Construction of microneedle transistor sensor and application of microneedle transistor sensor in histamine in-situ detection

Abstract

The invention relates to the technical field of electrochemical sensing, in particular to construction of a microneedle transistor sensor and application of the microneedle transistor sensor in histamine in-situ detection. According to the expanded microneedle transistor sensor provided by the invention, a molecularly imprinted polymer (MIPs) microneedle sensing electrode of which the surface is modified with specific recognition histamine is taken as a transistor expanded grid electrode, and high-sensitivity and high-selectivity in-situ detection of histamine molecules in mouse skin interstitial fluid is realized by utilizing a signal amplification function of a transistor.

Description

Technical Field

[0001] The invention relates to the technical field of electrochemical sensing, and in particular to the construction of a microneedle transistor sensor and its application in in-situ detection of histamine. Background Art

[0002] With the improvement of living standards and the aggravation of environmental pollution, allergic diseases, tumors, cardiovascular diseases and diabetes are listed as the four major common and difficult diseases by the World Health Organization. Among them, allergic diseases are immune response diseases with complex mechanisms, and multiple factors can cause allergic reactions. Histamine, as an important biologically active amine substance, plays a vital role in allergic reactions. It not only participates in regulating immune responses, but also can induce a series of physiological changes such as vasodilation, itching and pain, and smooth muscle contraction. Therefore, the rapid and accurate detection of histamine is extremely important for the early diagnosis, accurate treatment and efficacy monitoring of allergic diseases. However, the content of histamine in human body fluids is low (≤5ng / mL), with large fluctuations, and significant differences in different tissues and different physiological states. Traditional histamine detection methods usually rely on blood samples, which are highly invasive and still have limitations in detecting allergic reactions in local areas. Therefore, real-time detection of histamine is extremely challenging.

[0003] Tingting Zhang et al. developed a Fe 3 O 4 and a highly sensitive sandwich structure substrate for Ag-based SERS nanotags. 3 O 4 @SiO 2 -COOH was used as a capture component to enrich histamine. After functionalizing Ag nanoparticles with glycine (Gly) and (3-Aminopheyonyl)boronic acid (APBA), they were used to attach histamine and act as SERS nanotags, respectively. -4 ~10 -8 A linear relationship between Raman intensity and histamine concentration was observed in the M range, with a detection limit of 7.24 × 10 -9 M. This method also showed good selectivity in the presence of other neurotransmitters.

[0004] Hui Dong et al. developed a series of genetically encoded G protein-coupled receptor activated (GRAB) HA (GRABHA) sensors with good photostability, subsecond kinetics, nanomolar affinity, and high specificity. Using these GRABHA sensors, the authors measured electrical stimulation-evoked HA release in acute brain slices with high spatiotemporal resolution. In addition, the authors recorded HA release in the hypothalamus and preoptic area of ​​the prefrontal cortex during the sleep-wake cycle of freely moving mice and found different HA dynamic patterns between these specific brain regions.

[0005] In summary, existing technologies can effectively detect histamine within a certain detection range, but these technologies cannot achieve in situ detection. In addition, commonly used biological recognition layers (enzymes, antibodies, etc.) are easily denatured and inactivated during storage and use due to their biological activity, and lose their recognition ability and have poor stability. Summary of the invention

[0006] In view of this, the present invention provides a construction of a microneedle transistor sensor and its application in in situ detection of histamine. The present invention provides an extended microneedle transistor sensor, which uses a microneedle sensing electrode with a surface modified with a molecular imprinting polymer (MIPs) that specifically recognizes histamine as a transistor extension gate, and utilizes the signal amplification function of the transistor to achieve high-sensitivity and high-selectivity in situ detection of histamine molecules in mouse skin interstitial fluid.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a method for preparing a microneedle sensing electrode, comprising the following steps:

[0009] Step 1, taking glycidyl methacrylate, trimethylolpropane trimethacrylate, triethylene glycol dimethacrylate, and 1-hydroxycyclohexyl phenone, mixing them evenly and then curing them by ultraviolet light to obtain polymer microneedles PGMA MNs;

[0010] Step 2, taking the polymer microneedle PGMA MNs, washing and sensitizing the MNs to obtain sensitized microneedles, and then mixing the MNs with a glucose solution and a metal plating solution to obtain conductive microneedles; connecting the copper wire to the conductive microneedles using a conductive metal paste, and curing the metal paste;

[0011] Step 3: Evenly mix the solution containing KCl, histamine dihydrochloride, KOH and pyrrole monomer, and use it as electrolyte solution after deoxygenation. The conductive microneedle after fixing the metal slurry is used as the working electrode, and the counter electrode and the reference electrode constitute a three-electrode system. In-situ electropolymerization is performed by an electrochemical method, and the microneedle sensing electrode is obtained by washing and drying.

