not produce blood-related pain or coagulation. Therefore, the lactate level of interstitial fluid is used as an auxiliary reference indicator for monitoring the course of sepsis.

[0005] Since interstitial fluid exists near the surface of the skin, microneedles can be applied to the outside of the skin in the form of traditional transdermal patches to sample and monitor skin ISF in a minimally invasive manner, making it easier to obtain biomarkers in ISF without causing blood-related pain or coagulation. At the same time, the micron-sized needle body pierces the micropores in the stratum corneum, which are difficult to reach capillaries and heal within a few minutes to avoid bacterial infection. Microneedle biosensors, which are integrated with microneedle technology and biosensor systems, have also become a new high-throughput, highly sensitive, rapid real-time detection technology that has recently become a hot topic internationally. Microneedle-based sensing has the advantages of ease of use and adaptability to in situ biomarker analysis.

[0006] In the integrated microneedle biosensor device, the electrochemical sensor, as a new type of analytical device, can convert the chemical changes of the target analyte into detectable electrical signals through specific bioaffinity or biocatalytic behavior, and use electrodes as adapters to output and amplify the signal for visualization. Electrochemical systems are widely used for accurate monitoring of trace analytes due to their automation, rapid response, long life, high sensitivity and high selectivity.

[0007] At present, medical acupuncture needles are often used directly as electrodes for microneedle array electrodes. The length of medical acupuncture needles is at the millimeter level, and professional techniques are required. In addition, silicon or metal materials are also used as array electrode substrates. However, the biocompatibility of the two is poor, and they are prone to breakage during the insertion process, resulting in infection. In addition, the cost of both is high, and the manufacturing process is complex. Among them, the metallization of microneedles is also prone to metal layer shedding, and the stability is poor. At the same time, microneedle sensors often use a two-step process of interstitial fluid extraction and post-extraction detection, which is complicated to operate, time-consuming, and difficult to meet the requirements of instant detection. Therefore, these problems need to be further resolved. Summary of the invention

[0008] The purpose of the present invention is to provide a microneedle array electrochemical sensor for in situ detection of lactic acid, a marker of sepsis, and a preparation method thereof. By constructing a three-electrode microneedle system to detect lactic acid in interstitial fluid, instant and continuous minimally invasive detection is achieved, thereby improving patient compliance. At the same time, the sensor uses a resin composite material as an electrode substrate, which has good mechanical properties and biocompatibility, avoiding problems such as breakage and infection after the microneedle is inserted into the skin; and the operation is simple, and batch production can be achieved, effectively reducing costs; and the sensitivity is improved by functionally modifying the electrode, which can meet the detection needs of lactic acid, a marker of sepsis patients, and achieve in situ monitoring.

[0009] To achieve the above-mentioned purpose, the present invention provides a microneedle array electrochemical sensor for in-situ detection of lactic acid, a sepsis marker, comprising a working electrode, a reference electrode and a counter electrode arranged on a base, wherein the three electrodes are all made of a conductive layer made of a conductive mixed material made of a resin as a matrix composite nanomaterial and directly used as a microneedle array electrode, and an insulating layer is provided, wherein the working electrode is additionally provided with an enzyme electrode modification layer and a protective layer. The three electrodes are all electrically connected through wires, and the wires are insulated by a catheter, and finally the wires are assembled into a sensor.

[0010] The resin composite material used in the present invention has good biocompatibility. The strength of the microneedle can be improved by modifying the material to prevent the electrode from breaking and breaking after being inserted into the skin, thereby affecting the sensor performance. In addition, the synthetic resin material also has the characteristics of easy processing, short processing cycle, low cost, etc., and is easy to achieve large-scale production.

[0011] The present invention uses resin composite carbon nanotubes as the electrode substrate material of the microneedle electrode array, which can improve the performance and stability of the sensor. At the same time, the functional modification of the microneedle electrode can further realize the detection of lactic acid, a sepsis marker in the interstitial fluid, to meet the needs of the human body for auxiliary monitoring of the course of sepsis. The present invention also introduces polyvinyl pyrrolidone with good biocompatibility as a protective layer of the sensor, and the detection of lactic acid in the interstitial fluid of the skin by the microneedle electrochemical sensor is restored to the maximum extent without causing damage to the skin. The base is a base made of photocurable resin, and the conductive layer is obtained by curing a conductive material mixed liquid made of epoxy resin as a matrix, multi-walled carbon nanotubes as conductive nanomaterials, and dichloromethane as a dispersing solvent. The energized wire is a silver wire, which is connected to the electrode through the photocurable resin and insulated with a polyvinyl chloride hose; the electrode insulation layer is a photocurable resin layer, and the protective layer is a polyvinyl pyrrolidone layer. The enzyme electrode modification layer is prepared by the following method: first modifying the polythiophene-citrate film, using the carbodiimide condensation method, and fixing the lactate oxidase to the working electrode by chemical crosslinking combined with the physical adsorption of gelatin.

[0012] Preferably, the microneedle electrochemical sensor has a base that is a cuboid with a length of 20 mm, a width of 9 mm, and a thickness of 2 mm; the microneedle portion of the electrode is arranged in three 3*3 arrays, i.e., three microneedle array electrodes, the base of each microneedle array electrode is a cuboid with a length of 4 mm, a width of 4 mm, and a thickness of 1.5 mm, and each microneedle array electrode is 2 mm apart; wherein the microneedles of each microneedle array electrode are all conical, with a needle length of 800 μm, a needle bottom diameter of 320 μm, and a needle tip distance of 600 μm between each microneedle. The base is a base made of photocurable resin, the conductive layer is obtained by curing a conductive material mixture obtained by composite multi-walled carbon nanotubes with epoxy resin as a matrix, the insulating layer is a photocurable resin layer, the protective layer is a polyvinyl pyrrolidone layer, the enzyme electrode modification layer is a polythiophene-citrate film, EDC-NHS, lactate oxidase, and gelatin, the energized wire is a silver wire, the three electrodes are connected by photocurable resin, and the conduit of the insulated wire is a polyvinyl chloride hose.

[0013] Preferably, the base of the microneedle electrochemical sensor is a rectangular parallelepiped with a length of 20 mm, a width of 9 mm, and a thickness of 2 mm; the microneedle part is arranged in three 3*3 arrays, i.e., three microneedle array electrodes, and the base of each microneedle array electrode is a rectangular parallelepiped with a length of 4 mm, a width of 4 mm, and a thickness of 1.5 mm, and is 2 mm apart from each other; wherein each microneedle is conical, with a needle length of 800 μm, a needle base diameter of 320 μm, and a needle tip distance of 600 μm between each microneedle.

[0014] The present invention also protects the electrochemical sensor for lactic acid detection using the functionalized microneedle electrode, comprising the following steps:

[0015] S1. Preparation of conductive material: using epoxy resin as matrix, dichloromethane as solvent, compounding multi-walled carbon nanotubes, and adding epoxy resin curing agent after uniform dispersion to obtain a conductive material mixed solution;

[0016] S2, preparing a microneedle array: filling a conductive material mixture into a microneedle array electrode negative mold made of polydimethylsiloxane, and curing to obtain a microneedle array;

[0017] S3, packaging electrodes: the bases of the three array microneedles are energized through wires respectively, the filled resin is cured to obtain a three-electrode base, and the wires are arranged, packaged and demolded to obtain the microneedle array electrode;

[0018] S4, insulation treatment: coating the microneedle array electrode obtained in step S3 with a photocurable resin on the area other than the microneedle tips, and insulating the exposed part of the wire with a PVC hose through the photocurable resin;

[0019] S5, preparing a working electrode: sequentially modifying the surface of the microneedle array electrode after the insulation treatment in S4 with polythiophene-citrate, EDC-NHS, lactate oxidase, and gelatin as an enzyme electrode modification layer to obtain a working electrode with an additional enzyme electrode modification layer;

[0020] S6. Prepare a protective layer: Fill polyvinyl pyrrolidone into a single array of the microneedle array electrode negative mold made of polydimethylsiloxane by centrifugation, reset the working electrode with the additional enzyme electrode modification layer obtained in S5 in the negative mold, put it in an oven at 40°C for overnight curing, and demold to obtain a microneedle electrochemical sensor with a polyvinyl pyrrolidone protective layer.

