A microneedle electrode for continuous sensitive physiological information monitoring and a preparation method and application thereof

Abstract

The application discloses a microneedle electrode for continuous sensitive physiological information monitoring and a preparation method and application thereof, and belongs to the technical field of electrochemistry. The microneedle electrode comprises a substrate and a liquid-like membrane layer covalently coupled to the surface of the substrate. The preparation raw material of the liquid-like membrane layer comprises a low-surface-energy polymer. The surface energy of the low-surface-energy polymer is less than or equal to 78 mJ / m 2 The application constructs a liquid-like membrane layer with a low-surface-energy polymer on the surface of the substrate. The flexibility, smoothness and fluidity of the low-surface-energy polymer are favorable to the sliding of various liquids, so that the interaction between the microneedle electrode and various biological substances can be reduced, the adhesion of proteins, bacteria and cells and the like can be significantly inhibited, and the microneedle electrode with good anti-adhesion performance is obtained, which has a good application prospect in blood glucose detection.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of electrochemistry, and particularly relates to a microneedle electrode for continuous and sensitive physiological information monitoring and a preparation method and application thereof. BACKGROUND

[0002] Diabetes is a major chronic disease in clinical practice. Currently, the routine blood glucose monitoring method in clinical practice mainly involves fingertip blood sampling or venous blood sampling, and then placing the blood in a blood glucose meter or blood glucose test paper for detection, which requires frequent operation 2-6 times per day. Frequent, invasive, and painful blood sampling and detection not only bring inconvenience to patients' life, but also increase the risk of wounds and reduce the detection compliance. In addition, for patients with severe diabetes, it is necessary to observe the dynamic blood glucose changes, which requires continuous and real-time monitoring of blood glucose. Therefore, the development of a full-chain system of continuous and sensitive monitoring sensing devices and integrated devices, and the research to break through the bottleneck of effective monitoring of chronic diseases such as diabetes, have great significance.

[0003] In recent years, micro-needle diagnosis and treatment technology (MN) is booming as a cutting-edge painless transdermal technology. The length of the micro-needle is usually 500-800 microns, which can just penetrate the stratum corneum to contact the dermis, so as to effectively avoid pain by not touching the blood vessels and nerves in the deep dermis. Moreover, the integration of the micro-needle and the sensor can effectively detect the biological information (ISF glucose level in the stable stage is highly correlated with blood) in the subcutaneous interstitial fluid (ISF) mainly existing in the dermis. However, there is still a major bottleneck in the current micro-needle electrochemical sensing. In complex biological fluids (such as whole plasma or blood), especially in the human body environment, the surface of the sensing electrode will usually cause non-specific biological adhesion due to a large number of coexisting background species (such as proteins, cells, etc.), thereby hindering the effective coupling of the target biomarker with the electrode surface, resulting in sensing failure and inability to achieve continuous and sensitive monitoring, which has become a major challenge for the rapid development and application of biological sensing.

[0004] Studies have shown that the interaction between biological substances and the substrate is an important reason for the adhesion. For example, for protein adhesion, the following four types of interactions are mainly involved, 1) ionic or electrostatic interaction, 2) hydrogen bond, 3) hydrophobic interaction (mainly entropy-driven), and 4) charge transfer or particle electron donor / acceptor type interaction. For the adhesion of biological substances such as cells and bacteria, it is mainly mediated by the protein ligand integrin or adhesion (composed of proteins and polysaccharide molecules) embedded in the membrane surface. Therefore, reducing the interaction between the surface and the biological substances such as proteins, inhibiting non-specific adhesion and contamination, has become an important strategy for real-time and stable monitoring of electrode sensors.

[0005] Current biosensor devices are often hindered by the non-specific adhesion of biological substances such as protein biomolecules, cells and bacteria in vivo, which prevents the transfer of electrons to the underlying electrode, resulting in sensor failure, and even causing tissue fibrosis. Techniques for achieving continuous and stable monitoring of sensors include physical porous or membrane filtration, biological engineering, chemical coating modification, etc. Compared with physical and biological engineering techniques, the chemical modification method, which only constructs an anti-adhesion surface by grafting or coating a layer of polymer, has the advantages of high universality, easy operation, simplicity and efficiency, and has become one of the most common strategies for designing continuous and stable monitoring sensors. However, there are the following problems in developing chemical modification to inhibit adhesion and achieve continuous and sensitive monitoring of sensors at the current stage: First, most current sensors are mainly limited to modification based on polyethylene glycol (PEG) or zwitterions and their derivatives. The anti-adhesion performance of these sensors usually depends on the steric hindrance effect, that is, when biological substances such as proteins approach the modified surface, the flexible molecular chains are severely squeezed to produce a large steric hindrance effect, thereby forming a physical and energy barrier to adhesion. This characteristic requires a long chain and a high surface grafting / coating density to effectively inhibit adhesion when biological substances such as proteins adhere to the surface, but it also causes the transfer of the signal to be measured to the underlying electrode to be severely hindered, thereby severely affecting the sensitivity. In addition, PEG and the like may be subject to oxidative damage in the presence of oxygen and transition metals, and may cause adverse immune reactions in vivo, thereby affecting their continuous action in high levels of blood and serum. Zwitterionic polymers not only require complex synthesis and purification of high-charge molecules, but also need to consider the influence of external pH and electric field on the charge state at the interface, and polypeptide or natural zwitterionic polymers may be easily hydrolyzed (degraded). Second, for transiently dissolved coatings, special environments are often required, and the universality is severely insufficient. Third, for lubricating liquid perfusion surfaces, the lubricating liquid is easily lost in a complex fluid environment, resulting in failure of the anti-pollution function. Furthermore, the use of lubricating liquid to coat the electrode will prevent the coupling of the target biomarker with the electrode surface, resulting in loss of sensing function. SUMMARY

[0006] To overcome at least one problem of the prior art described above, one of the purposes of the present application is to provide a microneedle electrode.