[0012] In some specific embodiments of the present invention, the UV curing time in step 1 is 10 to 50 minutes. Preferably, the UV curing time is 10 minutes.

[0013] In some specific embodiments of the present invention, the metal in step 2 includes one or more of silver, platinum or gold.

[0014] In some specific embodiments of the present invention, the sensitizing solution used in the sensitization treatment includes SnCl 2 ·2H 2 O, HCl, and water.

[0015] In some specific embodiments of the present invention, the method for preparing the metal plating solution comprises: taking 0.06 M to 0.08 M AgNO 3 The solution is stirred and 25% to 40% ammonia water is added dropwise until the solution changes from turbid to clear again, thereby obtaining the metal plating solution.

[0016] In some specific embodiments of the present invention, the concentration of the pyrrole monomer in step 3 is 0.05 mol / L to 0.2 mol / L.

[0017] Preferably, the concentration of the pyrrole monomer is 0.12 mol / L.

[0018] In some specific embodiments of the present invention, the counter electrode comprises a platinum sheet electrode; and the reference electrode comprises an Ag / AgCl electrode.

[0019] The invention also provides a microneedle sensing electrode microneedle prepared by the preparation method.

[0020] The present invention also provides a microneedle transistor sensor, including using the microneedle sensing electrode as a transistor extension gate to obtain the microneedle transistor sensor.

[0021] The present invention also provides a histamine detection method, comprising: using an instrument with electrical signal measurement and analysis functions, the connection ends of which are respectively connected to the source and drain of the microneedle transistor sensor and the Ag / AgCl reference electrode;

[0022] The Ag / AgCl reference electrode and the microneedle sensing electrode of the microneedle transistor sensor are placed in a solution containing histamine, and the output characteristic curve and the transfer characteristic curve of the transistor are measured to detect histamine.

[0023] In some specific embodiments of the present invention, the instrument includes a semiconductor tester; when the semiconductor tester measures a transistor output characteristic curve, the VDS interval is set to include 0~-1 V, and the VGS includes 0.4 V; when measuring a transfer characteristic curve, the VGS interval is set to include 0.3~1.7 V, and the VDS includes 0.2 V.

[0024] The present invention also provides the use of any of the following items in preparing a device for detecting histamine:

[0025] (I), the microneedle sensing electrode; and / or

[0026] (II), the microneedle transistor sensor.

[0027] The present invention also provides a histamine detection device, comprising any of the following items:

[0028] (I), the microneedle sensing electrode; and / or

[0029] (II), the microneedle transistor sensor.

[0030] The present invention includes but is not limited to providing the following beneficial effects:

[0031] (1) Highly sensitive and highly selective detection: The present invention achieves highly sensitive and highly selective detection of histamine by designing an extended microneedle gate transistor sensor combined with the highly selective recognition ability of molecular imprinted polymers (MIPs). This detection method can provide faster and more accurate results than traditional detection technologies (such as ELISA and mass spectrometry), especially in the detection of low-concentration histamine.

[0032] (2) Real-time in-situ detection capability: The microneedle sensing system of the present invention can directly contact the interstitial fluid of the skin to achieve real-time in-situ detection of histamine. This real-time monitoring capability is crucial for the early diagnosis and treatment of allergic diseases because it can provide immediate feedback and help doctors adjust treatment plans in a timely manner.

[0033] (3) Minimally invasive and biocompatibility: The minimally invasive nature of the microneedle sensing system reduces the pain and discomfort of patients, while its good biocompatibility ensures the safety of in vivo application. This makes the system suitable for long-term monitoring and personal health management. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0035] Figure 1 The load-displacement curves of PGMA MNs at different photocuring times are shown;

[0036] Figure 2 Optical images of microneedles prepared at different photocuring times after compression testing: (a) photocuring for 10 min; (b) photocuring for 15 min; (c) photocuring for 20 min;

[0037] Figure 3 The infrared curves of electropolymerized MIPs before and after elution are shown;

[0038] Figure 4 Impedance test in PBS buffer with different histamine concentrations: (a) 10~10 6 Hz impedance curve; (b) Linear fitting of impedance modulus value and histamine concentration at 10000 Hz;