[0021] Preferably, the conductive material mixed solution described in step S1 is prepared by the following steps: epoxy resin E51 is used as a matrix, multi-walled carbon nanotubes are compounded, the mass concentration of multi-walled carbon nanotubes is 15%, dichloromethane is used as a dispersing solvent, and the material is obtained by ultrasonic oscillation at a power of more than 95% for 20 minutes to ensure that the multi-walled carbon nanotubes in the material are completely dissolved and evenly dispersed in a semi-viscous state. Based on this, epoxy resin curing agent is added at a mass ratio of 1:2 to epoxy resin, and after thorough vortex mixing, ultrasonic oscillation is performed for 5 minutes to obtain a conductive material mixed solution;

[0022] Preferably, the array microneedle described in step S2 is obtained by the following steps: transferring the microneedle array negative mold made of polydimethylsiloxane to a 50 mL centrifuge tube, adding the conductive composite material to three arrays of the microneedle array electrode negative mold in a volume of 25 μL three times, covering with a lid made of polydimethylsiloxane, transferring the mold to a 50 mL centrifuge tube, balancing two by two, centrifuging at a speed of 4000 rpm for 12 minutes, taking out the negative mold and curing at room temperature for 6 hours to obtain a microneedle array;

[0023] Preferably, the packaged sensor described in step S3 is obtained by the following steps: applying the photocurable resin to the base of the three microneedle arrays in the negative mold of the microneedle array electrode with a watercolor pen, bending the silver wire into an arc shape to increase the contact surface, connecting it to the microneedle through the photocurable resin, and curing it under a 405nm ultraviolet lamp for 30 seconds, which is carried out three times to ensure that the silver wire is fixed on the base. Finally, pour the photocurable resin on the last layer of the negative mold, and cure it under a 405nm ultraviolet lamp for 30 seconds each to obtain a base that supports the three microneedle arrays. The microneedle array electrode obtained after demolding;

[0024] Preferably, the insulation treatment described in step S4 is obtained by the following steps: coating the area of ​​the microneedle array electrode except the microneedle tips with a small hook pen with a photocurable resin, and curing it under a 405nm ultraviolet lamp for 30 seconds to form an insulation layer on the electrode surface; covering the exposed part of the silver wire with a polyvinyl chloride hose for 2 / 3 of the length, and bonding the silver wire and the catheter with a photocurable resin after curing for 30 seconds under a 405nm ultraviolet lamp, thereby further completing the insulation treatment on the back of the electrode;

[0025] Preferably, the working electrode modification described in step S5 is obtained by the following steps: the microneedle working electrode is placed in 10 mL of a polythiophene mixed solution (0.02 mol / L poly(3,4-ethylenedioxythiophene) and 86.6 mg of sodium citrate, ultrasonicated at 30°C for 30 min), and electroplated at 1.2 V for 600 s to obtain a polythiophene-citrate film; 15 μL of a solution containing 0.2 mol / L EDC and 0.1 mol / L NHS is dripped onto the surface of the polythiophene-citrate modified electrode for 1 h, and then gently washed with ultrapure water; finally, 15 μL of a mixed enzyme solution containing 1.5 U of lactate oxidase and 2.5 mg / mL of gelatin (mixed by volume in a ratio of 1:1) is dripped onto the surface of the modified electrode and incubated at 4°C for 6 h.

[0026] The present invention also protects the use of a microneedle array electrochemical sensor for in-situ detection of lactic acid, a sepsis marker, in lactic acid monitoring. The resin composite material used in the present invention has good biocompatibility, and the mechanical properties of the microneedles are improved by modifying the material to avoid breakage after insertion into the skin, thereby affecting the sensor performance. At the same time, the size and interface shape of the microneedles can be flexibly adjusted to match the dynamic lactic acid detection requirements of different application scenarios, and the electrodes can be modified according to different materials to further improve the response to lactic acid.

[0027] Compared with the prior art, the present invention has the following advantages:

[0028] 1. At present, microneedle array electrodes often use medical acupuncture needles directly as electrodes. The length of medical acupuncture needles is at the millimeter level, which requires professional skills. The length of the microneedle array electrode proposed in the present invention is at the micrometer level, which can ensure that it enters the skin surface. At the same time, by changing the needle length to control the penetration depth to reduce damage, it is convenient for daily use, and can be designed according to various materials and templates to obtain microneedles with different functions and configurations to meet the needs of different detection objects and detection scenarios.

[0029] 2. Currently, microneedle array electrodes are often based on silicon or metal materials. The biocompatibility of the two is poor, and they are prone to breakage during the insertion process, resulting in infection. In addition, the cost of the two is high, and the manufacturing process is complicated. The metallization is also prone to metal layer shedding, and the stability is poor. The present invention uses epoxy resin as the matrix and composite carbon nanotubes. By adjusting the solvent, material ratio, and curing time, microneedles with complete morphology, good mechanical properties, skin puncture properties, and biocompatibility are obtained and directly used as array electrodes. Microneedles with the required performance and safety are prepared under mild conditions using cheap and conventional materials, and their applicability as penetrable skin biosensors is determined. It is cost-effective and can be mass-produced without additional conditions.

[0030] 3. At present, microneedle array electrochemical sensors usually use two steps: interstitial fluid extraction and post-extraction detection, which is complicated and time-consuming. The microneedle sensor proposed in the present invention uses a photocurable resin to insulate and expose the needle tip as a conductive interface, and modifies different materials according to the detected object to further improve the sensitivity. The corresponding biomarker is directly sensed through the conductive tip to achieve in-situ sensing. In-situ monitoring has the advantages of simple operation and short time consumption.

[0031] 4. The present invention introduces polyvinyl pyrrolidone (PVP) with good biocompatibility into the microneedle array electrochemical sensor to wrap the microneedles to avoid wear of the sensor interface and consumption of the modification material layer. When the microneedles pierce the skin and enter the interstitial fluid, the PVP dissolves, and the functionalized microneedle array reacts after contacting the interstitial fluid to generate a response current, thereby protecting the sensor interface.

[0032] 5. Currently, microneedle sensors are combined with a variety of detection methods. The present invention directly uses microneedles with conductive properties as electrodes to construct a conductive system, combines microneedles with traditional electrochemical systems, and combines skin puncture to sense interstitial fluid-related markers and electrochemical measurement of corresponding electrical signals. It has both the low trauma of microneedles and the high sensitivity of electrochemistry, and realizes signal amplification and data visualization, which is used for continuous monitoring of sepsis patients, providing new possibilities for tracking disease progression and reference for drug use. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a schematic diagram of a microneedle array electrochemical sensor for in situ detection of sepsis marker lactate according to the present invention;

[0034] Figure 2 This is a scanning electron microscope characterization image of a conductive microneedle of a microneedle array electrochemical sensor for in situ detection of lactic acid, a sepsis marker, according to the present invention;

[0035] Figure 3 A force-displacement curve of a conductive microneedle of a microneedle array electrochemical sensor for in situ detection of lactic acid, a sepsis marker, according to the present invention;

[0036] Figure 4 A stress-strain curve of a conductive microneedle of a microneedle array electrochemical sensor for in situ detection of lactic acid, a sepsis marker, according to the present invention;

[0037] Figure 5 This is a test diagram of a conductive microneedle sealing membrane of a microneedle array electrochemical sensor for in situ detection of sepsis marker lactate according to the present invention;

[0038] Figure 6 This is a test diagram of the conductive microneedle mouse skin of a microneedle array electrochemical sensor for in situ detection of lactic acid, a sepsis marker, according to the present invention;

[0039] Figure 7 A cell viability diagram of the material used for the microneedles of a microneedle array electrochemical sensor for in situ detection of lactic acid, a sepsis marker, according to the present invention;

