Background
At present, a commercialized Continuous Glucose Monitor (CGMS) adopts an electrochemical method based on enzyme reaction to convert the glucose concentration into an electric signal in real time. The CGMS products all contain glucose oxidase, and the glucose oxidase can specifically recognize glucose and is insensitive to other saccharides in organisms. Technologies used in currently commercially available CGMS can be classified into first-generation enzyme technologies and second-generation wired enzyme technologies.
The first generation of enzyme technology is the indirect detection of glucose concentration by detecting the concentration of hydrogen peroxide generated during the oxidation of glucose, the working electrode used being typically a noble metal electrode. The generation of hydrogen peroxide needs to consume oxygen, and the oxygen concentration (0.2-0.3 mM) in human blood is far lower than the glucose concentration (5-10 mM) in blood, so a specially designed biocompatible outer membrane capable of controlling oxygen and glucose fluxes simultaneously needs to be covered on the surface of the glucose sensor. Such outer film materials have complicated structures, and similar technologies are disclosed in patent application documents with patent application numbers CN201080053713.4, CN201080062485.7, CN201980030243.0, etc. In addition, the generated hydrogen peroxide is not limited in the diffusion direction of the sensing layer, and only the hydrogen peroxide diffused to the surface of the electrode can generate current for sensing, so that high requirements are imposed on the preparation process of the electrode. In addition, the detection potential used in the first generation enzyme technology is usually 0.4V or more. At this potential, some interfering substances, such as ascorbic acid and acetaminophen, are oxidized, producing an interfering current. Therefore, the effective working time of CGMS based on the first generation enzyme technology generally varies from 7 days to 10 days.
The second generation of the wired enzyme technology is to realize the electron transfer between the glucose oxidase and the electrode by replacing oxygen with an artificially synthesized mediator. The electron mediator is grafted to the side chains of the polymer by a long flexible chain, as described in detail in U.S. patent application publication No. US 6605200B 1. The redox center Flavin Adenine Dinucleotide (FAD) of the glucose oxidase is wrapped by a thicker insulating layer, and a mediator with a long flexible chain can realize effective contact with the FAD to transfer electrons out. And form carriers such as electrons or holes through rapid reduction and rapid oxidation, and conduct current through self-exchange. The reduced mediator collides with the oxidized mediator, and the reduced mediator transfers electrons, or the oxidized mediator transfers holes. Although in theory, electrons or holes can also propagate by hopping between fixed position mediators, the solid-state physical trap-trap hopping phenomenon is rarely seen in redox hydrogels. The long flexible chains transfer electrons faster than the short chain mediators because they increase the displacement amplitude of the tethered mediator, significantly increasing the contact collision frequency. In addition, in the patent application with the patent application number CN200980139400.8, the electronic mediators with different redox potentials are synthesized by adjusting the ligands of the synthesized mediators, and the redox potentials of the electronic mediators are generally less than 0.3V, thereby improving the anti-interference capability of CGMS. Although the artificial synthetic mediator replaces oxygen to realize electron transfer between the glucose oxidase and the electrode, the oxygen still generates electron transfer with the glucose oxidase. There is a competing relationship between oxygen and the synthetic media. Thus, in documents CN 113521399A, US 6932894B 2 and the like, a biocompatible outer membrane is prepared to reduce the permeation of oxygen into the sensing layer while controlling the flux of glucose into the sensing layer. By this outer membrane, the interference of oxygen is reduced. However, oxygen, as a small molecule dissolved in body fluid, can still transfer the electrons of glucose oxidase in competition with an electron mediator in the slightly swollen pores along with the diffusion of water molecules to the sensing layer. When the degree of crosslinking of the membrane is increased to reduce the inflow of oxygen, the detection range of CGMS is further increased, but the sensitivity is lowered and the detected current value is also increased by external disturbance.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a special material for intelligent glucose monitoring and application thereof, so as to solve the problems of low sensitivity, weak anti-interference capability and the like caused by the interference of oxygen in the process of detecting glucose by the existing second generation CGMS (China general microbiological culture Collection center) cable enzyme technology.