[0039] Figure 5 Selectivity test of the electrochemical detection system of the microneedle sensing electrode: (a) at a concentration of 10 -8 Electrochemical impedance spectroscopy curves of the microneedle sensing electrode in PBS buffer with various biogenic amines at a concentration of 10 -8 M The change rate of the interface impedance modulus of the microneedle sensing electrode in various biogenic amine PBS buffers;

[0040] Figure 6 The extended microneedle gate transistor sensor for in vitro histamine detection is shown: (a) The output curve of the extended microneedle gate transistor sensor in PBS buffer with different histamine concentrations; (b) The change of the saturation current of the extended microneedle gate transistor sensor with the change of histamine concentration;

[0041] Figure 7 Figure 2 shows the selectivity test of the extended microneedle gate transistor sensor: (a) at 10 -8 The saturation channel current change rate of the transistor output characteristic curve in PBS buffer with different biogenic amines at 10 -8 Output characteristic curves of the extended microneedle gate transistor sensor in PBS buffer with different biogenic amines of M;

[0042] Figure 8 The measured values ​​of each group of cells using the microplate reader (photometer) at different culture times are shown;

[0043] Fig. 9 Schematic diagram of in situ detection of histamine in mouse skin interstitial fluid;

[0044] Fig.10 The output characteristic curves of the extended microneedle gate transistor sensor at different time points after sensitization are shown;

[0045] Fig.11The stained images of mouse skin tissue sections are shown: (a) before puncture; (b) 0 min after the microneedle electrode is removed; (c) 20 min after the microneedle electrode is removed;

[0046] Fig.12 Optical images of vaporized gold conductive microneedles: (a) actual image; (b) optical microscope image magnified 50 times; (c) optical microscope image magnified 100 times. DETAILED DESCRIPTION

[0047] The present invention discloses the construction of a microneedle transistor sensor and its application in the in situ detection of histamine. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar substitutions and modifications are obvious to those skilled in the art and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0048] The present invention designs and prepares an extended microneedle gate transistor sensor, uses a microneedle sensing electrode whose surface is modified with a molecular imprinting polymer (MIPs) that specifically recognizes histamine as a transistor extended gate, and utilizes the signal amplification function of the transistor to achieve high-sensitivity and high-selectivity in situ detection of histamine molecules in mouse skin interstitial fluid.

[0049] (1) Design, preparation and characterization of microneedle sensing electrodes.

[0050] Microneedle sensing electrodes that can specifically recognize histamine molecules were prepared through three steps: solution casting, chemical silver plating, and in-situ electropolymerization of MIPs. Polymer microneedles (PGMA MNs) were prepared by solution casting photocurable materials. The silver plating layer was used for electrical signal conduction, and then the MIPs recognition layer with polypyrrole as the main body was in-situ electropolymerized on the surface of the conductive microneedle electrode using histamine molecules as templates. The specific cavity formed by the template molecule during the synthesis of MIPs can be specifically adsorbed with histamine molecules through hydrogen bonds and π-π bond interactions. Thus, a microneedle sensing electrode with histamine-specific recognition ability was obtained.

[0051] (2) In vitro detection study of microneedle sensing system.

[0052] The prepared microneedle sensing electrode is used as the working electrode of the electrochemical detection system and the extended gate electrode of the transistor detection system, respectively, to achieve specific detection of histamine in PBS buffer. In the electrochemical detection system, when the MIPs modified on the surface of the microneedle sensing electrode specifically adsorbs histamine molecules, the reduction of the surface conductive sites will cause the increase of the electrode interface impedance. In order to further improve the detection performance of the sensor, the present invention continues to construct an extended microneedle gate transistor sensor. When the MIPs modified on the extended microneedle gate capture negatively charged histamine molecules, the gate potential is reduced, causing the sensor to transfer and the channel current of the output characteristic curve to shift. The extended microneedle gate transistor sensor has a wider linear detection range and a lower detection limit, and has good selectivity and stability, and can achieve specific and sensitive detection of histamine molecules.

[0053] (3) Expand the in vivo detection research of microneedle gate transistor sensors.

[0054] Biocompatibility was demonstrated in cell culture experiments using microneedle sensing electrode leaching solution. Mouse skin tissue sections before and after microneedle puncture demonstrated its low invasiveness and the successful construction of the skin detection channel. The ability of the extended microneedle gate transistor sensor to detect histamine in situ under the mouse skin was further studied by establishing a mouse sensitization model. The results showed that compared with the control group, as the skin allergic reaction of the experimental group mice intensified, a large amount of histamine molecules were induced to be released. These histamine molecules were captured by the MIPs on the extended microneedle gate, resulting in a significant increase in the rate of change of the channel saturation current of the transistor sensing system, realizing the in situ detection of histamine.