[0040] Figure 8 A cyclic voltammogram of a conductive microneedle ferrocyanide electrode solution of a microneedle array electrochemical sensor for in situ detection of sepsis marker lactic acid according to the present invention;

[0041] Fig. 9 It is a time-current diagram of a microneedle array electrochemical sensor for in-situ detection of lactic acid, a sepsis marker, of the present invention, detecting lactic acid of different concentrations using conductive microneedles;

[0042] Fig.10 This is a diagram showing the results of quantitative detection and analysis of lactic acid by conductive microneedles of a microneedle array electrochemical sensor for in situ detection of lactic acid, a sepsis marker, according to the present invention;

[0043] Fig.11 This is a test diagram of a microneedle three-electrode sealing membrane of a microneedle array electrochemical sensor for in situ detection of sepsis marker lactate according to the present invention;

[0044] Fig.12 This is a diagram showing the test analysis results of a microneedle three-electrode sealing membrane of a microneedle array electrochemical sensor for in situ detection of sepsis marker lactate according to the present invention;

[0045] Fig.13 This is a microneedle three-electrode mouse skin test diagram of a microneedle array electrochemical sensor for in situ detection of sepsis marker lactate according to the present invention;

[0046] Fig.14 A cyclic voltammogram of a solution of a microneedle three-electrode ferrocyanide electrode of a microneedle array electrochemical sensor for in situ detection of sepsis marker lactic acid according to the present invention;

[0047] Fig.15 This is a feasibility result diagram of detecting lactic acid using a microneedle array electrochemical sensor for in situ detection of lactic acid, a sepsis marker, according to the present invention;

[0048] Fig.16 This is a scanning electron microscope characterization image of the interface modification of a microneedle array electrochemical sensor for in situ detection of sepsis marker lactate according to the present invention;

[0049] Fig.17 A cyclic voltammetric characterization diagram of a microneedle array electrochemical sensor assembly for in situ detection of sepsis marker lactate according to the present invention;

[0050] Fig.18 This is a diagram showing the specific analysis results of lactic acid detected by a microneedle array electrochemical sensor for in situ detection of lactic acid, a sepsis marker, according to the present invention;

[0051] Fig.19 This is a diagram showing the analysis results of a microneedle array electrochemical sensor for in-situ detection of lactic acid, a sepsis marker, in phosphate buffer for quantitative detection of lactic acid;

[0052] Fig. 20 This is a diagram showing the analysis results of a microneedle array electrochemical sensor for in situ detection of lactic acid, a sepsis marker, in an interstitial fluid skin simulation model for quantitative detection of lactic acid;

[0053] Fig.21 This is a diagram showing the reproducibility analysis results of the microneedle array electrochemical sensor for in-situ detection of lactic acid, a sepsis marker, in the present invention;

[0054] Fig. 22 This is a diagram showing the results of intra-group reproducibility analysis of lactate detected by a microneedle array electrochemical sensor for in situ detection of lactate, a sepsis marker, according to the present invention;

[0055] Fig.23 This is a diagram showing the reproducibility analysis results of lactate detection among groups using a microneedle array electrochemical sensor for in situ detection of lactate, a sepsis marker, according to the present invention;

[0056] Fig.24 This is a diagram showing the reversible analysis results of a microneedle array electrochemical sensor for in-situ detection of lactic acid, a sepsis marker, in a skin simulation model;

[0057] Fig.25 This is a diagram showing the long-term stable analysis results of a microneedle array electrochemical sensor for in-situ detection of lactic acid, a sepsis marker, in a skin simulation model;

[0058] Fig.26 This is a microscopic characterization image of the PVP protective layer of a microneedle array electrochemical sensor for in situ detection of sepsis marker lactate according to the present invention;

[0059] Fig. 27 It is a CV graph of a ferrocyanide electrode pair solution of a microneedle electrochemical sensor based on a PVP protective layer of a microneedle array electrochemical sensor for in situ detection of lactic acid, a sepsis marker, according to the present invention;

[0060] Fig.28 A real-time IT diagram of a lactic acid solution of a microneedle electrochemical sensor based on a PVP protective layer of a microneedle array electrochemical sensor for in situ detection of lactic acid, a sepsis marker, according to the present invention;

[0061] Fig.29 This is an IT comparison diagram of lactic acid detection based on a PVP protective layer and an original microneedle electrochemical sensor of a microneedle array electrochemical sensor for in situ detection of lactic acid, a sepsis marker, according to the present invention;

[0062] Fig.30 A microneedle array electrochemical sensor for in situ detection of lactic acid, a sepsis marker, continuously detects signal changes of different concentrations of lactic acid (0, 0.25, 0.5, 1, 2, 4, 8, 16, 32, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0 mmol / L) in a skin simulation model every minute;

[0063] Fig.31 The present invention discloses a microneedle array electrochemical sensor for in situ detection of lactate, a sepsis marker, which simulates the real-time signal response of lactate changes during the course of disease progression in sepsis patients in a skin simulation model. DETAILED DESCRIPTION

[0064] The following examples are further explanations of the present invention, rather than limitations of the present invention. Unless otherwise defined, all technical terms used hereinafter have the same meaning as those generally understood by those skilled in the art. The technical terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention. Unless otherwise specified, the experimental materials and reagents herein are conventional commercial products in the art.

[0065] A method for preparing a microneedle array electrochemical sensor for in situ detection of sepsis marker lactate comprises the following steps:

[0066] S1. Preparation of conductive material: using epoxy resin as matrix, dichloromethane as solvent, compounding multi-walled carbon nanotubes, and adding epoxy resin curing agent after uniform dispersion to obtain a conductive material mixed solution;

[0067] S2, preparing a microneedle array: filling a conductive material mixture into a microneedle array electrode negative mold made of polydimethylsiloxane, and obtaining a microneedle array electrode after curing;

[0068] S3, packaging electrodes: three array microneedle substrates are energized through wires respectively, the filling resin is cured to obtain a base for receiving three electrodes, and the wiring is arranged, packaged and demolded to obtain the microneedle array electrode;

[0069] S4, insulation treatment: the microneedle array electrode obtained in step S3 is coated with a photocurable resin in the area other than the microneedle tips, and the exposed part of the wire is insulated by a polyvinyl chloride hose through the photocurable resin. After the insulation treatment, three electrodes are obtained, namely, a reference electrode and a counter electrode, wherein the working electrode needs to be modified with lactase;

[0070] S5, preparing a working electrode: sequentially modifying the surface of the microneedle array electrode after the insulation treatment in S4 with polythiophene-sodium citrate, EDC-NHS, lactate oxidase, and gelatin as an enzyme electrode modification layer to obtain a working electrode with an additional enzyme electrode modification layer;

[0071] S6. Prepare a protective layer: fill polyvinyl pyrrolidone into a single array of a microneedle array electrode negative mold made of polydimethylsiloxane by centrifugation, reset the working electrode with the additional enzyme electrode modification layer obtained in S5 in the negative mold, place it in an oven at 40°C for overnight curing, and demold to obtain a microneedle electrochemical sensor with a polyvinyl pyrrolidone protective layer.

[0072] The following embodiment is preferred. In step S1, the conductive material mixture is prepared by the following steps: epoxy resin E51 is used as a matrix, composite multi-walled carbon nanotubes (≥95%, inner diameter: 5-12nm, outer diameter: 30-50nm, length: 10-20um), the mass concentration of multi-walled carbon nanotubes is 15%, dichloromethane is used as a solvent, and the material is obtained by ultrasonic oscillation at a power of more than 95% for 20 minutes, and the temperature is kept below 37°C to ensure that the multi-walled carbon nanotubes in the material are completely dissolved and evenly dispersed in a semi-viscous state. Based on this, epoxy resin curing agent is added, and the mass ratio of epoxy resin is 1:2, and after thorough vortex mixing, ultrasonic oscillation is performed for 5 minutes to obtain a conductive material mixture.

