WO2019218562A1 - 一种葡萄糖传感器电极及其制备方法与应用 - Google Patents

一种葡萄糖传感器电极及其制备方法与应用 Download PDF

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WO2019218562A1
WO2019218562A1 PCT/CN2018/106842 CN2018106842W WO2019218562A1 WO 2019218562 A1 WO2019218562 A1 WO 2019218562A1 CN 2018106842 W CN2018106842 W CN 2018106842W WO 2019218562 A1 WO2019218562 A1 WO 2019218562A1
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nano
polypyrrole
electrode
titanium
glucose
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PCT/CN2018/106842
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French (fr)
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宁成云
王珍高
于鹏
钱磊
周正难
邢君
陈俊琪
周蕾
谭帼馨
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华南理工大学
广东工业大学
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Priority to SG11202007810VA priority Critical patent/SG11202007810VA/en
Priority to US17/054,790 priority patent/US11788984B2/en
Publication of WO2019218562A1 publication Critical patent/WO2019218562A1/zh

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • G01N27/3271—Amperometric enzyme electrodes for analytes in body fluids, e.g. glucose in blood
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
    • G01N27/3278—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction involving nanosized elements, e.g. nanogaps or nanoparticles

Definitions

  • the invention belongs to the technical field of medical instruments, and in particular relates to a glucose sensor electrode and a preparation method and application thereof.
  • the glucose sensor electrode is used to detect glucose quickly and highly sensitively.
  • electrochemical glucose sensors Compared with acoustic, optical and chemiluminescence methods, electrochemical methods have the advantages of high sensitivity, low cost, easy operation, faster response time, and portable miniaturization.
  • the core technology of electrochemical glucose sensors is electrode design and processing.
  • Various types of nanomaterials such as metal oxides, carbonaceous materials (such as carbon nanotubes and graphene) have been widely used in glucose sensors due to excellent properties such as size effect of nanomaterials and high reactivity.
  • material development has focused on enhancing sensitivity and detection limits, glucose sensor electrode materials have many disadvantages, such as insufficient preparation process of electrodes, high preparation cost, and poor coating adhesion.
  • the invention constructs a glucose sensor electrode of a semiconductor nano-needle titania and a nano-cone structure conductive polypyrrole, and a micro-area electric field is also formed between the nano-needle titania and the nano-cone polypyrrole in the electrode.
  • the glucose sensor electrode of the invention has stable structure, and the nano-needle titania and the nano-cone polypyrrole have strong binding force to the conductive substrate, good adsorption to the enzyme and a very good promoting effect on the oxidation reaction of glucose.
  • the electrode of the present invention is capable of efficiently detecting glucose.
  • the invention constructs a high-efficiency glucose sensor coating on the surface of the titanium electrode by alkali heat treatment, laser etching and electrochemical method, and the coating has the micro-region characteristics of the conductive nano-cone structure polypyrrole and the semiconductor nano-needle structure titanium dioxide. .
  • the nano-needle titanium dioxide in the electrode of the invention has good catalytic properties, and a micro-area electric field is formed between the nano-needle titania and the nano-cone polypyrrole, which can further promote protein adhesion and efficiently graft the protease on the electrode surface ( Glucose oxidase).
  • the coating on the surface of the electrode of the invention (nano-needle titanium dioxide and nano-cone polypyrrole) has high protease adhesion characteristics and high detection sensitivity of the nano-cone structure, and achieves high sensitivity glucose detection.
  • Another object of the present invention is to provide an application of the above glucose sensor electrode.
  • the glucose sensor electrode is used to efficiently detect glucose.
  • a glucose sensor electrode comprising a titanium electrode, a surface of the titanium electrode formed of nano-needle titanium dioxide and a region formed by a nano-cone structure polypyrrole, each formed by a nano-cone structure polypyrrole and formed by nano-needle titanium dioxide
  • the regions are adjacent to each other, and each region formed by the nanopyram structure polypyrrole is a region formed by nanoneedle titania; the region formed by the nanopyram structure polypyrrole is grafted with an enzyme capable of detecting glucose.
  • the enzyme capable of detecting glucose is glucose oxidase; a region formed by nanoneedle titania and a region formed by a nanopyram structure polypyrrole are at least one region which is a microdomain.
  • the microdomains refer to the width (including all widths), diameter and/or area of the regions on the order of microns.
  • the nano-cone structure polypyrrole is a doped or undoped nano-cone structure polypyrrole, preferably a doped nano-cone structure polypyrrole, and the doped nano-cone structure polypyrrole is a citric acid doped nano cone Structure polypyrrole.
  • the region formed by the nanoneedle titania and the region formed by the nanopyram structure polypyrrole are at least one region which is a plurality of independent regions; each of the independent regions is a microdomain (ie, the width, diameter and/or area of the region is micrometer) Grade ( ⁇ 1000 ⁇ m), such as: the area is rectangular, the width is micron; the area is circular, the diameter is micron, etc.).
  • the region formed by the nanocone structure polypyrrole forms a micro-area potential difference with the region formed by the nanoneedle titania.
  • the region formed by the nano-cone structure polypyrrole is of any shape such as a circle, an ellipse, a rectangle, a loop shape or the like.
  • the region formed by the nanoneedle titanium dioxide and the region formed by the nanopyram structure polypyrrole are a plurality of independent regions, the region formed by the nanoneedle titania and the region formed by the nanopyram structure polypyrrole are arranged or periodically arranged. .
  • the region formed by the nano-needle titania is a whole region, and the region formed by the nano-cone structure polypyrrole is a plurality of independent regions, the region formed by the nano-cone structure polypyrrole is periodically arranged, and the region formed by each nano-cone structure polypyrrole The circumference is surrounded by nano-needle titanium dioxide.