[0007] A second purpose of the present application is to provide a preparation method of the microneedle electrode described above.

[0008] A third purpose of the present application is to provide a microneedle sensor.

[0009] A fourth purpose of the present application is to provide a blood glucose monitor.

[0010] To achieve the above purposes, the technical solution adopted by the present application is:

[0011] The first aspect of the present application provides a microneedle electrode comprising a substrate and a liquid-like film layer covalently coupled to the surface of the substrate; the preparation raw material of the liquid-like film layer comprises a low surface energy polymer; the surface energy of the low surface energy polymer is ≤78 mJ / m 2 .

[0012] The present application is based on surface modification by lubrication strategy, and constructs a lubrication surface with full wettability and liquid flow state, specifically, a low surface energy polymer is covalently coupled to the surface of the substrate, thereby realizing "liquid-like" molecular modification of the lubrication surface, so that the surface morphology of the substrate only changes at the nanoscale, and due to the flexibility, smoothness and flowability of the low surface energy polymer, the modified surface often has little ion / electric, hydrogen bond and donor / acceptor interaction with proteins; and due to the ability to repel most oil-like and hydrophobic solvents and molecules, the hydrophobic interaction between the modified surface and protein molecules is also small. Therefore, benefiting from the reduction of various interaction forces, proteins and other biological substances are easily sheared or diffused into the medium by the dynamic flow of the liquid in a dynamic or even static environment, thereby facilitating the sliding of the microneedle electrode in various liquids, and thereby significantly inhibiting the adhesion of proteins, bacteria and cells and other biological substances.

[0013] In some embodiments of the present application, the surface energy of the low surface energy polymer is 1-78 mJ / m 2 ; for example, any value or a range value between any two values in 10, 20, 30, 40, 50, 60, 70, 78 mJ / m 2 , such as 10-40 mJ / m 2 .

[0014] Preferably, the low surface energy polymer comprises at least one of polydimethylsiloxane (PDMS), polytetrafluoroethylene (PTFE), polyvinylidene fluoride (PVDF), polystyrene (PS) or polytrifluoroethylene (PF3E); further preferably, the low surface energy polymer comprises polydimethylsiloxane (PDMS), polytetrafluoroethylene (PTFE) or a combination thereof; more preferably, the low surface energy polymer is selected from polydimethylsiloxane (PDMS).

[0015] The surface energy of polydimethylsiloxane (PDMS), polytetrafluoroethylene (PTFE), polyvinylidene fluoride (PVDF), polystyrene (PS) and polytrifluoroethylene (PF3E) is between 18-35 mJ / m 2 , which can achieve good anti-adhesion effect in the present application.

[0016] Preferably, the thickness of the liquid-like film layer is 1-10 nm; further preferably, 1.5-5 nm; more preferably, 1.8-2.5 nm.

[0017] Preferably, the substrate has a planar microneedle array; the planar microneedle array has 3-7 microneedles; each microneedle has a length of 600-1000 μm; further preferably, each microneedle has a length of 700-900 μm; more preferably, each microneedle has a length of 750-850 μm.

[0018] With the specific microneedle length, the microneedle electrode can just pierce the stratum corneum and contact the dermis without touching the blood vessels and nerves in the deep dermis, thus effectively avoiding pain.

[0019] Preferably, a catalytic substance is further arranged between the substrate and the liquid-like membrane layer; the catalytic substance comprises glucose oxidase and platinum nanoparticles.

[0020] The glucose oxidase can catalyze the oxidation reaction of glucose to generate an electrical signal, thus the blood glucose level can be detected; the platinum nanoparticles can improve the catalytic efficiency of the glucose oxidase.

[0021] The second aspect of the present application provides a preparation method of the microneedle electrode of the first aspect of the present application, comprising the following steps: preparing a liquid-like membrane layer on the surface of a substrate to obtain the microneedle electrode.

[0022] Alternatively, a catalytic substance is arranged on the surface of the substrate, and then a liquid-like membrane layer is prepared to obtain the microneedle electrode.

[0023] Preferably, before the liquid-like membrane layer is prepared, the surface of the substrate is further subjected to a hydroxyl modification step.

[0024] The surface of the substrate is subjected to a hydroxyl modification, so that the modified substrate can be coupled with a low-surface-energy polymer to prepare the liquid-like membrane layer.

[0025] In some specific embodiments of the present application, a mercaptoethanol solution is used to modify the surface of the substrate.

[0026] Preferably, the method for preparing the liquid-like membrane layer is selected from atomic deposition (ALD).

[0027] Preferably, the deposition time of the atomic deposition is 0.5 min-5 h.

[0028] Preferably, the preparation of the liquid-like membrane layer is carried out in an atmosphere of an acid gas and a protective gas; further preferably, the acid gas comprises hydrochloric acid gas; and the protective gas comprises nitrogen.