[0073] The following embodiments are preferred, in step S2, the microneedle array electrode is obtained by the following steps: adding the conductive material in a volume of 25 μL three times to the three arrays of the microneedle array electrode negative mold made of polydimethylsiloxane, ensuring that there are no bubbles in the liquid in the mold, and then transferring the negative mold to a 50 mL centrifuge tube (with a wooden column at the bottom), covering the mold cover, balancing the two and centrifuging at 4000 rpm for 12 minutes, taking out the negative mold, adding the conductive material a second time until it is flush with the top of the negative mold, and curing at room temperature for 6 hours to obtain the microneedle array electrode (at this time, the array microneedles corresponding to the three negative molds are directly used as the three electrodes for subsequent work, namely the working electrode, the reference electrode, and the counter electrode. The three electrodes are all insulated, and the working electrode is additionally provided with an enzyme electrode modification layer and a protective layer).

[0074] The packaged sensor described in step S3 is obtained by the following steps: applying the photocurable resin to the base of the three microneedle arrays through a watercolor pen, bending the silver wire to an arc shape to increase the contact area, connecting it to the base of the microneedle array through the photocurable resin, and curing it under a 405nm ultraviolet lamp for 30s to ensure that the silver wire is fixed to the base. Finally, pour the photocurable resin on the last layer of the mold, and cure it up and down under a 405nm ultraviolet lamp for 30s each to obtain a base for the three microneedle electrodes. The microneedle array electrode obtained after demoulding has a base layer of a rectangular parallelepiped with a length of 20mm, a width of 9mm, and a thickness of 2mm; the electrode microneedle part is arranged in 3 3*3 arrays, that is, 3 microneedle array electrodes, and the base of each microneedle array electrode is a rectangular parallelepiped with a length of 4mm, a width of 4mm, and a thickness of 1.5mm, and is 2mm apart from each other; wherein each microneedle is conical, with a needle length of 800μm, a needle bottom diameter of 320μm, and a needle tip distance of 600μm between each microneedle.

[0075] The modification of the working electrode with the additional enzyme electrode modification layer described in step S5 is obtained by the following steps:

[0076] a. Prepare polythiophene mixed solution: 50 mL 0.02 mol / L polythiophene mixed solution (49.893 mL ultrapure water, 0.433 g sodium citrate, 107 μL poly(3,4-ethylenedioxythiophene)), ultrasonicate for 30 min at below 30°C in an ultrasonic machine, and store in a 4°C refrigerator.

[0077] b. Place the microneedle working electrode in 10 mL of polythiophene mixed solution and electrodeposit at 1.2 V for 600 s to obtain a polythiophene-citrate film;

[0078] c. 15 μL of a solution containing 0.2 mol / L EDC and 0.1 mol / L NHS was dripped onto the surface of the polythiophene-citrate film modified electrode, stored at room temperature for 1 h, and then gently washed with ultrapure water;

[0079] d. Finally, 15 μL of a mixed enzyme solution containing 1.5 U of lactate oxidase and 2.5 mg / mL of gelatin was dripped onto the modified electrode surface and incubated at 4°C for 6 h. Finally, the portion not bound to the electrode surface was gently washed away with a phosphate buffer solution.

[0080] Example 1: Preparation of a conductive microneedle

[0081] (1) Using epoxy resin E51 as the matrix, compound multi-walled carbon nanotubes (≥95%, inner diameter: 5-12nm, outer diameter: 30-50nm, length: 10-20μm), the mass concentration of multi-walled carbon nanotubes is 15%, using dichloromethane as the solvent, using 95% or more power ultrasonic oscillation for 30 minutes to obtain the material, keeping the temperature below 37°C to ensure that the multi-walled carbon nanotubes in the material are completely dissolved and evenly dispersed. Based on this, epoxy resin curing agent W93 is added, the mass ratio of epoxy resin curing agent W93 to epoxy resin is 1:2, and vortex mixing is thorough to obtain a conductive material mixture.

[0082] (2) Add the conductive material mixture obtained in step (1) in a volume of 200 μL to the negative mold of the microneedle array electrode made of polydimethylsiloxane, ensuring that there are no bubbles in the liquid in the negative mold, and then transfer the negative mold to a 50 mL centrifuge tube (with a wooden column at the bottom), cover the mold cover, balance the two, and centrifuge at 4000 rpm for 10 min. Remove the mold, add the conductive composite material again until it is flush with the top of the mold, and cure at room temperature for 6 h before demolding to obtain the conductive microneedles (also called microneedle array).

[0083] Example 2: Preparation of a microneedle array electrode

[0084] (1) Based on Example 1, a photocurable resin was applied to the substrate of a microneedle array (also called conductive microneedle) of Example 1 using a liner pen, the silver wire was bent into an arc shape to increase the contact area to adhere to the photocurable resin, and cured under a 405 nm ultraviolet lamp for 15 seconds. The photocurable resin was connected to a microneedle array to ensure that the silver wire was fixed to the substrate, thereby obtaining a microneedle array electrode;

[0085] (2) coating the microneedle array electrode obtained in step (1) with a photocurable resin except for the microneedle tips, and curing it under a 405 nm ultraviolet lamp until the conductive pen does not emit light when in contact with it; covering the exposed silver wire with a polyvinyl chloride hose by 2 / 3, and curing the photocurable resin under a 405 nm ultraviolet lamp for 15 seconds for insulation treatment to obtain a microneedle array electrode.

[0086] Example 3: Preparation of a microneedle electrochemical sensor for lactic acid detection

[0087] (1) Prepare a polythiophene mixed solution: 50 mL of a 0.02 mol / L polythiophene mixed solution (49.893 mL of ultrapure water, 0.433 g of sodium citrate, and 107 μL of poly(3,4-ethylenedioxythiophene)), ultrasonicate the solution in an ultrasonic machine at a temperature below 30° C. for 40 min, and store the solution at 4° C.

[0088] (2) The microneedle working electrode obtained in Example 2 was placed in 10 mL of the polythiophene mixed solution prepared in step (1) and electrodeposited at 1.2 V using the time-current method for 600 s, then washed with ultrapure water and dried with nitrogen to obtain a polythiophene-citrate modified electrode;

[0089] (3) dripping 25 μL of a solution containing 0.2 mol / L EDC and 0.1 mol / L NHS onto the surface of the polythiophene-citrate modified electrode obtained in step (2), storing it at room temperature for 1 h, and then gently washing it with ultrapure water to obtain a modified electrode;

[0090] (4) Finally, 25 μL of a mixed enzyme solution containing 1.5 U of lactate oxidase and 2.5 mg / mL of gelatin was dripped onto the modified electrode surface obtained in step (3), and incubated at 4°C for 6 h. Finally, a phosphate buffer solution was gently washed to remove the portion not bound to the electrode surface, thereby obtaining a working electrode with an additional enzyme electrode modification layer (i.e., a microneedle array electrochemical sensor for lactate detection).

