An integrated interstitial fluid sampling and electrochemical detection system, and methods of making and using the same

Abstract

The application belongs to the technical field of sensor preparation, and particularly relates to an integrated interstitial fluid sampling and electrochemical detection system and a preparation method and application thereof. The integrated interstitial fluid sampling and electrochemical detection system comprises a microneedle array unit, an electrochemical sensor and a micro negative pressure pump. The microneedle array unit has good biocompatibility, is safe and reliable, and will not damage blood vessels and nerves of a patient; the microneedle is short and small, has weak discomfort, does not affect normal activities of a user, and is more conducive to continuous long-term monitoring; the electrochemical sensor can accurately identify a template molecule and transmit a signal; and the micro negative pressure pump can generate vacuum pressure to collect a sample amount meeting detection. The integrated interstitial fluid sampling and electrochemical detection system can be used by being inserted into a subcutaneous or intradermal position of a human body, can realize real-time and integrated sampling-detection, and can detect various biomarkers.

Description

Technical Field

[0001] This invention belongs to the field of sensor fabrication technology, specifically relating to an integrated interstitial fluid sampling and electrochemical detection system and its fabrication method. Background Technology

[0002] Biomarkers (such as glucose, β-hydroxybutyrate, and lactate) are key indicators reflecting the body's physiological metabolic state and disease-related changes. Their accurate detection is of great significance in health monitoring, disease early warning, and metabolic status assessment. Interstitial fluid (ISF), as an important carrier of biomarkers, accounts for approximately 15% of the total body fluids and directly participates in cellular metabolism and substance exchange. It is rich in various biomarkers closely related to the body's state; therefore, the detection of biomarkers using ISF as a sample has become a research hotspot in the biomedical field.

[0003] Traditional biomarker detection methods (such as microdialysis, open-flow microperfusion, biopsy sampling, and reverse iontophoresis) are either highly invasive, prone to causing tissue damage / bleeding, and causing pain, or suffer from sample dilution and distorted results due to the use of perfusion solutions, or have limited applicability (e.g., unable to detect small molecule biomarkers), require frequent calibration, and have extremely poor user compliance. Traditional biomarker detection requires separate steps for sampling, sample transfer, and laboratory testing, which is redundant and prone to sample contamination and loss. It cannot achieve real-time detection and fails to meet the needs of dynamic monitoring. Furthermore, the operation is complex, dependent on professional personnel and equipment, and its size and operation method are incompatible with the application scenarios of wearable devices.

[0004] Given the technical problems with the above technologies, traditional biomarker detection is difficult to meet the application needs of daily continuous monitoring and convenient use. Summary of the Invention

[0005] The purpose of this invention is to provide an integrated interstitial fluid sampling and electrochemical detection system, its preparation method, and its application. The integrated interstitial fluid sampling and electrochemical detection system provided by this invention is simple to operate, highly applicable, and provides accurate detection results. It can achieve real-time, integrated sampling and detection and can be used for daily continuous detection in wearable devices.

[0006] To achieve the above objectives, the present invention provides the following technical solution: This invention provides an integrated interstitial fluid sampling and electrochemical detection system, including a microneedle array unit, an electrochemical sensor, and a micro negative pressure pump; The microneedle array unit includes a base and a hollow microneedle array located on the surface of the base; the hollow microneedle array has a through sampling channel; The electrochemical sensor includes a screen-printed electrode, a signal transduction layer sequentially modified on the surface of the screen-printed electrode, and a template molecularly imprinted film; the signal transduction layer comprises MXene and a conductive polymer. The micro negative pressure pump includes a vacuum generating chamber and a sampling chamber; the vacuum generating chamber and the sampling chamber are connected by a hollow conduit, and the sampling chamber is used to accommodate the base of the microneedle array unit and the electrochemical sensor; at least the tip portion of the hollow microneedle array of the microneedle array unit is exposed outside the sampling chamber.

[0007] Preferably, the base of the microneedle array unit is connected to the electrochemical sensor via a slot; the electrochemical sensor has a through hole.

[0008] Preferably, the thickness of the signal transduction layer is 20~200 nm; the conductive polymer includes poly(3,4-ethylenedioxythiophene) (PEDOT) and / or polypyrrole (PPy).

[0009] Preferably, the template molecule includes one of glucose, β-hydroxybutyrate, melatonin, or a small molecule protein; the thickness of the template molecule imprinted membrane is 30~100 nm.

[0010] Preferably, the material of the micro negative pressure pump includes polydimethylsiloxane (PDMS) and / or polymethyl methacrylate (PMMA).

[0011] This invention also provides a method for preparing the above-mentioned integrated interstitial fluid sampling and electrochemical detection system, comprising the following steps: A solution containing MXene and conductive polymer monomers is applied to the surface of a screen-printed electrode to perform a first electrochemical deposition, thereby obtaining a screen-printed electrode modified with a signal transduction layer. The screen-printed electrode modified with the signal transduction layer is placed in a prepolymerization solution containing template molecules, functional monomers, beacon molecules, and dispersion medium for a second electrochemical deposition. The template molecules are then washed away to obtain an electrochemical sensor. The electrochemical sensor and microneedle array unit are assembled into the sampling chamber of a micro negative pressure pump to obtain an integrated interstitial fluid sampling and electrochemical detection system.