[0029] The preparation of the liquid-like membrane layer in the acid gas atmosphere is also conducive to the hydrolysis-condensation reaction between the low-surface-energy polymer and the hydroxyl group on the surface of the substrate; in some embodiments of the present application, the acid gas is also conducive to ensuring the activity of the glucose oxidase. The protective gas is helpful for stabilizing and cleaning the chamber.

[0030] By controlling the deposition time, liquid film-like layers with different thicknesses can be obtained, thereby realizing the modification of liquid film-like layers with precise thicknesses.

[0031] The third aspect of the present application provides a microneedle sensor comprising the microneedle electrode of the first aspect of the present application.

[0032] The fourth aspect of the present application provides a blood glucose monitor comprising the microneedle electrode of the first aspect of the present application or the microneedle sensor of the third aspect of the present application.

[0033] Preferably, the blood glucose monitor further comprises at least one of a wireless data transmission module, an artificial intelligence analysis algorithm module or a user interaction module.

[0034] By adding a wireless data transmission module, the sensor can wirelessly transmit monitoring data to nearby receiving devices such as smartphones or medical monitoring stations, thereby realizing remote monitoring; by adding an artificial intelligence analysis algorithm module, the collected physiological data can be subjected to deep learning and pattern recognition using artificial intelligence analysis algorithms, which can provide more accurate analysis results and health advice; by adding a user interaction module, users can easily view monitoring data, health reports and alerts.

[0035] The present application has the following beneficial effects: the present application constructs a liquid film-like layer with a low surface energy polymer on the surface of the substrate, which is flexible, smooth and fluid, and is beneficial to the sliding of various liquids, thereby reducing the interaction of the microneedle electrode with various biological substances, significantly inhibiting the adhesion of proteins, bacteria and cells, and obtaining a microneedle electrode with good anti-adhesion performance, which has good application prospects in blood glucose detection. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 It is the physical map of the microneedle electrode of Example 1 and its SEM map in the modification process.

[0037] Figure 2 It is a film thickness change graph obtained by using different hydrolysis-condensation reaction times in Examples 1-8.

[0038] Figure 3 It is an optical microscope image of the dynamic sliding behavior of water on the surface of the electrode of Example 4 and the surface of the unmodified electrode.

[0039] Figure 4 It is the sliding angle of various liquids on the surface of the modified electrode with different film thicknesses.

[0040] Figure 5 It is an electrochemical performance graph of the microneedle electrode.

[0041] Figure 6 The fluorescence image for showing the adhesion of fluorescent fibrinogen on the surface of the modified electrode with different thickness of the liquid-like membrane layer.

[0042] Figure 7 The statistical analysis chart for showing the adhesion of fluorescent fibrinogen on the surface of the modified electrode with different thickness of the liquid-like membrane layer.

[0043] Figure 8 The statistical analysis chart for showing the adhesion of NIH-3T3 cells on the surface of the modified electrode with different thickness of the liquid-like membrane layer.

[0044] Figure 9 The statistical analysis chart for showing the influence of the modified electrode on cell viability.

[0045] Figure 10 The fluorescence image for showing the adhesion of NIH-3T3 cells on the surface of the modified electrode with different thickness of the liquid-like membrane layer and the fluorescence image for showing the influence of the modified electrode on cell viability.

[0046] Figure 11 The schematic diagram for detecting blood glucose according to the present application. DETAILED DESCRIPTION

[0047] The present application will be further described in details by the following specific examples. It should be understood that the following examples are only used to further illustrate the present application and should not be construed as limiting the scope of the present application. Some non-essential improvements and adjustments made by those skilled in the art according to the principles set forth in the present application are within the scope of the present application. The following examples specifically illustrate the process parameters, which are only one example in the appropriate range. Those skilled in the art can make appropriate selection within the range according to the description herein, but not limited to the specific data in the following examples. The raw materials, reagents or devices used in the following examples and comparative examples can be obtained from conventional commercial channels, or can be obtained by known methods, unless otherwise specified.

[0048] In some embodiments of the present application, the substrate has a planar microneedle array; each array has 5 microneedles, and the microneedle size is: length 800 μm, width 200 μm, thickness 200 μm, and spacing 800 μm.

[0049] In some embodiments of the present application, the tip of each microneedle is thinned by laser to form a sharp cone, and the sharp cone is designed as a micro arrow structure, the tip of each micro arrow is processed into a pinch angle of about 30°, and both sides of the tip have a micro arrow structure, each barb angle has a sharp arrow, and the exposed edge is about 20 μm. This design can mechanically interlock the arrow tip with the tissue, so that the microneedle array can better adhere to the skin surface after penetrating the skin, and the biocompatibility is improved.

[0050] In some embodiments of the present application, a wireless data transmission module can be added to enable the sensor to wirelessly transmit monitoring data to nearby receiving devices such as smartphones or medical monitoring stations, thus enabling remote monitoring. A set of artificial intelligence analysis algorithms is developed to perform deep learning and pattern recognition on the collected physiological data, providing more accurate analysis results and health recommendations. A user-friendly interface and interaction system is designed to allow users to easily view monitoring data, health reports, and alerts.

