A microneedle electrode for detecting citrullinated histone h3 and a preparation method and application thereof

Abstract

The application discloses a lignin carbon fiber-based microneedle electrode, which comprises a working microneedle electrode, a counter microneedle electrode and a reference microneedle electrode; the working microneedle electrode is a specific biological recognition interface constructed by depositing gold on a microneedle substrate, bridging through a mercapto-polyethylene glycol-carboxyl self-assembled molecular layer and covalently coupling H3Cit antibodies by using an NHS / EDC·HCl activation method; the microneedle substrate is prepared by the following steps: ball milling and ultrasonic dispersion of lignin carbon fibers in a dichloromethane solution of polylactic acid-glycolic acid copolymer to obtain a slurry, and injecting the slurry into a microneedle array mold and removing bubbles in vacuum. The application discloses a three-electrode sensor, which comprises a working electrode, a reference electrode and a counter electrode, and the working microneedle electrode is used as the working electrode. The application discloses an application of the three-electrode sensor in detection of H3Cit. The lignin carbon fiber-based microneedle electrode has regular arrangement and good mechanical properties, and can effectively recognize H3Cit.

Description

Technical Field

[0001] This invention belongs to the field of biomedical sensing technology and relates to an electrochemical sensor, specifically to a microneedle electrode and its preparation method and application, particularly to a microneedle electrode based on lignin-based carbon fiber (LCF) and its preparation method, and its application in detecting citrullinated histone H3 (H3Cit), a biomarker for metastasis after breast cancer surgery. Background Technology

[0002] Breast cancer is the most common malignant tumor among women worldwide, and surgical resection remains the core treatment method. However, a recurrence and metastasis rate as high as 30% after surgery is a leading cause of death for patients. While removing the tumor, surgery can reshape the microenvironment, promoting the spread of residual cancer cells.

[0003] Recent studies have found that postoperative neutrophils are massively activated and form extracellular neutrophil traps (NETs), which directly promote metastasis through mechanisms such as capturing circulating tumor cells and disrupting the vascular barrier. Citrullinated histone H3, a core component of NETs, ​​has the characteristics of high specificity, early appearance, and dynamic monitoring, making it an ideal liquid biopsy biomarker for early warning of postoperative metastasis.

[0004] However, traditional H3Cit detection methods (such as ELISA) lack sufficient sensitivity in the early stages of H3Cit transfer and cannot achieve continuous dynamic monitoring, making it difficult to meet clinical early warning needs. While electrochemical sensors offer advantages such as high sensitivity and rapid response, their performance is highly dependent on electrode materials. Currently used materials such as graphene and carbon nanotubes are expensive, have poor biocompatibility, and require complex surface modification processes, severely hindering their clinical translation.

[0005] Therefore, developing a novel electrode material that combines high performance, low cost, good biocompatibility, and ease of functionalization to construct an electrochemical sensor capable of real-time, dynamic monitoring of H3Cit has become a critical issue that urgently needs to be addressed in the field of post-operative management of breast cancer. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies by providing a low-cost, highly sensitive, highly specific, and biocompatible microneedle electrode for real-time, dynamic detection of trace amounts of H3Cit.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] A lignin-carbon fiber-based microneedle electrode includes a working microneedle electrode, a counter microneedle electrode, and a reference microneedle electrode. The working microneedle electrode is constructed by depositing gold (Au) on a microneedle substrate and bridging it with a self-assembled molecular layer of thiol-poly(ethylene glycol)-carboxylic acid (HS-PEG-COOH), utilizing N-hydroxysuccinimide (NHS) / 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride. The specific biorecognition interface is constructed by covalently coupling H3Cit antibody using the hydrochloride (EDC·HCl) activation method; the microneedle electrode is prepared by depositing a platinum (Pt) conductive layer on a microneedle substrate; the reference microneedle electrode is prepared by depositing an Ag / AgCl conductive layer on a microneedle substrate; the microneedle substrate is prepared by ball milling and ultrasonically dispersing lignin carbon fiber (LCF) in a dichloromethane solution of poly(lactic-co-glycolic acid, PLGA) to obtain a slurry, injecting the slurry into a microneedle array mold, and degassing under vacuum.

[0009] The lignin carbon fiber (LCF) is obtained by granulating and melt-blowing lignin to obtain lignin fiber, and then pre-oxidizing and carbonizing the lignin fiber.

[0010] Specifically, the lignin-carbon fiber (LCF) is prepared by the following method, including the following steps:

[0011] Step (1): The lignin is granulated and melt-blown to obtain lignin fibers;

[0012] Step (2): The lignin fibers are subjected to pre-oxidation treatment and carbonization treatment in sequence to obtain lignin carbon fibers.

[0013] In step (1), a twin-screw granulator is used to granulate lignin to obtain lignin particles.

[0014] The granulation temperature is 150–190°C, and the screw speed is 50–100 rpm.

[0015] Preferably, the granulation temperature can be 180°C and the screw rotation speed can be 80 rpm.

[0016] In some implementations, lignin is granulated to form lignin powder into lignin particles with uniform particle size (e.g., 0.5–2 mm), reducing the difference in raw material bulk density, ensuring uniform feeding during meltblowing, and avoiding spinneret blockage or fiber diameter fluctuations.

[0017] Lignin particles are placed in a meltblown testing machine to obtain lignin fibers. The meltblown temperature is 230–250℃, the screw speed is 200–350 rpm, the hot air frequency is 30–50 Hz, and the receiving distance is 30–50 cm.

[0018] Preferably, the meltblown temperature can be 250°C, the screw rotation speed can be 250 rpm, the hot air frequency can be 45 Hz, and the receiving distance can be 40 cm.

[0019] In some implementations, lignin particles are melt-blown to stretch molten lignin into fibers using a high-speed airflow, serving as a precursor for carbon fibers.

[0020] In step (2), lignin fibers are subjected to pre-oxidation and carbonization treatments in a tube furnace to obtain lignin carbon fibers.

[0021] In some implementations, the lignin fibers are pre-oxidized and carbonized to remove non-carbon atoms in order to prepare lignin carbon fibers.

[0022] The atmosphere for the pre-oxidation treatment is air, the temperature for the pre-oxidation treatment is 250-300℃, and the holding time for the pre-oxidation treatment is 1-2 hours.

[0023] Preferably, the pre-oxidation treatment involves heating from room temperature to 250-300°C in an air atmosphere at a heating rate of 0.1-0.4°C / min, and then holding at 250-300°C for 1-2 hours.

[0024] More preferably, the pre-oxidation treatment involves heating from room temperature to 280°C in an air atmosphere at a heating rate of 0.25°C / min, and holding at 280°C for 1 hour.

[0025] The carbonization process is carried out in a nitrogen or argon atmosphere, at a temperature of 800–1500°C, and for 1–2 hours.

[0026] Preferably, the carbonization process involves heating from room temperature to 800–1500°C at a rate of 3–6°C / min under a nitrogen or argon atmosphere, and holding at 800–1500°C for 1–2 hours.

[0027] More preferably, the carbonization treatment is carried out in a nitrogen or argon atmosphere, with the temperature increased from room temperature to 1000°C at a heating rate of 3°C / min, and held at 1000°C for 1 hour; or in a nitrogen or argon atmosphere, with the temperature increased from room temperature to 1200°C at a heating rate of 3°C / min, and held at 1200°C for 1 hour.

[0028] The microneedle array mold includes an array of hollow conical molds, each mold having a height of 600-800 μm, a bottom circle diameter of 200-300 μm, a top spacing of 500-600 μm between two adjacent molds, and an array number of 10-20*10-20.