  • the nano-cone structure polypyrrole is disposed on the chlorine-doped polypyrrole, that is, a chlorine-doped polypyrrole is deposited between the polypyrrole of the nano-cone structure and the titanium electrode.
  • the preparation method of the glucose sensor electrode comprises the following steps:
  • the oxide film on the surface of the titanium electrode is removed to obtain a pretreated titanium electrode; the pretreated titanium electrode is placed in a hydrothermal reaction vessel containing a NaOH solution to perform an alkali thermal reaction, and a nanoneedle structure titanium dioxide film layer is formed on the surface of the titanium electrode;
  • the depth of the groove is the same as the thickness of the nano-needle structure titanium dioxide film layer; specifically, forming a nanometer on the surface of the titanium electrode
  • the titanium dioxide film layer of the needle structure is laser etched, and part of the nano-needle structure titanium dioxide is etched to form a nano-needle structure titanium dioxide film layer having a micro pattern;
  • the three-electrode mode is selected, the conductive metal is the counter electrode, the titanium electrode of the nano-needle structure titanium dioxide film layer having the micro-pattern in the step (2) is the working electrode, and the electrolyte solution is an aqueous solution containing pyrrole and chloride ions, and the electric potential is controlled by the chronopotentiometry. a chemical reaction, chlorine-doped polypyrrole is deposited in a groove pattern of a nano-needle structure titanium dioxide film layer having a micropattern;
  • the conductive metal is the counter electrode
  • the titanium electrode in which the chlorine-doped polypyrrole is deposited in the step (3) is the working electrode
  • the electrolyte is a buffer solution containing pyrrole and citric acid
  • the electrochemical reaction is controlled by the chronopotentiometry.
  • a chlorine-doped polypyrrole forms a nano-cone structure
  • a citric acid-doped polypyrrole a nano-cone structure
  • a citric acid-doped polypyrrole is deposited in a groove pattern
  • a nano-needle structure titanium dioxide and a nano-cone structure polypyrrole are obtained.
  • the enzyme capable of detecting glucose is grafted onto the titanium electrode prepared in the step (4) to obtain a glucose sensor electrode.
  • the temperature of the alkali thermal reaction in the step (1) is 70 to 110 ° C; and the concentration of the sodium hydroxide solution in the step (1) is 1 to 4 mol/L, preferably 2 mol/L.
  • the alkali heat treatment in the step (1) is carried out for 12 to 36 hours, preferably 24 hours.
  • the removal of the oxide film on the surface of the titanium electrode in the step (1) means that the titanium electrode is subjected to pickling, and the pickling solution is a mixed solution of hydrofluoric acid and nitric acid.
  • the pickling time is 5 to 10 min, preferably 7 min.
  • the drying temperature in the step (1) is ⁇ 100 °C.
  • the laser light of the laser etching in the step (2) is 1 to 5 W; and the laser scanning speed is 50 to 300 mm/s.
  • the grooves are one or more, preferably a plurality. When there are a plurality of grooves, the groove array is distributed. In summary, the grooves form a plurality of domains.
  • the electrochemical reaction current in the step (3) is 10 to 30 mA/cm 2 ; the electrochemical reaction time is 10 s to 40 s;
  • the concentration of the chloride ion in the step (3) is 0.1 to 0.4 mol/L, and the concentration of the pyrrole is 0.1 to 0.3 mol/L.
  • the electrochemical reaction current in the step (4) is 0.5 to 2.0 mA/cm 2 ; the electrochemical reaction time is 10 to 50 s, preferably 20 s; the conductive metal is platinum, copper, etc.; Phosphate buffer solution;
  • the pH of the buffer solution in the step (4) is 6.8 to 7.2.
  • the concentration of the pyrrole in the step (4) is 0.1 to 0.3 mol/L, and the concentration of the citric acid is 0.05 to 0.3 mol/L.
  • the enzyme capable of detecting glucose in the step (5) is glucose oxidase; the specific step of the step (5) is: immersing the titanium electrode prepared in the step (4) in a PBS solution containing bovine serum albumin and glucose oxidase (phosphoric acid) The reaction was carried out in a salt buffer solution to obtain a glucose sensor electrode.
  • the bovine serum albumin (bovine serum egg-modified glucose oxidase, which improves the thermostability of the enzyme and the resistance to proteases) and the glucose oxidase concentration in the step (5) are 30 to 80 g/L and 10 to 50 g, respectively. /mL.
  • the temperature of the reaction in the step (5) is 4 to 8 ° C; the reaction time is 20 to 50 min, preferably 30 min.
  • the glucose sensor electrode is used for glucose concentration detection.
  • the laser etching in the step (2) means etching the titanium dioxide film layer on the surface of the titanium electrode using an infrared fiber laser printer at room temperature.
  • the laser path is set by software, the power of the laser and the scanning speed are set, and the nano-needle titanium dioxide film layer is patterned to prepare a micro-area array structure.
  • the present invention has the following advantages and beneficial effects:
  • the process of the invention is simple, stable and low in cost, and the coating (nano-needle titanium dioxide film layer and nano-cone structure polypyrrole) is grown in situ on the surface of the conductive substrate, and has strong bonding force and low interface resistance. ;
  • the electrode of the invention consists of nano-needle titania and nano-cone structure polypyrrole, the nano-cone structure polypyrrole forms a micro-region, the nano-needle titania and the nano-cone structure polypyrrole form a potential difference, and the micro-area electric field promotes protein adhesion, Glucose oxidase can be efficiently grafted on the surface of the electrode;
  • the electrode material of the present invention has high reliability in detecting glucose.
