NIR light-controlled carbon nanotube-enzyme-loaded hydrogel composite and its application in enzyme-based electrochemical biosensing technology

Abstract

This invention discloses a temperature-responsive hydrogel with a low critical solution temperature 37°C higher than physiological temperature. It is formed by the polymerization reaction of comonomers N-isopropylacrylamide, 1-pyrene methacrylate, and acrylic acid with a crosslinking agent initiated by a free radical initiator. This invention further provides an enzyme-carrying hydrogel prepared from the aforementioned temperature-responsive hydrogel and a carbon nanotube-enzyme-carrying hydrogel composite material. The carbon nanotube-enzyme-carrying hydrogel composite material of this invention is in a swollen state at physiological temperature, and its reversible swelling-contraction changes, regulated by near-infrared light, dynamically regulate the catalytic activity of biological enzymes. Furthermore, carbon nanotubes possess excellent conductivity, and changes in the microgel morphology promote electron transfer within the three-dimensional conductive framework of the composite material, thereby enhancing the electrochemical response. The carbon nanotube-enzyme-carrying hydrogel composite material of this invention can be used to construct intelligent sensors suitable for in vivo controlled analysis.

Description

Technical Field

[0001] This invention belongs to the field of biosensing technology, specifically relating to a temperature-responsive hydrogel, an enzyme-carrying hydrogel and a carbon nanotube-enzyme-carrying hydrogel composite material prepared therefrom, and their application in the preparation of enzyme-based electrochemical biosensors. Background Technology

[0002] The nervous system contains a variety of chemical substances, and imbalances in these neurochemicals can cause behavioral disorders and neurological diseases. Therefore, studying neurochemical molecules is of paramount importance for understanding their role in brain function and major neurological disorders.

[0003] The analysis of neurochemicals in the brain is generally divided into different levels, such as single vesicles, single cells, brain slices, and in vivo. Among them, the detection of chemical substances at the single vesicle, single cell, and brain slice levels is separated from the real environment of in vivo life, making it difficult to maintain the inherent connections and interactions between cells. In comparison, the analysis of neurochemicals at the in vivo level can more realistically and directly reflect the response of the nervous system to external stimuli in various physiological and pathological processes. In vivo electrochemical analysis method uses microelectrodes implanted in specific brain regions to monitor the dynamic changes of neurochemicals in the brain in situ. Due to its advantages such as high spatiotemporal resolution, real-time, and in vivo, it has attracted much attention in neurochemical research. In recent years, enzyme-based electrochemical biosensors have been widely used. However, there are still two challenges in in vivo analysis. (1) Poor enzyme controllability: Enzymes have high specificity and efficient catalytic activity for specific substrates, but biological enzymes have problems such as poor stability and difficulty in controlling catalytic activity. (2) Difficulty in direct electron transfer: Direct electron transfer of biological enzymes on the electrode surface is the basis of bioelectrochemical devices. Direct electron transfer at the electrode surface refers to the catalytic active site of an oxidoreductase (which involves electrons in its catalytic process) establishing an electronic connection directly with the solid electrode surface, achieved through electron tunneling. Therefore, the enzyme's active site needs to be sufficiently close to the electrode surface, typically less than 2 nm. However, the redox sites of most biological enzymes are deeply embedded within the molecule, making direct electron transfer at the electrode surface difficult.

[0004] Hydrogels are hydrophilic polymers with a three-dimensional network structure, possessing soft properties similar to living tissues, making them ideal biomaterials. Stimulus-responsive hydrogels, as a class of smart materials, not only possess the advantages of traditional hydrogels but also can sense minute external stimuli (temperature, pH, magnetic field strength, etc.), and their physical / chemical properties (such as volume, wettability, and surface charge) can dynamically change. Among various smart hydrogels, PNIPAAm, as a typical temperature-responsive hydrogel, has gained considerable favor among researchers. When the temperature changes near its lower critical solution temperature (LCST), the hydrogel morphology undergoes reversible swelling-shrinkage changes. Poly(N-isopropylacrylamide) (PNIPAAm) exhibits temperature responsiveness, undergoing a phase transition with temperature changes, and can be applied in fields such as drug delivery, tissue engineering, and bioimaging. However, PNIPAAm's LCST is 32°C, lower than the physiological temperature (37°C), limiting its application range in the biotechnology field.

[0005] In recent years, smart biocatalysts combining enzymes with stimulus-responsive groups have attracted widespread attention. Depending on the presence or absence of external stimuli, the stimulus-responsive groups attached to the enzyme can regulate enzyme activity by changing their conformation or other properties, thus acting as molecular switches. Temperature-responsive smart enzymes have been reported. Palai (Palai T, Kumar A, Bhattacharya P K. Synthesis and characterization of thermo-responsive poly-N-isopropylacrylamide bioconjugates for application in the formation of galacto-oligosaccharides[J]. Enzyme Microb Technol, 2014, 55:40-49.) conjugated PNIPAMAm with β-galactosidase. This bioconjugate had a LCST of 26.5℃ and retained approximately 90% of its enzyme activity after 24 precipitation-dissolution cycles. Although the reversible conformational change of thermosensitive copolymers can achieve the "on-off" effect of enzyme activity, in practical applications, especially in complex biological systems, it is not possible to directly use a heat source as an external control element to control the material's performance through direct heating.