[0029] Preferably, in the microneedle array mold, each mold has a height of 700 μm, a bottom circle diameter of 250 μm, a top spacing of 550 μm between two adjacent molds, and an array size of 10*10.

[0030] The microneedle array mold is a silicon-based microneedle array mold.

[0031] The curing and demolding process involves solvent evaporation at room temperature, drying, and demolding. The slurry solidifies through solvent evaporation, ultimately yielding the microneedle substrate upon demolding. Solvent evaporation occurs at room temperature and typically takes 24 hours. Drying is performed at 60°C for 8 hours.

[0032] Another object of the present invention is to provide a green preparation method for the lignin carbon fiber-based microneedle substrate, comprising the following steps:

[0033] Step (1): Ball mill the lignin carbon fiber and ultrasonically disperse it in a dichloromethane solution of PLGA to obtain a slurry. Inject the slurry into a microneedle array mold, vacuum degas it, solidify and demold it to obtain a microneedle substrate.

[0034] Step (2): Au is deposited on the microneedle substrate and bridged by a self-assembled molecular layer of HS-PEG-COOH. The specific biorecognition interface constructed by covalently coupling H3Cit antibody using the NHS / EDC activation method is then obtained to obtain the working microneedle electrode. A Pt conductive layer and an Ag / AgCl conductive layer are deposited on the microneedle substrate to obtain the counter microneedle electrode and the reference microneedle electrode, respectively.

[0035] In step (1), in some embodiments, ball milling of lignin-fiber carbon is used to prepare micron-sized LCF, making it easier to disperse uniformly in the subsequent polymer solution, forming a stable suspension and avoiding performance inhomogeneity due to agglomeration. PLGA acts as a binder, providing formability; at the same time, it is also a biodegradable material, making the final product usable in the biomedical field.

[0036] The mass ratio of the lignin carbon fiber (LCF) to polylactic acid-glycolic acid copolymer (PLGA) is 20:80 to 80:20.

[0037] The LCF is ball-milled to a particle size ≤10 μm.

[0038] Preferably, the LCF is ball-milled to a particle size ≤ 5 μm.

[0039] The PLGA mentioned is PLGA 75:25 (molar ratio of lactic acid to glycolic acid = 3:1), Mw > 100000 Da.

[0040] Preferably, the PLGA has a Mw≈30000.

[0041] The working microneedle electrode is formed by ion sputtering Au onto a microneedle substrate using an ion sputtering instrument, followed by immersion in HS-PEG-COOH solution (or local drop addition), and placed in a humidified chamber for 4–8 hours at room temperature. The substrate is then removed, rinsed with anhydrous ethanol to remove unbound HS-PEG-COOH, and dried with nitrogen. An activation solution of EDC·HCl and NHS is prepared using PBS buffer and uniformly applied to the microneedle substrate surface, reacting at room temperature for 30–60 minutes. The activated microneedle substrate is then immersed in a solution of H3Cit antibody and incubated with slow shaking at -4–8°C for 12–24 hours. Finally, the microneedle substrate is immersed in a tris-hydroxymethylaminomethane-hydrochloric acid buffer (Tris-HCl) containing bovine serum albumin (BSA) and incubated at room temperature for 1–3 hours to block unbound sites. Polysorbate 20 is then used to further seal the unbound sites. Washing with 20, Tween-20 solution 3 to 6 times removes unbound antibodies, yielding a working microneedle electrode grafted with H3Cit antibody.

[0042] The method for preparing the Au film is as follows: The microneedle substrate is placed in the high-vacuum sputtering chamber of an ion sputtering instrument. Under continuous rotation, a high-purity gold target is used to sputter for 90 to 180 seconds in an argon atmosphere at a working pressure of 0.02 to 0.05 mBar and a sputtering current of 20 to 40 mA, thereby forming a continuous gold film on the microneedle substrate.

[0043] The thickness of the Au film is 10–200 nm, preferably 10–30 nm, and more preferably 20 nm.

[0044] The HS-PEG-COOH solution is a 0.1–10 mM solution prepared with anhydrous ethanol. The molecular weight of the HS-PEG-COOH is 5000.

[0045] The activation solution is a mixture of EDC·HCl and NHS prepared with PBS buffer at pH 5.5, with a molar ratio of EDC·HCl to NHS of 5:1 to 1:1 and a concentration of EDC·HCl of 0.1 to 1 mM. This invention utilizes a PBS buffer environment at pH 5.5 to maximize the activation efficiency of the carboxyl groups. Under this weakly acidic condition, sufficient deprotonated carboxyl groups can be provided for efficient reaction with EDC·HCl, while significantly inhibiting hydrolysis side reactions of EDC·HCl molecules and their active intermediates, thus ensuring sufficient interaction with NHS to generate a stable amine-reactive ester (NHS ester). This optimization step lays a solid foundation for subsequent efficient and stable coupling with antibody molecules under physiological pH conditions.

[0046] The Tris-HCl buffer solution has a pH of 8.5 and a BSA concentration of 5–20 mg / mL.

[0047] The volume percentage of the Tween-20 solution is 0.01 to 0.1%.

[0048] The Tween-20 solution was prepared using a PBS buffer solution at pH 7.4.

[0049] Preferably, the microneedle electrode is obtained by depositing a Pt conductive layer on a microneedle substrate using an ion sputtering apparatus.

[0050] Specifically, the microneedle electrode is prepared by the following method: placing the microneedle substrate in the high-vacuum sputtering chamber of an ion sputtering instrument, using a high-purity gold target under an argon atmosphere while in a continuous rotating state, sputtering Pt to form a Pt conductive layer on the microneedle substrate.

[0051] The high-vacuum sputtering chamber operates at a pressure of 0.02–0.05 mBar, with a sputtering current of 20–40 mA and a sputtering time of 90–180 seconds.

[0052] The thickness of the Pt conductive layer is 10-200 nm, preferably 10-30 nm, and more preferably 20 nm.

[0053] The reference microneedle electrode is prepared by the following method: the microneedle substrate is sequentially treated with a sensitizing solution containing 8-12 g / L SiCl2 and 40 mL / L hydrochloric acid for 25-35 min, and then treated with an activating solution containing 0.2-0.3 g / L PdCl2 for 25-35 min; the substrate is then immersed in a chemical plating solution consisting of an equal volume mixture of 2-4 g / L AgNO3 silver ammonia solution and 2.3-2.7 g / L glucose-1.8-2.2 g / L NaOH reducing solution, and reacted at 45-55℃ for 5-10 min to deposit a uniform silver layer; then, it is oxidized in the dark using a 0.1-0.2 mol / L FeCl3 solution for 5-10 min to convert the silver layer surface in situ into stable AgCl to form an Ag / AgCl conductive layer, thus obtaining the reference microneedle electrode.

[0054] The sensitizing solution is prepared with water as the solvent, and the concentration of hydrochloric acid is 40 mL / L, which means that 1L of sensitizing solution contains 40 mL of concentrated hydrochloric acid with a mass fraction of approximately 36% to 38%.

[0055] The activation solution is a 0.2-0.3 g / L PdCl2 solution prepared with water as the solvent.

[0056] Preferably, at room temperature, the microneedle substrate is sequentially treated with a sensitization solution containing 8–12 g / L SnCl2 and 40 mL / L hydrochloric acid for 25–35 min, and then treated with an activation solution containing 0.2–0.3 g / L PdCl2 for 25–35 min.