  • Example 1 is a schematic view showing the preparation of a glucose sensor electrode (not grafted enzyme) in Example 1;
  • Example 2 is a scanning electron image of a glucose sensor electrode (non-grafted enzyme) prepared in Example 1; a is nanoneedle titania, b is a glucose sensor electrode, and c is a nanocone polypyrrole (citric acid doped);
  • Example 3 is an elemental distribution diagram of a glucose sensor electrode (non-grafted enzyme) prepared in Example 1; left: scanning electron micrograph, medium: surface distribution map of Ti element, right: surface distribution map of C element;
  • Example 4 is a graph showing the relationship between the current response detected by the glucose sensor electrode and the concentration of glucose in Example 5.
  • Examples 1 to 4 are preparations of glucose sensor electrodes without grafting glucose oxidase; and Example 5 is preparation of glucose sensor electrodes grafted with glucose oxidase.
  • the glucose sensor electrodes of Examples 1 to 4 include a titanium electrode, the surface of the titanium electrode is composed of a region formed by nanoneedle titania and a region formed by a nanopyram structure polypyrrole, and the region formed by the nanoneedle titania and the polypyrrole formed by the nanocone structure
  • the regions are all a plurality of independent regions, each of which is rectangular, and the region formed by the nano-titanium dioxide is arranged with the region formed by the nano-cone structure polypyrrole (ie, alternately arranged on the surface of the titanium electrode).
  • Titanium surface alkali heat treatment titanium sheet (0.5 mm ⁇ 10 mm ⁇ 10 mm) was ultrasonically cleaned in acetone, ethanol and deionized water for 10 min; then hydrofluoric acid (concentration of 0.54 mol/L in mixed acid) and nitric acid ( The concentration of 0.25mol/L in the mixed acid was used to clean the oxide layer on the surface of the titanium sheet. The cleaning time was 7 min.
  • the acid-treated material was ultrasonically cleaned in deionized water for 10 min, and dried for use to obtain a pretreated titanium sheet;
  • the pretreated titanium sheet was immersed in a hydrothermal reaction vessel containing 70 mL of a 2 mol/L NaOH solution, and reacted at a constant temperature of 100 ° C for 24 hours. After the reaction was completed, it was taken out, rinsed with deionized water, and dried in a vacuum drying oven. Drying in use, forming a titanium dioxide layer of nanoneedle structure on the surface of the titanium sheet;
  • the titanium dioxide layer of the nanoneedle structure is laser-etched away to form a plurality of grooves or grooves having the same depth as the thickness of the titanium dioxide layer of the nanoneedle structure; the titanium dioxide layer of the nanoneedle structure is divided into grooves by the groove a parallel rectangular area (60 ⁇ m in width), the cross section of the groove is also rectangular (width 50 ⁇ m); the groove is alternately arranged with the titanium dioxide layer of the etched nanoneedle structure; the nanoneedle of the micropattern is obtained Structure of titanium dioxide layer;
  • the three-electrode mode is adopted, the titanium plate of the titanium dioxide layer with the nano-needle structure on the surface is the working electrode, the copper piece is the counter electrode, the saturated calomel electrode is the reference electrode, and the concentration of the pyrrole in the electrolyte solution is 0.2 mol/L.
  • the concentration of hydrochloric acid was 0.25 mol/L, and the electrochemical reaction was controlled by chronopotentiometry.
  • the reaction current was 20 mA/cm 2 and the reaction time was 20 seconds.
  • a layer of chlorine-doped poly was deposited in the groove pattern (by laser etching). Pyrrole, after the reaction, immersed in deionized water to remove pyrrole and hydrochloric acid which are not reacted on the surface, to obtain a titanium electrode deposited with chlorine-doped polypyrrole;
  • the titanium electrode deposited with chlorine-doped polypyrrole is the working electrode
  • the copper plate is the counter electrode
  • the saturated calomel electrode is the reference electrode
  • the electrolyte solution is a buffer solution of pyrrole and citric acid (the pH of the solution is 6.8, PBS)
  • the concentration of pyrrole in the electrolyte solution was 0.2 mol/L
  • the concentration of citric acid was 0.1 mol/L.
  • the electrochemical reaction was controlled by chronopotentiometry, the reaction current was 1.5 mA/cm 2 , and the reaction time was 20 seconds.
  • a polypyrrole/citric acid complex having a nano-cone structure deposited on the chlorine-doped polypyrrole (a nano-cone structure citrate-doped polypyrrole is deposited in a laser-etched groove) to obtain a nano-cone structure
  • a working electrode (glucose sensor electrode of ungrafted glucose oxidase) in which the polypyrrole/citric acid and the nanoneedle structure of the titanium dioxide microdomain are arranged.
  • FIG. 2 A schematic diagram of the preparation of a glucose sensor electrode for preparing an ungrafted glucose oxidase in this example is shown in FIG. 2 is an SEM image of the glucose sensor electrode of the non-grafted glucose oxidase prepared in Example 1.
  • the nanopyram structure of the polypyrrole/citric acid and the nano-needle structure of the titanium dioxide microdomains are arranged in the electrode.
  • the titanium dioxide nano-needle formed by the alkali heat treatment grows perpendicular to the surface of the titanium.
  • the non-conductive titanium dioxide film layer is partially etched away to expose the conductive titanium substrate, which provides a basis for further polypyrrole polymerization.
  • the polypyrrole nanocone grows perpendicular to the surface of the conductive titanium substrate by templateless electrochemical polymerization.
  • 3 is an elemental distribution diagram of a glucose sensor electrode of ungrafted glucose oxidase prepared in Example 1; left: scanning electron micrograph, medium: surface distribution map of Ti element, right: surface distribution map of C element, semiconductor titanium dioxide
  • the structure and the conductive polypyrrole structure are arranged in phase. As shown in Fig.