[0006] Photo-regulated smart enzymes offer advantages such as non-invasiveness, spatiotemporal specificity, and pollution-free operation. Shimoboji et al. (Shimoboji T, Larenas E, Fowler T, et al. Photoresponsive polymer-enzyme switches[J]. Proc Natl Acad Sci USA, 2002, 99(26):16592-16596.) synthesized two photosensitive polymers by polymerizing N,N-dimethylacrylamide with 4-phenylazophenylacrylate and N-4-phenylazophenylacrylamide, respectively, and then linked them to dextranase. Under specific light irradiation, the polymer aggregation state changed, and enzyme activity was controlled by adjusting the contact between the enzyme's active site and the substrate. However, most current photo-regulated smart enzymes are constructed by combining azobenzene analogs, spiropyran groups, or photosensitive protein domains with the enzyme, typically requiring high-energy lasers such as ultraviolet or blue light to control the photosensitive groups. Their preparation process is complex, costly, and difficult to achieve precise on / off control. In addition, ultraviolet or blue light has low biosafety and poor tissue penetration, making it unsuitable for live animal analysis. Summary of the Invention

[0007] This invention addresses the shortcomings of existing technologies by providing a temperature-responsive enzyme-carrying hydrogel and, combined with the concept of light modulation, further offers a near-infrared light-controlled carbon nanotube-enzyme-carrying hydrogel composite material, which is a new intelligent sensing technology suitable for in vivo controllable analysis.

[0008] To achieve the objectives of this invention, the specific technical solution is as follows:

[0009] A temperature-responsive hydrogel is formed by the polymerization reaction of comonomers N-isopropylacrylamide, 1-pyrene methacrylate and acrylic acid with a crosslinking agent initiated by a free radical initiator.

[0010] Preferably, the molar ratio of N-isopropylacrylamide, 1-pyrene methacrylate and acrylic acid is 13-16:1-4:1-4.

[0011] In a specific example of the present invention, the molar ratio of N-isopropylacrylamide, 1-pyrene methacrylate and acrylic acid is 15:2:2.

[0012] Preferably, the crosslinking agent is selected from one or more of N,N-methylenebisacrylamide, ethylene glycol dimethacrylate, and polyethylene glycol diacrylate.

[0013] In a specific example of the present invention, the crosslinking agent is selected from N,N-methylenebisacrylamide.

[0014] Preferably, the free radical initiator is selected from one or more of ammonium persulfate, potassium persulfate, and azobisisobutyronitrile.

[0015] Preferably, the molar ratio of the comonomer, crosslinking agent, and free radical initiator is 19:0.75:0.45.

[0016] In a specific example of the present invention, the temperature-responsive hydrogel is prepared by the following method: N-isopropylacrylamide, 1-pyrene methacrylate and acrylic acid are dissolved in water, N,N-methylenebisacrylamide and sodium dodecyl sulfate are added sequentially, the above mixed solution is heated under nitrogen atmosphere, ammonium persulfate is added to initiate the polymerization reaction, the reaction is completed and cooled to room temperature, and unreacted monomers are removed by dialysis to obtain the hydrogel.

[0017] Another objective of this invention is to provide an enzyme-loaded hydrogel, wherein the temperature-responsive hydrogel of this invention is loaded with biological enzymes through one or more of the following methods: physical adsorption, chemical cross-linking, and covalent bonding.

[0018] The enzyme-carrying hydrogel of the present invention can load any type of biological enzyme involved in the art, such as glucose oxidase and horseradish peroxidase.

[0019] Preferably, a cross-linking agent can be used to attach the bio-enzyme molecules to the temperature-responsive hydrogel. The cross-linking agent is selected from 1-ethyl-(3-dimethylaminopropyl)carbodiimide and / or N-hydroxysuccinimide.

[0020] The enzyme-carrying hydrogel of this invention has a mass ratio of the biological enzyme to the temperature-responsive hydrogel of 0.2–1.4:1. In a specific example of this invention, the mass ratio of the biological enzyme to the temperature-responsive hydrogel is 1:1.

[0021] In a specific example of the present invention, the temperature-responsive hydrogel is suspended in a buffer solution containing 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), and the EDC-activated hydrogel is mixed and reacted with a biological enzyme (such as glucose oxidase) at 4°C to prepare an enzyme-loaded hydrogel.

[0022] Another objective of this invention is to provide a carbon nanotube-enzyme-carrying hydrogel composite material, wherein the enzyme-carrying hydrogel of this invention is doped with carbon nanotubes.

