CN108760858B - Nano-silver modified titanium dioxide nano-column array enzyme electrode and preparation method and application thereof - Google Patents

Nano-silver modified titanium dioxide nano-column array enzyme electrode and preparation method and application thereof Download PDF

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CN108760858B
CN108760858B CN201810556846.1A CN201810556846A CN108760858B CN 108760858 B CN108760858 B CN 108760858B CN 201810556846 A CN201810556846 A CN 201810556846A CN 108760858 B CN108760858 B CN 108760858B
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王永
时梅林
左凤梅
徐凯
庄银苹
韩翠平
吴长宇
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Xuzhou Medical University
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Abstract

The invention relates to a nano-silver modified titanium dioxide nano-column array enzyme electrode and a preparation method and application thereof2A nanopillar array; will then grow with TiO2Soaking the carbon paper of the nano-column array in the silver sol for 3-15 hours, taking out, washing with deionized water, and drying to obtain a nano-silver modified titanium dioxide nano-column array; and finally, taking bovine serum albumin-glutaraldehyde as a cross-linking agent, and fixing the glucose oxidase on the surface of the nano-silver modified titanium dioxide nano-column array by adopting an improved cross-linking method to form the nano-silver modified titanium dioxide nano-column array enzyme electrode. The titanium dioxide nano-column array enzyme electrode modified by nano-silver prepared by the invention has better electrocatalytic activity and can be well applied to glucose biosensors or enzyme biofuel cells.

Description

Nano-silver modified titanium dioxide nano-column array enzyme electrode and preparation method and application thereof
Technical Field
The invention relates to the technical field of biosensors, in particular to a titanium dioxide nano-column array enzyme electrode modified by nano-silver and a preparation method and application thereof.
Background
The glucose oxidase electrode is a biosensor for measuring the glucose content in a solution to be measured, an immobilized enzyme membrane is arranged on the sensitive surface of a basic electrode, when the electrode is inserted into the solution to be measured, the glucose oxidase in the enzyme membrane generates catalytic reaction to generate electrode active substances, and response current changes along with the concentration of the active substances, so that the concentration of reactants or reaction products in the reaction catalyzed by the glucose oxidase is measured.
The composition and surface structure of the electrode material and the fixation of the glucose oxidase on the surface of the electrode are important factors for determining the electrocatalytic activity of the glucose oxidase electrode biosensor. The nano-structure material has a large specific surface area, and can provide more active sites for the fixation of glucose oxidase, thereby improving the fixation efficiency of the glucose oxidase and enhancing the electrocatalytic activity of the glucose oxidase electrode biosensor. A wide variety of nanomaterials have been used in glucose oxidase electrodes, such as carbon nanoparticles, Au nanoparticles, Pt nanoparticles, and SiO2、Al2O3、MnO2And TiO2Etc. oxide nanoparticles. Wherein, due to TiO2The nanometer material has the advantages of good biocompatibility, relatively high conductivity, no toxicity, stability, low cost, various structures of TiO, etc2Nanomaterials have been used for enzyme immobilization during the fabrication of glucolase electrode biosensors.
Highly ordered TiO compared to other structures2The nano array can provide a one-way channel for electron transmission, has high electron conduction efficiency, and the existing TiO2Nanotube arrays have been widely used as carrier materials for glucose oxidase. However, due to the action of surface tension, it is difficult for either glucose oxidase to be immobilized on the electrode surface or electrolyte solution to diffuse on the electrode surface to reach TiO2The interior of the nanotube is not fully made TiO2The advantage of large specific surface area of the nanotube array influences the improvement of the fixation efficiency of the glucose oxidase. In addition, it is made of TiO only2Enzyme electrodes made of nanomaterials also often exhibit low bioelectrocatalytic activity.
Disclosure of Invention
In view of the above problems in the prior art, the present invention aims to provide a nano-silver modified titanium dioxide nano-pillar array enzyme electrode with better electrocatalytic performance, and a preparation method and an application of the enzyme electrode.
In order to solve the above technical problems, a first aspect of the present invention provides a method for preparing a titanium dioxide nanopillar array enzyme electrode modified by nano silver, the method comprising the following steps:
(1) growing TiO on carbon paper by taking the carbon paper as a substrate material2A nanopillar array;
(2) growing TiO in the step (1)2Soaking the carbon paper of the nano-column array in the silver sol for 3-15 hours, then taking out, washing with deionized water, and drying to obtain a nano-silver modified titanium dioxide nano-column array;
(3) and (3) taking bovine serum albumin-glutaraldehyde as a cross-linking agent, and fixing glucose oxidase on the surface of the nano-silver modified titanium dioxide nano-column array in the step (2) by adopting an improved cross-linking method to form the nano-silver modified titanium dioxide nano-column array enzyme electrode.