[0012] Preferably, in the solution containing MXene and conductive polymer monomers, the volume ratio of the MXene solution to the conductive polymer monomers is 500~2000:1.

[0013] Preferably, the functional monomer includes one of 3-aminophenylboronic acid, o-phenylenediamine, or polypyrrole; The beacon molecule includes one of thionine, Prussian blue, or methylene blue; The molar ratio of the template molecule to the functional monomer is 1:2~8; Preferably, the first electrochemical deposition is a constant voltage electrochemical deposition; the voltage of the first electrochemical deposition is 1~1.5 V, and the deposition time is 30~60 s; The second electrochemical deposition is a cyclic voltammetric electrochemical deposition; the voltage of the second electrochemical deposition is -0.2~1.0 V, the scan rate is 20~100 mV / s, and the number of scan cycles is 10~30 cycles; the scan is stopped when there is no obvious redox peak.

[0014] This invention also provides the application of the above-mentioned integrated interstitial fluid sampling and electrochemical detection system in the detection of biomarkers for non-disease diagnostic purposes.

[0015] This invention provides an integrated interstitial fluid sampling and electrochemical detection system, comprising a microneedle array unit, an electrochemical sensor, and a micro negative pressure pump. The microneedle array unit includes a base and a hollow microneedle array located on the surface of the base. The hollow microneedle array has a through sampling channel. The electrochemical sensor includes a screen-printed electrode, a signal transduction layer sequentially modified on the surface of the screen-printed electrode, and a template molecular imprinted film. The signal transduction layer comprises MXene and a conductive polymer. The micro negative pressure pump includes a vacuum generating chamber and a sampling chamber. The vacuum generating chamber and the sampling chamber are connected by a hollow conduit. The sampling chamber is used to accommodate the base of the microneedle array unit and the electrochemical sensor. At least the tip portion of the hollow microneedle array of the microneedle array unit is exposed outside the sampling chamber. This invention involves inserting a hollow microneedle array into the surface layer to be tested during sampling. By pressing the vacuum chamber of a micro-needle pump, negative pressure is generated. The collected interstitial fluid, driven by this negative pressure, flows through the sampling channels of the hollow microneedle array to the surface of the electrochemical sensor. Recognition and electrical signal conversion are achieved via a molecularly imprinted membrane and signal transduction layer, completing real-time integrated sampling and detection. This invention uses a hollow microneedle array as the sampling unit. The microneedles have good biocompatibility, are painless or minimally invasive, cause very little discomfort, do not affect user activity, and are beneficial for continuous long-term monitoring (not less than 1 hour).

[0016] The MXene of this invention is stacked in layers, exhibiting strong conductivity and abundant functional groups on its surface, enabling the construction of a continuous conductive network. A conductive polymer serves as a crosslinking agent, enhancing the adhesion between the MXene and the screen-printed electrode while further improving electron transport efficiency, thus providing better conductivity for the screen-printed electrode. This invention uses the analyte molecule as a template molecule and the template molecule imprinted membrane as a recognition layer, exhibiting highly specific recognition capabilities and high detection accuracy. The electrochemical sensor modified with the signal transduction layer and recognition layer of this invention possesses high sensitivity, enabling the rapid conversion of the specific binding signal between the biomarker and the template molecule imprinted membrane into a directly detectable electrical signal. This invention allows for the detection of different biomarkers by replacing the electrochemical sensor.

[0017] The miniature negative pressure pump used in this invention has the ability to generate vacuum pressure, which can provide sampling pressure, thereby collecting a sample volume sufficient for detection (≈1.5 μL / needle / h). This invention integrates sampling and electrochemical detection, eliminating the need for additional sampling and transfer steps, simplifying operation, and enabling real-time, integrated sampling and detection.

[0018] This invention also provides a method for preparing the integrated interstitial fluid sampling and electrochemical detection system described above. The preparation method provided by this invention is simple to operate, low in cost, and easy to implement for large-scale industrial production. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 This is a schematic diagram of an integrated interstitial fluid sampling and electrochemical detection system. Figure 2 This is a physical image of an integrated interstitial fluid sampling and electrochemical detection system; Figure 3 This is a schematic diagram of a microneedle array unit; Figure 4 This is a picture of a miniature negative pressure pump. Figure 5 SEM images of MXene modified for screen-printed electrodes; Figure 6 SEM image of glucose molecularly imprinted membrane / MXene / screen printed electrode; Figure 7 This is a current-time graph for glucose detection in Example 1; Figure 8SEM image of β-hydroxybutyric acid molecularly imprinted membrane / MXene / screen-printed electrode; Figure 9 The current-time graph for detecting β-hydroxybutyric acid in Example 2 is shown. Detailed Implementation

[0021] This invention provides an integrated interstitial fluid sampling and electrochemical detection system, including a microneedle array unit, an electrochemical sensor, and a micro negative pressure pump; The microneedle array unit includes a base and a hollow microneedle array located on the surface of the base; the hollow microneedle array has a through sampling channel; The electrochemical sensor includes a screen-printed electrode, a signal transduction layer sequentially modified on the surface of the screen-printed electrode, and a template molecular imprinted film. The signal transduction layer comprises MXene and a conductive polymer. The micro negative pressure pump includes a vacuum generating chamber and a sampling chamber; the vacuum generating chamber and the sampling chamber are connected by a hollow conduit; the sampling chamber is used to accommodate the base of the microneedle array unit and the electrochemical sensor; at least the tip portion of the hollow microneedle array of the microneedle array unit is exposed outside the sampling chamber.