  • the distribution of the C element and the Ti element on the electrode surface showed that the conductive polypyrrole and the titanium dioxide were interphase-distributed, the width of the titanium oxide portion was 60 ⁇ m, and the width of the polypyrrole portion was 50 ⁇ m.
  • the film layer on the surface of the material is deposited in an in-situ manner, and the bonding force is good.
  • Titanium surface alkali heat treatment titanium sheet (0.5 mm ⁇ 10 mm ⁇ 10 mm) was ultrasonically cleaned in acetone, ethanol and deionized water for 10 min; then hydrofluoric acid (concentration of 0.54 mol/L in mixed acid) and nitric acid ( The concentration of 0.25mol/L in the mixed acid was used to clean the oxide layer on the surface of the titanium sheet. The cleaning time was 7 min.
  • the acid-treated material was ultrasonically cleaned in deionized water for 10 min, and dried for use to obtain a pretreated titanium sheet;
  • the pretreated titanium sheet was immersed in a hydrothermal reaction vessel containing 70 mL of a 2 mol/L NaOH solution, and reacted at a constant temperature of 100 ° C for 24 hours. After the reaction was completed, it was taken out, rinsed with deionized water, and dried in a vacuum drying oven. Drying in use, forming a titanium dioxide layer of nanoneedle structure on the surface of the titanium sheet;
  • the three-electrode mode is adopted, the titanium plate of the titanium dioxide layer with the nano-needle structure on the surface is the working electrode, the copper piece is the counter electrode, the saturated calomel electrode is the reference electrode, and the concentration of the pyrrole in the electrolyte solution is 0.2 mol/L.
  • the concentration of hydrochloric acid was 0.25 mol/L, and the electrochemical reaction was controlled by chronopotentiometry.
  • the reaction current was 20 mA/cm 2 and the reaction time was 20 seconds.
  • a layer of chlorine-doped poly was deposited in the groove pattern (by laser etching). Pyrrole, after the reaction, immersed in deionized water to remove pyrrole and hydrochloric acid which are not reacted on the surface, to obtain a titanium electrode deposited with chlorine-doped polypyrrole;
  • the titanium electrode deposited with chlorine-doped polypyrrole is the working electrode
  • the copper plate is the counter electrode
  • the saturated calomel electrode is the reference electrode
  • the electrolyte solution is a buffer solution of pyrrole and citric acid (the pH of the solution is 6.8, PBS)
  • the concentration of pyrrole in the electrolyte solution was 0.2 mol/L
  • the concentration of citric acid was 0.1 mol/L.
  • the electrochemical reaction was controlled by chronopotentiometry, the reaction current was 1.5 mA/cm 2 , and the reaction time was 20 seconds.
  • a polypyrrole/citric acid complex having a nano-cone structure deposited on the chlorine-doped polypyrrole (a nano-cone structure citrate-doped polypyrrole is deposited in a laser-etched groove) to obtain a nano-cone structure
  • a working electrode (glucose sensor electrode of ungrafted glucose oxidase) in which the polypyrrole/citric acid and the nanoneedle structure of the titanium dioxide microdomain are arranged.
  • the composite material structure prepared in this example was similar to that of Example 1, and the detection performance was similar to that of Example 1.
  • Titanium surface alkali heat treatment Titanium sheets (0.5 mm ⁇ 10 mm ⁇ 10 mm) were ultrasonically cleaned in acetone, ethanol and deionized water for 10 min. Then, the oxide layer on the surface of the titanium sheet was washed with hydrofluoric acid (0.54 mol/L) and nitric acid (0.25 mol/L) mixed acid for 7 min; the acid-treated material was ultrasonically cleaned in deionized water for 10 min, and dried for use.
  • the pretreated titanium piece is obtained; the pretreated titanium piece is immersed in a hydrothermal reaction kettle containing 70 mL of a 2 mol/L NaOH solution, and reacted at a constant temperature of 100 ° C for 24 hours; after the reaction is completed, it is taken out and used. Immersion washing in ionized water, drying in a vacuum drying oven for use, forming a titanium dioxide layer of nanoneedle structure on the surface of the titanium sheet;
  • the three-electrode mode is adopted, the titanium plate of the titanium dioxide layer with the nano-needle structure on the surface is the working electrode, the copper piece is the counter electrode, the saturated calomel electrode is the reference electrode, and the concentration of the pyrrole in the electrolyte solution is 0.2 mol/L.
  • the concentration of hydrochloric acid was 0.25 mol/L, and the electrochemical reaction was controlled by chronopotentiometry.
  • the reaction current was 30 mA/cm 2 and the reaction time was 20 seconds.
  • a layer of chlorine-doped poly was deposited in the groove pattern (by laser etching). Pyrrole, after the reaction, immersed in deionized water to remove pyrrole and hydrochloric acid which are not reacted on the surface, to obtain a titanium electrode deposited with chlorine-doped polypyrrole;
  • the titanium electrode deposited with chlorine-doped polypyrrole is the working electrode
  • the copper plate is the counter electrode
  • the saturated calomel electrode is the reference electrode
  • the electrolyte solution is a buffer solution of pyrrole and citric acid (the pH of the solution is 6.8, PBS)
  • the concentration of pyrrole in the electrolyte solution was 0.2 mol/L
  • the concentration of citric acid was 0.1 mol/L.
  • the electrochemical reaction was controlled by chronopotentiometry, the reaction current was 1.5 mA/cm 2 , and the reaction time was 20 seconds.
  • a polypyrrole/citric acid complex having a nano-cone structure deposited on the chlorine-doped polypyrrole (a nano-cone structure citrate-doped polypyrrole is deposited in a laser-etched groove) to obtain a nano-cone structure
  • a working electrode (glucose sensor electrode of ungrafted glucose oxidase) in which the polypyrrole/citric acid and the nanoneedle structure of the titanium dioxide microdomain are arranged.