[0023] Preferably, the carbon nanotubes are multi-walled carbon nanotubes.

[0024] Preferably, the mass ratio of carbon nanotubes to enzyme-carrying hydrogel is 0.6 to 4.2:4, more preferably 3:4.

[0025] Another objective of this invention is to provide the application of the temperature-responsive hydrogel, enzyme-carrying hydrogel, or carbon nanotube-enzyme-carrying hydrogel composite material described herein in the preparation of enzyme-based electrochemical biosensors.

[0026] The temperature-responsive hydrogel of this invention has a low critical dissolution temperature higher than the physiological temperature of 37°C, and can undergo reversible swelling-contraction changes under temperature or light regulation to dynamically regulate the catalytic activity of biological enzymes.

[0027] In a specific example of the present invention, the carbon nanotube-enzyme-carrying hydrogel undergoes reversible swelling-shrinkage changes under near-infrared light irradiation, dynamically regulating the catalytic activity of the biological enzyme.

[0028] In the application described in this invention, the carbon nanotube-enzyme-carrying hydrogel is subjected to near-infrared light irradiation under the following conditions: excitation wavelength 700-1700 nm, power 0.2-2.0 W / cm². 2 The illumination time is 0.1-5 min, preferably 1-4 min. In a specific example of this invention, the excitation light source is 1064 nm with a power of 1 W / cm². 2 Illumination time: 1-2 minutes.

[0029] Another objective of this invention is to provide a near-infrared light-controlled enzyme-based electrochemical biosensor, wherein the carbon electrode surface of the biosensor is coated with the enzyme-carrying hydrogel or carbon nanotube-enzyme-carrying hydrogel composite material described in this invention.

[0030] Advantages of this invention:

[0031] 1. This invention selects N-isopropylacrylamide, 1-pyrene methacrylate and acrylic acid as comonomers by screening and adjusting the types and proportions of comonomers. The resulting temperature-responsive hydrogel has a lower critical dissolution temperature that is 37°C higher than the physiological temperature, which allows it to dynamically regulate enzyme catalytic activity under physiological conditions by being affected by temperature changes.

[0032] 2. The temperature-responsive hydrogel of the present invention exhibits essentially unchanged light transmittance after multiple heating-cooling cycles, demonstrating excellent temperature responsiveness and reversible phase transition capability.

[0033] 3. The temperature-responsive hydrogel structure described in this invention can protect the bioactivity of the loaded biological enzymes.

[0034] 4. This invention prepares a carbon nanotube-enzyme-loaded hydrogel composite material by doping an enzyme-loaded hydrogel with multi-walled carbon nanotubes. Based on the excellent photothermal conversion efficiency of carbon nanotubes, this composite material can convert near-infrared light energy into heat energy, thereby regulating the microgel morphology and further controlling enzyme catalytic activity. Electrochemical studies show that, using the above composite material-modified carbon fiber electrodes as the research object and near-infrared light as the external control element, reversible regulation of enzyme catalytic activity on the electrode surface can be achieved.

[0035] 5. This invention utilizes the excellent conductivity of carbon nanotubes, and the change in microgel morphology promotes electron transfer in the three-dimensional conductive framework of the composite material, thereby improving the electrochemical response.

[0036] 6. In this invention, 1-pyrene methacrylate is selected as the comonomer of the hydrogel. The 1-pyrene methacrylate unit can easily form a π-π conjugated system with carbon nanotubes, thereby promoting the stable modification of the material on the surface of the carbon electrode.

[0037] 7. The electrochemical research results of this invention show that the oxidation peak current remains essentially unchanged after multiple on-off cycles of near-infrared light. This indicates that near-infrared light can be used to control enzyme catalytic reactions on the electrode surface. Based on this, using near-infrared light as a control element, a new intelligent sensing technology suitable for in vivo controllable analysis can be constructed, promoting clinical diagnosis and treatment, and possessing significant biological application value. Attached Figure Description

[0038] Figure 1 Infrared spectrum (A) and nuclear magnetic resonance (NMR) spectrum (B) of temperature-responsive hydrogel PNI-co-AA-co-Py.

[0039] Figure 2 The transmittance of the temperature-responsive hydrogel PNI-co-AA-co-Py varies with temperature. (A) and (B) are cyclic graphs of the transmittance of the hydrogel under temperature control at 25℃ and 55℃.

[0040] Figure 3 Thermal stability of the free enzyme and the enzyme-carrying hydrogel GOD@PNI-co-AA-co-Py described in this invention at 45°C.

[0041] Figure 4 Carbon nanotube-enzyme-carrying hydrogel composites (GOD@PNI-co-AA-co-Py / MWCNTs) with different multi-walled carbon nanotube doping concentrations and pure water were compared under near-infrared light (1064 nm, 1.0 W / cm²). 2 Temperature changes under irradiation.