[0022] In this invention, unless otherwise specified, all raw materials are commercially available products well known to those skilled in the art.

[0023] The integrated interstitial fluid sampling and electrochemical detection system provided by this invention includes a microneedle array unit, which comprises a base and a hollow microneedle array located on the surface of the base; the hollow microneedle array has a through sampling channel. In this invention, the base and the hollow microneedle array are preferably made of the same material; the material is preferably one of photosensitive resin, transparent resin, and high-temperature resistant resin, more preferably photosensitive resin, and the photosensitive resin is preferably HTL yellow resin (Shenzhen Mofang New Material Technology Co., Ltd.). This invention uses HTL yellow resin as the raw material for the microneedle array, which has the advantages of low cytotoxicity and no skin sensitization. It does not leach harmful substances upon contact with interstitial fluid or skin tissue, thus avoiding sample contamination and inflammatory reactions, and has better biocompatibility.

[0024] In this invention, the base preferably has a slot to facilitate connection between the base of the microneedle array unit and the electrochemical sensor. The slot also allows for easy replacement of electrochemical sensors modified with different template molecularly imprinted membranes, meeting the detection needs of various biomarkers.

[0025] This invention does not impose special requirements on the dimensions of the microneedle array unit; it can be designed according to actual conditions. In one specific embodiment, the overall height of the microneedle array unit is 2.4 mm; the length and width of the base are both 10 mm, and the height is 1.4 mm; the thickness of the plate layer in the slot of the base is 0.2 mm; each hollow microneedle is a square pyramid shape with a base side length of 0.5 mm, a hollow diameter of 0.2 mm, and a height of 1 mm; the distance between the tips of adjacent hollow microneedles is 1.2 mm. This invention uses square pyramid-shaped hollow microneedles, resulting in higher puncture efficiency and stronger channel retention capability.

[0026] In this invention, the number of hollow microneedles can be set according to actual needs, preferably one of 6×6, 8×8, and 10×10, and more preferably 6×6. In this invention, the hollow microneedle array preferably covers 3 / 4 of the base area, and is preferably 2 mm away from the edge of the base.

[0027] In this invention, the microneedle array unit is preferably fabricated using 3D printing technology. This invention does not specify a particular 3D printing method; any 3D printing method well-known in the art can be used. This invention uses 3D printing technology to fabricate the microneedle array unit, which can precisely print the hollow through-channels and pyramidal structures of hollow microneedles, meeting the structural accuracy requirements for sampling.

[0028] The integrated interstitial fluid sampling and electrochemical detection system provided by this invention includes an electrochemical sensor. The electrochemical sensor comprises a screen-printed electrode, a signal transduction layer sequentially modified on the surface of the screen-printed electrode, and a template molecularly imprinted film. The signal transduction layer comprises MXene and a conductive polymer. In this invention, the screen-printed electrode is a three-electrode system, comprising a working electrode, a reference electrode, and a counter electrode. The working electrode is preferably a printed carbon electrode or a printed gold electrode, the reference electrode is preferably a silver chloride electrode, and the counter electrode is preferably a platinum electrode. During detection, the screen-printed electrode is connected to an external electrochemical workstation, and a current-time method is used for scanning to achieve signal measurement.

[0029] In this invention, the thickness of the signal transduction layer is preferably 20-200 nm, more preferably 20-100 nm; the conductive polymer is preferably PEDOT and / or PPy, more preferably PEDOT. This invention ensures optimal conductivity, detection reliability, and controlled manufacturing costs by controlling the thickness of the signal transduction layer.

[0030] In this invention, the template molecule in the template molecular imprinting membrane is preferably one of glucose, β-hydroxybutyrate, melatonin, or a small molecule protein; the thickness of the template molecular imprinting membrane is preferably 30-100 nm, more preferably 30-50 nm. By controlling the thickness of the template molecular imprinting membrane, this invention can optimize performance in terms of the effectiveness of recognition sites, mass transfer efficiency, binding capacity, and detection sensitivity and selectivity, while avoiding detection defects caused by excessively thick or thin imprinting layers.

[0031] In this invention, the electrochemical sensor is provided with a through hole to facilitate the discharge of gas when the vacuum negative pressure pump is pressed and the transfer of the sample from the through channel of the microneedle array unit to the sampling chamber.