[0051] Embodiment 1

[0052] A microneedle electrode is prepared according to the following steps:

[0053] 1) Using SUS304 stainless steel sheet as the substrate, laser micro-etching technology is used to etch the stainless steel substrate to obtain a planar microneedle array. Each array has 5 microneedles, with a size of 800 μm in length, 200 μm in width, and 200 μm in thickness, and a spacing of 800 μm. The tip of the microneedle is thinned by laser to form a sharp cone. The cone is designed as a micro-arrow structure, and the tip of each micro-arrow is processed into a pinch angle of about 30°. The tip of each micro-arrow has a sharp arrow on both sides, and each barb angle has a sharp arrow. The exposed edge is about 20 μm. Then, the surface of the microneedle array is cleaned by plasma, and then a layer of gold is electroplated using electrochemical deposition technology. Subsequently, the microneedle electrode is placed in a mercaptoethanol solution to introduce hydroxyl groups on the electrode surface, thereby enabling the electrode to be coupled with a liquid-like molecule (PDMS) for functionalization. In a platinum sulfite solution, platinum nanoparticles are electroplated using electrochemical deposition technology to improve the sensing sensitivity, and then washed with deionized water and dried. Subsequently, BSA (80 mg / mL) and glutaraldehyde (2.5%) are mixed as an adhesive solution with glucose oxidase (GOx, 50 mg / mL) at a volume ratio of 5:2:1, and mixed well on a shaker. Then, the mixture is uniformly coated on the electrode surface using a spray coating technique and dried overnight. Ag is sputtered on the electrode surface using a mask, and then the microneedle is placed in a 1 M KCl / HCl buffer solution and a current of 1 μA is applied for 1 min to prepare an Ag / AgCl reference electrode. The above hydroxylated gold electrode is directly used as the counter electrode.

[0054] 2) The above microneedle array electrode is placed in an ALD reactor, and the reactor is evacuated to a vacuum state. Inert gas nitrogen (N2) is used to stabilize and clean the chamber. Then, in a 37°C chamber temperature environment, weakly acidic water vapor (H2O / HCl), N2, and PDMS (Mw is 550, 2000-6000, and 350000, respectively) are sequentially introduced into the chamber at a dose of 50±10 Pa, and a hydrolysis-condensation reaction occurs on the electrode surface. The reaction time is controlled to be 1 min, thereby achieving precise thickness of the liquid-like film layer modification on the surface of the microneedle electrode.

[0055] Example 2

[0056] A microneedle electrode, the preparation steps of which are different from those of Example 1 in that the reaction time for controlling the hydrolysis-condensation reaction on the electrode surface is 2 min. The other raw materials and steps are the same as those of Example 1.

[0057] Example 3

[0058] A microneedle electrode, the preparation steps of which are different from those of Example 1 in that the reaction time for controlling the hydrolysis-condensation reaction on the electrode surface is 5 min. The other raw materials and steps are the same as those of Example 1.

[0059] Example 4

[0060] A microneedle electrode, the preparation steps of which are different from those of Example 1 in that the reaction time for controlling the hydrolysis-condensation reaction on the electrode surface is 10 min. The other raw materials and steps are the same as those of Example 1.

[0061] Example 5

[0062] A microneedle electrode, the preparation steps of which are different from those of Example 1 in that the reaction time for controlling the hydrolysis-condensation reaction on the electrode surface is 20 min. The other raw materials and steps are the same as those of Example 1.

[0063] Example 6

[0064] A microneedle electrode, the preparation steps of which are different from those of Example 1 in that the reaction time for controlling the hydrolysis-condensation reaction on the electrode surface is 1 h. The other raw materials and steps are the same as those of Example 1.

[0065] Example 7

[0066] A microneedle electrode, the preparation steps of which are different from those of Example 1 in that the reaction time for controlling the hydrolysis-condensation reaction on the electrode surface is 2 h. The other raw materials and steps are the same as those of Example 1.

[0067] Example 8

[0068] A microneedle electrode, the preparation steps of which are different from those of Example 1 in that the reaction time for controlling the hydrolysis-condensation reaction on the electrode surface is 4 h. The other raw materials and steps are the same as those of Example 1.

[0069] Comparative Example 1

[0070] A microneedle electrode, the preparation steps of which are different from those of Example 1 in that step 2) is to place the microneedle array electrode in an ALD reactor and evacuate the reactor to a vacuum state to remove impurities that may affect the deposition process; without using H2O / HCl and N2, directly evaporate PDMS into the chamber at a dose of 50±10 Pa, control the reaction time (1 min, 2 min, 5 min, 10 min, 20 min, 1 h, 2 h, 4 h). Step 1) is the same as Example 1. The microneedle electrode in this example is obtained.

[0071] Performance test

[0072] (1) The electrode surface film thickness, coverage and other properties are characterized by XPS, ellipsometer, SEM, AFM and other methods.

[0073] Figure 1 The physical map of the microneedle electrode of Example 1 and its SEM map in the modification process, wherein (a) is the physical map of the microneedle electrode, (b) is the SEM map of the microneedle electrode, (c) is the SEM map of the material after plating a layer of gold on the substrate, (d) is the SEM map of the material after plating platinum nanoparticles and spraying glucose oxidase, (e) is the SEM map of the material after atomic deposition of PDMS. From Figure 1 It can be seen that the physical map directly shows the physical form and structure of the microneedle electrode, including the layout and size of the microneedle array on the substrate; and the SEM map provides the microscopic details of the microneedle electrode surface, including the shape of the microneedle, the surface texture, and the uniformity and coverage of the liquid-like film layer. Through the SEM image, the distribution of the liquid-like film layer on the microneedle surface and the change of the surface morphology after modification can be observed, which is crucial for evaluating the quality and performance of the liquid-like film layer.