[0042] Figure 5Scanning electron microscope (SEM) images of a multi-walled carbon nanotube carbon fiber electrode (A), an enzyme-loaded hydrogel carbon fiber electrode (B), and a carbon nanotube-enzyme-loaded hydrogel carbon fiber electrode (C).

[0043] Figure 6 A carbon nanotube-enzyme-carrying hydrogel composite material (GOD@PNI-co-AA-co-Py / MWCNTs) was used to modify an electrode to control the catalytic activity of glucose oxidase. (A) Cyclic voltammograms of the composite material-modified electrode before and after near-infrared irradiation; (B) On-off cycle of oxidation peak current under near-infrared light control.

[0044] Figure 7 The effect of multi-walled carbon nanotube doping on the control of glucose oxidase catalytic activity by an electrode modified with carbon nanotube-enzyme-carrying hydrogel composite material (GOD@PNI-co-AA-co-Py / MWCNTs).

[0045] Figure 8 The effect of enzyme loading on the control of glucose oxidase catalytic activity by an electrode modified with carbon nanotube-enzyme-loaded hydrogel composite material (GOD@PNI-co-AA-co-Py / MWCNTs).

[0046] Figure 9 A carbon nanotube-enzyme-carrying hydrogel composite material (GOD-HRP@PNI-co-AA-co-Py / MWCNTs) modified electrode controls the catalytic activity of the glucose oxidase-horseradish peroxidase cascade reaction. (A) Electrochemical response of the modified electrode to glucose molecules before and after near-infrared light irradiation. (B) Electrochemical response of different concentrations of glucose on the composite material modified electrode under near-infrared light irradiation.

[0047] Figure 10 Electrodes modified with carbon nanotube-enzyme-carrying hydrogel composite material (GOD-HRP@PNI-co-AA-co-Py / MWCNTs) before and after near-infrared light irradiation were used to control the electrochemical response of glucose oxidase-horseradish peroxidase cascade reaction to glucose molecules in the mouse brain. Detailed Implementation

[0048] The following detailed embodiments further illustrate the above-mentioned content of the present invention. However, the embodiments should not be construed as any limitation on the present invention. The scope of protection of the present invention is determined by the claims. Unless otherwise specified, the following embodiments are all carried out using conventional prior art.

[0049] Example 1: Preparation of temperature-responsive hydrogel PNI-co-AA-co-Py

[0050] 0.170 g of N-isopropylacrylamide (NIPAAm), 0.063 g of 1-pyrene methacrylate (Py-Ac), and 0.014 g of acrylic acid (AA) were dissolved in 95 mL of deionized water. Under stirring, 0.012 g of N,N-methylenebisacrylamide (MBAA) and 0.0017 g of sodium dodecyl sulfate (SDS) were added sequentially. The mixture was heated to 70 °C under nitrogen atmosphere, and then ammonium persulfate (APS) (0.01 g dissolved in 5 mL) was added to initiate the polymerization reaction (the concentrations of N-isopropylacrylamide, 1-pyrene methacrylate, and acrylic acid in the reaction system were 15 mM, 2 mM, and 2 mM, respectively). After reacting for 7 hours, the reaction solution was cooled to room temperature and then dialyzed for 7 days to prepare the PNI-co-AA-co-Py hydrogel.

[0051] Performance characterization:

[0052] 1. Composition and structural characterization of PNI-co-AA-co-Py hydrogel

[0053] Infrared spectrum ( Figure 1 A) and 1H NMR spectrum ( Figure 1 B) Both indicate that the PNI-co-AA-co-Py hydrogel contains three monomers, confirming the successful polymerization of the monomers.

[0054] 2. Study on the temperature response properties of PNI-co-AA-co-Py hydrogel

[0055] A 0.1% (w / w) hydrogel was placed in a cuvette, and the transmittance of the sample at different temperatures was measured using a UV-Vis spectrophotometer. The temperature range was 25–55 °C, and the sample was kept stable at the desired temperature for 10 min before each reading. A temperature-transmittance curve of the hydrogel was plotted based on the results. Figure 2 A).

[0056] Experimental data showed that the phase transition temperature of the gel was 39℃, which is higher than the physiological temperature (37℃). To verify the temperature repeatability of the PNI-co-AA-co-Py hydrogel, the change in transmittance of PNI-co-AA-co-Py after 5 cycles at 25℃ and 55℃ was measured. The results are as follows: Figure 2 As shown in Figure B, the transmittance of the hydrogel remained essentially unchanged after multiple heating-cooling cycles. These data demonstrate that the hydrogel exhibits excellent temperature responsiveness and reversible phase transition capability.

[0057] Example 2: Effect of comonomer ratio on the temperature response properties of hydrogels

[0058] Hydrogels with different comonomer molar ratios were prepared according to the preparation method of Example 1, and the effect of these ratios on the temperature response performance of the hydrogels was investigated.