[0032] The integrated interstitial fluid sampling and electrochemical detection system provided by this invention includes a micro-negative pressure pump, which comprises a vacuum generating chamber and a sampling chamber connected by a hollow conduit. The sampling chamber accommodates the base of the microneedle array unit and the electrochemical sensor. At least the tip portion of the hollow microneedle array of the microneedle array unit is exposed outside the sampling chamber. In this invention, the micro-needle pump is preferably made of PDMS and / or PMMA, more preferably PDMS. In this invention, the vacuum generating chamber has a cavity, and air in the cavity is discharged by pressing the vacuum generating chamber to generate sufficient negative pressure for sampling. In this invention, the sampling chamber has a recess for accommodating the base of the microneedle array unit and the electrochemical sensor.

[0033] This invention does not impose any special requirements on the specific dimensions of the micro negative pressure pump; it can be designed according to actual conditions. In one specific embodiment of this invention, the overall length of the micro negative pressure pump is 58 mm and the height is 9 mm; the length of the vacuum generating chamber is 25 mm, the width is 25 mm, and the height is 5 mm; the vacuum generating chamber has a square cavity with a side length of 16 mm; the sampling chamber has a length of 14.4 mm, a width of 14.4 mm, and a height of 4 mm; the sampling chamber has a square recess with a side length of 10.4 mm.

[0034] In this invention, the method for preparing the micro negative pressure pump preferably includes the following steps: The miniature negative pressure pump mold was obtained by 3D printing according to the mold dimensions of the miniature negative pressure pump. The casting liquid is poured into a miniature negative pressure pump mold and cured. After curing, the miniature negative pressure pump component is obtained by demolding. A miniature negative pressure pump is obtained by assembling the components of a miniature negative pressure pump.

[0035] This invention does not impose special requirements on the dimensions of the micro negative pressure pump mold; it can be designed to match the dimensions of the micro negative pressure pump. In this invention, the micro negative pressure pump mold is preferably obtained using 3D printing. As a specific embodiment of this invention, the mold for the micro negative pressure pump includes a bottom mold and a top mold.

[0036] In one specific embodiment of the present invention, the casting liquid is preferably obtained by mixing PDMS base adhesive and PDMS curing agent; the PDMS base adhesive is preferably polydimethylsiloxane prepolymer, and the PDMS curing agent is preferably a crosslinking agent; the volume ratio of the PDMS base adhesive to the PDMS curing agent is preferably 5~20:1, more preferably 10:1. In the present invention, the casting step is preferably: after the uniformly mixed casting liquid is allowed to stand to remove air bubbles, it is slowly poured into the bottom mold and the top mold.

[0037] In this invention, the curing temperature is preferably 80~120℃, more preferably 100℃; the curing time is preferably 1~3 h, more preferably 2 h.

[0038] In this invention, the assembly is preferably performed by bonding, and the adhesive used for bonding is preferably medical-grade Antekol.

[0039] This invention provides a method for preparing the above-mentioned integrated interstitial fluid sampling and electrochemical detection system, comprising the following steps: A solution containing MXene and conductive polymer monomers is applied to the surface of a screen-printed electrode to perform a first electrochemical deposition, thereby obtaining a screen-printed electrode modified with a signal transduction layer. The screen-printed electrode modified with the signal transduction layer is placed in a prepolymerization solution containing template molecules, functional monomers, beacon molecules, and dispersion medium for a second electrochemical deposition. The template molecules are then washed away to obtain an electrochemical sensor. The electrochemical sensor and microneedle array unit are assembled into the sampling chamber of a micro negative pressure pump to obtain an integrated interstitial fluid sampling and electrochemical detection system.

[0040] This invention involves applying a solution containing MXene and a conductive polymer monomer to the surface of a screen-printed electrode for a first electrochemical deposition, thereby obtaining a screen-printed electrode modified with a signal transduction layer. In this invention, the preparation method of the solution containing MXene and the conductive polymer monomer preferably includes the following steps: The MXene solution was ultrasonically mixed with the conductive polymer monomer.

[0041] In this invention, the MXene solution is preferably obtained by mixing an MXene dispersion with a buffer solution. The concentration of the MXene dispersion is preferably 6 mg / mL, and the buffer solution is preferably PBS buffer (pH 7.45). The concentration of the MXene solution is preferably 2-5 mg / mL, more preferably 3 mg / mL. In this invention, the conductive polymer monomer is preferably one of EDOT or pyrrole. In this invention, the volume ratio of the MXene solution to the conductive polymer monomer is preferably 500-2000:1, more preferably 1000:1. The ultrasonic mixing temperature is preferably 24-28°C, more preferably 25-27°C; the ultrasonic mixing time is preferably 1-2 h, more preferably 1 h; and the ultrasonic power is preferably 30-100 W, more preferably 50 W.

[0042] In this invention, the first electrochemical deposition is preferably constant voltage electrochemical deposition, and the voltage of the first electrochemical deposition is preferably 1~1.5 V, more preferably 1.2 V; the first electrochemical deposition time is preferably 30~60 s, more preferably 60 s. In this invention, after the first electrochemical deposition, the surface of the screen-printed electrode is dark black.