The technical scheme adopted by the invention is as follows:
a conductive nano material glucose special material is obtained by covalent crosslinking of a conductive nano material containing a redox ligand and glucose oxidase through a crosslinking agent.
The preparation method of the conductive nano material glucose special material comprises the following steps:
(1) firstly, carrying out surface modification treatment on the surface of a conductive nano material by using a silane coupling agent to enable the surface of the conductive nano material to have active groups;
(2) connecting the modified conductive nano material with a redox ligand through a covalent bond;
(3) and carrying out covalent crosslinking on the conductive nano material connected with the redox ligand and the glucose oxidase through a crosslinking agent.
Preferably, the conductive nanomaterial in step (1) is one or more of graphdiyne, nanogold, nanosilver, nanoplatinum, nano conductive carbon black, carbon nanotube, football, graphene or reduced graphene oxide.
Preferably, the silane coupling agent described in step (1) has the general formula Y (CH)2)nSiX3Wherein n is 0-3, and X is one or more of methoxy, ethoxy, methoxyethoxy and acetoxyl; and Y is one of vinyl, amino, epoxy, methacryloxy, sulfydryl or carbamido.
Preferably, the redox ligand described in step (2) has the general formula Os (L)2RClnOr Ru (L)2RClnWherein L is a ligand containing two nitrogen heterocycles, R is a mono-or bis-heterocyclic ligand containing a reactive group, if R is a mono-heterocyclic ligand containing a reactive group, then n is 1, and if R is a bis-heterocyclic ligand containing a reactive group, then n is 0.
Further, the L is one of N, N ' -dimethyl-2, 2 ' -biimidazole, 2 ' -bipyridine, 4 ' -dimethyl-2, 2 ' -bipyridine, 4 ' -dimethoxy-2, 2 ' -bipyridine, 4 ' -dichloro-2, 2 ' -bipyridine and 4,4 ' -diamino-2, 2 ' -bipyridine;
further, R is one of imidazole, pyridine, bipyridine, biimidazole and pyridine biimidazole with amino, carboxyl or aldehyde group.
Preferably, the crosslinking agent in step (3) is one or more of glutaraldehyde, polyethylene glycol diglycidyl ether, poly (propylene glycol) diglycidyl ether, 1, 4-butanediol diglycidyl ether, 1, 6-hexanediol diglycidyl ether, neopentyl glycol diglycidyl ether, trimethylolpropane (ethane) triglycidyl ether, glycerol triglycidyl ether, pentaerythritol tetraglycidyl ether, and genipin.
Preferably, the crosslinking reaction of step (3) is: mixing the conductive nano material connected with the redox ligand, glucose oxidase and a cross-linking agent solution according to a cross-linking reaction feeding ratio, and reacting for 45 min-2 d at the temperature of 20-45 ℃.
The invention also provides application of the conductive nano material glucose special material in blood glucose monitoring and diabetes management instruments.
In summary, compared with the prior art, the invention has the following advantages and beneficial effects:
1. the invention increases the speed of electron transfer of the electron mediator through the conductive nano material with the redox ligand, thereby overcoming the oxygen effect in the second generation of wired enzyme technology; the electrochemical glucose sensor prepared by the sensing material provided by the invention can specifically detect glucose, has high linear correlation coefficient of a stable current signal and the glucose concentration, and can eliminate the influence of an interference substance on the detection of glucose by acetaminophenol, so that the conductive nano glucose sensing material based on the modification of the redox ligand has wide application value in the intelligent detection of glucose;
2. according to the invention, the redox ligand serving as the electron mediator is in covalent connection with the conductive nano material, so that the electron mediator directly transfers electrons to the next electron mediator through collision, or transfers the electrons to a plurality of electron mediators connected to the surface of the conductive nano material, or directly transfers the electrons to an electrode through the highly conductive nano material, and multi-channel transfer is realized;
3. the conductive nano-material glucose sensing material provided by the invention has long effective working time due to multi-channel electronic transmission and structural stability, and the working performance of the conductive nano-material glucose sensing material cannot be influenced along with the change of time.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.