[0074] Figure 2 The film thickness change graph obtained by using different hydrolysis-condensation reaction times in Examples 1-8. As can be seen from the graph, the change of hydrolysis-condensation reaction time is crucial for controlling the accurate thickness of the liquid-like film layer, because the film thickness directly affects the anti-adhesion and electrochemical performance of the microneedle electrode. The graph shows a plurality of data points, each point representing the film thickness measured at a certain reaction time, so that the change of the film thickness with the increase of the reaction time can be observed; the blue curve array in the graph represents the film layer, showing the change of the film thickness with the reaction time.

[0075] Σ represents the number of free non-overlapping polymer chains filling the fixed area, when 1<Σ<5, the "mushroom" state transitions to the "brush" state, the grafted chains are moderately packed, the substrate surface is uniformly covered and more easily free to stretch and rotate, thereby significantly increasing flexibility and fluidity, and when the grafted thickness is about 2 nm, Σ≈1.8, which promotes the sliding of various liquids.

[0076] (2) Anti-adhesion test: Fluorescent fibrinogen (Fg) (0.1 mg / mL) was selected as a representative protein and incubated with the microneedle electrode modified with different thickness of the liquid-like film at 37 °C for 24 h (short-term) and 7 d (long-term), respectively. Meanwhile, the unmodified electrode was used as a control group. After incubation, the experimental and control electrodes were washed once with PBS buffer (0.01 M, pH = 7.4), and then the protein adhesion images were recorded by fluorescence microscopy at the same exposure time, and the relative fluorescence intensity on the substrate surface was quantitatively analyzed by ImageJ to evaluate the anti-protein adhesion level on the surface of the electrode modified with different thickness of the liquid-like film.

[0077] RAW cells and NIH / 3T3 cells were incubated with the microneedle electrode modified with different thickness of the liquid-like film at 37 °C, 5% CO2 incubator for 24 h (short-term) and 7 d (long-term), respectively. Meanwhile, the unmodified electrode was used as a control group. After incubation, the cells were stained with 10 μg / mL Hoechst 33342. Then the cell adhesion images were recorded by fluorescence microscopy, and the anti-cell adhesion level on the surface of the electrode modified with different thickness of the liquid-like film was evaluated by cell counting statistics.

[0078] The microneedle electrodes with different thickness (including working electrode, reference electrode, counter electrode) were placed horizontally on the measuring table, and the unmodified electrode was used as a control group. 3 μL of probe liquid (deionized water, ethanol, toluene and Dulbecco modified Eagle medium with fetal bovine serum (DMEM-FBS)) was added to the surface at 3 random points by the sessile drop method, and the drop images were collected within 5 s. The contact angle of different probe liquids was calculated by contact angle analysis software combined with statistical analysis, and the hydrophilic and hydrophobic properties of each electrode were evaluated. For the sliding angle test, 10 μL of the above probe liquid was dropped through the inclined base to record the angle until the liquid drop started to slide along the surface, and the sliding adhesion was recorded.

[0079] Figure 3 Optical microscope images of the dynamic sliding behavior of water on the surface of the electrode of Example 4 and the surface of the unmodified electrode, wherein (a1) is the state of water on the unmodified electrode (i.e. SUS304 stainless steel sheet) before sliding, (a2) is the state of water on the unmodified electrode after sliding, (a3) is the wetting area left by water on the unmodified electrode after sliding; (b1) is the state of water on the modified electrode (i.e. the microneedle electrode obtained in Example 4) before sliding, (b2) is the state of water on the modified electrode after sliding, (b3) is the wetting area left by water on the modified electrode after sliding, and the black part in (a3) and (b3) represents the area contaminated on the surface after the liquid slides, i.e. the wetting area. From the above, it can be seen that the wetting area of the unmodified electrode is larger than that of the modified electrode, and the wetting area of the modified electrode is smaller than that of the unmodified electrode. Figure 3See, with proper inclination, droplets slide off the modified electrode surface without leaving any residue, while the unmodified electrode is wetted, and the images demonstrate the sliding process of water droplets when inclined, especially on the modified electrode surface, water droplets can quickly slide off without leaving any residue, which indicates that the modified surface has excellent anti-adhesion performance and superhydrophobic properties. In contrast, the unmodified electrode surface shows wetting of water droplets, indicating that water droplets are more likely to spread and adhere on the unmodified surface.

[0080] Figure 4 The sliding angles of various liquid droplets, including water, ethanol, toluene, and DMEM-FBS, on the modified electrode surfaces with different film layer thicknesses were measured. The sliding angle is an important parameter for measuring the anti-adhesion performance of a surface, which reflects the angle required for a droplet to start sliding on an inclined surface. A smaller sliding angle indicates that the surface has better anti-adhesion performance, and the droplet is more likely to slide on the surface. In Examples 1-8, the film layer thicknesses of Examples 4-8 were in the range of 1-4 nm, all of which had smaller contact angles, good hydrophobicity, and good anti-sliding adhesion.