[0057] The reference microneedle electrode was fabricated through a three-step chemical process. First, in the sensitization step, an acidic environment prevented the hydrolysis of SnCl2, ensuring the stability of Sn. 2+ The Sn adsorbed on the surface of the microneedle substrate is stably adsorbed; subsequently, during the activation process, the Sn adsorbed on the surface of the microneedle substrate... 2+ Pd in ​​the activation solution 2+ In-situ reduction to uniformly distributed palladium nanoparticles (Sn) 2+ + Pd 2+ → Sn 4+ + Pd↓), forming highly efficient catalytic centers. Next, electroless silver plating is performed to construct a conductive silver layer: the activated microneedle substrate is immersed in the electroless plating solution, where surface palladium nanoparticles strongly catalyze silver ammonia complex ions ([Ag(NH3)2)). + The glucose reduction reaction causes silver atoms to preferentially deposit and connect at the catalytic site, forming a uniform, dense, and bright conductive silver layer. Finally, the silver layer is converted into a functional Ag / AgCl reference layer through chemical oxidation: under light-protected conditions, silver-plated microneedles are immersed in a FeCl3 solution, Fe... 3+ As a mild oxidizing agent, it selectively oxidizes surface silver atoms and binds Cl in the solution. -In-situ formation of a dense AgCl film (Ag + Fe) 3+ + Cl - → AgCl↓ + Fe 2+ Ultimately, a stable Ag / AgCl solid / solid contact system is formed on the surface of the microneedles, and its potential is determined by Cl. - Concentration determines (AgCl(s) + e) - Ag(s) + Cl - Thus, a reference microneedle electrode is prepared.

[0058] The thickness of the Ag / AgCl conductive layer is 10–15 μm.

[0059] Another object of the present invention is to provide a three-electrode sensor, including a working electrode, a reference electrode and a counter electrode, wherein the working microneedle electrode described in the present invention is used as the working electrode, the counter microneedle electrode, Pt wire or carbon rod described in the present invention is used as the counter electrode, the reference microneedle electrode or Ag / AgCl electrode described in the present invention is used as the reference electrode, and PBS buffer or fetal bovine serum at pH 7.4 is used as the electrolyte.

[0060] The three-electrode sensor described in this invention is used in the detection of H3Cit.

[0061] The application is as follows:

[0062] The three-electrode sensor was incubated in PBS solution (pH 7.4) containing different concentrations of citrullinated histone H3. After incubation, electrochemical impedance spectroscopy was performed, and the impedance spectra were recorded and stored. The charge transfer resistance was obtained from the impedance spectra. A standard curve was plotted with the logarithm of the antigen concentration on the x-axis and the charge transfer resistance (Rct) on the y-axis.

[0063] Electrochemical impedance spectroscopy (EIS) of the three-electrode sensor was tested using an electrochemical workstation: The three-electrode sensor was immersed in the test solution containing the target citrullinated histone H3 for incubation, allowing the citrullinated histone H3 to bind sufficiently and specifically to the H3Cit antibody on the working microneedle electrode; after incubation, electrochemical impedance spectroscopy was performed; the impedance spectrum was recorded and stored, and the charge transfer resistance (Rct) was obtained from the impedance spectrum; the charge transfer resistance (Rct) was then substituted into a standard curve to obtain the concentration of citrullinated histone H3 in the test solution.

[0064] Preferably, the concentration of the citrullinated histone H3 is 0.001, 0.01, 0.1, 1, 10, 50, or 100 ng / mL. Of course, it is understood that the concentration gradient is not limited to this.

[0065] Preferably, the three-electrode sensor is immersed in PBS solution (pH 7.4) of different concentrations of citrullinated histone H3 and incubated at 25–37°C for 0.5–4 h.

[0066] More preferably, the three-electrode sensor is immersed in PBS solution (pH 7.4) of different concentrations of citrullinated histone H3 and incubated at 37°C for 30–60 min.

[0067] When performing electrochemical impedance spectroscopy, the applied DC bias voltage is the open-circuit potential of the system, the AC perturbation amplitude is 1 mV to 20 mV, preferably 5 mV or 10 mV, and the frequency scan range is 0.1 Hz to 100 kHz.

[0068] Preferably, the three-electrode sensor is immersed in the test solution containing the target citrullinated histone H3 and incubated at 25–37°C for 0.5–4 h.

[0069] More preferably, the incubation temperature is 37°C and the incubation time is 30–60 min.

[0070] Those skilled in the art can obtain the charge transfer resistance from the impedance spectrum: The measured impedance data at different frequencies are plotted with Z' (real part) on the x-axis and -Z'' (imaginary part) on the y-axis to obtain the Nyquist plot; an equivalent circuit diagram is constructed; after antigen binding, an "insulating layer" that hinders electron transfer is formed on the surface of the working microneedle electrode, resulting in a significant increase in Rct. Using ZView software, the Nyquist plot data is imported, the constructed equivalent circuit diagram is selected, fitted, and Rct is read.

[0071] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0072] (1) The present invention adopts the demolding and molding microneedle preparation technology, which has a short synthesis cycle and uses lignin waste as raw material, resulting in low cost and high environmental friendliness, and the microneedles have good mechanical properties.

[0073] (2) The present invention uses HS-PEG-COOH self-assembled molecular layer as bridging arm, and utilizes thiol (-SH) to form a stable gold-sulfur (Au-S) covalent bond with the gold surface to construct a highly ordered monolayer, which enables the antibody to be firmly and densely grafted onto the microneedle surface, so that more target antigens can be captured per unit area, thereby amplifying the detection signal, increasing the proportion of effective antibody, and combined with the improvement of anti-interference ability, enabling the sensor to detect lower concentrations of target substances.

[0074] (3) This invention utilizes the NHS / EDC activation method to efficiently activate the carboxyl group (-COOH) at the end of the PEG, thereby forming a stable amide covalent bond with the amino group (-NH2) on the H3Cit antibody. This method overcomes the shortcomings of physical adsorption methods, such as random antibody orientation and easy detachment, and achieves directional, firm, and high-density fixation of antibody molecules, maximizing the exposure of their antigen binding sites.

[0075] (4) The three-electrode sensor of the present invention is used to detect H3Cit, which can effectively identify H3Cit and ensure the high stability and reproducibility of the detection results. Attached Figure Description

[0076] Figure 1 This is a flowchart of the preparation method of lignin carbon fiber based microneedle electrode.

[0077] Figure 2 This is a scanning electron microscope image of the microneedle substrate prepared in Example 1.

[0078] Figure 3 The results show the mechanical properties of the microneedle substrate prepared in Example 2.

[0079] Figure 4 This is an H&E staining image of mouse epidermis after puncture using the microneedle base prepared in Example 2 (the punctured epidermis can be clearly seen within the red circle).

[0080] Figure 5 This is an XPSN 1s image of the working microneedle electrode before and after treatment with HS-PEG-COOH, EDC / NHS, and H3Cit antibodies in Example 4.

[0081] Figure 6 These are laser confocal microscopy images of the microneedle substrate cross-section of the H3Cit antibody and goat anti-rabbit IgG H&L (PE) antibody grafted onto the surface in Example 4; where a is a laser confocal microscopy image under dark field and b is a laser confocal microscopy image under bright field.

[0082] Figure 7 The images show the EIS plot (a) and the corresponding Rct-lg[H3Cit] plot (b) of the three-electrode system for detecting H3Cit in Example 4.

[0083] Figure 8 The images show the EIS plot (a) and the corresponding Rct-lg[H3Cit] plot (b) of the three-electrode system for detecting H3Cit in Example 5.

[0084] Figure 9The images are laser confocal microscopy images of the working microneedle electrode prepared in Comparative Example 1 grafted with goat anti-rabbit IgG H&L (PE) antibody; where a is a laser confocal microscopy image under dark field and b is a laser confocal microscopy image under bright field. Detailed Implementation

[0085] The present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. Unless otherwise specified, the experimental methods described in the following embodiments are all routine operations, and the reagents used are commercially available.