  • the composite material structure prepared in this example was similar to that of Example 1, and the detection performance was similar to that of Example 1.
  • Titanium surface alkali heat treatment titanium sheet (0.5mm ⁇ 10mm ⁇ 10mm) was ultrasonically cleaned in acetone, ethanol and deionized water for 10min; then hydrofluoric acid (0.54mol / L) and nitric acid (0.25mol / L) The acid layer on the surface of the titanium sheet was washed with mixed acid for 7 min; the acid-treated material was ultrasonically cleaned in deionized water for 10 min, dried and used to obtain a pretreated titanium sheet; the pretreated titanium sheet was immersed in a concentration of 70 mL. The reaction was carried out in a hydrothermal reaction vessel of 2 mol/L NaOH solution at a constant temperature of 100 ° C for 24 h. After the reaction was completed, the sample was taken out and rinsed with deionized water, dried in a vacuum drying oven, and the surface of the titanium sheet was nanometer-formed. a titanium dioxide layer of a needle structure;
  • the three-electrode mode is adopted, the titanium plate of the titanium dioxide layer with the nano-needle structure on the surface is the working electrode, the copper piece is the counter electrode, the saturated calomel electrode is the reference electrode, and the concentration of the pyrrole in the electrolyte solution is 0.2 mol/L.
  • the concentration of hydrochloric acid was 0.25 mol/L, and the electrochemical reaction was controlled by chronopotentiometry.
  • the reaction current was 20 mA/cm 2 and the reaction time was 20 seconds.
  • a layer of chlorine-doped poly was deposited in the groove pattern (by laser etching). Pyrrole, after the reaction, immersed in deionized water to remove pyrrole and hydrochloric acid which are not reacted on the surface, to obtain a titanium electrode deposited with chlorine-doped polypyrrole;
  • the titanium electrode deposited with chlorine-doped polypyrrole is the working electrode
  • the copper plate is the counter electrode
  • the saturated calomel electrode is the reference electrode
  • the electrolyte solution is a buffer solution of pyrrole and citric acid (the pH of the solution is 6.8, PBS)
  • the concentration of pyrrole in the electrolyte solution is 0.2 mol/L
  • the concentration of citric acid is 0.2 mol/L.
  • the electrochemical reaction is controlled by chronopotentiometry, the reaction current is 0.9 mA/cm 2 , and the reaction time is 20 seconds.