The invention adopts TiO2The nano-column array is used as an enzyme electrode carrier material because of being mixed with TiO2Nanotube array phase, TiO2The nano-pillar array not only has large specific surface area and high electron conduction efficiency, but also is beneficial to the diffusion of electrolyte solution to the surface of an electrode and the diffusion of electrolyte solution to the surface of an electrode through TiO2The surface of the nano-pillar array is modified with nano-silver particles, so that the electrocatalytic activity of the nano-pillar array is greatly enhanced.
The carbon paper is used as a substrate material and is formed by bonding a carbon fiber framework and a carbon material, and has the advantages of good conductivity, stable property, low price and the like. The carbon paper has rough surface, a large number of holes inside and larger specific surface, and is grown with TiO2Ideal substrate material for nanopillar arrays.
Further, in the step (1), the carbon paper is used as a substrate material, and TiO grows on the carbon paper2The nano-pillar array specifically comprises the following steps:
(1.1) taking carbon paper with a certain size, cleaning and drying for later use;
(1.2) mixing a titanium source and absolute ethyl alcohol according to the volume ratio of 1: 4-1: 20, then dropwise adding glacial acetic acid into the mixed solution, wherein the volume ratio of the glacial acetic acid to the titanium source is 1: 50-1: 250, and stirring at room temperature to react to obtain TiO2A colloidal solution;
(1.3) dipping the carbon paper dried and reserved in the step (1.1) into the TiO in the step (1.2)2Taking out the colloid solution, drying, treating at the temperature of 300-400 ℃ for 10-30 min, and cooling to room temperature to obtain adsorbed TiO2Carbon paper of nanoparticles;
(1.4) subjecting the adsorbed TiO to2Putting the carbon paper of the nano particles into a mixed solution of hydrochloric acid and a titanium source, reacting for 4-20 hours at 150-200 ℃, washing a product obtained by the reaction with deionized water to remove residual reaction liquid on the surface, and drying to obtain the grown amorphous TiO2Carbon paper of nano-pillar array;
(1.5) placing the product dried in the step (1.4) in a high-temperature furnace in an inert gas atmosphere for calcination treatment, wherein the calcination temperature is 400-600 ℃, the calcination time is 1-3 hours, the heating rate is 3-10 ℃/min, and then controlling the temperature to be reduced to obtain the crystal-phase-grown TiO2Carbon paper with nano-pillar array.
Further, the preparation process of the mixed solution of the hydrochloric acid and the titanium source is as follows: mixing concentrated hydrochloric acid with the mass concentration of 36-38% with deionized water according to the volume ratio of 1:1, then adding a titanium source, and uniformly stirring the titanium source and the concentrated hydrochloric acid according to the volume ratio of 1: 25-1: 60 to obtain the mixed solution of the hydrochloric acid and the titanium source.
Further, the titanium source is any one of butyl titanate, ethyl titanate, isopropyl titanate and titanium tetrachloride.
Further, the calcination treatment in the step (1.5) specifically includes: under the protection of inert gas, heating to 250-300 ℃ at a heating rate of 3-10 ℃/min, keeping the temperature constant at 250-300 ℃ for 10-15 min, heating to 400-600 ℃ at a heating rate of 3-10 ℃/min, calcining at 400-600 ℃ for 1-3 hours at constant temperature, and then controlling the temperature to decrease.
Further, in the step (2), the preparation process of the silver sol comprises: uniformly mixing ethylene glycol and deionized water according to the volume ratio of 1:1, then respectively adding polyvinylpyrrolidone-K30 and sodium borohydride into the mixed solution, stirring for 1-2 min, adding silver nitrate while stirring for reaction, wherein the molar ratio of the silver nitrate to the polyvinylpyrrolidone-K30 to the sodium borohydride is 1 (0.01-0.03) to 0.5-1, and obtaining the silver sol after reaction.