[0055] The beneficial effects of the present invention are as follows:

[0056] (1) Highly sensitive and highly selective detection: The present invention achieves highly sensitive and highly selective detection of histamine by designing and extending the microneedle gate transistor sensor and combining it with the highly selective recognition ability of molecular imprinting polymers (MIPs). This detection method can provide faster and more accurate results than traditional detection technologies (such as ELISA and mass spectrometry) (Table 1), especially in the detection of low-concentration histamine.

[0057] Table 1 Performance comparison of several histamine detection technologies

[0058]

[0059] (2) Real-time in-situ detection capability: The microneedle sensing system of the present invention can directly contact the interstitial fluid of the skin to achieve real-time in-situ detection of histamine. This real-time monitoring capability is crucial for the early diagnosis and treatment of allergic diseases because it can provide immediate feedback and help doctors adjust treatment plans in a timely manner.

[0060] (3) Minimally invasive and biocompatibility: The minimally invasive nature of the microneedle sensing system reduces the pain and discomfort of patients, while its good biocompatibility ensures the safety of in vivo application. This makes the system suitable for long-term monitoring and personal health management.

[0061] In particular, other types of conductive materials (such as gold, platinum, etc.) are used instead of silver to construct the conductive layer on the surface of the microneedle to improve the stability and durability of the sensor. When evaporating gold on the surface of the microneedle, a high vacuum thermal evaporation coating device (DH350E Hangzhou Dahua Instrument Manufacturing Co., Ltd.) is used to evaporate a gold conductive layer with a thickness of 80 nm on the surface of the microneedle at a speed of 0.3 A / s. The resistance of the evaporated gold microneedle is in the range of 0.9-2 Ω. The optical image of the evaporated gold conductive microneedle is shown in Figure 1. Fig.12 shown.

[0062] Different molecular imprinting techniques, such as surface imprinting or nanoimprinting techniques, are used to achieve specific recognition of histamine.

[0063] Other types of field effect transistors (such as silicon-based FETs) are used as signal amplification units to improve detection sensitivity.

[0064] These alternatives can be adjusted according to the requirements of different application scenarios to achieve the best detection effect. Improvements and modifications based on the technical solution of the present invention should also be considered as the protection scope of the present invention.

[0065] Unless otherwise specified, the raw materials and reagents used in the construction of the microneedle transistor sensor provided by the present invention and its application in the in situ detection of histamine can be purchased from the market.

[0066] The present invention will be further described below in conjunction with embodiments:

[0067] Example 1 Design, preparation and characterization of microneedle sensing electrodes

[0068] Specific content: A microneedle sensing electrode that can specifically recognize histamine molecules was prepared through three steps: solution casting, chemical silver plating and in situ electropolymerization of MIPs.

[0069] Step 1: Solution pouring.

[0070] 2 mL of glycidyl methacrylate (GMA), 1.5 mL of trimethylolpropane trimethacrylate (TMPTA), 3.5 mL of triethylene glycol dimethacrylate (TEGDMA) and 0.04 g of 1-hydroxycyclohexyl phenone were mixed and poured into the PDMS microneedle mold. The microneedles were cured by UV light for 5 min, 10 min, 15 min, 20 min, 25 min and 30 min respectively. The optical images of the microneedles prepared at different photocuring times after compression test were observed to prepare polymer microneedle arrays with suitable mechanical strength to obtain polymer microneedles (PGMA MNs).

[0071] The load-displacement curves of PGMA MNs at different photocuring times and the optical images of microneedles prepared at different photocuring times after compression testing are shown in Figure 2. Figure 1 , 2 As shown. With the increase of UV curing time, the deformation amount produced by the same load decreases, indicating that the cross-linking reaction between polymer molecules inside PGMA MNs is still continuing, forming more cross-linking points and network structures, and the microneedle strength gradually increases. The load-displacement curves of microneedles with UV irradiation time of 25 min and 30 min are almost the same, indicating that the cross-linking between polymer molecules inside PGMA MNs has reached stability at this time, and the microneedle strength no longer continues to increase. The UV curing time of PGMA MNs in subsequent experiments was selected as 10 min.