[0086] Example 1

[0087] like Figure 1 As shown, a lignin-carbon fiber-based microneedle electrode is prepared by the following method, including the following steps:

[0088] Step (1), Granulation: Vacuum heat treatment of lignin powder until the moisture content is ≤0.2%, then place it in a twin-screw granulator, adjust the granulation temperature to 180℃ and the screw speed to 80 rpm, and granulate to obtain lignin particles;

[0089] Step (2), meltblowing: Place the lignin particles in a meltblowing test machine, adjust the meltblowing temperature to 250℃, the screw speed to 250 rpm, the hot air frequency to 45 Hz, and the receiving distance to 40 cm, and obtain lignin fibers by meltblowing;

[0090] Step (3), Pre-oxidation treatment: Place lignin fibers in a tube furnace, introduce air, adjust the heating rate to 0.25℃ / min, heat from room temperature to 280℃, hold at 280℃ for 1 h, and cool naturally to room temperature to obtain lignin carbon fiber precursor.

[0091] Step (4), carbonization treatment: Place the lignin carbon fiber precursor in a tube furnace, introduce argon gas, adjust the heating rate to 3℃ / min, heat from room temperature to 1000℃, hold at 1000℃ for 1h, and cool naturally to room temperature to obtain lignin carbon fiber (LCF).

[0092] Step (5): 1.5 g LCF was ball-milled to a particle size ≤ 5 μm and ultrasonically dispersed in 15 mL of PLGA (acid-terminated, lactide:glycolide 75:25 (molar ratio), Mw≈30000, concentration 0.1 g / mL) dichloromethane solution to obtain a slurry; Silicon mold casting: The obtained slurry was injected into a silicon-based microneedle array mold (the silicon-based microneedle array mold consists of multiple arrays of hollow cones, each cone has a needle length (i.e., height) of 700 μm, a bottom diameter of 250 μm, a top spacing of 550 μm between adjacent cones, and a cone array number of 10×10), and vacuum degassing was performed; Curing and demolding: The solvent was evaporated at room temperature for 24 h, dried at 60 ℃ for 8 h, and demolded under vacuum to obtain the microneedle substrate; The scanning electron microscope (SEM) image of the microneedle substrate is shown below. Figure 2 As shown, the microneedle substrate array is neat, and each microneedle substrate is nearly conical in shape. The length (i.e., height) of the microneedle substrate is 700 μm, the tip diameter is 50 μm, and the bottom diameter is 250 μm.

[0093] Step (6): Deposit a 20 nm Pt film on the microneedle substrate obtained in step (5). Specific operation: Place the microneedle substrate in the high vacuum sputtering chamber of an ion sputtering instrument. Under continuous rotation, use a high-purity gold target and sputter for 90 s at a working pressure of 0.02 mBar and a sputtering current of 20 mA in an argon atmosphere to form a continuous Pt film (i.e., a Pt conductive layer) with a thickness of 20 nm on the microneedle substrate, thus obtaining the microneedle electrode.

[0094] Step (7): At room temperature, the microneedle substrate prepared in step (5) is sequentially treated with 10 mL of sensitization solution (prepared with water) containing 10 g / L SnCl2 and 40 mL / L concentrated hydrochloric acid (37%) and activation solution (prepared with water) containing 0.25 g / L PdCl2. The treatment time in both the sensitization solution and the activation solution is 30 min, so that the nano-palladium catalytic centers are adsorbed on the surface of the microneedle substrate. Then, the microneedle substrate is immersed in 10 mL of chemical plating solution mixed with equal volumes of 3.0 g / L AgNO3 silver ammonia solution, 2.5 g / L glucose and 2.0 g / L NaOH reducing solution, and reacted at 50°C for 8 minutes to deposit a uniform silver layer. After that, it is immersed in 0.1 mol / L FeCl3 solution for light-protected oxidation reaction for 5 minutes, so that the surface of the silver layer is converted in situ into stable AgCl (i.e., Ag / AgCl conductive layer, about 10 μm thick), and a reference microneedle electrode is obtained.

[0095] Step (8): Deposit a 20 nm Au film on the microneedle substrate obtained in step (5). Specific procedures: Place the microneedle substrate in the high-vacuum sputtering chamber of an ion sputtering instrument. Under continuous rotation, use a high-purity gold target and sputter for 90 seconds at a working pressure of 0.02 mBar and a sputtering current of 20 mA under an argon atmosphere, thereby forming a continuous gold film with a thickness of 20 nm on the microneedle substrate. Immerse the microneedle substrate with the deposited Au film in 10 mL of HS-PEG-COOH (Mw≈5000) solution (10 mL, 5 mg / mL, prepared with anhydrous ethanol), place it in a humidified chamber, and let it stand at room temperature for 4 h. Remove the substrate, rinse it three times with anhydrous ethanol to remove unbound thiol molecules, and dry it with nitrogen. Then immerse it in 10 mL of activation solution (using a mixed solvent of EDC·HCl and NHS prepared in PBS buffer at pH 5.5, with concentrations of 0.4 M and 0.1 M of EDC·HCl and NHS, respectively). In step M), the activation solution is uniformly applied to the surface of the microneedle substrate, and the reaction is carried out at room temperature for 30 minutes. The H3Cit antibody (Citrullinated Histone H3 (human, recombinant), Cayman Chemical, Item No. 17926) is diluted with PBS buffer (pH 7.4) to a final concentration of 50 μg / mL. The activated microneedle substrate is immersed in the H3Cit antibody solution and incubated with slow shaking at 4°C for 12 hours. The incubated microneedle substrate is then immersed in Tris-HCl buffer (pH 8.5) containing 10 mg / mL BSA and allowed to stand at room temperature for 1 hour to block unbound sites. The substrate is then removed and rinsed three times with PBS buffer (pH 7.4) containing 0.05% Tween-20 for 5 minutes each time to remove unbound antibody and obtain the working microneedle electrode.

[0096] Step (9): Using the microneedle electrode, reference microneedle electrode, and working microneedle electrode prepared in steps (6), (7), and (8), construct a three-electrode system. Immerse the three electrodes in PBS solution (pH 7.4) with H3 concentration gradients of (0, 1 pg / mL, 10 pg / mL, 100 pg / mL, 1 ng / mL, 10 ng / mL) for incubation (incubation temperature: 37 ℃, incubation time: 30 min). After incubation, perform electrochemical impedance spectroscopy (EIS) measurements. The applied DC bias voltage is the open-circuit potential of the system, the AC perturbation amplitude is 10 mV, and the frequency scan range is 0.1 Hz to 100 kHz. Record and store the impedance spectrum at this time as the detection spectrum, and the corresponding impedance value is denoted as Z. i The equivalent circuit was fitted using ZView software to analyze the change in charge transfer resistance (Rct).

[0097] Example 2

[0098] A lignin-carbon fiber-based microneedle electrode is prepared by the following method, including the following steps:

[0099] Step (1), Granulation: Vacuum heat-treated lignin powder to a moisture content ≤0.2% is placed in a twin-screw granulator, the granulation temperature is adjusted to 180℃ and the screw speed is 80rpm, and lignin particles are obtained.

[0100] Step (2), meltblowing: Place the lignin particles in a meltblowing test machine, adjust the meltblowing temperature to 250℃, the screw speed to 250 rpm, the hot air frequency to 45 Hz, and the receiving distance to 40 cm, and obtain lignin fibers by meltblowing;

[0101] Step (3), Pre-oxidation treatment: Place lignin fibers in a tube furnace, introduce air, adjust the heating rate to 0.25℃ / min, heat from room temperature to 280℃, hold at 280℃ for 1 h, and cool naturally to room temperature to obtain lignin carbon fiber precursor.