[0091] Example 4: Preparation of a microneedle array electrochemical sensor for in situ detection of sepsis marker lactate ( Figure 1 Schematic diagram of the preparation of the microneedle electrochemical sensor)

[0092] (1) Using epoxy resin E51 as the matrix, composite multi-walled carbon nanotubes (purity ≥ 95%, inner diameter: 5-12nm, outer diameter: 30-50nm, length: 10-20μm), the mass concentration of multi-walled carbon nanotubes is 15%, using dichloromethane as the solvent, using 95% or more power ultrasonic oscillation for 30 minutes to obtain the material, keeping the temperature below 37°C to ensure that the multi-walled carbon nanotubes in the material are completely dissolved and evenly dispersed. Based on this, epoxy resin curing agent W93 is added, 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 composite material (at this time it is in a mixed liquid state);

[0093] (2) Add the conductive composite material in a volume of 25 μL three times to three arrays of the microneedle array electrode negative mold made of polydimethylsiloxane, ensuring that there are no bubbles in the liquid in the negative mold, and then transfer the negative mold to a 50 mL centrifuge tube (with a wooden column at the bottom), cover the mold cover, balance the two at a speed of 4000 rpm for 10 minutes, remove the mold, add the conductive material again until it is flush with the top of the groove, and cure at room temperature for 6 hours to obtain three microneedle arrays, also called three conductive microneedles;

[0094] (3) Use a watercolor pen to apply photocurable resin to the bases of the three microneedle arrays in the negative mold of the microneedle array electrode. Bend the silver wire into an arc shape to increase the contact surface, and connect it to the three microneedle arrays through the photocurable resin. Curing it under a 405nm ultraviolet lamp for 30 seconds, divided into three times, ensure that the silver wire is fixed to the base of the microneedle array. Finally, pour the photocurable resin on the top layer of the negative mold, and cure it under a 405nm ultraviolet lamp for 30 seconds each to obtain a base for the three microneedle arrays. After demolding, the microneedle array electrode is obtained; the array microneedles corresponding to the three negative molds are directly used as the three electrodes for subsequent work, namely the working electrode, the reference electrode, and the counter electrode. The three electrodes are all insulated, and the working electrode is subsequently additionally provided with an enzyme electrode modification layer and a protective layer;

[0095] (4) The microneedle array electrode obtained in step (3) is completely coated with a photocurable resin in the area except the microneedle tips, and cured under a 405nm ultraviolet lamp for 60s to form an insulating layer. The exposed part of the silver wire connected to the base of the microneedle array electrode is covered with a polyvinyl chloride hose, and the photocurable resin is cured under a 405nm ultraviolet lamp for 30s to perform insulation treatment to obtain a microneedle array electrode with an insulating layer;

[0096] (5) Functional modification of the working electrode

[0097] a. Prepare a polythiophene mixed solution: 50 mL of 0.02 mol / L polythiophene mixed solution (49.893 mL of ultrapure water, 0.433 g of sodium citrate, 107 μL of poly(3,4-ethylenedioxythiophene)), ultrasonicate for 30 min at a temperature below 30°C in an ultrasonic machine to obtain a polythiophene mixed solution, and store it in a refrigerator at 4°C.

[0098] b. The microneedle working electrode with the insulating layer obtained in step (4) was placed in 10 mL of the polythiophene mixed solution obtained in step a, and electrodeposited for 600 s at 1.2 V using the time-current method to obtain a polythiophene-citrate modified electrode;

[0099] c. 15 μL of a solution containing 0.2 mol / L EDC and 0.1 mol / L NHS was dripped onto the surface of the polythiophene-citrate film modified electrode, stored at room temperature for 1 h, and then gently washed with ultrapure water to obtain a modified electrode;

[0100] d. Lactate oxidase (3 U of lactate oxidase dissolved in 15 μL phosphate buffer solution) and gelatin (2.5 mg / mL) were mixed in a volume ratio of 1:1, and finally 15 μL of 1.5 U lactate oxidase-gelatin mixture was dripped on the surface of the modified electrode obtained in step c, and incubated at 4°C for 6 h. Finally, the phosphate buffer solution was used to gently wash away the enzyme that was not bound to the electrode surface to obtain a working electrode with an additional enzyme electrode modification layer (i.e., a microneedle array electrochemical sensor for lactate detection).

[0101] (6) Filling polyvinyl pyrrolidone into a single array of a microneedle array electrode negative mold made of polydimethylsiloxane by centrifugation, resetting the microneedle array electrode obtained in step (5) in the negative mold, curing in an oven at 40° C. overnight, and demolding to obtain a microneedle electrochemical sensor with a polyvinyl pyrrolidone protective layer.

[0102] Effect verification

[0103] Observe the surface morphology of the conductive microneedles (also called microneedle arrays) prepared in Example 1

[0104] The prepared conductive microneedles were placed under a scanning electron microscope to observe the surface morphology of the microneedles, including the shape, height, bottom diameter, and spacing of the microneedles. At the same time, the scanning electron microscope was used to determine whether the prepared microneedle array was complete and whether the needle tip was broken. Figure 2 As shown, Figure A is a top view of a 10*10 array, Figure B is a top view of a 2*2 array, and Figure C is a top view of a single microneedle; Figure D is a side view of a 10*10 array, Figure E is a side view of a 2*2 array, and Figure F is a side view of a single microneedle. The microneedles are all conical in shape, each needle is 800μm high, the bottom diameter is 320μm, and the distance between the needle tips is 600μm; the microneedle array is intact and there is no breakage.

[0105] Mechanical properties of the conductive microneedles (also called microneedle arrays) prepared in Example 1 were characterized

[0106] The prepared conductive microneedle is placed under an electronic universal testing machine, with the tip of the microneedle facing upward. The machine moves downward at a speed of 0.1mm / min. After the machine touches the microneedle, it continues to move downward, applying a certain force to the microneedle. At the same time, the instrument records the force and displacement of the microneedle. When the applied pressure reaches the maximum value of 20N, the data is stopped, and the instrument stops moving downward to obtain the force-displacement curve. At the same time, under the same conditions, the mechanical properties of the microneedle are tested when it is compressed and deformed until 80% deformation occurs. Figure 3 As shown in Figure 2, the microneedles can withstand forces greater than 7 N / needle without breaking, which is greater than the commonly reported breaking force of 0.3-0.4 N / needle. Figure 4 As shown, the microneedle has no obvious breaking point after 80% deformation, which satisfies the force required for the microneedle to enter the interstitial fluid in the body for detection.

[0107] The skin puncture performance of the conductive microneedles (also called microneedle arrays) prepared in Example 1 was characterized

[0108] In order to test whether the conductive microneedles can pierce the skin, the puncture performance was tested using parafilm and mouse skin. Figure 5 , Figure 6 The test diagram of the conductive microneedle of the present invention using sealing film and mouse skin is shown in FIG. Figure 5 As shown in the figure, the first three layers of sealing film all have 10×10 array holes, and each layer of sealing film is about 126μm. The results show that the conductive microneedle can pierce through the three layers of sealing film, proving that the penetration depth of the conductive microneedle is about 400μm. Figure 6 As shown in the figure, after the conductive microneedle was inserted into the mouse skin for 5 minutes and then pulled out, a complete 10×10 micropore array was visible in the mouse skin, and these micropores generated by the conductive microneedle were almost re-sealed within 20 minutes, returning to their original state, with little damage to the skin. Therefore, the conductive microneedle can puncture the skin, contact the interstitial fluid, and realize the continuous monitoring of human lactate.

[0109] Biocompatibility characterization of the materials used in the conductive microneedles (also called microneedle arrays) prepared in Example 1

[0110] Mouse embryonic osteoblasts were seeded in 96-well plates, with 100 μL per well, and each well contained approximately 1.0×10 4Cells were placed in a cell culture incubator at 37°C and 5% carbon dioxide for 24 hours. After the cells adhered to the wall and grew, different materials were added (epoxy resin-multi-walled carbon nanotubes-dichloromethane, epoxy resin-multi-walled carbon nanotubes-dichloromethane / photocuring resin, epoxy resin-multi-walled carbon nanotubes-dichloromethane / photocuring resin / polyvinyl pyrrolidone, polyvinyl pyrrolidone). Three replicate wells were set for each material, and cells cultured in high-glucose medium without any materials were used as a blank control group. After 24 hours of cell culture, 10 μL of cell viability detection reagent was added, and the 96-well plate was gently shaken to mix the solution evenly, then placed in the incubator for another 3 hours. Finally, the absorbance of each well was measured with an enzyme-labeled instrument at a wavelength of 450nm, and the cell activity of the experimental group was calculated. Figure 7 As shown, the cell activity of each group was greater than 90%, and the cytotoxicity level was level 1, indicating that the materials used to prepare the conductive microneedles were almost non-toxic and had good biocompatibility, providing a safety reference for the conductive microneedles to enter the interstitial fluid in the body for lactic acid detection.