Further, in the step (3), the improved crosslinking method specifically includes the following steps:
(3.1) dissolving 20-30 mg of bovine serum albumin in 1.0mL of phosphate buffer solution with the pH value of 6.8, then adding 80-150 mu L of glutaraldehyde into the phosphate buffer solution to prepare a bovine serum albumin-glutaraldehyde crosslinking agent solution, and storing the bovine serum albumin-glutaraldehyde crosslinking agent solution at the temperature of 4 ℃;
(3.2) dissolving 1-3 mg of glucose oxidase in 100 mu L of phosphate buffer solution with the pH value of 6.8 to prepare a glucose oxidase solution, and storing the glucose oxidase solution at the temperature of 4 ℃;
(3.3) soaking the nano-silver modified titanium dioxide nano-column array in a phosphate buffer solution so that the nano-silver modified titanium dioxide nano-column array has a wet surface;
(3.4) coating 10-30 mu L of bovine serum albumin-glutaraldehyde crosslinking agent solution on the surface of the wet nano-silver modified titanium dioxide nano-column array, and naturally standing for 5-20 min at room temperature for surface adsorption to prepare the nano-silver modified titanium dioxide nano-column array adsorbed with the bovine serum albumin-glutaraldehyde crosslinking agent;
(3.5) coating 10-30 mu L of glucose oxidase solution on the surface of the nano-silver modified titanium dioxide nano-column array adsorbed with the bovine serum albumin-glutaraldehyde crosslinking agent, standing at 4 ℃ for 8-24 hours to fix the glucose oxidase through a crosslinking reaction, then soaking in a phosphate buffer solution to remove the glucose oxidase which is not fixed, and finally drying to obtain the nano-silver modified titanium dioxide nano-column array enzyme electrode.
The second aspect of the invention provides a nano-silver modified titanium dioxide nano-column array enzyme electrode, which is prepared by any one of the methods in the first aspect of the invention, wherein the height of the titanium dioxide nano-column is 1-3 μm, and the width of the titanium dioxide nano-column is 50-150 nm.
Further, the crystal form of the titanium dioxide nano column array is a rutile crystal form.
The third aspect of the invention provides an application of the titanium dioxide nano-column array enzyme electrode modified by nano-silver in the second aspect in a glucose biosensor or an enzyme biofuel cell.
The titanium dioxide nano-column array enzyme electrode modified by nano-silver and the preparation method and the application thereof have the following beneficial effects:
the invention takes carbon paper as a substrate material, firstly, TiO is constructed on the carbon paper2Array of nanopillars, then on TiO2Modifying the surface of the nano-column array with nano-silver particles to obtain nano-silver modified TiO2Nano column array and nano silver modified TiO2The surface of the nano-column array adopts an improved cross-linking method to adsorb and fix glucose oxidase, thereby obtaining the TiO modified by nano-silver2The nano-column array enzyme electrode has simple preparation process, not only utilizes the large specific surface of the carbon paper to grow more TiO2Nano-pillar array, and uses TiO2The nano-column array has the characteristics of large specific surface area, high electron conduction efficiency, favorable diffusion of electrolyte solution to the surface of the electrode and high electrocatalytic activity of nano-silver particles, thereby greatly improving the electrocatalytic activity of the prepared enzyme electrode.
The nano-silver modified titanium dioxide nano-column array enzyme electrode has low preparation cost, is easy for large-scale production, and can be well applied to glucose biosensors and enzyme biofuel cells.
Drawings
In order to more clearly illustrate the technical solution of the present invention, the drawings used in the description of the embodiment or the prior art will be briefly described below. It is obvious that the drawings in the following description are only some embodiments of the invention, and that for a person skilled in the art, other drawings can be derived from them without inventive effort.
FIG. 1 is a process flow diagram of a preparation method of a nano-silver modified titanium dioxide nano-column array enzyme electrode provided by the invention, wherein A is carbon paper, and B is adsorbed TiO2Carbon paper of nanoparticles, C being grown with amorphous TiO2Carbon paper of the nano-column array, D is a titanium dioxide nano-column array/carbon paper, and E is a nano-silver modified titanium dioxide nano-column array;
FIG. 2 is an SEM photograph of a titania nanorod array/carbon paper provided in the present invention, wherein (2a) is a front view and (2b) is a cross-sectional view;
FIG. 3 is an XRD pattern of a titanium dioxide nanopillar array/carbon paper provided by the present invention;
FIG. 4 is an SEM photograph of a nano silver/titanium dioxide nano column array/carbon paper provided by the present invention;
FIG. 5 is a pair H2O2A current-time curve of response, wherein (5a) is a carbon paper electrode, (5b) is a titanium dioxide nanopillar array/carbon paper electrode, and (5c) is a nanosilver/titanium dioxide nanopillar array/carbon paper electrode;
FIG. 6 shows the response current of carbon paper electrode (a), titanium dioxide nanopillar array/carbon paper electrode (b) and nano silver/titanium dioxide nanopillar array/carbon paper electrode (c) with H2O2A linear fitting curve of the concentration variation relation;
FIG. 7 is a current-time curve for the response to glucose, where (7a) is a glucose oxidase/carbohydrase electrode, (7b) is a glucose oxidase/titanium dioxide nanopillar array/carbohydrase electrode, and (7c) is a glucose oxidase/nanosilver/titanium dioxide nanopillar array/carbohydrase electrode;
FIG. 8 is a linear fitting curve of response current of glucose oxidase/carbon paper enzyme electrode (a), glucose oxidase/titanium dioxide nano-column array/carbon paper enzyme electrode (b) and glucose oxidase/nano-silver/titanium dioxide nano-column array/carbon paper enzyme electrode (c) in relation to change of glucose concentration.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
Example 1
The embodiment provides a preparation method of a titanium dioxide nanorod array enzyme electrode modified by nano silver, as shown in fig. 1, the method comprises the following specific steps:
(1) growing TiO on carbon paper by using carbon paper as substrate material2Nanopillar array
The carbon paper is cut into the following length, width and thickness: 50mm by 20mm by 0.2mm pieces were washed with acetone and deionized water in sequence by ultrasonic for 10min, and then dried at 80 ℃ for use, as shown in A of FIG. 1.