[0102] Step (4), carbonization treatment: Place the lignin carbon fiber precursor in a tube furnace, introduce argon gas, adjust the heating rate to 3℃ / min, heat from room temperature to 1000℃, hold at 1000℃ for 1h, and cool naturally to room temperature to obtain lignin carbon fiber (LCF).

[0103] Step (5): 1.5 g LCF was ball-milled to a particle size ≤ 5 μm and ultrasonically dispersed in 15 mL of PLGA (0.167 g / mL) in dichloromethane solution; the resulting slurry was injected into a silicon-based microneedle array mold (same as in Example 1) and vacuum defoamed; curing and demolding: the solvent was evaporated at room temperature for 24 h, dried at 60 ℃ for 8 h, and demolded to obtain the microneedle substrate; Figure 3 These are the results of the mechanical property evaluation of the microneedle substrate used in this embodiment. Figure 4 This is an H&E staining image of mouse epidermis after microneedle base puncture in this embodiment. It can be seen that the single needle breaking strength is greater than 2N, which can penetrate the mouse epidermis.

[0104] Step (6): Deposit a 20 nm Pt film on the microneedle substrate obtained in step (5). Specific operation: Place the microneedle substrate in the high vacuum sputtering chamber of an ion sputtering instrument. Under continuous rotation, use a high-purity gold target and sputter for 90 seconds at a working pressure of 0.02 mBar and a sputtering current of 20 mA in an argon atmosphere to form a continuous Pt film with a thickness of 20 nm on the microneedle substrate, thus obtaining the microneedle electrode.

[0105] Step (7): At room temperature, the microneedle substrate prepared in step (5) is sequentially treated with 10 mL of sensitization solution containing 10 g / L SnCl2 and 40 mL / L concentrated hydrochloric acid (37%) and activation solution containing 0.25 g / L PdCl2. The treatment time in both the sensitization solution and the activation solution is 30 min, so that the nano-palladium catalytic centers are adsorbed on the surface of the microneedle substrate. Then, the microneedle substrate is immersed in 10 mL of chemical plating solution containing equal volumes of 3.0 g / L AgNO3 silver ammonia solution and 2.5 g / L glucose-2.0 g / L NaOH reducing solution. The reaction is carried out at 50 °C for 8 minutes to deposit a uniform silver layer. Then, a light-protected oxidation reaction is carried out using 0.1 mol / L FeCl3 solution for 5 minutes to convert the silver layer surface in situ into stable AgCl (thickness of about 10 μm), thus obtaining the reference microneedle electrode.

[0106] Step (8): Referring to step (8) of Example 1, a 20 nm Au film was deposited on the microneedle substrate prepared in step (5) using an ion sputtering instrument. The microneedle substrate with the Au film deposited was immersed in HS-PEG-COOH (Mw≈5000) solution (10 mL, 0.05 g / mL, prepared with anhydrous ethanol), placed in a humidified chamber, and allowed to stand at room temperature for 4 h. The substrate was then removed, rinsed three times with anhydrous ethanol to remove unbound thiol molecules, and dried with nitrogen. It was then immersed in 10 mL of activation solution (same as in Example 1) to ensure that the activation solution evenly covered the probe surface, and reacted at room temperature for 30 minutes. The H3Cit antibody (same as in Example 1) was diluted with PBS (pH 7.4) to a final concentration of 50 μg / mL. The activated microneedle substrate was immersed in the H3Cit antibody solution and incubated with slow shaking at 4°C for 12 h. The incubated microneedle substrate was then immersed in 10 mg / mL BSA Tris-HCl buffer (pH 7.4). In step 8.5), the mixture was allowed to stand at room temperature for 1 hour to block unbound sites; then it was washed three times with PBS containing 0.05% Tween-20 for 5 minutes each time to remove unbound antibodies and obtain the working microneedle electrode.

[0107] Step (9): Using the microneedle electrode, reference microneedle electrode, and working microneedle electrode prepared in steps (6), (7), and (8), a three-electrode system was constructed. The three electrodes were immersed in PBS solution (pH 7.4) with a citrullinated histone H3 concentration gradient of (0, 1 pg / mL, 10 pg / mL, 100 pg / mL, 1 ng / mL, 10 ng / mL) for incubation (incubation temperature: 37 ℃, incubation time: 30 min). After incubation, electrochemical impedance spectroscopy was performed. The applied DC bias voltage was the open-circuit potential of the system, the AC perturbation amplitude was 10 mV, and the frequency scan range was 0.1 Hz to 100 kHz. The impedance spectrum at this time was recorded and stored as the detection spectrum, and the corresponding impedance value was denoted as Z. iThe equivalent circuit was fitted using ZView software to analyze the change in charge transfer resistance (Rct).

[0108] Example 3

[0109] A lignin-carbon fiber-based microneedle electrode is prepared by the following method, including the following steps:

[0110] Step (1), Granulation: Vacuum heat treatment of lignin powder until the moisture content is ≤0.2%, then place it in a twin-screw granulator, adjust the granulation temperature to 180℃ and the screw speed to 80 rpm, and granulate to obtain lignin particles;

[0111] Step (2), meltblowing: Place the lignin particles in a meltblowing test machine, adjust the meltblowing temperature to 250℃, the screw speed to 250 rpm, the hot air frequency to 45 Hz, and the receiving distance to 40 cm, and obtain lignin fibers by meltblowing;

[0112] Step (3), Pre-oxidation treatment: Place lignin fibers in a tube furnace, introduce air, adjust the heating rate to 0.25℃ / min, heat from room temperature to 280℃, keep at 280℃ for 1 hour, and cool naturally to room temperature to obtain lignin carbon fiber precursor.

[0113] Step (4), carbonization treatment: Place the lignin carbon fiber precursor in a tube furnace, introduce argon gas, adjust the heating rate to 3℃ / min, heat from room temperature to 1000℃, hold at 1000℃ for 1 h, and cool naturally to room temperature to obtain lignin carbon fiber (LCF).

[0114] Step (5): 1.5 g LCF was ball-milled to a particle size ≤ 5 μm and ultrasonically dispersed in 15 mL of PLGA (0.233 g / mL) dichloromethane solution to obtain a slurry; the obtained slurry was injected into a silicon-based microneedle array mold (same as in Example 1), and vacuum degassing was performed; curing and demolding: the solvent was evaporated at room temperature for 24 h, dried at 60 ℃ for 8 h, and demolded to obtain the microneedle substrate;

[0115] Step (6): Deposit a 20 nm Pt film on the microneedle substrate prepared in step (5) using an ion sputtering instrument. Specifically, place the microneedle substrate in the high vacuum sputtering chamber of the ion sputtering instrument, and use a high-purity gold target under continuous rotation. Sputter for 90 seconds at a working pressure of 0.02 mBar and a sputtering current of 20 mA under an argon atmosphere to form a continuous Pt film with a thickness of 20 nm on the microneedle substrate, thus obtaining the microneedle electrode.

[0116] Step (7): At room temperature, the microneedle substrate prepared in step (5) is sequentially treated with 10 mL of sensitization solution containing 10 g / L SnCl2 and 40 mL / L concentrated hydrochloric acid (37%) and activation solution containing 0.25 g / L PdCl2. The treatment time in both the sensitization solution and the activation solution is 30 min, so that the nano-palladium catalytic centers are adsorbed on the surface. Then, the substrate is immersed in 10 mL of chemical plating solution mixed with an equal volume of 3.0 g / L AgNO3 silver ammonia solution and 2.5 g / L glucose-2.0 g / L NaOH reducing solution, and reacted at 50°C for 8 minutes to deposit a uniform silver layer. Then, a light-protected oxidation reaction is carried out using 0.1 mol / L FeCl3 solution for 5 minutes to convert the silver layer surface in situ into stable AgCl (thickness of about 10 μm), thus obtaining the reference microneedle electrode.