[0111] Electrochemical performance characterization of the microneedle array electrode prepared in Example 2

[0112] The microneedle array electrode was placed in a 10mmol / L potassium ferrocyanide, 0.1mmol / L potassium chloride solution system, with the microneedle array electrode as the working electrode, the silver / silver chloride reference electrode, and the platinum counter electrode. The cyclic voltammetry method in the electrochemical workstation (initial potential of -0.5V, end point potential of 0.8V, scan rate of 0.1V / s, sampling interval of 0.001V) was selected to verify the redox performance of the microneedle array electrode. Figure 8 The microneedle array electrode has good electrochemical properties, with an oxidation potential of about 0.37 V, a reduction potential of about 0.13 V, a potential difference of about 0.24 V, and redox peak currents tending to be symmetrical, with the peak current values ​​being basically the same.

[0113] Performance characterization of the microneedle electrochemical sensor for lactic acid detection prepared in Example 3

[0114] L-lactic acid was dissolved in phosphate buffer (0.01mol / L, pH 7.4) to prepare a phosphate buffer of lactic acid. The microneedle electrochemical sensor prepared in Example 3 (i.e., the working electrode with an additional enzyme electrode modification layer) was tested in blank phosphate buffer and lactic acid solution for electrochemical characterization. Cyclic voltammetry showed that the microneedle electrochemical sensor of the present invention had a peak current response to hydrogen peroxide produced by lactic acid catalyzed lactate oxidase, and the peak position was about 0.7V. With 0.7V as the detection potential, the time current method was used to detect the real-time current of lactic acid solutions of different concentrations. After 200s, when the blank phosphate buffer signal was stable, different concentrations of lactic acid were added in sequence every 100s, and the final concentrations were 0.5mmol / L, 1.0mmol / L, 2mmol / L, 4mmol / L, 8mmol / L, and 16mmol / L, respectively. Fig. 9 It shows that the current changes obviously and rapidly each time lactate is added, and the current increases with the increase of lactate concentration. Fig.10 It is shown that in the range of 0.5mmol / L to 16mmol / L, there is a good linear relationship between the current difference and the lactic acid concentration. The above results prove that the sensor of the present invention has a good real-time response to the detection of lactic acid.

[0115] The microneedle array electrochemical sensor prepared in Example 4 was used for lactic acid detection

[0116] (1) Due to the change in the array, the microneedle three electrodes (i.e., the microneedle array electrode obtained in Example 4, i.e., the working electrode, the reference electrode, and the counter electrode) were also tested for skin puncture performance through sealing film and mouse skin. Fig.11 , Fig.12 It shows that three holes of 3×3 array appear in the first three layers of sealing film. The microneedle array electrode obtained in Example 4 can completely pierce the three layers of sealing film, the piercing depth is about 400μm, and the piercing rate reaches 100%. At the same time, Fig.13 The results show that after the microneedle array electrode obtained in Example 4 pierced the mouse skin, three complete 3×3 micropore arrays were visible in the mouse skin, and these micropores generated by the microneedle array electrode obtained in Example 4 were almost re-sealed within 20 minutes and restored to the original state, with little damage to the skin. Therefore, the microneedle three-electrode can also pierce the skin, contact the interstitial fluid, and realize continuous monitoring of human lactate.

[0117] (2) The microneedle three electrodes were placed in a 10 mmol / L potassium ferrocyanide, 0.1 mmol / L potassium chloride solution system, and the cyclic voltammetry method (initial potential of -0.5 V, end point potential of 0.8 V, scan rate of 0.1 V / s, sampling interval of 0.001 V) in the electrochemical workstation was used to verify the redox performance of the microneedle three electrodes. Fig.14As shown, the microneedle array electrode obtained in Example 4 has good electrochemical performance, with an oxidation potential of about 0.15 V, a reduction potential of about -0.1 V, a potential difference of about 0.25 V, and redox peak currents tending to be symmetrical, with substantially the same peak current values.

[0118] (3) L-lactic acid was dissolved in phosphate buffer (0.01 mol / L, pH 7.4) to prepare a lactic acid solution. The cyclic voltammetry results showed that the microneedle electrochemical sensor of the present invention (microneedle array electrochemical sensor for lactic acid detection obtained in Example 4) had a peak current response to hydrogen peroxide produced by lactic acid catalyzed by lactate oxidase, and the peak position was about 0.7 V. With 0.7 V as the detection potential, the lactic acid solution and the blank phosphate solution were detected by the time current method. Fig.15 As shown, when the lactic acid concentration was 4 mmol / L, the difference between the experimental group and the control group was △I = 17.26 μA. The results showed that the lactate oxidase-gelatin / polythiophene-citrate / epoxy resin-multi-walled carbon nanotube-dichloromethane microneedle array electrochemical sensor of the present invention is feasible for detecting lactic acid.

[0119] (4) Fig.16 These are SEM characterization images of microneedle array electrochemical sensors. Figure A is a SEM image of epoxy resin-multi-walled carbon nanotube-dichloromethane microneedle array electrode, with a rough surface and clearly visible tubular structure of multi-walled carbon nanotubes, and obvious pores; Figure B is a SEM image of polythiophene-citrate / epoxy resin-multi-walled carbon nanotube-dichloromethane microneedle array electrode, with a layer of smooth film with fine particles dispersed on the surface of the microneedle array electrode, so that the rough protrusions of the bottom layer are covered, but still retain a certain porous structure; Figure C is a SEM image of gelatin / polythiophene-citrate / epoxy resin-multi-walled carbon nanotube Figure 1 is a lactate oxidase-gelatin / polythiophene-citrate / epoxy resin-multi-walled carbon nanotube-dichloromethane microneedle array electrode, showing that gelatin makes the electrode surface blurred; Figure D is a lactate oxidase-gelatin / polythiophene-citrate / epoxy resin-multi-walled carbon nanotube-dichloromethane microneedle array electrode, in which the lactate oxidase protein is dispersed on the polythiophene film, and the cross-linking adsorption of the enzyme further covers the porous structure. At the same time, a membrane structure is formed on the electrode surface due to the presence of gelatin. The results show that the lactate oxidase-gelatin / polythiophene-citrate / epoxy resin-multi-walled carbon nanotube-dichloromethane microneedle array electrode is successfully assembled.

[0120] (5) Fig.17The CV characterization diagram of the assembly process of the microneedle array electrochemical sensor for lactic acid detection. When polythiophene-citrate is electrodeposited on the microneedle electrode, polythiophene has good conductivity and the current value increases. Since lactate oxidase has good catalytic activity for lactic acid, the current value increases further, but the current difference is low. When lactate oxidase is bound to the electrode surface through the EDC-NHS chemical bond, the current value is significantly improved. The combination of physical adsorption of gelatin and EDC-NHS chemical cross-linking fixes lactate oxidase on the surface of the microneedle electrode modified with polythiophene-citrate, and the current value reaches the maximum. At the same time, the modification of gelatin to the polythiophene-citrate surface has no change in the current value. Therefore, the CV characterization results of these different modified electrodes prove that the electrochemical biosensor is successfully assembled.

[0121] (6) Fig.18 It was further investigated whether the presence of 20mmol / L glucose, 1mmol / L ascorbic acid, 1mmol / L uric acid, 1mmol / L urea, 0.2mmol / L dopamine, and 0.2mmol / L cysteine ​​had an effect on the detection of the constructed microneedle electrochemical sensor (microneedle array electrochemical sensor for lactic acid detection obtained in Example 4). The results showed that the current signals of glucose, ascorbic acid, urea, urea, dopamine, and cysteine ​​were all close to the background value, and the current difference ΔI was small, which was significantly different from the current signal of lactic acid, indicating that the microneedle electrochemical sensor for lactic acid detection of the present invention has good specificity.