[0072] Step 2: Chemical silver plating.

[0073] Preparation of silver plating solution: Take 20 mL of 0.08 M and 0.06 M AgNO 3 The solution was placed in a 40 mL centrifuge tube and stirred while adding AgNO 3 Add 25% ammonia water dropwise to the solution until the turbid solution turns clear again. Place the prepared mixed solution in an ice water bath to cool for later use.

[0074] The front and back surfaces of the prepared PGMA MNs were treated with a plasma cleaner (YZD08-2C, Beijing Zhongke Jiming Technology Co., Ltd.) at 45% power for 5 min and then immediately placed in a solution of 0.4 g SnCl 2 ·2H 2 O, 148 μL 37% HCl and 10 mL water sensitizer solution was shaken for 15 min, and the sensitized microneedles were rinsed with ultrapure water. Finally, the sensitized microneedles and 5 mL 110 g / L and 25 g / L glucose solutions were added to the silver plating solution in an ice water bath for 50 min to obtain conductive microneedles (PGMA / Ag MNs). After silver plating, ultrapure water was used to rinse the microneedles. 2Blow dry and set aside. Use conductive silver paste (particle size 100 nm, 60%, SPI Company, USA) to connect the copper wire and the conductive microneedle, and heat on a hot plate at 60℃ for 6 h to solidify the silver paste.

[0075] Step 3: In situ electropolymerization of MIPs.

[0076] PGMA / Ag MNs were used as working electrodes, and the preparation conditions of electropolymerized MIPs were investigated in the electrode system using an electrochemical workstation. 145 mg KCl, 0.5 g histamine dihydrochloride, 105 mg KOH, and different volumes of pyrrole monomers were mixed in water and N 2 The electropolymerization solution was prepared by bubbling and deoxygenating for 10 min. The above solution was used as the electrolyte solution, the conductive microneedle was used as the working electrode, the platinum electrode was used as the counter electrode, and the Ag / AgCl electrode was used as the reference electrode to form a three-electrode system. The electrochemical workstation was subjected to cyclic voltammetry for in-situ electropolymerization, with a voltage range of -0.4 V~0.5 V, a scan rate of 0.01 V / s, and 7 cycles. The conductive microneedles were placed in electrolytes with pyrrole monomer concentrations of 0.07 mol / L, 0.12 mol / L, and 0.18 mol / L, respectively, and electropolymerization was carried out according to the above cyclic voltammetry steps. In the electrolyte containing 0.07 mol / L pyrrole monomer concentration, MIPs were only observed at the bottom of the microneedle. In the electrolyte containing 0.12 mol / L pyrrole monomer concentration, MIPs evenly covered the entire microneedle surface. When electropolymerized in an electrolyte containing 0.18 mol / L pyrrole monomer concentration, the MIPs electropolymerized on the microneedle surface were locally thick and unevenly distributed. Therefore, the pyrrole monomer concentration in the electrolyte in subsequent experiments was 0.12 mol / L. After the polymerization was completed, the microneedles were placed in 0.05 MH 2 SO 4 , 0.08 M NaOH and ultrapure water for 15 min, 15 min, and 20 min, and then washed with N 2 The surface of the microneedles was blown dry to obtain electropolymerized MIP microneedles (PGMA / Ag / MIPs MNs).

[0077] The infrared curves of electropolymerized MIPs before and after elution are as follows Figure 3 1530 cm -1 The absorption peak at 1300 cm corresponds to the stretching vibration of C=C. -1 The peak at 770 cm corresponds to the stretching vibration of CN / CC. -1 and 1530 cm -1 The peak at 890-1030 cm corresponds to the bending vibration of CH and −1The peaks in the range correspond to the "ring breathing" of the polymer. These absorption peaks correspond to the infrared characteristic peaks of polypyrrole, further confirming the successful electropolymerization of pyrrole monomer on the surface of the microneedle. In addition, by comparing the infrared spectra of the microneedle samples before and after elution, it was found that in the infrared spectra of the eluted samples, the 770 cm-1 peak representing the stretching vibration of the primary amino group NH -1 and 1530 cm -1 The absorption peak at 1480 cm represents the C=N stretching vibration. -1 The absorption peak at 4° weakened and disappeared, indicating that the template molecule (histamine molecule) in the process of synthesizing MIPs was successfully eluted.

[0078] Example 2 In vitro detection study of microneedle sensing system

[0079] Content 1: Study on in vitro histamine detection using electrochemical microneedle working electrodes

[0080] ① Sensitivity of the electrochemical detection system.