[0117] Step (8): Referring to step (8) of Example 1, a 20 nm Au film was deposited on the microneedle substrate prepared in step (5) using an ion sputtering instrument. The microneedle substrate with the deposited Au film was immersed in HS-PEG-COOH (Mw≈5000) solution (10 mL, 0.05 g / mL, prepared with anhydrous ethanol), placed in a humidified chamber, and allowed to stand at room temperature for 4 h. The substrate was then removed, rinsed three times with anhydrous ethanol to remove unbound thiol molecules, and dried with nitrogen. It was then immersed in 10 mL of activation solution (same as in Example 1) to ensure that the activation solution evenly covered the probe surface, and reacted at room temperature for 30 minutes. The H3Cit antibody was diluted with PBS (pH 7.4) to a final concentration of 50 μg / mL. The activated microneedle substrate was immersed in the H3Cit antibody solution and incubated with slow shaking at 4°C for 12 h. The incubated microneedle substrate was then immersed in 10 mg / mL BSA Tris-HCl buffer (pH 7.4). In step 8.5), the mixture was allowed to stand at room temperature for 1 hour to block unbound sites; it was then washed three times with PBS containing 0.05% Tween-20 for 5 minutes each time to remove unbound antibodies and obtain the working microneedle electrode.

[0118] Step (9): Using the microneedle electrode, reference microneedle electrode, and working microneedle electrode prepared in steps (6), (7), and (8), a three-electrode system was constructed. The three electrodes were immersed in PBS solution (pH 7.4) with a citrullinated histone H3 concentration gradient of (0, 1 pg / mL, 10 pg / mL, 100 pg / mL, 1 ng / mL, 10 ng / mL) for incubation (incubation temperature: 37 ℃, incubation time: 30 min). After incubation, electrochemical impedance spectroscopy was performed. The applied DC bias voltage was the open-circuit potential of the system, the AC perturbation amplitude was 10 mV, and the frequency scan range was 0.1 Hz to 100 kHz. The impedance spectrum at this time was recorded and stored as the detection spectrum, and the corresponding impedance value was denoted as Z. iThe equivalent circuit was fitted using ZView software to analyze the change in charge transfer resistance (Rct).

[0119] Example 4

[0120] A lignin-carbon fiber-based microneedle electrode is prepared by the following method, including the following steps:

[0121] Step (1), Granulation: Vacuum heat treatment of lignin powder until the moisture content is ≤0.2%, then place it in a twin-screw granulator, adjust the granulation temperature to 180℃ and the screw speed to 80 rpm, and granulate to obtain lignin particles;

[0122] Step (2), meltblowing: Place the lignin particles in a meltblowing test machine, adjust the meltblowing temperature to 250℃, the screw speed to 250 rpm, the hot air frequency to 45 Hz, and the receiving distance to 40 cm, and obtain lignin fibers by meltblowing;

[0123] Step (3), Pre-oxidation treatment: Place lignin fibers in a tube furnace, introduce air, adjust the heating rate to 0.25℃ / min, heat from room temperature to 280℃, keep at 280℃ for 1 hour, and cool naturally to room temperature to obtain lignin carbon fiber precursor.

[0124] Step (4), carbonization treatment: Place the lignin carbon fiber precursor in a tube furnace, introduce argon gas, adjust the heating rate to 3℃ / min, heat from room temperature to 1000℃, hold at 1000℃ for 1 h, and cool naturally to room temperature to obtain lignin carbon fiber (LCF).

[0125] Step (5): 1.5 g LCF was ball-milled to a particle size ≤ 5 μm and ultrasonically dispersed in 15 mL of PLGA (0.233 g / mL) dichloromethane solution to obtain a slurry; the obtained slurry was injected into a silicon-based microneedle array mold (same as in Example 1), and vacuum degassing was performed; curing and demolding: the solvent was evaporated at room temperature for 24 h, dried at 60 ℃ for 8 h, and demolded to obtain the microneedle substrate;

[0126] Step (6): Referring to step (8) of Example 1, a 20 nm Au film was deposited on the microneedle substrate prepared in step (5) using an ion sputtering instrument. The microneedle substrate with the deposited Au film was immersed in HS-PEG-COOH (Mw≈5000) solution (10 mL, 0.05 g / mL, prepared with anhydrous ethanol), placed in a humidified chamber, and allowed to stand at room temperature for 4 h. The substrate was then removed, rinsed three times with anhydrous ethanol to remove unbound thiol molecules, and dried with nitrogen. Then, 10 mL of activation solution (same as in Example 1) was immersed to ensure the activation solution evenly covered the probe surface, and the reaction was carried out at room temperature for 30 minutes. The H3Cit antibody was diluted with PBS (pH 7.4) to a final concentration of 50 μg / mL. The activated microneedle substrate was immersed in the H3Cit antibody solution and incubated with slow shaking at 4 °C for 12 h. The incubated microneedle substrate was then immersed in 10 mg / mL BSA Tris-HCl buffer (pH 7.4). In step 8.5), the unbound sites were blocked by standing at room temperature for 1 hour. The unbound antibodies were removed by rinsing three times with PBS containing 0.05% Tween-20 for 5 minutes each time, and the working microneedle electrode was obtained. Figure 5 XPS N 1s images of the working microneedle electrode before and after treatment with HS-PEG-COOH, EDC / NHS, and H3Cit antibodies show that the intensity of the amide carbonyl peak (-NC=O, 287.8 eV) increases after antibody conjugation, indicating successful antibody grafting. The antibody-grafted working microneedle electrode was incubated with goat anti-rabbit IgG H&L (PE) antibody solution (1:200 dilution in PBS) at room temperature for 2 h. Observation using a laser confocal microscope revealed significant fluorescence on the microneedle surface. Figure 6 This demonstrates that the antibody was successfully grafted onto the surface of the microneedle substrate.

[0127] Step (7): Using the working microneedle electrode prepared in step (6), a three-electrode system was constructed with platinum wire and a commercial Ag / AgCl electrode. The three electrodes were immersed in PBS solution (pH 7.4) with citrullinated histone H3 concentration gradients of (0, 1 pg / mL, 10 pg / mL, 100 pg / mL, 1 ng / mL, 10 ng / mL) for incubation (incubation temperature: 37 ℃, incubation time: 30 min). After incubation, electrochemical impedance spectroscopy was performed. The applied DC bias voltage was the open-circuit potential of the system, the AC perturbation amplitude was 10 mV, and the frequency scan range was 0.1 Hz to 100 kHz. The impedance spectrum at this time was recorded and stored as the detection spectrum, and the corresponding impedance value was denoted as Z. i The equivalent circuit was fitted using ZView software to analyze the change in charge transfer resistance (Rct). Figure 7Figures a and b show the EIS plot and the corresponding Rct-lg[H3Cit] plot for the detection of H3 by the three-electrode system, respectively. It can be seen that as the concentration of H3 increases, the capacitive arc radius of the electrochemical impedance spectroscopy (EIS) increases significantly, and the charge transfer resistance (Rct) shows a good linear relationship with the logarithm of the antigen concentration. This is because the antigen-antibody binding effectively hinders the interfacial electron transfer, confirming that the three-electrode system can effectively detect H3Cit.