[0059] The results are shown in Table 1. The results indicate that the lower critical temperature (LCT) of the hydrogel gradually increases with the increase of acrylic acid content. When the molar ratio of N-isopropylacrylamide, acrylic acid, and 1-pyrene methacrylate is 13–16:2:1–4, the LCT of the PNI-co-AA-co-Py hydrogel is close to the human physiological body temperature of 36.0–37.0 °C. The hydrogel prepared with a preferred molar ratio of N-isopropylacrylamide, acrylic acid, and 1-pyrene methacrylate of 15:2:2 has an LCT of 39 °C, which is more suitable for in vivo analysis.

[0060] Table 1

[0061]

[0062] Example 3: Preparation of enzyme-loaded hydrogel GOD@PNI-co-AA-co-Py

[0063] The PNI-co-AA-co-Py hydrogel (2 mg) prepared in Example 1 was suspended in a buffer solution (5 mL) containing 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC, 4 mg). The EDC-activated hydrogel was mixed with glucose oxidase (GOD, 2 mg) and reacted at 4 °C for 10 hours. After the reaction, the mixture was purified to prepare the enzyme-loaded hydrogel (GOD@PNI-co-AA-co-Py).

[0064] Performance characterization: Investigating the thermal stability of enzymes in enzyme-loaded hydrogels.

[0065] A hydrogel solution immobilized with glucose oxidase (GOD) (GOD@PNI-co-AA-co-Py) and a solution of free glucose oxidase (GOD) were incubated at 45°C for 150 min. Samples were taken every 30 min for activity assay. The bioactivity of GOD was evaluated based on the HRP-H2O2-TMB enzymatic reaction. The concentrations of GOD in the enzyme-loaded hydrogel and free GOD were kept consistent (e.g., 20 μg / mL). Free GOD or the enzyme-loaded hydrogel was dispersed in 0.1 mL of Tris buffer (pH 7.5, 50 mM) containing 10 μg / mL horseradish peroxidase (HRP), and then 0.2 mL of prepared TMB solution was added as a hydrogen donor. Glucose solution (5 mM, 0.2 mL) was immediately added to activate the catalytic reaction. The generated H2O2 oxidized TMB, and the absorbance at 650 nm was recorded using a UV-Vis spectrophotometer.

[0066] The results are as follows Figure 3As shown, compared with naked enzymes, the enzyme-carrying hydrogel (GOD@PNI-co-AA-co-Py) of this invention exhibits less enzyme activity loss. This demonstrates that the hydrogel structure can protect enzyme activity.

[0067] Example 4: Preparation of carbon nanotube-enzyme-carrying hydrogel composite material (GOD@PNI-co-AA-co-Py / MWCNTs)

[0068] The enzyme-carrying hydrogel (GOD@PNI-co-AA-co-Py) prepared in Example 3 was mixed with different amounts of multi-walled carbon nanotubes (MWCNTs). The concentration of the enzyme-carrying hydrogel (GOD@PNI-co-AA-co-Py) was 2 mg / mL, and the concentrations of the multi-walled carbon nanotubes were 1.5 mg / mL, 0.8 mg / mL, 0.4 mg / mL, and 0.2 mg / mL, respectively. The mixture was thoroughly mixed by sonication in an ice-water bath for 30 min. Carbon nanotube-enzyme-carrying hydrogel composite materials (GOD@PNI-co-AA-co-Py / MWCNTs) with different carbon nanotube contents were prepared by self-assembly through π-π interactions.

[0069] Example 5 examines the photothermal conversion effect of the carbon nanotube-enzyme-carrying hydrogel composite material.

[0070] The composite material solution prepared in Example 4 was irradiated with near-infrared light. A 1064nm laser with a power of 1W / cm² was used in this experiment. 2 The illumination time was 4 minutes, and ultrapure water was used as a blank control group. The temperature changes of different composite material solutions were measured under the same conditions.

[0071] The results are as follows Figure 4 As shown, the solution temperature of the carbon nanotube-enzyme-carrying hydrogel composite material (GOD@PNI-co-AA-co-Py / MWCNTs) gradually increased with increasing light exposure time. Furthermore, the heating rate accelerated with increasing MWCNT content in the composite material. However, under the same experimental conditions, the temperature of pure water remained almost unchanged. These results indicate that MWCNTs in the composite material can effectively absorb near-infrared light and generate sufficient heat. When the solution temperature is below the critical temperature, the gel is in a swollen state; when the solution temperature is above the critical temperature, the gel is in a contracted state. Based on this, the properties of the composite material can be modulated using near-infrared light to further regulate enzyme catalytic activity.

[0072] Example 6: Investigating the reversible regulation of enzyme catalytic activity in composite materials using electrochemical techniques.