[0043] This invention employs an MXene layer as a signal transduction layer. MXene possesses high electronic conductivity, stability, and good biocompatibility, and can form a layered conductive layer. A conductive polymer is used as a crosslinking agent, which provides efficient electron transport for electrochemical detection, exhibiting higher biomarker-electron conversion capability and higher detection sensitivity. The conductive polymer monomer can undergo physical adsorption and chemical bonding with the screen-printed electrode surface during polymerization, ensuring a firm bond between the MXene layer and the screen-printed electrode, making the modification of the MXene layer more robust, and ensuring the structural stability of the electrochemical sensor.

[0044] In this invention, the screen-printed electrode modified with the signal transduction layer is placed in a prepolymerization solution containing template molecules, functional monomers, beacon molecules, and dispersion medium for a second electrochemical deposition; the template molecules are then washed away to obtain an electrochemical sensor.

[0045] In this invention, the dispersion medium is preferably a buffer solution, and the buffer solution is preferably one of phosphate-buffered saline (PBS, pH=9.0), acetate buffer (pH=5.0), or phosphate-buffered saline (PBS, pH=7.4); the functional monomer is preferably one of 3-aminophenylboronic acid, o-phenylenediamine, or polypyrrole. In this invention, the target molecule to be tested is used as the template molecule, and the template molecule is preferably one of glucose, β-hydroxybutyrate, melatonin, or a small molecule protein; the beacon molecule is preferably one of thionine, Prussian blue, or methylene blue.

[0046] In this invention, the molar ratio of the template molecule to the functional monomer is preferably 1:2 to 8, more preferably 1:5. In this invention, the amounts of the dispersion medium and the beacon molecule can be adjusted according to actual production needs. As a specific embodiment of this invention, when the template molecule is glucose, the molar ratio of the template molecule to the dispersion medium is preferably 1:10 to 1:50, more preferably 1:20; the molar ratio of the template molecule to the beacon molecule is preferably 50:1 to 10:1, more preferably 20:1; the molar ratio of the functional monomer to the dispersion medium is preferably 1:1.25 to 1:25, more preferably 1:4; when the template molecule is β-hydroxybutyric acid, the molar ratio of the template molecule to the dispersion medium is preferably 1:1 to 1:10, more preferably 1:5; the molar ratio of the template molecule to the beacon molecule is preferably 100:1 to 20:1, more preferably 53:1; the molar ratio of the functional monomer to the dispersion medium is preferably 1:0.125 to 1:5, more preferably 1:1.

[0047] In this invention, the second electrochemical deposition is preferably cyclic voltammetry electrochemical deposition; the voltage of the second electrochemical deposition is preferably -0.2~1.0 V, more preferably -0.2~0.6 V; the scan rate is preferably 20~100 mV / s, more preferably 50 mV / s, and the number of scan cycles is preferably 10~30 cycles, more preferably 20 cycles; the scan is stopped when there is no obvious redox peak.

[0048] In this invention, the elution medium used to wash away the template molecule is preferably compatible with the template molecule; when the template molecule is glucose, the washing reagent is preferably an anhydrous ethanol-acetic acid mixture, wherein the volume ratio of anhydrous ethanol to acetic acid in the anhydrous ethanol-acetic acid mixture is preferably 10:1; when the template molecule is β-hydroxybutyric acid, the washing reagent is preferably anhydrous ethanol; the washing is carried out under magnetic stirring conditions, and the stirring time is 10-15 min. This invention does not have special requirements for the drying method; any drying method well known to those skilled in the art can be used.

[0049] This invention assembles the electrochemical sensor and microneedle array unit into the sampling chamber of a micro negative pressure pump to obtain an integrated interstitial fluid sampling and electrochemical detection system. In this invention, the assembly of the integrated interstitial fluid sampling and electrochemical detection system preferably includes the following steps: drilling a hole in the electrochemical sensor; aligning the drilled electrochemical sensor with a pre-set slot on the back of the microneedle array unit, inserting it along the slot guide, and securing it in place; aligning the concave surface of the sampling chamber of the micro negative pressure pump with the back of the microneedle array unit, and uniformly applying medical adhesive to the bonding surface to obtain the integrated interstitial fluid sampling and electrochemical detection system.

[0050] In this invention, the drilling process preferably includes the following steps: using a micro-driller to drill holes in the electrochemical sensor, with the drilling positions designed according to actual conditions, to obtain an electrochemical sensor with through holes. As a specific embodiment of this invention, the hole diameter is preferably 1-3 mm, more preferably 2 mm; the number of holes is preferably 2-8, more preferably 5.

[0051] To further illustrate the present invention, the following detailed description of the invention's solutions, in conjunction with the accompanying drawings and embodiments, is provided, but should not be construed as limiting the scope of protection of the present invention.

[0052] Example 1 Detection of glucose (Glu): A schematic diagram of the integrated interstitial fluid sampling and electrochemical detection system used in this embodiment is shown below. Figure 1 As shown in the picture, the actual product is as follows. Figure 2 As shown, it includes a microneedle array unit, an electrochemical sensor, and a micro negative pressure pump.