  • a polypyrrole/citric acid complex having a nano-cone structure deposited on the chlorine-doped polypyrrole (a nano-cone structure citrate-doped polypyrrole is deposited in a laser-etched groove) to obtain a nano-cone structure
  • a working electrode (glucose sensor electrode of ungrafted glucose oxidase) in which the polypyrrole/citric acid and the nanoneedle structure of the titanium dioxide microdomain are arranged.
  • the composite material structure prepared in this example was similar to that of Example 1, and the detection performance was similar to that of Example 1.
  • the glucose sensor electrode (grafted glucose oxidase) prepared in Example 1 was immersed in PBS containing bovine serum albumin (50 mg/mL) and glucose oxidase (25 mg/mL), and reacted at 4 ° C for 30 min. Immerse in PBS and store in a refrigerator at 4 °C.
  • the glucose detection test was performed using an electrochemical workstation. Three-electrode mode is adopted.
  • the micro-pattern glucose sensor electrode (grafted glucose oxidase) is used as the working electrode, the copper plate is the counter electrode, the saturated calomel electrode is the reference electrode, and the PBS solution containing different concentrations of glucose is used as the electrolyte solution.
  • the concentration of glucose ranges from 0.1 to 4.0 mmol/L.
  • the electrodes prepared in Examples 2 to 4 were grafted with glucose oxidase, and the detection of glucose also had high reliability.

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Abstract

本发明属于医疗器械的技术领域，公开了一种葡萄糖传感器电极及其制备方法与应用。方法为(1)碱热处理，钛电极表面形成纳米针结构二氧化钛；(2)在纳米针结构二氧化钛膜层上形成微区凹槽，获得具有微图案的纳米针结构二氧化钛；(3)采用计时电位法，氯掺杂的聚吡咯沉积在凹槽中；(4)采用计时电位法，氯掺杂的聚吡咯上形成纳米锥结构柠檬酸掺杂的聚吡咯；(5)接枝能够检测葡萄糖的酶，得到葡萄糖传感器电极。本发明的葡萄糖传感器电极用于快速高灵敏地检测葡萄糖。本发明工艺简单、稳定，成本低，在电极表面高效地接枝能够检测葡萄糖的酶，对葡萄糖的检测具有较高的可靠性；电极的稳定好。

Description

一种葡萄糖传感器电极及其制备方法与应用 技术领域
本发明属于医疗器械的技术领域，具体涉及一种葡萄糖传感器电极及其制备方法与应用。所述葡萄糖传感器电极用于快速高灵敏地检测葡萄糖。
背景技术
糖尿病日益受到关注，血液中的葡萄糖检测已成为一种潜在的诊断和控制糖尿病的趋势，用于临床诊断和个人护理的精确的灵敏的血糖检测装置需求量成指数增长。检测血糖最广泛的方法是电化学方法。电化学方法相对于声学、光学和化学发光方法，具有灵敏度高、成本低、操作容易、响应时间更快以及便携式小型化等优点。电化学葡萄糖传感器的核心技术是电极设计与加工。由于纳米材料的尺寸效应及其高的反应活性等优异的性质，各种类型的纳米材料例如金属氧化物、碳质材料(如：碳纳米管和石墨烯)等已经广泛应用于葡萄糖传感器。虽然曾经的材料开发集中于增强灵敏度和检测极限，但是葡萄糖传感器电极材料还存在很多缺点，比如电极的制备工艺欠稳定、制备成本较高、涂层结合力较差等缺点。
本发明构建了一种半导体纳米针二氧化钛和纳米锥结构导电聚吡咯的葡萄糖传感器电极，并且电极中纳米针二氧化钛与纳米锥聚吡咯之间还形成微区电场。本发明的葡萄糖传感器电极结构稳定，纳米针二氧化钛和纳米锥聚吡咯与导电基材结合力强、对酶的吸附性好而且对葡萄糖的氧化反应具有非常好的促进作用。本发明的电极能够高效地检测葡萄糖。
发明内容
为了克服现有技术的缺点和不足，本发明的目的在于提供一种葡萄糖传感器电极及其制备方法。本发明通过碱热处理、激光刻蚀和电化学方法在钛电极表面构建了一层具有高效应的葡萄糖传感器涂层，该涂层具有导电纳米锥结构聚吡咯和半导体纳米针结构二氧化钛的微区特征。本发明的电极中纳米针二氧化钛具有较好的催化特性，同时纳米针二氧化钛与纳米锥聚吡咯之间还形成微 区电场，能够进一步促进蛋白质的粘附，在电极表面能够高效地接枝蛋白酶(葡萄糖氧化酶)。本发明电极表面的涂层(纳米针二氧化钛和纳米锥聚吡咯)具有高的蛋白酶粘附特性和纳米锥结构的高检测灵敏性，实现了高灵敏度的葡萄糖检测。
本发明的另一目的在于提供上述葡萄糖传感器电极的应用。所述葡萄糖传感器电极用于高效地检测葡萄糖。