[0081] The modified MIPs microneedle electrode (PGMA / Ag / MIPs MNs) was used as a working electrode (WE) to form a three-electrode system with a platinum electrode (counter electrode, CE) and an Ag / AgCl electrode (reference electrode, RE). The alternating current signal was used to measure the microneedle sensing electrode in the presence of different concentrations of histamine (10 -10 ~10 -7 The electrochemical impedance spectroscopy was tested in PBS buffer solutions with different concentrations of 50 mV and a frequency range of 10 Hz to 10 Hz. 6 Hz.

[0082] Different concentrations of histamine (10 -10 ~10 -7 The impedance test results in PBS buffer (M) are as follows Figure 4 As shown in Figure 2, with the increase of histamine molecule concentration in PBS buffer, the impedance modulus |Z| also increases, especially at 10 3 Hz~10 5 In the frequency range of Hz, the impedance curves under different concentrations change significantly. -10 ~10 -7 In the concentration range of 1000 M, the impedance modulus change was linearly correlated with the histamine concentration, with a sensitivity of 2.743 Ω / decade. The detection limit was calculated to be 7.06×10 -11 M.

[0083] ②Selectivity of electrochemical detection system.

[0084] The modified MIPs microneedle electrode (PGMA / Ag / MIPs MNs) was used as the working electrode, and together with the platinum electrode (counter electrode) and the Ag / AgCl electrode (reference electrode) formed a three-electrode system. The microneedle sensing electrode was used to detect the presence of the same concentration (10 -8 Electrochemical impedance spectroscopy tests were carried out in PBS buffer solutions containing histamine, tryptamine, β-phenylethylamine, spermine, agmatine sulfate and other biogenic amines in water (pH 7.0), to observe the responses of the microneedle sensing electrode to different types of biogenic amine molecules, to explore the specific detection of histamine by the electrochemical detection system of the microneedle working electrode, and its anti-interference ability in detection in organisms.

[0085] Selectivity test results of microneedle sensing electrode electrochemical detection system Figure 5 As shown in the figure, only the impedance modulus value in the histamine PBS buffer was significantly increased, and the impedance modulus values ​​tested in other biogenic amine PBS buffers did not change much. The impedance modulus value tested in the blank PBS buffer was |Z 0 |, at concentrations of 10 -8 The impedance modulus change Δ|Z| (Δ|Z| = |Z|-|Z 0 |), calculate the impedance modulus change rate in each solution. Microneedle sensing electrode pair 10 -8 The impedance modulus change rate of the PBS buffer solution with 100 M histamine can reach about 11%, but the impedance modulus change rate of the PBS buffer solution with other biogenic amines of the same concentration is only about 2% at most. The test results show that the microneedle sensing electrode has good selectivity for histamine molecules.

[0086] Content 2: Extending the microneedle gate transistor in vitro histamine detection research

[0087] ① The sensitivity of the microneedle transistor detection system.

[0088] The microneedle sensing electrode was connected to the gate of the 2SJ103GR transistor through a copper wire and a copper clip. The three terminals of the semiconductor tester were connected to the source and drain of the transistor and the Ag / AgCl reference electrode respectively. The reference electrode and the microneedle sensing electrode were placed in a liquid containing different concentrations (10 -12 ~10 -7 The output characteristic curve and transfer characteristic curve were tested in PBS buffer containing 10 M histamine. -12 The PBS buffer was replaced until the histamine concentration reached 10 -7M PBS buffer, and set the VDS range to 0~-1 V, VGS to 0.4 V, and test the output characteristic curves of the transistors at different concentrations. At the same time, set the VGS range to 0.3~1.7 V, VDS to 0.2 V, and test the transfer characteristic curves of the transistors at different concentrations.

[0089] Expanded microneedle gate transistor sensor for in vitro histamine detection results Figure 6 As shown in Figure 2, as the concentration of histamine molecules in the PBS buffer increases, the channel saturation current of the transistor also increases. -12 ~10 -7 In the concentration range of M, the saturated channel current of the transistor is linearly correlated with the histamine concentration, with a sensitivity of 18.1 μA / decade. The detection limit is 8.02×10 -15 M.

[0090] ②Selectivity of microneedle transistor detection system.