Taking 20mL of butyl titanate in a 200mL beaker, slowly adding 100mL of absolute ethyl alcohol while stirring, then dropwise adding 0.2mL of glacial acetic acid, and continuously stirring at room temperature until the reaction solution is milky white to prepare the TiO2A colloidal solution.
Dipping the spare carbon paper after treatment in the TiO2Adding into colloidal solution for 5min, taking out, oven drying at 80 deg.C, treating at 350 deg.C for 20min, naturally cooling to room temperature to obtain adsorbed TiO2Nanoparticle carbon paper, as shown in fig. 1B.
Take 18mL of H2O in a 50mL beaker, adding 18mL of concentrated hydrochloric acid with the mass concentration of 36-38%, stirring for 5min, then adding 0.54mL of butyl titanate into the hydrochloric acid solution, stirring rapidly for 10min,to obtain the mixed solution of hydrochloric acid and butyl titanate.
Will adsorb TiO2The carbon paper of nanoparticles was put into a 50mL teflon-lined reaction vessel, and then the above-mentioned mixed solution of hydrochloric acid and butyl titanate was added to the reaction vessel to react at 180 ℃ for 10 hours. After the reaction is finished, naturally cooling to room temperature, forming a layer of white substance on the front and back surfaces of the carbon paper, repeatedly washing the obtained sample by deionized water to remove residual reaction liquid on the surface of the material, airing, and preparing the amorphous TiO2A carbon paper of nanopillar array, as shown in fig. 1C.
Drying the dried TiO grown in an amorphous form2Placing the carbon paper of the nano-column array in an integrated program-controlled high-temperature furnace for calcination treatment, heating to 300 ℃ at a heating rate of 5 ℃/min under the protection of nitrogen, keeping the temperature at the environment of 300 ℃ for 10min, heating to 500 ℃ at a heating rate of 5 ℃/min, then calcining for 1.5 hours at the environment of 500 ℃, controlling the temperature reduction after the calcination is finished, finishing the program, and calcining the treated TiO2The nano-pillar array is converted into a more orderly and regular crystal phase from an amorphous state, and the TiO with the crystal phase growing thereon is obtained2The carbon paper of the nanopillar array, hereinafter referred to as titanium dioxide nanopillar array/carbon paper, is shown as D in fig. 1.
The SEM photograph of the titania nanorod array/carbon paper is shown in fig. 2, wherein (2a) is a front view thereof, and (2b) is a cross-sectional view thereof. As shown in fig. 2a, the surface of the carbon paper and the surface of the holes inside the carbon paper are uniformly covered by the titanium dioxide nano-pillar array, the titanium dioxide nano-pillars are quadrangular, the cylindrical surfaces are smooth and flat, the width of the titanium dioxide nano-pillars is about 50-150 nm, and each titanium dioxide nano-pillar is formed by orderly and tightly arranging a plurality of fine titanium dioxide nano-pillars in height. As shown in FIG. 2b, the height of the titania nanocolumn is about 1 to 3 μm.
FIG. 3 shows the XRD pattern of the titanium dioxide nanopillar array/carbon paper, which is compared to rutile phase TiO2Comparing the standard card (JCPDS No.21-1276) with the standard card (JCPDS No.26-1076) of carbon, the diffraction peak of the titanium dioxide nano-column is compared with that of rutile phase TiO2Is consistent with the standard cardObvious mixed peaks appear, and the titanium dioxide nano-column growing on the carbon paper is in a rutile crystal form.