本发明的目的通过以下技术方案实现：
一种葡萄糖传感器电极，包括钛电极，钛电极的表面由纳米针二氧化钛形成的区域与由纳米锥结构聚吡咯形成的区域组成，每一个由纳米锥结构聚吡咯形成的区域与由纳米针二氧化钛形成的区域相邻，每一由纳米锥结构聚吡咯形成的区域与区域之间为由纳米针二氧化钛形成的区域；纳米锥结构聚吡咯形成的区域接枝有能够检测葡萄糖的酶。所述能够检测葡萄糖的酶为葡萄糖氧化酶；由纳米针二氧化钛形成的区域和由纳米锥结构聚吡咯形成的区域其中至少有一种区域为微区。所述微区是指区域的宽度(包括所有宽度)、直径和/或面积为微米级。
所述纳米锥结构聚吡咯为掺杂或未掺杂的纳米锥结构聚吡咯，优选为掺杂的纳米锥结构聚吡咯，所述掺杂的纳米锥结构聚吡咯为柠檬酸掺杂的纳米锥结构聚吡咯。
所述由纳米针二氧化钛形成的区域和由纳米锥结构聚吡咯形成的区域其中至少有一种区域为多个独立区域；每一个独立区域为微区(即区域的宽度、直径和/或面积为微米级(≤1000μm)，如：区域为长方形，则宽为微米级；区域为圆形，则直径为微米级等等)。
所述由纳米锥结构聚吡咯形成的区域与由纳米针二氧化钛形成的区域形成微区电势差。
所述由纳米锥结构聚吡咯形成的区域为任意形状，如：圆形，椭圆形，长方形，回路形等。当由纳米针二氧化钛形成的区域和由纳米锥结构聚吡咯形成的区域都为多个独立区域时，由纳米针二氧化钛形成的区域和由纳米锥结构聚吡咯形成的区域相间排列或周期性相间排列。当由纳米针二氧化钛形成的区域为整体区域，由纳米锥结构聚吡咯形成的区域为多个独立区域时，纳米锥结构 聚吡咯形成的区域周期性排列，每一纳米锥结构聚吡咯形成的区域的四周为由纳米针二氧化钛形成的区域。
所述纳米锥结构聚吡咯设置在氯掺杂的聚吡咯上，即纳米锥结构的聚吡咯与钛电极之间沉积有氯掺杂的聚吡咯。
所述葡萄糖传感器电极的制备方法，包括以下步骤：
(1)钛表面碱热处理
去除钛电极表面的氧化膜，得到预处理的钛电极；将预处理的钛电极置于装有NaOH溶液的水热反应釜中进行碱热反应，钛电极表面形成纳米针结构二氧化钛膜层；
(2)微图案的纳米针二氧化钛膜层构建
在纳米针结构二氧化钛膜层上形成凹槽，获得具有微图案的纳米针结构二氧化钛膜层；所述凹槽的深度与纳米针结构二氧化钛膜层的厚度相同；具体是指对钛电极表面形成纳米针结构二氧化钛膜层进行激光刻蚀，刻蚀掉部分纳米针结构二氧化钛，形成具有微图案的纳米针结构二氧化钛膜层；
(3)电沉积氯掺杂的聚吡咯
选用三电极模式，导电金属为对电极，步骤(2)中具有微图案的纳米针结构二氧化钛膜层的钛电极为工作电极，电解质溶液为包含吡咯和氯离子的水溶液，采用计时电位法控制电化学反应，氯掺杂的聚吡咯沉积在具有微图案的纳米针结构二氧化钛膜层的凹槽图案中；
(4)纳米锥结构聚吡咯构建
选用三电极模式，导电金属为对电极，步骤(3)中沉积有氯掺杂的聚吡咯的钛电极为工作电极，电解质为包含吡咯和柠檬酸的缓冲溶液，采用计时电位法控制电化学反应，氯掺杂的聚吡咯上形成纳米锥结构柠檬酸掺杂的聚吡咯即纳米锥结构柠檬酸掺杂的聚吡咯沉积在凹槽图案中，获得具有纳米针结构二氧化钛和纳米锥结构聚吡咯的钛电极；
(5)接枝能够检测葡萄糖的酶：
将步骤(4)制备的钛电极上接枝能够检测葡萄糖的酶，得到得到葡萄糖传感器电极。
步骤(1)中所述碱热反应的温度为70～110℃；步骤(1)所述氢氧化钠溶 液浓度为1～4mol/L，优选2mol/L。
步骤(1)所述碱热处理的时间为12～36h，优选24h。
步骤(1)中去除钛电极表面的氧化膜是指将钛电极进行酸洗，所述酸洗溶液为氢氟酸和硝酸混合溶液。
所述酸洗时间为5～10min，优选7min。
步骤(1)中所述干燥的温度≤100℃。
步骤(2)中所述激光刻蚀的激光功率1～5W；激光扫描速度为50～300mm/s。所述凹槽为1个或多个，优选为多个。凹槽为多个时，凹槽阵列分布。总之，凹槽形成多个微区。
步骤(3)中所述电化学反应的电流为10～30mA/cm 2；电化学反应的时间为10s～40s；
步骤(3)中所述氯离子的浓度为0.1～0.4mol/L，吡咯的浓度为0.1～0.3mol/L。
步骤(4)中所述电化学反应的电流为0.5～2.0mA/cm 2；电化学反应的时间为10～50s，优选为20s；所述导电金属为铂、铜等；所述缓冲溶液为磷酸盐缓冲溶液；
步骤(4)中所述缓冲溶液的pH为6.8～7.2。
步骤(4)中所述吡咯的浓度为0.1～0.3mol/L，柠檬酸的浓度为0.05～0.3mol/L。
步骤(5)中所述能够检测葡萄糖的酶为葡萄糖氧化酶；步骤(5)的具体步骤为：将步骤(4)制备的钛电极浸泡于含有牛血清蛋白和葡萄糖氧化酶的PBS溶液(磷酸盐缓冲溶液)中进行反应，得到葡萄糖传感器电极。
步骤(5)中所述牛血清蛋白(牛血清蛋修饰葡萄糖氧化酶，提高酶的热稳定性和对蛋白酶等的抗性)和葡萄糖氧化酶的浓度分别为30～80g/L和10～50g/mL。
步骤(5)中所述反应的温度为4～8℃；所述反应的时间为20～50min，优选30min。
所述葡萄糖传感器电极用于葡萄糖浓度检测。
步骤(2)中所述激光刻蚀是指在室温条件下，使用红外光纤激光打印机 对钛电极表面的二氧化钛膜层进行刻蚀。通过软件设置激光路径，设置激光的功率和扫描速度，将纳米针二氧化钛膜层进行图案化加工，制备微区阵列结构。
与现有技术相比，本发明具有以下优点及有益效果：
(1)本发明工艺简单、稳定，成本低，并且涂层(纳米针二氧化钛膜层和纳米锥结构聚吡咯)原位生长于导电基材表面，具有较强的结合力、较低的界面电阻；
(2)本发明的电极由纳米针二氧化钛和纳米锥结构聚吡咯组成，纳米锥结构聚吡咯形成微区，纳米针二氧化钛和纳米锥结构聚吡咯间形成电势差，微区电场促进蛋白质的粘附，在电极表面能够高效地接枝葡萄糖氧化酶；
(3)本发明的电极材料对葡萄糖的检测具有较高的可靠性。
附图说明
图1为实施例1中葡萄糖传感器电极(未接枝酶)的制备示意图；
图2为实施例1制备的葡萄糖传感器电极(未接枝酶)的扫面电子图像；a为纳米针二氧化钛，b为葡萄糖传感器电极，c为纳米锥聚吡咯(柠檬酸掺杂)；