[0091] The microneedle sensing electrode was connected to the gate of the 2SJ103GR transistor through a copper wire and a copper clip. The three terminals of the semiconductor tester were connected to the source and drain of the transistor and the Ag / AgCl reference electrode respectively. The reference electrode and the microneedle sensing electrode were placed in the same concentration (10 -8 The output characteristic curves were tested in PBS buffer containing histamine, tryptamine, β-phenylethylamine, spermine and agmatine sulfate. By observing the response of the extended microneedle gate transistor sensor to different types of biogenic amine molecules, the specific detection ability of the microneedle sensing electrode for histamine and its anti-interference ability in detection in organisms were explored.

[0092] The selectivity test results of the extended microneedle gate transistor sensor are shown in Figure 7 As shown in the figure, only the saturated channel current of the transistor output curve in the histamine PBS buffer solution is greatly increased. In other biogenic amine PBS buffer solutions, the saturated channel current value of the transistor does not change much. The saturated channel current of the transistor output characteristic curve tested in the blank PBS buffer solution is I DS,0 , at concentrations of 10 -8 The saturated channel current of the transistor output characteristic curve measured in PBS buffer containing various biogenic amines is I DS , the change is ΔI DS = I DS -I DS,0 , so the saturated channel current change rate (ΔI DS / I DS,0Although the extended microneedle gate transistor sensor also has signal responses to some of the biogenic amine molecules (about 5%), it is quite different from the transistor saturation channel current change rate of histamine molecules (about 20%). The test results show that the extended microneedle gate transistor sensor has good selectivity for histamine molecules.

[0093] Example 3 Extended in vivo detection study of microneedle gate transistor sensor

[0094] Specific content: It starts from three aspects: biocompatibility experimental research, in situ detection research of histamine in mouse skin interstitial fluid, and minimally invasive healing test and evaluation of mouse skin tissue.

[0095] ① Biocompatibility test.

[0096] HUVEC was used as a cell model to verify the biocompatibility of the microneedle material. After the sample was disinfected, PGMA MNs, PGMA / Ag MNs and PGMA / Ag / MIPs MNs materials prepared in Example 1 were soaked in 2 mL of complete culture medium (6123030 Gibco, USA) for 24 h, and the extract was collected. Next, the cells were inoculated, and the specific operation steps were as follows: HUVEC cells in the logarithmic growth phase were taken, 5 mL of PBS solution was added, and the cells were shaken repeatedly to complete the cleaning of the residual culture medium between the cells. Then, the PBS solution was poured out and 1 mL of trypsin was added. After observing under an optical microscope that the cells changed from attached spindles to reduced circles, the bottle body was tapped to make the cells completely free in the culture bottle, and then 1 mL of complete culture medium was added to terminate the digestion. After the digestion was completed, all the cell suspensions in the culture bottle were extracted, transferred to a 15 mL centrifuge tube, centrifuged at 1000 rpm for 5 min, the upper suspension was extracted, the above-mentioned extract was added, and the cell suspension to be inoculated was formed by blowing evenly with a pipette. After completing the cell counting using a counting chamber, the cells were counted at 20,000 / cm 2 Endothelial cells were inoculated into 24-well plates at a density of 1.5 μg / cm2 and then transferred to a 37°C incubator for culture ( Figure 8 ). When the culture time was 1 day, the absorbance values ​​of each experimental group were not much different from those of the control group, indicating that the compounds leached from the microneedle device had no inhibitory effect on cells in a short period of time. With the increase of culture time (3 days), the green fluorescence area increased significantly, and the cell survival rate of each experimental group remained at a high level. When the culture time was 5 days, the absorbance of each experimental group decreased slightly compared with the control group, but it was generally maintained at a high level, with a cell survival rate of >85%. At the same time, there was no significant cell death in the experimental group, indicating that the compounds leached from the microneedle device had little effect on the activity and proliferation of cells under long-term action, indicating that the biocompatibility of the microneedle was good.

[0097] ②In situ detection and analysis of mouse skin interstitial fluid

[0098] An in situ detection experiment of histamine in mouse skin interstitial fluid was carried out by extending the microneedle gate transistor sensor ( Fig. 9 ). During the test, the semiconductor tester first connected the transistor source and drain normally, and then used two microneedle electrodes to pierce the mouse skin adjacently at the same time, one of which was PGMA / Ag / MIPs MNs (EG), and the other was a conductive microneedle electrode (RE) treated with Ag / AgCl conductive silver paste (the RE was obtained by evenly applying Ag / AgCl conductive silver paste on the surface of the microneedle and drying and curing it). The voltage Vref output by the semiconductor tester to RE was applied to the gate electrode of the transistor after passing through the mouse skin interstitial fluid and EG. When EG specifically binds to histamine in the mouse skin interstitial fluid, the negatively charged histamine molecules will cause the voltage applied to the transistor gate to decrease, thereby causing the change of the transistor channel current, realizing the process of histamine concentration change causing electrical signal change. By using the extended microneedle gate transistor sensor, the histamine in the mouse skin interstitial fluid was repeatedly detected at continuous time points (0 min, 5 min, 10 min, 15 min, 20 min, 25 min and 30 min) after sensitization was completed, and the sensor output characteristic curves obtained by detection at different time points.