[0073] Preparation of composite material modified electrodes: A 0.5 mL solution of the enzyme-loaded hydrogel and multi-walled carbon nanotubes (4:3 mass ratio) prepared in Example 4, a 0.5 mL solution of the enzyme-loaded hydrogel prepared in Example 3 (2 mg / mL, 0.5 mL), and a 0.5 mL solution of multi-walled carbon nanotubes (1.5 mg / mL, 0.5 mL) were dropped onto a flat glass plate. Under a microscope, a carbon fiber electrode (length: 1 mm, diameter: 8 μm) was immersed in the droplet and repeatedly rotated. After drying, composite material functionalized carbon fiber electrodes, multi-walled carbon nanotube carbon fiber electrodes, and enzyme-loaded hydrogel carbon fiber electrodes were prepared. Scanning electron microscope images of the three electrodes are shown below. Figure 5 As shown, the surface of the multi-walled carbon nanotube carbon electrode is coated with multi-walled carbon nanotube fibers and has a smooth surface; the surface of the enzyme-loaded hydrogel carbon electrode presents hydrogel microspheres; the carbon nanotubes on the surface of the composite functionalized carbon electrode are well embedded in the enzyme-loaded hydrogel microspheres.

[0074] This embodiment utilizes electrochemical techniques to further investigate the reversible regulation of enzyme catalytic activity by near-infrared light. The electrochemical assay employs a three-electrode system, where a composite functionalized carbon fiber electrode serves as the working electrode, a platinum wire as the counter electrode, and a silver / silver chloride electrode as the reference electrode. A 1064 nm laser is used as the light source. In the electrochemical measurements, the electrolyte solution is artificial cerebrospinal fluid (pH = 7.4). The working, counter, and reference electrodes are inserted into the artificial cerebrospinal fluid, and high-purity nitrogen is used for aeration. The scanning potential range of the cyclic voltammogram is -0.7 to 0.4 V, and the scan rate is 50 mV / s. Figure 6 As shown in Figure A, the composite material-modified electrode did not produce a significant peak current without near-infrared light irradiation. However, under near-infrared light (1064 nm, 1.0 W / cm²), the peak current was significantly reduced. 2 After irradiation for 2 minutes, the current increased, and a pair of distinct redox peaks were observed on the electrode surface, demonstrating that glucose oxidase in the composite material can achieve direct electron transfer on the electrode surface. This is because under near-infrared light irradiation, the hydrogel changes from a swollen to a contracted state, thereby promoting the proximity of the active sites of glucose oxidase to the electrode surface. Furthermore, the excellent conductivity of carbon nanotubes and the change in hydrogel morphology promote electron transfer within the three-dimensional conductive framework of the composite material, thus enhancing the electrochemical response. Figure 6 As shown in Figure B, the oxidation peak current remained essentially unchanged after multiple on-off cycles of near-infrared light. These results indicate that near-infrared light can be used to control enzyme-catalyzed reactions on the electrode surface.

[0075] Example 7: Effect of comonomer 1-pyrene methacrylate (Py-Ac) on the enzyme catalytic activity of carbon nanotube-enzyme-carrying hydrogel composite material

[0076] The Py-Ac unit in the gel PNI-co-AA-co-Py of this invention has a conjugated aromatic ring structure, which readily forms a π-π conjugated system with MWCNTs, thereby helping the material to be stably modified on the carbon electrode surface, promoting direct electron transfer between the enzyme and the electrode, and further improving the electrochemical response signal.

[0077] Based on this, referring to the methods of Examples 3, 4, and 6, carbon nanotube-enzyme-carrying hydrogel composite materials with different Py-Ac molar ratios (mass ratio of hydrogel to glucose oxidase is 1:1, and mass ratio of enzyme-carrying hydrogel to multi-walled carbon nanotube is 4:3) were prepared, and corresponding carbon fiber electrodes were prepared to investigate the effect on the enzyme catalytic activity of the carbon nanotube-enzyme-carrying hydrogel composite materials.

[0078] The results are shown in Table 2. The results indicate that the oxidation peak current increases with the increase of the proportion of 1-pyrene methacrylate. The oxidation peak current reaches its maximum value when the molar ratio of N-isopropylacrylamide, acrylic acid, and 1-pyrene methacrylate is 15:2:2, and then gradually decreases. The preferred molar ratio of N-isopropylacrylamide, acrylic acid, and 1-pyrene methacrylate is 15:2:2.

[0079] Table 2

[0080]

[0081] Example 8: Effect of Multi-walled Carbon Nanotube Content on Catalytic Activity

[0082] Following the method of Example 3, an enzyme-carrying hydrogel with a molar ratio of N-isopropylacrylamide, acrylic acid, and 1-pyrene methacrylate of 15:2:2 and a mass ratio of hydrogel to glucose oxidase of 1:1 was prepared. This hydrogel was further mixed with different amounts of multi-walled carbon nanotubes (MWCNTs) at concentrations of 0.3 mg / mL, 0.6 mg / mL, 0.9 mg / mL, 1.2 mg / mL, 1.5 mg / mL, 1.8 mg / mL, and 2.1 mg / mL. The solutions were then sonicated in an ice-water bath for 30 min to obtain a carbon nanotube-enzyme-carrying hydrogel composite material (GOD@PNI-co-AA-co-Py / MWCNTs). Following the method of Example 6, a carbon fiber electrode modified with the carbon nanotube-enzyme-carrying hydrogel composite material was prepared. The effect on enzyme catalytic activity was investigated.