[0081] (3) Electrochemical performance test: The performance of the sensor was evaluated by cyclic voltammetry, and the electrode was cycled between the oxidation and reduction potentials of the electrochemically active [Fe(CN)6] 3- / 4- solution. The current density and peak-to-peak potential (ΔEp) of the redox process were used to evaluate the electron transfer kinetics between the electrode surface and the solution, reflecting the overall quality and state of the solid-liquid interface. That is, the standardized gold electrode modified with different types of liquid film layer thicknesses was studied in 5mM [Fe(CN)6] 3- / 4- (i.e., [Fe(CN)6] 3- and [Fe(CN)6] 4- ) and 0.1mM KCl solution by cyclic voltammetry (fixed scan rate of 50mV / s, scan voltage range of -0.5V-0.5V) to study the effect of coating thickness on current density and peak-to-peak potential.

[0082] EIS was used to analyze the electron transfer resistance characteristics at the electrode interface. That is, the EIS of the sensor system modified with different types of liquid film layer thicknesses was studied in 5mM [Fe(CN)6] 3- / 4- and 0.1mM KCl solution in the frequency range of 0.1-106Hz.

[0083] The effect of the thickness of the liquid-like membrane layer on the sensitivity of the sensor was analyzed using electrochemical detection (amperometric current method). Glucose simulation liquid detection test. In different concentration gradients of glucose simulation liquid, the voltage corresponding to the characteristic peak was obtained according to the cyclic voltammetry method. Then the voltage was used as the preset bias voltage (the potential difference between the working electrode and the reference electrode) of the amperometric current method, and the response current of glucose of different concentrations was obtained, so as to quantitatively analyze the sensitivity and linear range of the working electrode. When analyzing the concentration of the analyte by electrochemical measurement, a constant bias voltage is applied to the working electrode, and after the current is stable for 2 min, the current-time (I-t) signal is recorded, and the concentration of the analyte is gradually increased within a certain time interval, and the signal-concentration correlation is established. Test and evaluate the response sensitivity of the glucose of the sensor system modified by different thicknesses of the liquid-like membrane layer, and use the unmodified sensor system as a control group.

[0084] Figure 5 The electrochemical performance and anti-adhesion performance of the microneedle electrode are shown in the figure, wherein (a1) is the CV curve of the microneedle electrode with different film layer thicknesses; (a2) is a statistical diagram of the current density and peak potential of the microneedle electrode with different film layer thicknesses; (b1) is the current-time curve of the glucose sensing performance of different sensors including a liquid-like membrane layer (~2 nm) before and after modification and before and after protein contamination; (b2) is the signal-concentration correlation of the glucose sensing performance of different sensors including a liquid-like membrane layer (~2 nm) before and after modification and before and after protein contamination; (c) is the sensitivity analysis of the glucose sensing performance of different sensors including a liquid-like membrane layer (~2 nm) before and after modification and before and after protein contamination; (d) is a fluorescence image of the glucose sensing performance of different sensors including a liquid-like membrane layer (~2 nm) before and after modification and before and after protein contamination; (e) is a statistical analysis of the glucose sensing performance of different sensors including a liquid-like membrane layer (~2 nm) before and after modification and before and after protein contamination, and the figure shows the adhesion of proteins (Fg, green fluorescence) on the surface of different electrodes (including working electrodes and reference electrodes) after 7 days of incubation. Figure 5 The microneedle electrode prepared in the embodiment has good electrochemical performance and anti-adhesion performance, and the modification using the liquid-like membrane layer can achieve good results.

[0085] (4) Continuous and sensitive monitoring: In vitro, the continuous and sensitive monitoring performance of the modified microneedle blood glucose sensor is evaluated. Specifically, as follows: a) microneedle array transdermal performance evaluation: the tip of the microneedle array is coated with a fluorescent dye (such as rhodamine B), and is inserted into the in vitro rat skin tissue and removed, and the transdermal performance is evaluated by fluorescence microscopy. b) In vitro subcutaneous glucose continuous and sensitive monitoring performance evaluation: In the present application, 0.1 mg / mL fibrinogen solution (Fg solution, with fluorescence, used for subsequent continuous and sensitive monitoring mechanism analysis) is selected as a complex biological fluid. The Fg solution is placed in a Franz diffusion device, and fresh rat skin is spread over the device, and the above-mentioned microneedle blood glucose sensor modified with the optimal liquid membrane layer is placed on the in vitro skin and transdermally, and the glucose concentration in the Fg solution is continuously monitored (7d). Among them, by adding a glucose solution at a certain time point to change the glucose concentration and form fluctuations, the electrical signal corresponding to the glucose level in the complex biological fluid is measured, and 1 reading is taken every 5 min. At the same time, the unmodified microneedle blood glucose sensor is used as a control group to evaluate the stability of continuous and sensitive monitoring.