Example 1
The embodiment provides a redox ligand modification-based conductive nano-material glucose sensing material, and the preparation process comprises the following steps:
A. weighing 1-100 mg of multi-walled carbon nanotube, placing the multi-walled carbon nanotube in a 10mL small bottle, adding 1-3 mL of deionized water and 0.01-1 mL of 3-aminopropyltriethoxysilane, performing ultrasonic treatment for 10-100 min to obtain a uniform dispersion, placing the dispersion at 25-50 ℃ for 1-24 h, adding 0.1-100 muL of 0.1M hydrochloric acid, placing the dispersion at room temperature for 6-48 h, dialyzing the obtained suspension, and performing centrifugal cleaning on the dialyzed suspension for multiple times to obtain the carbon nanotube with amino on the surface.
B. Mixing 10-100 mg of Os (bpy)2ClIm(CH2)11-COOH was dissolved in PBS at pH 5 to 8 with stirring, and then 150mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and 200mg of N-hydroxysuccinimide were added and stirred at room temperature for 15 to 60 min. 100. mu.L of an aminated carbon nanotube dispersion (pH 7.4) having a concentration of 200mg/mL was added dropwise, and the mixture was stirred at room temperature for 12 to 72 hours. And after the reaction is finished, centrifugally cleaning the obtained product by using deionized water, dialyzing by using the deionized water, and removing the free micromolecules to obtain the carbon nano tube with the surface modified with the redox ligand.
C. And D, mixing 10 mu L of the carbon nanotube dispersion liquid (10mg/mL) of the surface modified redox ligand obtained in the step B and 10 mu L of glucose oxidase solution (10mg/mL) through 10 mu L of 5% glutaraldehyde solution for 45min, and dripping the mixture on the surface of the electrode to obtain the glucose sensing layer reagent based on the conductive nano material modified by the redox ligand.
Example 2
The embodiment provides a redox ligand modification-based conductive nano-material glucose sensing material, and the preparation process comprises the following steps:
A. weighing 1-100 mg of graphene, placing the graphene in a 10mL small bottle, adding 1-3 mL of deionized water and 0.01-1 mL of methacryloxypropyltrimethoxysilane, carrying out ultrasonic treatment for 10-100 min to obtain a uniform dispersion, placing the dispersion at 25-50 ℃ for 1-24 h, adding 0.1-100 mu L of 0.1M hydrochloric acid, placing the dispersion at room temperature for 6-48 h, dialyzing the obtained suspension, and carrying out centrifugal cleaning on the dialyzed suspension for multiple times to obtain the graphene with acryloxy on the surface.
B. Mixing 10-100 mg of Os (diamino-bpy)2ClIm(CH2)11-NH2Dissolving in ethanol under stirring, adding 100 μ L of acryloxy modified stone with concentration of 200mg/mLAnd stirring the graphene dispersion liquid for 1-12 hours at 40-90 ℃. And after the reaction is finished, centrifugally cleaning the obtained product by using ethanol, dialyzing the product by using deionized water, and removing free small molecules to obtain the graphene with the surface modified with the redox ligand.
C. And D, mixing 10 mu L of graphene dispersion liquid (10mg/mL) of the surface modified redox ligand obtained in the step B and 10 mu L of glucose oxidase solution (10mg/mL) through 10 mu L of polyethylene glycol diglycidyl ether solution (10mg/mL), dripping the mixture on the surface of the electrode, and drying in vacuum at room temperature for 48h to obtain the conductive nano-material glucose sensing layer reagent modified based on the redox ligand.