[0122] (7) Dissolve 140 mg of agarose powder in artificial interstitial fluid (107.7 mmol / L NaCl, 3.5 mmol / L KCl, 1.5 mmol / L CaCl2, 0.7 mmol / LMgSO4, 26.2 mmol / LNaHCO3, 1.7 mmol / L Na3PO4, 9.6 mmol / L glucose, 10 mg / mL BSA), stir continuously at 120°C, heat until the liquid is uniform and transparent, pour the solution into a culture dish to solidify it, inject different concentrations of lactic acid into the agarose gel, diffuse into the gel at room temperature for 60 minutes, and cover with a layer of sealing film after overnight storage in a refrigerator at 4°C as an interstitial fluid skin simulation model. The relationship between lactic acid concentration and the detected electrical signal value in PBS solution and interstitial fluid skin simulation model was investigated respectively. Fig.19 In the range of 0.25mmol / L to 32mmol / L, the current difference ΔI detected in the PBS solution has a good linear relationship with the lactic acid concentration, ΔI=3.0690C+6.2645, and the detection limit is 0.1172mmol / L. Fig. 20It shows that the current difference ΔI detected by the interstitial fluid skin simulation model also has a good linear relationship with the lactic acid concentration, ΔI=1.6012C+1.4672, with a detection limit of 0.0319mmol / L. The linear range and detection limit are basically consistent with the detection results of PBS solution.

[0123] (8) Fig.21 The same microneedle array electrode (microneedle array electrochemical sensor for lactic acid detection obtained in Example 4) was tested five times. Fig. 22 Select five microneedle array electrochemical sensors from the same batch but obtained from different Examples 4, Fig.23 Five different batches and different microneedle array electrochemical sensors were selected to detect 4mmol / L lactic acid samples. The results showed that the intra-chip RSD was 1.17%, the intra-group RSD was 4.44%, and the inter-group RSD was 4.67%, indicating that the microneedle array electrochemical biosensor for lactic acid detection of the present invention has good stability.

[0124] (9) The microneedle array electrochemical sensor for lactic acid detection obtained in Example 4 was repeatedly inserted into skin simulation models with different concentrations for 5 times, with 2 mmol / L lactic acid and 4 mmol / L lactic acid as the two concentrations of skin simulation models. Fig.24 It shows that inserting different concentrations of skin models can restore the signal value to the corresponding concentration, and the signal reduction is not significant, which has good reversibility.

[0125] (10) The microneedle array electrochemical sensor for lactic acid detection obtained in Example 4 was repeatedly inserted into the skin simulation model at different time points of 0, 1, 2, 4, 6, 18, and 30 h to measure and compare the response of lactic acid. Fig.25 The response was shown to remain relatively constant (<10% variation), indicating that the microneedle array electrochemical sensor for lactate detection has good long-term stability.

[0126] Protective layer verification of a microneedle array electrochemical sensor for in situ detection of sepsis marker lactate prepared in Example 4

[0127] The overall signal reduction of the microneedle array electrochemical sensor in the skin model is not only due to the fact that the diffusion of lactic acid in the hydrogel matrix is ​​hindered, but also due to the fact that the microneedles of the microneedle array electrochemical sensor for lactic acid detection obtained in Example 4 are greatly affected by the wear of the sensing interface when piercing the skin model. Therefore, the PVP protective layer is introduced into the microneedle array electrochemical sensor for further verification. From the morphological analysis, as shown in FIG. Fig.26As shown, the microneedle without core-shell structure is shown in A, while the microneedle based on PVP protective layer is higher and wider than the original microneedle. On the basis of retaining the original microneedle, it has an additional transparent shell with microneedle morphology, as shown in B. When it enters the skin model, it gradually dissolves, the shell almost disappears, and the microneedle returns to its original state, as shown in C, finally exposing the interface required for microneedle detection.

[0128] From electrochemical analysis, such as Fig. 27 As shown in the figure, the microneedle array electrode based on the PVP protective layer in the ferrocyanide electrode solution went from the initial insulating state, that is, no electrochemical signal, to the gradual dissolution current response. In 10 minutes, it had basically recovered to the peak current value of the microneedle array electrode without the PVP protective layer. In addition, when the polyvinyl pyrrolidone / lactate oxidase-gelatin / polythiophene-citrate / epoxy resin-multi-walled carbon nanotubes-dichloromethane microneedle array electrochemical sensor was applied to the skin model, the real-time current response to lactic acid also appeared from nothing to something. When the microneedle electrochemical sensor with the corresponding PVP protective layer punctured the skin, the PVP gradually dissolved and the lactic acid sensing interface was gradually exposed, until it was infinitely close to the electrochemical signal in the solution. Fig.28 , Fig.29 It is also proved that when applied to lactate detection, the ΔI obtained by the polyvinyl pyrrolidone / lactate oxidase-gelatin / polythiophene-citrate / epoxy resin-multi-walled carbon nanotube-dichloromethane microneedle array electrochemical sensor in the skin model is 17.92μA, which is greater than the ΔI (10.12μA) of the original lactate oxidase-gelatin / polythiophene-citrate / epoxy resin-multi-walled carbon nanotube-dichloromethane microneedle array electrochemical sensor in the skin model, and is close to the lactate signal value ΔI (19.98μA) obtained in the solution.

[0129] Therefore, the present invention introduces a polyvinyl pyrrolidone (PVP) protective layer into the microneedle array electrochemical sensor to maximize the reduction of the response of the sensing interface to lactic acid. At the same time, it has been verified in the previous article that polyvinyl pyrrolidone has good biocompatibility. Therefore, the microneedle array electrochemical sensor based on the PVP core-shell structure can complete the detection of interstitial fluid lactic acid by puncturing the skin.

[0130] The microneedle array electrochemical sensor for in situ detection of sepsis marker lactate prepared in Example 4 was verified in a skin simulation model

[0131] Since the concentration of lactic acid in the skin is in a dynamic change process, the microneedle array electrochemical sensor is sequentially inserted into a skin simulation model filled with different concentrations of lactic acid, and the lactic acid signal is continuously detected every one minute. Fig.30The results show that the polyvinyl pyrrolidone / lactate oxidase-gelatin / polythiophene-citrate / epoxy resin-multi-walled carbon nanotubes-dichloromethane microneedle array electrochemical sensor can meet the detection of lactic acid from low concentration to high concentration (0, 0.25, 0.5, 1, 2, 4, 8, 16, 32 mmol / L), and the signal gradually increases; it can also meet the detection of lactic acid from high concentration to low concentration (32, 16, 8, 4, 2, 1, 0.5, 0.25, 0 mmol / L), and the signal gradually decreases; that is, the response current signal tends to be consistent with the change of lactic acid concentration, and in the rising and falling stages, the current signals of the same lactic acid concentration are basically the same, and the difference can be ignored, indicating that the electrochemical sensor of the microneedle array has the ability to continuously monitor lactic acid in a skin simulation model, which is helpful to continuously and real-time respond to lactic acid signals in the skin.

[0132] Since lactate concentration is related to the progression of sepsis, with blood lactate level as a reference, when the lactate concentration is 0.5-1.5mmol / L, sepsis is in the early stage of development; when the lactate concentration increases to 1.5mmol / L, sepsis enters the acute phase, which is an important early warning signal; when the blood lactate level is >2mmol / L, it is used to identify sepsis patients with a high risk of death. It is an important indicator of septic shock and requires antibiotics. At the same time, the lactate level in the interstitial fluid is slightly higher than the blood lactate level. Therefore, the polyvinyl pyrrolidone / lactate oxidase-gelatin / polythiophene-citrate / epoxy resin-multi-walled carbon nanotubes-dichloromethane microneedle array electrochemical sensor was first inserted into a skin model filled with 0.5mmol / L lactic acid, and then a low volume of high concentration lactic acid was added to make the final concentrations reach 1.5mmol / L, 2mmol / L, and 4mmol / L, respectively, and then most of the lactate in the lactate oxidase consumption model was added to simulate drug treatment for real-time current detection. Fig.31 As shown, the signal of the microneedle array electrochemical sensor is low at 0.5mmol / L, and gradually increases at 1.5mmol / L, 2mmol / L, and 4mmol / L. After adding lactate oxidase, the concentration is further reduced to a level close to the initial 0.5mmol / L lactic acid signal. Therefore, the microneedle array electrochemical sensor for in situ detection of sepsis marker lactate of the present invention can achieve continuous monitoring in a skin simulation model, and preliminarily meet the needs of sepsis course monitoring.