(2) Preparation of nano-silver modified titanium dioxide nano-column array
Take 15mL of H2And mixing O and 15mL of ethylene glycol in a 50mL beaker, uniformly stirring, adding 1.2g of polyvinylpyrrolidone-K30, uniformly stirring, adding 0.0296g of sodium borohydride, stirring for 1-2 min, adding 0.1784g of silver nitrate while stirring, and changing the reaction liquid into dark brown to obtain the silver sol.
And (2) soaking the titanium dioxide nano-column array/carbon paper prepared in the step (1) in the dark brown silver sol, soaking for 10 hours at a constant temperature of 40 ℃, taking out a sample, repeatedly washing with deionized water, and airing to obtain the nano-silver modified titanium dioxide nano-column array, which is hereinafter referred to as nano-silver/titanium dioxide nano-column array/carbon paper, as shown in E in figure 1.
Fig. 4 is an SEM photograph of the nano silver/titanium dioxide nano-pillar array/carbon paper, and it can be seen that silver nano-particles are uniformly deposited on the surface of the titanium dioxide nano-pillars, the particle size of a single Ag particle is about 10nm, and a small amount of Ag particles are aggregated on the surface of the titanium dioxide nano-pillars to form particles of 30 to 40 nm.
(3) Preparation of nano-silver modified titanium dioxide nano-column array enzyme electrode
Fixing glucose oxidase on the surface of the nano-silver/titanium dioxide nano-column array/carbon paper prepared in the step (2) by adopting an improved cross-linking method to form a nano-silver modified titanium dioxide nano-column array enzyme electrode, which is referred to as glucose oxidase/nano-silver/titanium dioxide nano-column array/carbon paper enzyme electrode for short, and the specific preparation process is as follows:
taking 25mg of bovine serum albumin in a small centrifuge tube, adding 1mL of phosphate buffer solution with the pH value of 6.8, uniformly mixing by oscillation, then adding 100 mu L of glutaraldehyde, uniformly mixing to obtain a bovine serum albumin-glutaraldehyde crosslinking agent solution, and keeping the bovine serum albumin-glutaraldehyde crosslinking agent solution at 4 ℃ for later use.
Weighing 2.5mg of glucose oxidase into a small centrifuge tube of 1.5mL, adding 100 mu L of phosphate buffer solution with the pH value of 6.8, uniformly mixing by oscillation to prepare glucose oxidase solution, and storing at the temperature of 4 ℃ for later use.
And (3) soaking the nano silver/titanium dioxide nano column array/carbon paper prepared in the step (2) in a phosphate buffer solution to wet the surface of the nano silver/titanium dioxide nano column array/carbon paper.
Coating 20 mu L of bovine serum albumin-glutaraldehyde crosslinking agent solution on the surface of the wet nano silver/titanium dioxide nano column array/carbon paper, and naturally standing for 15min at room temperature to prepare nano silver/titanium dioxide nano column array/carbon paper adsorbed with the bovine serum albumin-glutaraldehyde crosslinking agent;
and (3) coating 20 mu L of glucose oxidase solution on the surface of the nano silver/titanium dioxide nano column array/carbon paper adsorbed with the bovine serum albumin-glutaraldehyde crosslinking agent, and standing for 12 hours at 4 ℃ to perform crosslinking reaction. Because two aldehyde groups of the glutaraldehyde react with amino groups in bovine serum albumin and glucose oxidase molecules respectively, the glucose oxidase is crosslinked with the bovine serum albumin through the glutaraldehyde, and then the glucose oxidase is fixed on the outer surface through the adsorption of the bovine serum albumin on the surface of the nano silver/titanium dioxide nano column array/carbon paper. And (3) after the crosslinking reaction, soaking the substrate in phosphate buffer solution with the pH value of 6.8 to remove the glucose oxidase which is not fixed, and finally drying the substrate to obtain the glucose oxidase/nano silver/titanium dioxide nano column array/carbon paper enzyme electrode.
Comparative example 1
The method of example 1 was used to directly immobilize glucose oxidase onto the surface of pure carbon paper to produce a glucose oxidase/carbon paper enzyme electrode.
Comparative example 2
Preparing a titanium dioxide nano-column array/carbon paper by adopting the method of example 1, and fixing glucose oxidase on the surface of the prepared titanium dioxide nano-column array/carbon paper to form a glucose oxidase/titanium dioxide nano-column array/carbon paper enzyme electrode.