Example 3
The embodiment provides a redox ligand modification-based conductive nano-material glucose sensing material, and the preparation process comprises the following steps:
A. weighing 1-100 mg of nano conductive carbon black, placing the nano conductive carbon black in a 10mL small bottle, adding 1-3 mL of deionized water and 0.01-1 mL of gamma-glycidoxypropyltrimethoxysilane, performing ultrasonic treatment for 10-100 min to obtain a uniform dispersion, placing the dispersion at 25-50 ℃ for 1-24 h, adding 0.1-100 mu L0.1M hydrochloric acid, placing the dispersion at room temperature for 6-48 h, dialyzing the obtained suspension, and performing centrifugal cleaning on the dialyzed suspension for multiple times to obtain the nano conductive carbon black with epoxy groups on the surface.
B. Mixing 10-100 mg of Os (dimethyl-bpy)2ClIm(CH2)11-NH2Fully dissolving the mixture in ethanol under stirring, adding 100 mu L of epoxy group modified conductive carbon black dispersion liquid with the concentration of 200mg/mL, and stirring for 1-12 h at the temperature of 40-90 ℃. And after the reaction is finished, centrifugally cleaning the obtained product by using ethanol, dialyzing the product by using deionized water, and removing free small molecules to obtain the nano conductive carbon black with the surface modified with the redox ligand.
C. And D, mixing 10 mu L of the nano conductive carbon black dispersion liquid (10mg/mL) of the surface modified redox ligand obtained in the step B and 10 mu L of glucose oxidase solution (10mg/mL) through 10 mu L of 1, 4-butanediol diglycidyl ether solution (10mg/mL), dripping the mixture on the surface of an electrode, and drying in vacuum at room temperature for 48h to obtain the conductive nano material glucose sensing layer reagent based on the redox ligand modification.
Example 4
The embodiment provides a redox ligand modification-based conductive nano-material glucose sensing material, and the preparation process comprises the following steps:
A. weighing 1-100 mg of nano conductive graphite alkyne, placing the nano conductive graphite alkyne in a 10mL small bottle, adding 1-3 mL of deionized water and 0.01-1 mL of 3- (2-aminoethylamino) propyltrimethoxysilane, carrying out ultrasonic treatment for 10-100 min to obtain a uniform dispersion, placing the dispersion at 25-50 ℃ for 1-24 h, adding 0.1-100 mu L of 0.1M hydrochloric acid, placing the dispersion at room temperature for 6-48 h, dialyzing the obtained suspension, and carrying out centrifugal cleaning on the dialyzed suspension for multiple times to obtain the nano conductive graphite alkyne with amino on the surface.
B. Mixing 10-100 mg of Os (dimethoxy-bpy)2ClIm(CH2)11-COOH was dissolved in PBS at pH 5 to 8 with stirring, and then 150mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and 200mg of N-hydroxysuccinimide were added and stirred at room temperature for 15 to 60 min. 100 mu L of aminated nano conductive graphite alkyne dispersion liquid with the concentration of 200mg/mL (pH 7.4) is added dropwise, and stirring is carried out for 12-72 h at room temperature. And after the reaction is finished, centrifugally cleaning the obtained product by using deionized water, dialyzing by using the deionized water, and removing free small molecules to obtain the nano conductive graphite alkyne with the surface modified with the redox ligand.
C. And D, mixing 10 mu L of the nano conductive graphdine dispersion liquid (10mg/mL) of the surface modified redox ligand obtained in the step B and 10 mu L of glucose oxidase solution (10mg/mL) through 10 mu L of 1, 6-hexanediol diglycidyl ether solution (10mg/mL), dripping the mixture on the surface of an electrode, and drying the mixture in vacuum at room temperature for 48 hours to obtain the conductive nano material glucose sensing layer reagent modified based on the redox ligand.
Example 5
The embodiment provides a redox ligand modification-based conductive nano-material glucose sensing material, and the preparation process comprises the following steps:
A. weighing 100 mu L of nano-gold dispersion, placing the nano-gold dispersion into a 10mL small bottle, adding 1-3 mL of deionized water and 0.01-1 mL of 3- (2-aminoethylamino) propyl trimethoxy silane, performing ultrasonic treatment for 10-100 min to obtain uniform dispersion, placing the uniform dispersion at 25-50 ℃ for 1-24 h, adding 0.1-100 mu L of 0.1M hydrochloric acid, placing the uniform dispersion at room temperature for 6-48 h, dialyzing the obtained suspension, and performing centrifugal cleaning on the dialyzed suspension for multiple times to obtain the nano-gold with amino groups on the surface.