Claims

1. A microneedle array electrochemical sensor for in situ detection of sepsis marker lactate, comprising a working electrode, a reference electrode and a counter electrode arranged on a microneedle portion of a base, characterized in that: The working electrode, reference electrode and counter electrode are conductive layers made of resin as a matrix and composite nanomaterials as conductive materials, which are electrically connected through wires and insulated by conduits. The surfaces of the working electrode, reference electrode and counter electrode are all provided with electrode insulating layers, wherein the working electrode is additionally provided with an enzyme electrode modification layer and a protective layer.

2. The microneedle array electrochemical sensor according to claim 1, characterized in that: The base is made of photocurable resin, the conductive layer is obtained by curing a conductive material mixture made of epoxy resin as a matrix, multi-walled carbon nanotubes as conductive nanomaterials, and dichloromethane as a dispersing solvent, the energized wire is a silver wire, connected to the electrode via the photocurable resin, and insulated with a polyvinyl chloride hose; the electrode insulation layer is a photocurable resin layer, and the protective layer is a polyvinyl pyrrolidone layer. The enzyme electrode modification layer is prepared by the following method: firstly, a polythiophene-citrate film is modified, and a carbodiimide condensation method is used to fix lactate oxidase to a working electrode through chemical crosslinking combined with physical adsorption of gelatin.

3. The microneedle array electrochemical sensor according to claim 1, characterized in that: The base is a rectangular parallelepiped with a length of 20 mm, a width of 9 mm, and a thickness of 2 mm; the microneedle part of the electrode is arranged in three 3*3 arrays, namely three microneedle array electrodes, and the base of each microneedle array electrode is a rectangular parallelepiped with a length of 4 mm, a width of 4 mm, and a thickness of 1.5 mm, and the microneedle array electrodes are spaced 2 mm apart from each other; the microneedles of each microneedle array electrode are all conical, with a needle length of 800 μm, a needle base diameter of 320 μm, and a needle tip distance of 600 μm between each microneedle.

4. The method for preparing a microneedle array electrochemical sensor for in situ detection of sepsis marker lactate according to claim 1, characterized in that: The steps include: S1. Preparation of conductive material: using epoxy resin as matrix, dichloromethane as solvent, compounding multi-walled carbon nanotubes, and adding epoxy resin curing agent after uniform dispersion to obtain a conductive material mixed solution; S2, preparing a microneedle array: filling the conductive material mixture prepared in step S1 into a microneedle array electrode negative mold made of polydimethylsiloxane, and curing to obtain a microneedle array, and a total of 3 microneedle arrays are prepared; S3, packaging electrodes: the bases of the three microneedle arrays are energized through wires respectively, the bases that receive the three microneedle arrays are obtained by filling and curing the resin, and the microneedle array electrodes are obtained by wiring, packaging and demolding; S4, insulation treatment: the microneedle array electrode obtained in step S3 is coated with a photocurable resin in the area other than the microneedle tips, and the exposed part of the wire is insulated by a polyvinyl chloride hose through the photocurable resin, and insulation treatment is performed to obtain three electrodes, namely, a working electrode, a reference electrode and a counter electrode, wherein the working electrode needs to be modified with lactase; S5, preparing a working electrode: sequentially modifying the surface of the microneedle array electrode after the insulation treatment in step S4 with polythiophene-citrate, EDC-NHS, lactate oxidase, and gelatin as an enzyme electrode modification layer to obtain a working electrode with an additional enzyme electrode modification layer; S6. Prepare a protective layer: Fill polyvinyl pyrrolidone into a single array of a microneedle array electrode negative mold made of polydimethylsiloxane by centrifugation, reset the working electrode with the additional enzyme electrode modification layer obtained in step S5 in the negative mold, place it in an oven at 40°C for overnight curing, and demold to obtain a microneedle array electrochemical sensor with a polyvinyl pyrrolidone protective layer.

5. The preparation method according to claim 4, characterized in that: The conductive material mixed liquid described in step S1 is prepared by the following steps: epoxy resin E51 is used as a matrix, composite multi-walled carbon nanotubes, the mass concentration of the multi-walled carbon nanotubes is 15%, and dichloromethane is used as a dispersing solvent. Ultrasonic oscillation with a power of more than 95% is used for 20 minutes to obtain the material, ensuring that the multi-walled carbon nanotubes in the material are completely dissolved and evenly dispersed in a semi-viscous state. Based on this, epoxy resin curing agent is added, and the mass ratio of epoxy resin curing agent to epoxy resin is 1:

2. After thorough vortex mixing, ultrasonic oscillation is performed for 5 minutes to obtain a conductive material mixed liquid.

6. The preparation method according to claim 4, characterized in that: The microneedle array described in step S2 is obtained by the following steps: transferring the microneedle array electrode negative mold made of polydimethylsiloxane to a 50 mL centrifuge tube, adding the conductive material to the three arrays of the microneedle array electrode negative mold in a volume of 25 μL three times, covering with a lid made of polydimethylsiloxane, balancing two by two, centrifuging at 4000 rpm for 12 min, taking out the negative mold and curing at room temperature for 6 h to obtain the microneedle array.

7. The preparation method according to claim 4, characterized in that: The packaged sensor described in step S3 is obtained by the following steps: applying photocurable resin to the bases of the three microneedle arrays in the negative mold of the microneedle array electrode with a watercolor pen, bending the silver wire into an arc shape to increase the contact surface, connecting it to the base of the microneedle array through the photocurable resin, and curing it under a 405 nm ultraviolet lamp for 30 seconds, which is performed three times to ensure that the silver wire is fixed on the base; finally, pouring photocurable resin into the last layer of the negative mold, curing it under a 405 nm ultraviolet lamp for 30 seconds each, obtaining a base for receiving the three microneedle arrays, and obtaining the microneedle array electrode after demolding.

8. The preparation method according to claim 4, characterized in that: The insulation treatment described in step S4 is obtained by the following steps: the area of ​​the microneedle array electrode except the microneedle tip is coated with a photocurable resin using a small hook pen, and cured under a 405 nm ultraviolet lamp for 30 seconds to form an insulating layer on the surface of the microneedle array electrode; the exposed part of the silver wire is covered with a polyvinyl chloride hose, and the silver wire and the hose are bonded after being cured under a 405 nm ultraviolet lamp for 30 seconds, thereby further completing the insulation treatment on the back of the electrode.

9. The preparation method according to claim 4, characterized in that: The modification of the working electrode of the additional enzyme electrode modification layer described in step S5 is obtained by the following steps: 10 mL of 0.02 mol / L poly(3,4-ethylenedioxythiophene) solution and 86.6 mg of sodium citrate are ultrasonicated at 30°C for 30 min, and the microneedle working electrode is electrodeposited in the solution at 1.2 V for 600 s to obtain a polythiophene-citrate modified electrode; 15 μL of a solution containing 0.2 mol / L EDC and 0.1 mol / L NHS was dripped onto the surface of the polythiophene-citrate film modified electrode for 1 h, and then gently washed with ultrapure water; finally, 15 μL of a mixed enzyme solution containing 1.5 U lactate oxidase and 2.5 mg / mL gelatin was dripped onto the surface of the modified electrode and incubated at 4°C for 6 h to obtain a working electrode with an additional enzyme electrode modification layer.

10. The microneedle array electrochemical sensor for in situ detection of sepsis marker lactate prepared by the preparation method according to any one of claims 4 to 9 is used for lactate detection, characterized in that: The microneedle array electrochemical sensor for in situ detection of sepsis marker lactate prepared by the preparation method described in any one of claims 4 to 9 is placed in a lactic acid solution prepared with a 10 mmol / L pH 7.4 phosphate buffer solution, and the peak position of hydrogen peroxide produced by lactate catalyzed by lactate oxidase is determined by cyclic voltammetry. Using this as the detection potential, electrochemical detection is performed using the time current method, and the changes in electrical signals are recorded.

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