[0053] The microneedle array unit includes a base and a hollow microneedle array located on the surface of the base. The overall height of the microneedle array unit is 2.4 mm. The base is 10 mm long and 10 mm wide, and 1.4 mm high. The plate thickness of the slot in the base is 0.2 mm. Each hollow microneedle is a square pyramid with a base side length of 0.5 mm, a hollow diameter of 0.2 mm, and a height of 1 mm. The distance between the tips of adjacent hollow microneedles is 1.2 mm. The hollow microneedle array consists of 6×6 square symmetrically arranged hollow microneedles. Each hollow microneedle is a square pyramid with a base side length of 0.5 mm, a hollow diameter of 0.2 mm, and a height of 1 mm. The distance between adjacent hollow microneedles is 0.7 mm. A schematic diagram of the microneedle array unit is shown below. Figure 3 As shown.

[0054] The electrochemical sensor comprises a screen-printed electrode (SPE), a signal transduction layer sequentially modified on the surface of the screen-printed electrode, and a glucose molecularly imprinted membrane; the signal transduction layer comprises PEDOT and MXene, and the thickness of the signal transduction layer is 20 nm; the thickness of the glucose molecularly imprinted membrane is 50 nm. The electrochemical sensor is located in a slot in the microneedle array unit base.

[0055] The miniature negative pressure pump includes a vacuum generating chamber and a sampling chamber. The overall length of the miniature negative pressure pump is 58 mm, and the height is 9 mm. The vacuum generating chamber is 25 mm long, 25 mm wide, and 5 mm high. The vacuum generating chamber contains a square cavity with a side length of 16 mm. The sampling chamber is 14.4 mm long, 14.4 mm wide, and 4 mm high. The sampling chamber contains a square recess with a side length of 10.4 mm. The sampling chamber houses the base of the microneedle array unit and the electrochemical sensor, with the hollow microneedle array of the microneedle array unit exposed outside the sampling chamber.

[0056] The preparation method of the integrated interstitial fluid sampling and electrochemical detection system in this embodiment includes the following steps: Using 3D printing technology and HTL yellow resin (Shenzhen Mofang New Material Technology Co., Ltd.) as raw material, a microneedle array unit with 36 hollow microneedles arranged in a 6×6 square was printed. The overall dimensions are 10 mm long × 10 mm wide × 2.4 mm high. The bottom edge length of a single microneedle cone is 0.5 mm, the hollow diameter is 0.2 mm, and the height is 1 mm. The spacing between adjacent microneedles is 0.7 mm. The base height of the microneedle array unit is 1.4 mm, and a slot is provided in the base. The plate thickness of the slot is 0.2 mm.

[0057] 6 mL of MXene dispersion (Jiangsu Xianfeng Nanomaterials Co., Ltd.) was dispersed in 4 mL of PBS buffer (pH 7.45) to prepare an MXene solution with a concentration of 3 mg / mL. 10 μL of EDOT was dispersed in the MXene solution and sonicated at 50 W for 1 h at 25 °C to obtain a mixed solution. The mixed solution was dropped onto the surface of a screen-printed electrode, and a signal transduction layer was electrochemically deposited on the surface of the SPE using a constant voltage method. The deposition voltage was 1.2 V and the deposition time was 60 s, forming a dark black signal transduction layer. The resulting MXene-modified screen-printed electrode was denoted as MXene / SPE. 3.42 mg of 3-aminophenylboronic acid, 0.9 mg of glucose, and 10 mL of 0.01 M phosphate buffer (pH 9.0) were mixed, and 5 μL of 0.5 mM thionine solution was added and mixed thoroughly. The MXene / SPE was placed in the above mixed solution, and cyclic voltammetry was used to scan for 20 cycles, with a scan range of -0.2 to 0.6 V and a scan rate of 50 mV / s, until no obvious redox peak was observed, and a polymer film was formed by electrodeposition. The electrode was immersed in an anhydrous ethanol-acetic acid mixture with a volume ratio of 10:1 and stirred and washed for 10 min to elute glucose molecules, thus obtaining a glucose molecularly imprinted membrane / MXene / SPE.

[0058] The dimensions of the miniature negative pressure pump mold were designed according to the size of the miniature negative pressure pump. The bottom and top molds were fabricated using 3D printing. PDMS base adhesive and PDMS curing agent were mixed evenly at a volume ratio of 10:1 and allowed to stand to remove air bubbles. The mixture was then poured into the bottom and top molds and cured at 100℃ for 2 hours. After demolding, the bottom and top layers of the miniature negative pressure pump were obtained. Medical adhesive was used to bond and assemble the bottom and top layers of the negative pressure pump into a semi-closed structure. The resulting miniature negative pressure pump is shown in the image below. Figure 4 As shown.

[0059] After obtaining the glucose molecularly imprinted membrane / MXene / SPE, five holes with a diameter of 2 mm were punched on the top of the glucose molecularly imprinted membrane / MXene / SPE. The perforated glucose molecularly imprinted membrane / MXene / SPE was then aligned with the preset slot on the back of the microneedle array unit, inserted and fixed along the slot guide. The concave surface of the micro negative pressure pump sampling chamber was aligned with the back of the microneedle array unit, and adhesive was evenly applied to the bonding surface. The micro negative pressure pump sampling chamber and the microneedle array were then pressurized and bonded together to obtain an integrated interstitial fluid sampling and electrochemical detection system for detecting glucose molecules.