Example 2
In the present example, pure carbon paper, the titanium dioxide nanorod array/carbon paper prepared in comparative example 2, and the nano silver/titanium dioxide nanorod array/carbon paper prepared in example 1 were used as working electrodes, and platinum sheet electrodes were used as working electrodesA counter electrode, a saturated calomel electrode is used as a reference electrode, a phosphate buffer solution with the pH value of 6.8 is used as a working electrolyte solution under the working potential of-0.6V, a current-time curve test is carried out, and H pair is detected by the counter electrode, the saturated calomel electrode and the phosphate buffer solution2O2Electrocatalytic performance of (a), wherein H2O2The glucose oxidase of (a) catalyzes the product of the reaction.
The specific detection process is that H is added every 50s2O2Solution, starting from 100s, with each increase in H2O2The concentration increased to 0.1mM (representing 0.1mmol ∙ L-1The same applies hereinafter); starting from 600s, each increment of H2O2The concentration is increased to 0.2 mM; starting from the 850 th s, each increasing H2O2The concentration was increased to 0.5mM until the end of the test, and the results of the test are shown in FIGS. 5 and 6. FIG. 5 is a pair H2O2A response current-time curve, wherein (5a) is a pure carbon paper electrode, (5b) is a titanium dioxide nano-column array/carbon paper electrode, and (5c) is a nano-silver/titanium dioxide nano-column array/carbon paper electrode; FIG. 6 shows the response current of carbon paper electrode (a), titanium dioxide nanopillar array/carbon paper electrode (b) and nano silver/titanium dioxide nanopillar array/carbon paper electrode (c) with H2O2A linear fit curve of the concentration variation relationship.
From the detection results, it can be seen that the carbon paper electrode pair H2O2The linear concentration range of response is 0-7 mM, and the titanium dioxide nano-column array/carbon paper electrode pair H2O2The linear concentration range of response is 0-3 mM, and the nano silver/titanium dioxide nano column array/carbon paper electrode pair H2O2The linear concentration range of response is 0-5 mM; carbon paper electrode pair H2O2The response current density of (2) was 0.0325mA ∙ mM-1∙cm-2Titanium dioxide nanorod array/carbon paper electrode pair H2O2Has a response current density of 1.138mA ∙ mM-1∙cm-2Nano silver/titanium dioxide nano column array/carbon paper electrode pair H2O2Has a response current density of 1.62mA ∙ mM-1∙cm-2Thus, the nano silver/titanium dioxide nano column array/carbon paper electrode pair H2O2The response current density of the carbon paper electrode is 50 times that of the carbon paper electrode, and is 1.42 times that of the titanium dioxide nano-column array/carbon paper electrode.
Therefore, compared with the carbon paper electrode and the titanium dioxide nano-column array/carbon paper electrode, the nano-silver/titanium dioxide nano-column array/carbon paper electrode pair H2O2Has better electrocatalytic performance.
Example 3
In the present example, the glucose oxidase/carbon paper prepared in comparative example 1, the glucose oxidase/titanium dioxide nanorod array/carbon paper prepared in comparative example 2, and the glucose oxidase/nanosilver/titanium dioxide nanorod array/carbon paper prepared in example 1 were respectively used as working electrodes, taking a platinum sheet electrode as a counter electrode, taking a saturated calomel electrode as a reference electrode, taking a phosphate buffer solution with the pH value of 6.8 as a working electrolyte solution, carrying out a current-time curve test, detecting the electrocatalytic performance of the three on glucose, wherein-0.5V is selected as a working potential when testing the glucose oxidase/carbon paper enzyme electrode, and-0.6V is selected as a working potential when testing the glucose oxidase/nano silver/titanium dioxide nano column array/carbon paper enzyme electrode.
The specific detection process is that glucose solution is added every 50s, and the glucose concentration increased every time is 0.05mM from the 100 th s; from the 400 th s, the glucose concentration increased to 0.1mM each time until the end of the test, and the results are shown in FIGS. 7 and 8. FIG. 7 is a current-time curve for the response to glucose, wherein (7a) is a glucose oxidase/carbohydrase electrode, (7b) is a glucose oxidase/titanium dioxide nanopillar array/carbohydrase electrode, and (7c) is a glucose oxidase/nanosilver/titanium dioxide nanopillar array/carbohydrase electrode; FIG. 8 is a linear fitting curve of response current of glucose oxidase/carbon paper enzyme electrode (a), glucose oxidase/titanium dioxide nano-column array/carbon paper enzyme electrode (b) and glucose oxidase/nano-silver/titanium dioxide nano-column array/carbon paper enzyme electrode (c) in relation to change of glucose concentration.