[0083] The results are as follows Figure 7 As shown, the oxidation peak current increases with increasing multi-walled carbon nanotube content; the oxidation peak current is highest when the mass ratio of enzyme-loaded hydrogel to multi-walled carbon nanotubes is 4:3; as the carbon nanotube content continues to increase, the oxidation peak current decreases. Therefore, a mass ratio of enzyme-loaded hydrogel to multi-walled carbon nanotubes of 4:3 is preferred.

[0084] Example 9: Effect of enzyme loading on catalytic activity of the gel

[0085] Enzyme-loaded hydrogels with different enzyme loading amounts were prepared according to the method in Example 3. Carbon nanotube-enzyme-loaded hydrogel composite carbon fiber electrodes were prepared according to Examples 4 and 6, with a molar ratio of N-isopropylacrylamide, acrylic acid, and 1-pyrene methacrylate of 15:2:2, and a mass ratio of enzyme-loaded hydrogel to multi-walled carbon nanotubes of 4:3. The effect on enzyme catalytic activity was investigated.

[0086] like Figure 8 As shown, the results indicate that the oxidation peak current increases with the increase of glucose oxidase content in the hydrogel, and the oxidation peak current reaches its maximum value when the enzyme-loaded hydrogel is prepared with glucose oxidase and hydrogel at a mass ratio of 1:1. Therefore, it is preferable to prepare the enzyme-loaded hydrogel with glucose oxidase and hydrogel at a mass ratio of 1:1.

[0087] Example 10: Exploring the reversible detection of glucose molecules using electrochemical techniques.

[0088] The catalytic activity of multi-enzyme cascade reactions is closely related to the inter-enzyme distance. Therefore, to further demonstrate the strategy of regulating enzyme catalytic activity using near-infrared light, this embodiment uses a glucose oxidase-horseradish peroxidase dual-enzyme system as a model for the controllable analysis of glucose molecules. The working principle of this enzyme cascade reaction is as follows: In the presence of glucose molecules, glucose oxidase catalyzes the production of gluconic acid and hydrogen peroxide from glucose; horseradish peroxidase further catalyzes the reduction of hydrogen peroxide to water, while simultaneously accelerating the oxidation of the electron mediator methylene blue, thereby generating an electrochemical signal.

[0089] Enzyme-loaded hydrogels containing glucose oxidase and horseradish peroxidase were prepared according to the method in Example 3. The PNI-co-AA-co-Py hydrogel (2 mg) prepared in Example 1 was suspended in a buffer solution (5 mL) containing 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC, 4 mg). The EDC-activated hydrogel was reacted with glucose oxidase (GOD, 1 mg), horseradish peroxidase (HRP, 1 mg), and methylene blue (0.04 mmol / L) at 4 °C for 10 hours. After the reaction, the mixture was purified to obtain the enzyme-loaded hydrogel (GOD-HRP@PNI-co-AA-co-Py). Carbon nanotube-enzyme-loaded hydrogel composite carbon fiber electrodes were prepared according to Examples 4 and 6, with a molar ratio of N-isopropylacrylamide, acrylic acid, and 1-pyrene methacrylate of 15:2:2, and a mass ratio of enzyme-loaded hydrogel to multi-walled carbon nanotubes of 4:3. The effect on enzyme catalytic activity was investigated.

[0090] like Figure 9As shown in Figure A, the electrochemical response is weak in the presence of glucose molecules without near-infrared light irradiation. However, after near-infrared light irradiation, the electrochemical signal is significantly enhanced, indicating that an enzyme cascade catalytic reaction has occurred. This is mainly because under near-infrared light irradiation, the hydrogel shrinks, the distance between enzymes decreases, thereby shortening the substrate diffusion path and further enhancing the electrochemical signal. In addition, the closer distance between the composite material and the electrode surface facilitates electron transfer to the electrode interface. Figure 9 As shown in Figure B, the electrochemical signal gradually increased with increasing glucose concentration (0, 1.0, 2.0, 3.0, 4.0, 5.0, and 6.0 mM). These results demonstrate that the composite material-modified electrode described above, using near-infrared light as the control element, can be used for the controllable analysis of biomolecules.

[0091] Example 11: The enzyme-based electrochemical biosensor of the present invention is applied to the controlled detection of glucose molecules in the mouse brain.

[0092] First, the mice were fixed on a stereotaxic apparatus. Then, the carbon fiber electrode, silver / silver chloride reference electrode, and platinum counter electrode prepared in Example 10 were implanted into the mouse brain. A 1064nm laser was used as the light source, with a wavelength of 1.0W / cm². 2 Irradiate for 1 min. Investigate the electrochemical response of the modified electrode to glucose molecules before and after near-infrared light irradiation.