[0060] The MXene / SPE prepared in this embodiment was characterized by SEM, and the results are as follows: Figure 5 As shown. According to Figure 5 As can be seen, a layered structure appears on the surface of the SPE after being covered with MXene. The glucose molecularly imprinted membrane / MXene / SPE prepared in this embodiment was characterized by SEM, and the results are as follows: Figure 6 As shown. According to Figure 6 As can be seen, a uniform film appears on the surface of MXene / SPE after modification with glucose molecular imprinted membrane.

[0061] Glucose detection was performed on the integrated interstitial fluid sampling and electrochemical detection system prepared in this embodiment. First, glucose solutions with concentrations of 0.1 mM, 0.5 mM, 2.5 mM, 5 mM, and 10 mM were prepared. The detection platform was immersed in each glucose solution for 10 min, and then electrochemical chronoamperometry (it) was performed in a PBS system at a constant potential of -0.3 V (vs Ag / AgCl). The glucose concentration gradient and real-time current response are shown below. Figure 7 As shown, in buffer solutions of different concentrations, the current response decreases with increasing glucose concentration. Within the range of 0–10 mM, there is a good linear relationship between glucose concentration and current change, with the fitted curve being ΔI = 0.47 + 0.07C. (Glu) (R 2 =0.996), and its limit of detection (LOD) is 45 μM.

[0062] Example 2 Detection of β-hydroxybutyric acid: The integrated interstitial fluid sampling and electrochemical detection system used in this embodiment includes a microneedle array unit, an electrochemical sensor, and a micro negative pressure pump. The structure and preparation method of the microneedle array unit and the micro negative pressure pump are the same as in Example 1.

[0063] The electrochemical sensor comprises a screen-printed electrode (SPE), a signal transduction layer sequentially modified on the surface of the screen-printed electrode, and a β-hydroxybutyrate molecularly imprinted film; the signal transduction layer comprises PEDOT and MXene, and the thickness of the signal transduction layer is 20 nm; the thickness of the β-hydroxybutyrate molecularly imprinted film is 30 nm. The electrochemical sensor is located in a slot in the microneedle array unit base.

[0064] The preparation method of the integrated interstitial fluid sampling and electrochemical detection system in this embodiment includes the following steps: 6 mL of MXene dispersion (Jiangsu Xianfeng Nanomaterials Co., Ltd.) was dispersed in 4 mL of PBS buffer (pH=7.35) to prepare an MXene solution with a concentration of 3 mg / mL. 10 μL of EDOT was dispersed in the MXene solution and sonicated at 50 W for 1 h at 25 °C to obtain a mixed solution containing MXene and PEDOT monomers. The mixed solution was dropped onto the surface of a screen-printed electrode, and a signal transduction layer was electrochemically deposited on the surface of the SPE using a constant voltage method. The deposition voltage was 1.2 V and the deposition time was 60 s, forming a dark black signal transduction layer. The MXene-modified screen-printed electrode was obtained and denoted as MXene / SPE.

[0065] A 0.75 mM Prussian blue (PB) solution was prepared by dissolving 3 mM FeCl3, 3 mM K3Fe(CN)6, and 100 mM KCl in 0.1 M hydrochloric acid. 2.1 mg β-hydroxybutyric acid and 10.8 mg o-phenylenediamine (molar ratio 1:5) were mixed with 10 mL of 0.1 M pH 5.0 acetate buffer solution, and 0.5 mL of the above PB solution was added to obtain a mixed solution. The MXene / SPE was placed in the mixed solution, and cyclic voltammetry was used to scan for 20 cycles at a range of 0.3–1.0 V and a scan rate of 50 mV / s until no obvious redox peak was observed, thus forming a polymeric film by electrodeposition. The electrode was magnetically stirred in anhydrous ethanol for 15 min to elute the template molecules, yielding a β-hydroxybutyric acid molecularly imprinted membrane / MXene / SPE.

[0066] After obtaining the β-hydroxybutyric acid molecularly imprinted membrane / MXene / SPE, five holes with a diameter of 2 mm were punched on the top of the β-hydroxybutyric acid molecularly imprinted membrane / MXene / SPE. The perforated β-hydroxybutyric acid molecularly imprinted membrane / MXene / SPE was aligned with the preset slot on the back of the microneedle array unit, inserted and fixed along the slot guide. The concave surface of the micro negative pressure pump sampling chamber was aligned with the back of the microneedle array unit, and medical adhesive was evenly applied to the bonding surface. The micro negative pressure pump sampling chamber and the microneedle array were then pressurized and bonded together to obtain an integrated interstitial fluid sampling and electrochemical detection system for detecting β-hydroxybutyric acid.