According to the detection result, the response current density of the glucose oxidase/carbon paper enzyme electrode to the glucose is 0.0276mA ∙ mM-1∙cm-2The response current density of the glucose oxidase/titanium dioxide nano-column array/carbon paper enzyme electrode to glucose is 0.13mA ∙ mM-1∙cm-2The response current density of the glucose oxidase/nano-silver/titanium dioxide nano-column array/carbon paper enzyme electrode to glucose is 0.182mA ∙ mM-1∙cm-2It can be seen that the response current density of the glucose oxidase/nano silver/titanium dioxide nano column array/carbon paper enzyme electrode to glucose is 6.6 times that of the glucose oxidase/carbon paper enzyme electrode and 1.4 times that of the glucose oxidase/titanium dioxide nano column array/carbon paper enzyme electrode.
Therefore, compared with a glucose oxidase/carbon paper enzyme electrode and a glucose oxidase/titanium dioxide nano-column array/carbon paper enzyme electrode, the glucose oxidase/nano-silver/titanium dioxide nano-column array/carbon paper enzyme electrode has better electrocatalytic performance on glucose.
The linear concentration range of the glucose oxidase/nano silver/titanium dioxide nano column array/carbon paper enzyme electrode to the glucose response is 0-0.9 mM, and the linear correlation coefficient is R2The detection limit was 1.6 μ M (signal to noise ratio of 3) 0.9996.
In summary, the present invention uses carbon paper as the substrate material, and first constructs TiO on the carbon paper2Array of nanopillars, then on TiO2Modifying the surface of the nano-column array with nano-silver particles to obtain nano-silver modified TiO2Nano column array and nano silver modified TiO2The surface of the nano-column array adopts an improved cross-linking method to adsorb and fix glucose oxidase, thereby obtaining the TiO modified by nano-silver2The nano-column array enzyme electrode has simple preparation process, not only utilizes the large specific surface of the carbon paper to grow more TiO2Nano-pillar array, and uses TiO2The nano-column array has the characteristics of large specific surface area, high electron conduction efficiency, favorable diffusion of electrolyte solution to the surface of the electrode and high electrocatalytic activity of nano-silver particles, so that the prepared titanium dioxide nano-column array enzyme electrode modified by nano-silver has better electrocatalytic activity. The nano-silver modified titanium dioxide nano-column array enzyme electrode has low preparation cost and is easy for large-scale production,can be well applied to glucose biosensors and enzyme biofuel cells.
The foregoing description has disclosed fully preferred embodiments of the present invention. It should be noted that those skilled in the art can make modifications to the embodiments of the present invention without departing from the scope of the appended claims. Accordingly, the scope of the appended claims is not to be limited to the specific embodiments described above.

Claims (8)

1. A preparation method of a titanium dioxide nano-column array enzyme electrode modified by nano-silver is characterized by comprising the following steps:
(1) growing TiO on carbon paper by taking the carbon paper as a substrate material2A nanopillar array; the TiO is2TiO in nano-pillar array2The nano-pillars are in the shape of a quadrangular prism, and each TiO is2The nano-pillar is composed of a plurality of tiny TiO in a quadrangular prism shape2The nano-columns are orderly and closely arranged in height;
(2) growing TiO in the step (1)2Soaking the carbon paper of the nano-column array in the silver sol for 3-15 hours, then taking out, washing with deionized water, and drying to obtain a nano-silver modified titanium dioxide nano-column array;
(3) fixing glucose oxidase on the surface of the nano-silver modified titanium dioxide nano-column array in the step (2) by using bovine serum albumin-glutaraldehyde as a cross-linking agent and adopting an improved cross-linking method to form the nano-silver modified titanium dioxide nano-column array enzyme electrode;
wherein, in the step (1), the carbon paper is used as a substrate material, and TiO grows on the carbon paper2The nano-pillar array specifically comprises the following steps:
(1.1) taking carbon paper with a certain size, cleaning and drying for later use;
(1.2) mixing a titanium source and absolute ethyl alcohol according to the volume ratio of 1: 4-1: 20, then dropwise adding glacial acetic acid into the mixed solution, wherein the volume ratio of the glacial acetic acid to the titanium source is 1: 50-1: 250, and stirring at room temperature to react to obtain TiO2A colloidal solution;
(1.3) dipping the carbon paper dried and reserved in the step (1.1) into the TiO in the step (1.2)2Taking out the colloid solution, drying, treating at 300-400 ℃ for 10-30 min, and cooling to room temperature to obtain adsorbed TiO2Carbon paper of nanoparticles;
(1.4) subjecting the adsorbed TiO to2Putting the carbon paper of the nano particles into a mixed solution of hydrochloric acid and a titanium source, reacting for 4-20 hours at 150-200 ℃, washing a product obtained by the reaction with deionized water to remove residual reaction liquid on the surface, and drying to obtain the grown amorphous TiO2Carbon paper of nano-pillar array;
(1.5) heating the product dried in the step (1.4) to 250-300 ℃ at a heating rate of 3-10 ℃/min under the protection of inert gas, keeping the temperature constant at 250-300 ℃ for 10-15 min, heating to 400-600 ℃ at a heating rate of 3-10 ℃/min, calcining at 400-600 ℃ for 1-3 hours at constant temperature, and controlling to cool to obtain the TiO with crystal phase growing2Carbon paper with nano-pillar array.