[0128] Example 5

[0129] A lignin-carbon fiber-based microneedle electrode is prepared by the following method, including the following steps:

[0130] Step (1), Granulation: Vacuum heat treatment of lignin powder until the moisture content is ≤0.2%, then place it in a twin-screw granulator, adjust the granulation temperature to 180℃ and the screw speed to 80 rpm, and granulate to obtain lignin particles;

[0131] Step (2), meltblowing: Place the lignin particles in a meltblowing test machine, adjust the meltblowing temperature to 250℃, the screw speed to 250 rpm, the hot air frequency to 45 Hz, and the receiving distance to 40 cm, and obtain lignin fibers by meltblowing;

[0132] Step (3), Pre-oxidation treatment: Place lignin fibers in a tube furnace, introduce air, adjust the heating rate to 0.25℃ / min, heat from room temperature to 280℃, keep at 280℃ for 1 hour, and cool naturally to room temperature to obtain lignin carbon fiber precursor.

[0133] Step (4), carbonization treatment: Place the lignin carbon fiber precursor in a tube furnace, introduce argon gas, adjust the heating rate to 3℃ / min, heat from room temperature to 1000℃, hold at 1000℃ for 1 h, and cool naturally to room temperature to obtain lignin carbon fiber (LCF).

[0134] Step (5): 1.5 g LCF was ball-milled to a particle size ≤ 5 μm and ultrasonically dispersed in 15 mL of PLGA (0.233 g / mL) dichloromethane solution to obtain a slurry; the obtained slurry was injected into a silicon-based microneedle array mold (same as in Example 1), and vacuum degassing was performed; curing and demolding: the solvent was evaporated at room temperature for 24 h, dried at 60 ℃ for 8 h, and demolded to obtain the microneedle substrate;

[0135] Step (6): Referring to step (8) of Example 1, a 20 nm Au film was deposited on the microneedle substrate prepared in step (5) using an ion sputtering instrument. The microneedle substrate with the deposited Au film was immersed in HS-PEG-COOH (Mw≈5000) solution (10 mL, 0.05 g / mL, prepared with anhydrous ethanol), placed in a humidified chamber, and allowed to stand at room temperature for 4 h. The substrate was then removed, rinsed three times with anhydrous ethanol to remove unbound thiol molecules, and dried with nitrogen. Then, 10 mL of activation solution (same as in Example 1) was immersed to ensure the activation solution evenly covered the probe surface, and the reaction was carried out at room temperature for 30 minutes. The H3Cit antibody was diluted with PBS (pH 7.4) to a final concentration of 50 μg / mL. The activated microneedle substrate was immersed in the H3Cit antibody solution and incubated with slow shaking at 4 °C for 12 h. The incubated microneedle substrate was then immersed in 10 mg / mL BSA Tris-HCl buffer (pH 7.4). In step 8.5), the mixture was allowed to stand at room temperature for 1 hour to block unbound sites; then it was washed three times with PBS containing 0.05% Tween-20 for 5 minutes each time to remove unbound antibodies and obtain the working microneedle electrode.

[0136] Step (7): Using the working microneedle electrode prepared in step (6), a three-electrode system was constructed with platinum wire and a commercial Ag / AgCl electrode. Fetal bovine serum was used as the electrolyte, and its electrochemical impedance spectroscopy (EIS) was tested using an electrochemical workstation. PBS solutions (pH 7.4) with citrullinated histone H3 concentration gradients (0, 1 pg / mL, 10 pg / mL, 100 pg / mL, 1 ng / mL, 10 ng / mL) were incubated with the three-electrode system (incubation temperature was 37 ℃, incubation time was 30 min) and then the EIS was tested. The equivalent circuit was fitted using ZView software, and the charge transfer resistance (Rct) was analyzed. Figure 8 Figures a and b show the EIS plot and the corresponding Rct-lg[H3Cit] plot for the detection of H3Cit using a three-electrode system, respectively. It can be seen that as the concentration of H3Cit increases (0~1 ng / mL), the capacitive arc radius of the electrochemical impedance spectroscopy (EIS) increases significantly, and the charge transfer resistance (Rct) shows a good linear relationship with the logarithm of the antigen concentration. However, when the concentration of H3Cit in serum rises to 10 ng / mL, the EIS plot changes from a semicircle to a slanted line representing diffusion control. This is because the high concentration of antigen and serum protein together form a dense insulating layer on the electrode surface.

[0137] Comparative Example 1

[0138] A lignin-carbon fiber-based microneedle electrode is prepared by the following method, including the following steps:

[0139] Step (1), Granulation: Vacuum heat treatment of lignin powder until the moisture content is ≤0.2%, then place it in a twin-screw granulator, adjust the granulation temperature to 180℃ and the screw speed to 80 rpm, and granulate to obtain lignin particles;

[0140] Step (2), meltblowing: Place the lignin particles in a meltblowing test machine, adjust the meltblowing temperature to 250℃, the screw speed to 250 rpm, the hot air frequency to 45 Hz, and the receiving distance to 40 cm, and obtain lignin fibers by meltblowing;

[0141] Step (3), Pre-oxidation treatment: Place lignin fibers in a tube furnace, introduce air, adjust the heating rate to 0.25℃ / min, heat from room temperature to 280℃, keep at 280℃ for 1 hour, and cool naturally to room temperature to obtain lignin carbon fiber precursor.

[0142] Step (4), carbonization treatment: Place the lignin carbon fiber precursor in a tube furnace, introduce argon gas, adjust the heating rate to 3℃ / min, heat from room temperature to 1000℃, hold at 1000℃ for 1 h, and cool naturally to room temperature to obtain lignin carbon fiber (LCF).

[0143] Step (5): 1.5 g of LCF was ball-milled to a particle size ≤ 5 μm, and ultrasonically dispersed in 15 mL of PLGA in dichloromethane solution (0.233 g / mL) to obtain a slurry; the obtained slurry was injected into a silicon-based microneedle array mold (same as in Example 1), and vacuum degassing was performed. Curing and demolding: the solvent was evaporated at room temperature for 24 h, and dried at 60 ℃ for 8 h, and the microneedle substrate was obtained after demolding;

[0144] Step (6): Referring to step (8) of Example 1, a 20 nm Au film was deposited on the microneedle substrate prepared in step (5) using an ion sputtering instrument. The microneedle substrate with the deposited Au film was immersed in HS-PEG-COOH (Mw≈5000) solution (10 mL, 0.05 g / mL, prepared with anhydrous ethanol), placed in a humidified chamber, and allowed to stand at room temperature for 4 h. The substrate was then removed, rinsed three times with anhydrous ethanol to remove unbound thiol molecules, and dried with nitrogen. It was then immersed in 10 mL of activation solution (same as in Example 1) to ensure that the activation solution uniformly covered the probe surface, and reacted at room temperature for 30 minutes to obtain the working microneedle electrode. The working microneedle electrode was co-incubated with goat anti-rabbit IgG H&L (PE) antibody solution (with PBS as solvent, diluted 1:200) at room temperature for 2 h. After observation using a laser confocal microscope, no obvious fluorescence was observed on the surface of the microneedle. Figure 9 By comparing Comparative Example 1 with Example 4 ( Figure 6 This further illustrates that the method in Example 4 successfully grafted the H3Cit antibody onto the surface of the microneedle.