[0086] The continuous and sensitive monitoring performance of the microneedle blood glucose sensor is evaluated using a type I diabetes model rat. Rats weighing 200-250 g are selected for diabetes modeling, and 12 h before modeling, the rats are subjected to fasting but not water deprivation treatment, and then the rats are injected intraperitoneally with streptozotocin (60 mg·kg -1 The above-mentioned microneedle blood glucose sensor modified with the optimal liquid membrane layer is placed on the skin of the diabetic rat back for continuous blood glucose monitoring (7d), and the electrical signal corresponding to the blood glucose in the interstitial fluid of the subcutaneous tissue is measured, and 1 reading is taken every 5 min, and the unmodified microneedle blood glucose sensor is used as a control group. During the entire recording period, blood is taken from the tail at a certain time point, and the actual blood glucose value is determined using a commercial blood glucose meter for comparison. In addition, during the entire test process, a certain amount of glucose solution is injected into the abdominal cavity of the rat at different time points to induce blood glucose fluctuations in vivo, thereby verifying the accuracy of blood glucose monitoring under different blood glucose states. The blood glucose values measured by the modified sensor and the unmodified sensor are error analyzed with the actual values to evaluate the sensitivity, stability and accuracy of the continuous blood glucose monitoring of the modified sensor.

[0087] The biological safety of the microneedle blood glucose sensor is evaluated by measuring the possible induced tissue inflammation, allergic reaction, tissue fibrosis, etc. during the transdermal monitoring process. The specific experiment is divided into 5 groups, group 1: the microneedle blood glucose sensor is implanted subcutaneously for 5 minutes and then removed; groups 2 and 3: the microneedle blood glucose sensor is implanted subcutaneously for 1 day and 7 days, respectively, and then removed; groups 4 and 5: the microneedle blood glucose sensor is implanted subcutaneously for 1 day and 7 days, respectively, but a bias voltage of 0.5-1V is continuously applied during the period to monitor the blood glucose fluctuation (every 5 minutes). After the microneedle sensor is removed, all rats are continuously fed for 7 days, and the skin at the sensor application site is fixed, embedded, sectioned, and stained with H&E. The inflammatory, allergic, and fibrotic conditions of the skin tissue are observed under a microscope, and the normal group (the same area of skin tissue without sensor application) is used as a control group to evaluate the biological safety of the sensor application.

[0088] Figure 6 The fluorescence image shows the adhesion of fluorescent fibrinogen on the surface of the electrode modified by the liquid-like membrane layer with different thicknesses. Green fluorescence shows fluorescent fibrinogen Fg. (a) is the fluorescence image of Fg adhesion for 1 day, and (b) is the fluorescence image of Fg adhesion for 7 days. This figure captures the adhesion of fibrinogen on the electrode modified by the liquid-like membrane layer with different thicknesses through fluorescence microscopy. The distribution and density of green fluorescence intensity can directly show the degree of protein adhesion. By comparing the images after 1 day and 7 days, the change of protein adhesion with time and the effect of the liquid-like membrane layer with different thicknesses on protein adhesion can be evaluated.

[0089] Figure 7 The statistical analysis chart shows the adhesion of fluorescent fibrinogen on the surface of the electrode modified by the liquid-like membrane layer with different thicknesses. Green fluorescence shows fluorescent fibrinogen Fg. (a) is the statistical analysis chart of Fg adhesion for 1 day, and (b) is the statistical analysis chart of Fg adhesion for 7 days. This chart provides Figure 6 The quantitative data of the fluorescence image in the middle, the fluorescence intensity is quantified by statistical analysis method, so as to obtain the accurate numerical value of the adhesion amount of fibrinogen on the surface of the electrode modified by the liquid-like membrane layer with different thicknesses. These data can be used to evaluate the trend of protein adhesion with time and the effect of the liquid-like membrane layer with different thicknesses on reducing protein adhesion. It can be seen from Figure 6-7 Compared with the unmodified electrode, the microneedle electrode prepared in the embodiment of the application has good anti-protein adhesion effect.

[0090] Figure 8Statistical analysis chart showing the adhesion of NIH-3T3 cells on the surface of the modified electrode with different thicknesses of the liquid-like film layer. The adhesion of NIH-3T3 cells is marked with green fluorescence, and the adhesion time is 1 day. This chart provides the adhesion of the cells on the modified electrode with different thicknesses of the liquid-like film layer after 1 day through statistical analysis. The statistical data of the number and distribution of green fluorescence-labeled cells can be used to evaluate the effect of different thicknesses of the liquid-like film layer on cell adhesion and the regulatory effect of the coating on cell adhesion behavior.

[0091] Figure 9 Statistical analysis chart showing the effect of the modified electrode on cell viability. The data of the effect of the modified electrode on cell viability are provided through statistical analysis methods. This may include the measurement results of cell survival rate, proliferation rate or other cell viability indicators. These data are crucial for evaluating the biocompatibility and potential cytotoxicity of the modified electrode.

[0092] Figure 10 Fluorescence images showing the adhesion of NIH-3T3 cells on the surface of the modified electrode with different thicknesses of the liquid-like film layer and the effect of the modified electrode on cell viability, wherein the green fluorescence shows NIH-3T3 cells,

[0093] (a) Fluorescence images of the adhesion of NIH-3T3 cells on the surface of the modified electrode with different thicknesses of the liquid-like film layer after 1 day, and (b) fluorescence images of the effect of the modified electrode on cell viability. This chart combines Figure 8 and Figure 9 information, and visually displays the adhesion of NIH-3T3 cells on the modified electrode with different thicknesses of the liquid-like film layer and the effect of the modified electrode on cell viability through fluorescence images. The distribution of green fluorescence-labeled cells and the viability indicators (such as the ratio of live cells and dead cells) can visually display the biocompatibility of the modified electrode and the effect on cell behavior. From Figure 8-10 it can be seen that, compared with the unmodified electrode, the microneedle electrode prepared in the embodiment of the present application has good anti-cell adhesion effect, good biocompatibility and low cytotoxicity.