2. The method for preparing the nano-silver modified titanium dioxide nano-column array enzyme electrode according to claim 1, wherein the preparation process of the mixed solution of hydrochloric acid and a titanium source is as follows: mixing concentrated hydrochloric acid with the mass concentration of 36-38% with deionized water according to the volume ratio of 1:1, then adding a titanium source, wherein the volume ratio of the titanium source to the concentrated hydrochloric acid is 1: 25-1: 60, and stirring and uniformly mixing to obtain the mixed solution of hydrochloric acid and the titanium source.
3. The method for preparing the nano-silver modified titanium dioxide nano-column array enzyme electrode according to claim 1 or 2, wherein the titanium source is any one of butyl titanate, ethyl titanate, isopropyl titanate and titanium tetrachloride.
4. The method for preparing the nano-silver modified titanium dioxide nano-column array enzyme electrode according to claim 1, wherein in the step (2), the preparation process of the silver sol comprises the following steps:
uniformly mixing ethylene glycol and deionized water according to the volume ratio of 1:1, then respectively adding polyvinylpyrrolidone-K30 and sodium borohydride into the mixed solution, stirring for 1-2 min, adding silver nitrate while stirring for reaction, wherein the molar ratio of the silver nitrate to the polyvinylpyrrolidone-K30 to the sodium borohydride is 1 (0.01-0.03) to 0.5-1, and obtaining the silver sol after reaction.
5. The method for preparing the nano-silver modified titanium dioxide nano-column array enzyme electrode according to claim 1, wherein in the step (3), the improved crosslinking method specifically comprises the following steps:
(3.1) dissolving 20-30 mg of bovine serum albumin in 1.0mL of phosphate buffer solution with the pH value of 6.8, then adding 80-150 mu L of glutaraldehyde into the phosphate buffer solution to prepare a bovine serum albumin-glutaraldehyde crosslinking agent solution, and storing the bovine serum albumin-glutaraldehyde crosslinking agent solution at the temperature of 4 ℃;
(3.2) dissolving 1-3 mg of glucose oxidase in 100 mu L of phosphate buffer solution with the pH value of 6.8 to prepare a glucose oxidase solution, and storing the glucose oxidase solution at the temperature of 4 ℃;
(3.3) soaking the nano-silver modified titanium dioxide nano-column array in a phosphate buffer solution so that the nano-silver modified titanium dioxide nano-column array has a wet surface;
(3.4) coating 10-30 mu L of bovine serum albumin-glutaraldehyde crosslinking agent solution on the surface of the wet nano-silver modified titanium dioxide nano-column array, and naturally standing for 5-20 min at room temperature for surface adsorption to prepare the nano-silver modified titanium dioxide nano-column array adsorbed with the bovine serum albumin-glutaraldehyde crosslinking agent;
(3.5) coating 10-30 mu L of glucose oxidase solution on the surface of the nano-silver modified titanium dioxide nano-column array adsorbed with the bovine serum albumin-glutaraldehyde crosslinking agent, standing at 4 ℃ for 8-24 hours to fix the glucose oxidase through a crosslinking reaction, then soaking in a phosphate buffer solution to remove the glucose oxidase which is not fixed, and finally drying to obtain the nano-silver modified titanium dioxide nano-column array enzyme electrode.
6. A nano-silver modified titanium dioxide nano-column array enzyme electrode is characterized by being prepared by the preparation method of the nano-silver modified titanium dioxide nano-column array enzyme electrode as claimed in any one of claims 1 to 5, wherein the height of the titanium dioxide nano-column is 1-3 μm, and the width of the titanium dioxide nano-column is 50-150 nm.
7. The nanosilver-modified titanium dioxide nanorod array enzyme electrode according to claim 6, wherein the crystal form of the titanium dioxide nanorod array is a rutile crystal form.
8. Use of the nanosilver-modified titanium dioxide nanopillar array enzyme electrode as claimed in claim 6 or 7 in a glucose biosensor or an enzyme biofuel cell.
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