Claims

1. A lignin-carbon fiber-based microneedle electrode, characterized in that: The system includes a working microneedle electrode, a counter microneedle electrode, and a reference microneedle electrode. The working microneedle electrode is constructed by depositing gold on a microneedle substrate and bridging it with a self-assembled thiol-polyethylene glycol-carboxyl molecular layer, using an N-hydroxysuccinimide / 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride activation method to covalently couple an H3Cit antibody to a specific biorecognition interface. The counter microneedle electrode is prepared by depositing a platinum conductive layer on a microneedle substrate. The reference microneedle electrode is prepared by depositing an Ag / AgCl conductive layer on a microneedle substrate. The microneedle substrate is prepared by ball milling and ultrasonically dispersing lignin carbon fibers in a dichloromethane solution of polylactic acid-glycolic acid copolymer to obtain a slurry, injecting the slurry into a microneedle array mold, and then vacuum degassing. The lignin carbon fibers are obtained by granulating and melt-blowing lignin to obtain lignin fibers, which are then pre-oxidized and carbonized. The mass ratio of the lignin carbon fibers to the polylactic acid-glycolic acid copolymer is 20:80 to 80: 20; The molecular weight of the thiol-polyethylene glycol-carboxyl group is 5000.

2. A method for preparing the lignin-carbon fiber-based microneedle electrode according to claim 1, characterized in that: Includes the following steps: Step (1): The lignin carbon fiber is ball-milled and ultrasonically dispersed in a dichloromethane solution of polylactic acid-glycolic acid copolymer to obtain a slurry. The slurry is injected into a microneedle array mold, vacuum degassing is performed, and the mold is solidified and demolded to obtain a microneedle substrate. Step (2): Au is deposited on the microneedle substrate and bridged by a self-assembled thiol-polyethylene glycol-carboxyl molecular layer. A specific biorecognition interface is constructed by covalently coupling H3Cit antibody using N-hydroxysuccinimide / 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride activation method, thus obtaining the working microneedle electrode. A Pt conductive layer and an Ag / AgCl conductive layer are deposited on the microneedle substrate to obtain the counter microneedle electrode and the reference microneedle electrode, respectively.

3. The method for preparing the lignin-carbon fiber-based microneedle electrode according to claim 2, characterized in that: The lignin-fiber cellulose is prepared by the following method, including the following steps: Step (1): The lignin is granulated and melt-blown to obtain lignin fiber; the granulation temperature is 150-190℃ and the screw speed is 50-100rpm; the melt-blowing temperature is 230-250℃, the screw speed is 200-350rpm, the hot air frequency is 30-50Hz, and the receiving distance is 30-50cm. Step (2): The lignin fibers are subjected to pre-oxidation treatment and carbonization treatment in sequence to obtain lignin carbon fibers; the atmosphere of the pre-oxidation treatment is air, the temperature of the pre-oxidation treatment is 250-300℃, and the holding time of the pre-oxidation treatment is 1-2h; the atmosphere of the carbonization treatment is nitrogen or argon, the temperature of the carbonization treatment is 800-1500℃, and the holding time of the carbonization treatment is 1-2h.

4. The method for preparing the lignin-carbon fiber-based microneedle electrode according to claim 2, characterized in that: The LCF is ball-milled to a particle size ≤10 μm; the polylactic acid-glycolic acid copolymer is PLGA 75:25, Mw > 100000 Da.

5. The method for preparing the lignin-carbon fiber-based microneedle electrode according to claim 2, characterized in that: The LCF is ball-milled to a particle size ≤ 5 μm.

6. The method for preparing the lignin-carbon fiber-based microneedle electrode according to claim 2, characterized in that: The working microneedle electrode is formed by ion sputtering Au onto the microneedle substrate using an ion sputtering instrument, immersing it in a mercapto-polyethylene glycol-carboxyl solution or locally adding a mercapto-polyethylene glycol-carboxyl solution, placing it in a humid chamber, and standing at room temperature for 4-8 hours; then removing the substrate, rinsing it with anhydrous ethanol, and drying it with nitrogen. A mixed solution of EDC·HCl and NHS was prepared using PBS buffer as the activation solution. This activation solution was used to evenly cover the surface of the microneedle substrate, and the reaction was carried out at room temperature for 30–60 minutes. The activated microneedle substrate was then immersed in a solution of H3Cit antibody at -4 to 8°C. o C. Incubate with oscillation for 12–24 h; then immerse the microneedle substrate in bovine serum albumin tris(hydroxymethyl)aminomethane-hydrochloric acid buffer, let stand at room temperature for 1–3 h, and wash with polysorbate 20 solution 3–6 times to obtain the working microneedle electrode grafted with H3Cit antibody. The microneedle electrode is obtained by depositing a Pt conductive layer on a microneedle substrate using an ion sputtering apparatus; the thickness of the Pt conductive layer is 10–200 nm. The reference microneedle electrode was prepared by the following method: the microneedle substrate was sequentially treated with a sensitizing solution containing 8–12 g / L SnCl2 and 40 mL / L concentrated hydrochloric acid for 25–35 min, and then treated with an activation solution containing 0.2–0.3 g / L PdCl2 for 25–35 min; the substrate was then immersed in a chemical plating solution consisting of an equal volume mixture of 2–4 g / L AgNO3 silver ammonia solution and 2.3–2.7 g / L glucose and 1.8–2.2 g / L NaOH reducing solution, at 45–55 °C. o The reaction proceeds for 5–10 minutes, depositing a uniform silver layer. Then, a 0.1–0.2 mol / L FeCl3 solution is used for light-protected oxidation for 5–10 minutes, causing the silver layer surface to be converted in situ into stable AgCl, forming an Ag / AgCl conductive layer, thus obtaining the reference microneedle electrode.

7. The method for preparing the lignin-carbon fiber-based microneedle electrode according to claim 6, characterized in that: The thickness of the Pt conductive layer is 10–30 nm.

8. The method for preparing the lignin-carbon fiber-based microneedle electrode according to claim 7, characterized in that: The thickness of the Pt conductive layer is 20 nm.

9. The method for preparing the lignin-carbon fiber-based microneedle electrode according to claim 6, characterized in that: The method for preparing the Au film is as follows: a microneedle substrate is placed in the high-vacuum sputtering chamber of an ion sputtering instrument. Under continuous rotation, a high-purity gold target is used to sputter for 90 to 180 seconds in an argon atmosphere at a working pressure of 0.02–0.05 mBar and a sputtering current of 20–40 mA, thereby forming a continuous gold film on the microneedle substrate. The thickness of the Au film is 10–200 nm. The mercapto-polyethylene glycol-carboxyl solution is a 0.1–10 mM solution prepared with anhydrous ethanol. The activation solution is a mixed solution of EDC·HCl and NHS prepared with PBS buffer at pH 5.5, with a molar ratio of EDC·HCl to NHS of 5:1 to 1:1 and a concentration of EDC·HCl of 0.1 to 1 mM. The pH of the tris(hydroxymethyl)aminomethane-hydrochloric acid buffer solution is 8.5; the BSA concentration is 5–20 mg / mL. The volume percentage of the polysorbate 20 solution is 0.01-0.1%; the polysorbate 20 solution is prepared using PBS buffer solution at pH 7.

4.

10. The method for preparing the lignin-carbon fiber-based microneedle electrode according to claim 9, characterized in that: The thickness of the Au film is 10–30 nm.

11. The method for preparing the lignin-carbon fiber-based microneedle electrode according to claim 10, characterized in that: The thickness of the Au film is 20 nm.

12. The method for preparing the lignin-carbon fiber-based microneedle electrode according to claim 6, characterized in that: The thickness of the Ag / AgCl conductive layer is 10–15 μm.

13. A three-electrode sensor, characterized in that: It includes a working electrode, a reference electrode, and a counter electrode, with the working microneedle electrode as described in claim 1 as the working electrode, the counter microneedle electrode, Pt wire, or carbon rod as described in claim 1 as the counter electrode, the reference microneedle electrode or Ag / AgCl electrode as described in claim 1 as the reference electrode, and PBS buffer or fetal bovine serum at pH 7.4 as the electrolyte.

14. The application of the three-electrode sensor of claim 13 in the detection of H3Cit for purposes other than disease diagnosis and / or treatment.

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