B. Mixing 10-100 mg of Os (N, N '-dialkylated-2, 2' -bi-imidazole)2Im-ImCOOH was dissolved in PBS (pH 5-8) with stirring, and then 150mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and 200mg of N-hydroxysuccinimide were added thereto, followed by stirring at room temperature for 15-60 min. And dropwise adding 100 mu L of aminated nano conductive nano gold dispersion liquid (pH is 7.4) with the concentration of 200mg/mL, and stirring at room temperature for 12-72 h. And after the reaction is finished, centrifugally cleaning the obtained product by using deionized water, dialyzing by using the deionized water, and removing free small molecules to obtain the nano conductive gold with the surface modified with the redox ligand.
C. And D, mixing 10 mu L of the nano conductive gold dispersion liquid (10mg/mL) with the surface modified redox ligand obtained in the step B and 10 mu L of glucose oxidase solution (10mg/mL) through 10 mu L of 1,6 polypropylene glycol diglycidyl ether solution (10mg/mL), dripping the mixture on the surface of an electrode, and drying in vacuum at room temperature for 48h to obtain the glucose sensing layer reagent based on the conductive nano material modified by the redox ligand.
The cyclic voltammograms of these working electrodes were tested in a phosphate buffered solution at pH 7.4, using the electrodes obtained as described in examples 1 to 5 as working electrodes, silver/silver chloride as reference electrode, and a platinum wire as reference electrode, respectively. As can be seen from FIG. 1, the redox peaks of these electrodes are below 0.3V, which is much lower than the voltage required for the catalytic oxidation of the interfering substance, acetamido. Therefore, the electrodes have better anti-interference capability.
Time-current curves in 0-40mM glucose PBS after coating a biocompatible layer with the glucose sensing layer reagents of examples 1-5, respectively, were performed, with increasing glucose concentrations of 5mM each time. As shown in fig. 2, as the glucose concentration increases, the current signal value also increases. And the glucose concentration and the current signal value have a better linear relation. The results show that the prepared electrodes can well test the glucose concentration in the glucose concentration range of 0-40 mM.
Anti-interference experiments were performed after wrapping a layer of biocompatible layer with the glucose sensing layer reagents of examples 1-5, respectively. As shown in FIG. 3, the current signal values at these electrodes showed a relatively good linear relationship with the glucose concentration, and then the PBS solution was replaced again, and 5mM acetaminophen was added, and the current values hardly changed. The results show that the prepared electrodes have no response to acetaminophen, which indicates that the electrodes have stronger anti-interference capability to acetaminophen.
The glucose sensing layer reagents of examples 1-5 were coated with a biocompatible layer and tested at different oxygen concentrations. As shown in FIG. 4, the current signal values of these electrodes are in a relatively good linear relationship in the range of 0-40mM glucose concentration in a 5% oxygen atmosphere. Subsequently, nitrogen gas was bubbled to change the oxygen concentration in the test environment, and the current value hardly changed. The results show that the prepared electrode is less disturbed by the oxygen concentration.
Long-term stability tests were performed after wrapping a biocompatible layer with the glucose sensing layer reagents of examples 1-5, respectively. After 15 days of continuous operation of these electrodes in the test solution, the sensitivity was measured daily and the results are shown in fig. 5. Where the sensitivity at day 0 is the unsterilized electrode test results and the reason that the sensitivity at day 1 is lower than the sensitivity at the subsequent 14 days is that the biocompatible layer is not fully swelling balanced. The sensitivity of these electrodes changed less over the following 14 days, indicating that the prepared electrodes had good long-term stability.
The above-mentioned embodiments only express the specific embodiments of the present application, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present application. It should be noted that, for those skilled in the art, without departing from the technical idea of the present application, several changes and modifications can be made, which are all within the protection scope of the present application.