[0067] The β-hydroxybutyric acid molecularly imprinted membrane / MXene / SPE prepared in this embodiment was characterized by SEM, and the results are as follows: Figure 8 As shown. According to Figure 8 It can be seen that after modification with β-hydroxybutyric acid molecularly imprinted film, a uniform film appears on the surface of the screen-printed electrode.

[0068] The integrated interstitial fluid sampling and electrochemical detection system prepared in this embodiment was used to detect β-hydroxybutyrate. First, β-hydroxybutyrate standard solutions with concentrations of 0.5 mM, 1.0 mM, 2.0 mM, 4.0 mM, and 6.0 mM were prepared. The electrochemical detection platform was incubated in each concentration of β-hydroxybutyrate standard solution for 15 min, followed by iterative detection at a constant potential of 0.15 V (vs Ag / AgCl). Response current data were collected, and the concentration gradient of β-hydroxybutyrate and the real-time current response are shown below. Figure 9 As shown, the current response value decreases with increasing β-hydroxybutyric acid concentration, confirming the sensor's ability to detect β-hydroxybutyric acid. Within the range of 0–6 mM, the β-hydroxybutyric acid concentration exhibits a good linear relationship with current change, ΔI = 0.24 + 0.24C. (β-HB) (R 2 =0.920), and its limit of detection (LOD) is 43 μM.

[0069] As can be seen from the above embodiments, the integrated interstitial fluid sampling and electrochemical detection system provided by the present invention has high sensitivity and can detect a variety of biomarkers.

[0070] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An integrated interstitial fluid sampling and electrochemical detection system, comprising a microneedle array unit, an electrochemical sensor, and a micro negative pressure pump; The microneedle array unit includes a base and a hollow microneedle array located on the surface of the base; the hollow microneedle array has a through sampling channel; The electrochemical sensor includes a screen-printed electrode, a signal transduction layer sequentially modified on the surface of the screen-printed electrode, and a template molecularly imprinted film; the signal transduction layer comprises MXene and a conductive polymer. The micro negative pressure pump includes a vacuum generating chamber and a sampling chamber; the vacuum generating chamber and the sampling chamber are connected by a hollow conduit, and the sampling chamber is used to accommodate the base of the microneedle array unit and the electrochemical sensor; at least the tip portion of the hollow microneedle array of the microneedle array unit is exposed outside the sampling chamber.

2. The integrated interstitial fluid sampling and electrochemical detection system according to claim 1, characterized in that, The base of the microneedle array unit is connected to the electrochemical sensor via a slot; The electrochemical sensor has a through hole.

3. The integrated interstitial fluid sampling and electrochemical detection system according to claim 1 or 2, characterized in that, The thickness of the signal transduction layer is 20~200 nm; The conductive polymer includes PEDOT and / or PPy.

4. The integrated interstitial fluid sampling and electrochemical detection system according to claim 1 or 2, characterized in that, The template molecule includes one of glucose, β-hydroxybutyrate, melatonin, or a small protein molecule. The thickness of the template molecularly imprinted film is 30~100 nm.

5. The integrated interstitial fluid sampling and electrochemical detection system according to claim 1 or 2, characterized in that, The materials used in the miniature negative pressure pump include PDMS and / or PMMA.

6. A method for preparing the integrated interstitial fluid sampling and electrochemical detection system according to any one of claims 1 to 5, characterized in that, Includes the following steps: A solution containing MXene and conductive polymer monomers is applied to the surface of a screen-printed electrode to perform a first electrochemical deposition, thereby obtaining a screen-printed electrode modified with a signal transduction layer. The screen-printed electrode modified with the signal transduction layer is placed in a prepolymerization solution containing template molecules, functional monomers, beacon molecules, and dispersion medium for a second electrochemical deposition. The template molecules are then washed away to obtain an electrochemical sensor. The electrochemical sensor and microneedle array unit are assembled into the sampling chamber of a micro negative pressure pump to obtain an integrated interstitial fluid sampling and electrochemical detection system.

7. The preparation method according to claim 6, characterized in that, In the solution containing MXene and conductive polymer monomers, the volume ratio of the MXene solution to the conductive polymer monomers is 500~2000:

1.

8. The preparation method according to claim 6, characterized in that, The functional monomer includes one of 3-aminophenylboronic acid, o-phenylenediamine, or polypyrrole; The beacon molecule includes one of thionine, Prussian blue, or methylene blue; The molar ratio of the template molecule to the functional monomer is 1:2~8.

9. The preparation method according to claim 6, characterized in that, The first electrochemical deposition is a constant voltage electrochemical deposition; the voltage of the first electrochemical deposition is 1~1.5 V, and the deposition time is 30~60 s; The second electrochemical deposition is a cyclic voltammetric electrochemical deposition; the voltage of the second electrochemical deposition is -0.2~1.0 V, the scan rate is 20~100 mV / s, and the number of scan cycles is 10~30 cycles; the scan is stopped when there is no obvious redox peak.

10. The application of the integrated interstitial fluid sampling and electrochemical detection system according to any one of claims 1 to 5 or the integrated interstitial fluid sampling and electrochemical detection system prepared by the preparation method according to any one of claims 6 to 9 in the detection of biomarkers for non-disease diagnostic purposes.

Images