[0099] The output characteristic curves of the extended microneedle gate transistor sensor at different time points after sensitization are shown in Figure 2. Fig.10 As shown in the figure, as time goes by, the saturated channel current of the output curve of the extended microneedle gate transistor continues to rise, indicating that the skin allergic reaction caused by the sensitizer causes the histamine concentration in the skin interstitial fluid to continue to rise. By comparing the output curves of the transistor sensor at different time points, the time-dependent change of histamine during the sensitization process is revealed. Within 30 minutes after the sensitization is completed, the output curve of the extended microneedle gate transistor can clearly distinguish the histamine concentration.

[0100] ③Test and analysis of minimally invasive healing of mouse skin tissue

[0101] The skin tissue samples of mice were quickly fixed after surgical resection, dehydrated and waxed, and then sliced. Finally, they were stained with hematoxylin-eosin (H&E) for microscopic observation. By comparing the tissue sections of normal skin, mouse skin at 0 min after microneedle removal, and 20 min after microneedle removal, the degree of damage to the skin tissue during the microneedle insertion process and the structural integrity of the skin tissue after 20 min of self-healing were evaluated ( Fig.11 ).

[0102] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a microneedle sensing electrode, characterized in that: The steps include: Step 1, taking glycidyl methacrylate, trimethylolpropane trimethacrylate, triethylene glycol dimethacrylate, and 1-hydroxycyclohexyl phenone, mixing them evenly and then curing them by ultraviolet light to obtain polymer microneedles PGMA MNs; Step 2, taking the polymer microneedle PGMA MNs, washing and sensitizing the MNs to obtain sensitized microneedles, and then mixing the MNs with a glucose solution and a metal plating solution to obtain conductive microneedles; connecting the copper wire to the conductive microneedles using a conductive metal paste, and curing the metal paste; Step 3: Evenly mix the solution containing KCl, histamine dihydrochloride, KOH and pyrrole monomer, and use it as electrolyte solution after deoxygenation. The conductive microneedle after fixing the metal slurry is used as the working electrode, and the counter electrode and the reference electrode constitute a three-electrode system. In-situ electropolymerization is performed by an electrochemical method, and the microneedle sensing electrode is obtained by washing and drying.

2. The preparation method according to claim 1, characterized in that The UV curing time in step 1 is 10 to 20 minutes.

3. The preparation method according to claim 1 or 2, characterized in that: The metal in step 2 includes one or more of silver, platinum or gold.

4. The preparation method according to any one of claims 1 to 3, characterized in that: The concentration of the pyrrole monomer in step 3 is 0.05 mol / L to 0.2 mol / L.

5. The preparation method according to any one of claims 1 to 4, characterized in that: The counter electrode comprises a platinum sheet electrode; the reference electrode comprises an Ag / AgCl electrode.

6. A microneedle sensing electrode prepared by the preparation method according to any one of claims 1 to 5.

7. Microneedle transistor sensor, characterized in that, The method comprises using the microneedle sensing electrode as described in claim 6 as a transistor extension gate to obtain the microneedle transistor sensor.

8. A method for detecting histamine, characterized in that: include: An instrument with electrical signal measurement and analysis function is used, and its connection ends are respectively connected to the source and drain of the microneedle transistor sensor as claimed in claim 7 and the Ag / AgCl reference electrode; The Ag / AgCl reference electrode and the microneedle sensing electrode of the microneedle transistor sensor are placed in a solution containing histamine, and the output characteristic curve and the transfer characteristic curve of the transistor are measured to detect histamine.

9. Use of any of the following in the preparation of a device for detecting histamine: (I) The microneedle sensing electrode as claimed in claim 6; and / or (II) The microneedle transistor sensor as described in claim 7.

10. A histamine detection device, characterized in that: Include any of the following: (I) The microneedle sensing electrode as claimed in claim 6; and / or (II) The microneedle transistor sensor as described in claim 7.

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