[0093] like Figure 10 As shown, the electrochemical response is weak without near-infrared light irradiation. After near-infrared light irradiation, the electrochemical signal is significantly enhanced. This demonstrates that near-infrared light can be used as an external control element to achieve controllable detection of glucose in the mouse brain.

Claims

1. A temperature-responsive hydrogel, characterized by Hydrogels are formed by the polymerization reaction of comonomers N-isopropylacrylamide, 1-pyrene methacrylate and acrylic acid with a crosslinking agent initiated by a free radical initiator. The molar ratio of N-isopropylacrylamide, 1-pyrene methacrylate and acrylic acid is 13~16:1~4:1~4.

2. The temperature-responsive hydrogel of claim 1, wherein The molar ratio of N-isopropylacrylamide, 1-pyrene methacrylate, and acrylic acid is 15:2:

2.

3. The temperature-responsive hydrogel of claim 1, wherein The crosslinking agent is selected from one or more of N,N-methylenebisacrylamide, ethylene glycol dimethacrylate, and polyethylene glycol diacrylate.

4. The temperature-responsive hydrogel as described in claim 1, characterized in that... The free radical initiator is selected from one or more of ammonium persulfate, potassium persulfate, and azobisisobutyronitrile.

5. The temperature-responsive hydrogel as described in claim 1, characterized in that... The molar ratio of comonomer, crosslinking agent and free radical initiator is 19:0.75:0.

45.

6. The temperature-responsive hydrogel as described in claim 1, characterized in that... The hydrogel was prepared by the following method: N-isopropylacrylamide, 1-pyrene methacrylate and acrylic acid were dissolved in water, and N,N-methylenebisacrylamide and sodium dodecyl sulfate were added in sequence. The above mixed solution was heated under nitrogen atmosphere, and ammonium persulfate was added to initiate the polymerization reaction. After the reaction was completed, the solution was cooled to room temperature, and unreacted monomers were removed by dialysis to obtain the hydrogel.

7. An enzyme-carrying hydrogel, characterized in that... The temperature-responsive hydrogel according to any one of claims 1-6 is loaded with biological enzymes by one or more of the following methods: physical adsorption, chemical crosslinking, and covalent bonding.

8. The enzyme-carrying hydrogel as described in claim 7, characterized in that... The bioenzyme is selected from glucose oxidase and / or horseradish peroxidase.

9. The enzyme-carrying hydrogel as described in claim 7, characterized in that... Bio-enzyme molecules are linked to temperature-responsive hydrogels using cross-linking agents.

10. The enzyme-carrying hydrogel according to claim 9, characterized in that... The crosslinking agent is selected from 1-ethyl-(3-dimethylaminopropyl)carbodiimide and / or N-hydroxysuccinimide.

11. The enzyme-carrying hydrogel as described in claim 9, characterized in that... The mass ratio of the bio-enzyme to the temperature-responsive hydrogel is 0.2~1.4:

1.

12. A carbon nanotube-enzyme-carrying hydrogel composite material, characterized in that... The enzyme-carrying hydrogel doped with carbon nanotubes according to any one of claims 7-11.

13. The carbon nanotube-enzyme-carrying hydrogel composite material as described in claim 12, characterized in that... The carbon nanotubes are multi-walled carbon nanotubes.

14. The carbon nanotube-enzyme-carrying hydrogel composite material as described in claim 12, characterized in that... The mass ratio of carbon nanotubes to enzyme-carrying hydrogels is 0.6~4.2:

4.

15. The application of the temperature-responsive hydrogel according to any one of claims 1-6, the enzyme-carrying hydrogel according to any one of claims 7-11, or the carbon nanotube-enzyme-carrying hydrogel composite material according to any one of claims 12-14 in the preparation of enzyme-based electrochemical biosensors.

16. The application as described in claim 15, characterized in that... The temperature-responsive hydrogel has a low critical dissolution temperature higher than the physiological temperature of 37°C, and undergoes reversible swelling-contraction changes under temperature or light regulation to dynamically control the catalytic activity of biological enzymes.

17. The application as described in claim 16, characterized in that... The carbon nanotube-enzyme-carrying hydrogel composite material according to any one of claims 12-14 undergoes reversible swelling-shrinkage changes under near-infrared light irradiation, dynamically regulating the catalytic activity of the biological enzyme.

18. The application as described in claim 17, characterized in that... The carbon nanotube-enzyme-loaded hydrogel is excited by near-infrared light with a wavelength of 700-1700 nm, a power of 0.2-2.0 W / cm 2 , and an illumination time of 0.1-5 min.

19. A near-infrared light-controlled enzyme-based electrochemical biosensor, characterized in that... The surface of the carbon electrode of the biosensor is coated with the enzyme-carrying hydrogel according to any one of claims 7-11 or the carbon nanotube-enzyme-carrying hydrogel composite material according to any one of claims 12-14.

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