[0094] Figure 11The principle diagram for detecting blood glucose, wherein (a) is a detailed principle diagram for detecting blood glucose, (b1) is a skin structure diagram of the process of detecting blood glucose, (b2) is a partial enlarged view of the skin structure, (c1) and (c2) respectively represent a blood glucose detection curve and an anti-adhesion curve obtained in the detection process. It can be seen that by adopting the microneedle electrode of the application, the thickness of the liquid-like film layer is controlled, the sliding is promoted, and high anti-adhesion is achieved; good electrochemical performance can also be maintained, and high sensitivity is achieved; and continuous and sensitive blood glucose monitoring is achieved. The microneedle sensor prepared by the microneedle electrode of the application has good blood glucose monitoring effect, and the specific process is as follows: 1) the microneedle blood glucose sensor is inserted into the skin, and the liquid-like film layer covers the microneedle; 2) glucose enters the sensor through the microneedle and reacts with glucose oxidase; 3) Pt nanoparticles act as catalysts to accelerate the oxidation reaction of glucose; 4) an electrical signal is generated by the oxidation reaction, which is converted into the actual blood glucose value through the change of current, so as to test the blood glucose concentration; 5) the anti-adhesion performance of the liquid-like film layer with a certain thickness reduces the adhesion of proteins and cells, and maintains the high sensitivity and electrochemical performance of the sensor; 6) the system can continuously monitor the blood glucose level and provide real-time data to help remote management of diabetes.

[0095] As can be seen from the embodiments of the application, by adopting the microneedle electrode of the application, long-term inhibition of biological substance adhesion (more than 7 days) can be achieved, and the inhibition efficiency of biological substance adhesion reaches more than 90%, ensuring the stability of continuous monitoring. The thickness of the liquid-like film layer can be controlled at the nanometer level, and the thickness is about 2 nm, which can efficiently inhibit biological substance adhesion and ensure that the sensitivity is not affected, and thus continuous and sensitive monitoring is theoretically feasible. The "liquid-like" molecules modify the lubricated surface, that is, a layer of low surface energy polymer (such as PDMS) is covalently coupled, and the surface topography only changes at the nanometer level. And the polymer is flexible and flowable, which is conducive to the sliding of various liquids, thereby reducing the interaction with biological substances and significantly inhibiting the adhesion of proteins, bacteria and cells; and further reducing adverse reactions and tissue damage and improving biocompatibility. The device developed by the microneedle electrode in the embodiments of the application can realize continuous and sensitive monitoring of blood glucose in an in-vitro complex biological liquid and an in-vivo environment, and will not cause inflammation. And through cell culture and animal models, it is proved that the level of inflammatory cytokines can be reduced.

[0096] In summary, the application constructs a liquid-like film layer with a low surface energy polymer on the surface of the substrate. Due to the flexibility, smoothness and flowability of the low surface energy polymer, the sliding of various liquids is facilitated, thereby reducing the interaction of the microneedle electrode with various biological substances, significantly inhibiting the adhesion of proteins, bacteria and cells, and obtaining a microneedle electrode with good anti-adhesion performance, which has good application prospects in blood glucose detection.

Claims

1. A microneedle electrode for detecting glucose, characterized by, The product comprises a substrate and a liquid-like film layer covalently coupled to the surface of the substrate; the raw material for preparing the liquid-like film layer includes a low surface energy polymer; the surface energy of the low surface energy polymer is ≤78mJ / m 2 The low surface energy polymer includes at least one of polydimethylsiloxane, polytetrafluoroethylene, polyvinylidene fluoride, polystyrene, or polytrifluoroethylene; the thickness of the liquid-like film layer is 1-10 nm; a catalytic substance is further disposed between the substrate and the liquid-like film layer; the catalytic substance includes glucose oxidase and platinum nanoparticles.

2. The microneedle electrode for detecting glucose according to claim 1, wherein, The base has a planar microneedle array; the planar microneedle array has 3-7 microneedles; each microneedle has a length of 600-1000 μm.

3. A method for preparing a microneedle electrode for detecting glucose according to any one of claims 1 to 2, characterized by, The method comprises the following steps: The base surface is provided with a catalytic substance, and then a quasi-liquid membrane layer is prepared to obtain the microneedle electrode.

4. The method for preparing a microneedle electrode for detecting glucose according to claim 3, characterized by, Before the quasi-liquid membrane layer is prepared, the base surface is further subjected to a hydroxyl modification step.

5. The method for preparing a microneedle electrode for detecting glucose according to claim 3, wherein, The method for preparing the quasi-liquid membrane layer is selected from an atomic deposition method.

6. The method for preparing the microneedle electrode for glucose detection according to claim 5, characterized in that, The deposition time of the atomic deposition method is 0.5 min-5 h.

7. A microneedle sensor for detecting glucose, characterized by, The microneedle electrode for detecting glucose according to any one of claims 1-2.

8. A blood glucose monitor for detecting glucose, characterized by, The microneedle electrode for detecting glucose according to any one of claims 1-2 or the microneedle sensor for detecting glucose according to claim 7.

9. The blood glucose monitor for detecting glucose according to claim 8, characterized by, At least one of a wireless data transmission module, an artificial intelligence analysis algorithm module, or a user interaction module is further included.

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