图3为实施例1制备的葡萄糖传感器电极(未接枝酶)的元素分布图；左：扫描电子显微图，中：Ti元素表面分布图，右：C元素表面分布图；
图4为实施例5中葡萄糖传感器电极检测的电流响应与葡萄糖的浓度关系曲线。
具体实施方式
下面结合实施例和附图对本发明作进一步详细的描述，但本发明的实施方式不限于此。实施例1～4为未接枝葡萄糖氧化酶的葡萄糖传感器电极的制备；实施例5为接枝葡萄糖氧化酶的葡萄糖传感器电极的制备。
实施例1～4中葡萄糖传感器电极包括钛电极，钛电极的表面由纳米针二氧化钛形成的区域与由纳米锥结构聚吡咯形成的区域组成，纳米针二氧化钛形成的区域与由纳米锥结构聚吡咯形成的区域都为多个独立的区域，每一独立的区域为长方形，纳米针二氧化钛形成的区域与由纳米锥结构聚吡咯形成的区域相间排列(即交替排布于钛电极的表面)。
实施例1
(1)钛表面碱热处理：钛片(0.5mm×10mm×10mm)依次在丙酮、乙醇和去离子水中超声清洗10min；然后采用氢氟酸(混合酸中的浓度0.54mol/L)和硝酸(混合酸中的浓度0.25mol/L)混合酸清洗钛片表面的氧化层，清洗时间为7min，酸处理后的材料在去离子水中超声清洗10min，干燥待用，得到预处理的钛片；将预处理的钛片浸入装有70mL浓度为2mol/L的NaOH溶液的水热反应釜中，在100℃恒温条件下反应24h，反应结束后，取出，用去离子水浸泡冲洗，在真空干燥箱中干燥待用，钛片表面形成纳米针结构的二氧化钛层；
(2)在室温条件下，使用红外光纤激光打印机对表面形成纳米针结构的二氧化钛层的钛片进行刻蚀，软件设置平行光路，设置激光器的功率为2.8W，扫描速度为100mm/s；部分纳米针结构的二氧化钛层被激光刻蚀掉，形成多个凹槽或沟槽，凹槽的深度与纳米针结构的二氧化钛层的厚度相同；所述纳米针结构的二氧化钛层被凹槽分割成多条平行长方形区域(宽为60μm)，所述凹槽的横截面也为长方形(宽为50μm)；凹槽与被刻蚀后的纳米针结构的二氧化钛层交替排布；获得微图案的纳米针结构的二氧化钛层；
(3)通过电化学方法在凹槽中电沉积氯掺杂的聚吡咯：
选用三电极模式，表面有微图案的纳米针结构的二氧化钛层的钛片为工作电极，铜片为对电极，饱和甘汞电极为参比电极，电解质溶液中吡咯的浓度为0.2mol/L，盐酸的浓度为0.25mol/L，采用计时电位法控制电化学反应，反应电流20mA/cm 2，反应时间为20秒，凹槽图案中(被激光刻蚀部分)沉积一层氯掺杂的聚吡咯，反应完后，浸泡在去离子水中以除去表面没有反应的吡咯和盐酸，得到沉积有氯掺杂聚吡咯的钛电极；
(4)纳米锥结构的聚吡咯构建：
选用三电极模式，沉积有氯掺杂的聚吡咯的钛电极为工作电极，铜片为对电极，饱和甘汞电极为参比电极，电解质溶液为吡咯和柠檬酸的缓冲溶液(溶液的pH为6.8，PBS)，电解质溶液中吡咯的浓度为0.2mol/L和柠檬酸的浓度为0.1mol/L，采用计时电位法控制电化学反应，反应电流为1.5mA/cm 2，反应时间为20秒，氯掺杂聚吡咯上沉积有纳米锥结构的聚吡咯/柠檬酸复合物(纳米锥结构柠檬酸掺杂的聚吡咯沉积在被激光刻蚀出的凹槽中)，得到即纳米锥 结构的聚吡咯/柠檬酸和纳米针结构的二氧化钛微区相间排列的的工作电极(未接枝葡糖氧化酶的葡萄糖传感器电极)。
本实施例制备未接枝葡糖氧化酶的葡萄糖传感器电极的制备示意图如图1所示。图2为实施例1制备的未接枝葡糖氧化酶的葡萄糖传感器电极的SEM图；如图2所示，电极中纳米锥结构的聚吡咯/柠檬酸和纳米针结构的二氧化钛微区相间排列。碱热处理形成的二氧化钛纳米针垂直于钛表面生长，激光刻蚀之后，不导电的二氧化钛膜层部分被刻蚀掉，露出导电的钛基材，为进一步聚吡咯聚合提供基础。通过无模板电化学聚合，聚吡咯纳米锥(柠檬酸掺杂)垂直于导电钛基材表面生长。图3为实施例1制备的未接枝葡糖氧化酶的葡萄糖传感器电极的元素分布图；左：扫描电子显微图，中：Ti元素表面分布图，右：C元素表面分布图，半导体二氧化钛结构和导电聚吡咯结构相间排列。如图3所示，电极表面(未接枝葡糖糖氧化酶)C元素和Ti元素的分布情况，结果说明导电聚吡咯和二氧化钛相间分布，二氧化钛部分宽度是60μm，聚吡咯部分宽度为50μm。
本实施例制备的电极中，材料表面的膜层是原位生长的方式沉积，结合力较好。
实施例2
(1)钛表面碱热处理：钛片(0.5mm×10mm×10mm)依次在丙酮、乙醇和去离子水中超声清洗10min；然后采用氢氟酸(混合酸中的浓度0.54mol/L)和硝酸(混合酸中的浓度0.25mol/L)混合酸清洗钛片表面的氧化层，清洗时间为7min，酸处理后的材料在去离子水中超声清洗10min，干燥待用，得到预处理的钛片；将预处理的钛片浸入装有70mL浓度为2mol/L的NaOH溶液的水热反应釜中，在100℃恒温条件下反应24h，反应结束后，取出，用去离子水浸泡冲洗，在真空干燥箱中干燥待用，钛片表面形成纳米针结构的二氧化钛层；
(2)在室温条件下，使用红外光纤激光打印机对表面形成纳米针结构的二氧化钛层的钛片进行刻蚀，软件设置平行光路(光路与光路之间的间隔是100μm，光斑的宽度是仪器固定的30μm)，设置激光器的功率为2.8W，扫描速度为300mm/s；部分纳米针结构的二氧化钛层被激光刻蚀掉，形成多个凹槽 或沟槽，凹槽的深度与纳米针结构的二氧化钛层的厚度相同；所述纳米针结构的二氧化钛层被凹槽分割成多条平行长方形区域，所述凹槽的横截面也为长方形；凹槽与被刻蚀后的纳米针结构的二氧化钛层交替排布；获得微图案的纳米针结构的二氧化钛层；
(3)通过电化学方法在凹槽中电沉积氯掺杂的聚吡咯：
选用三电极模式，表面有微图案的纳米针结构的二氧化钛层的钛片为工作电极，铜片为对电极，饱和甘汞电极为参比电极，电解质溶液中吡咯的浓度为0.2mol/L，盐酸的浓度为0.25mol/L，采用计时电位法控制电化学反应，反应电流20mA/cm 2，反应时间为20秒，凹槽图案中(被激光刻蚀部分)沉积一层氯掺杂的聚吡咯，反应完后，浸泡在去离子水中以除去表面没有反应的吡咯和盐酸，得到沉积有氯掺杂聚吡咯的钛电极；
(4)纳米锥结构的聚吡咯构建：
选用三电极模式，沉积有氯掺杂的聚吡咯的钛电极为工作电极，铜片为对电极，饱和甘汞电极为参比电极，电解质溶液为吡咯和柠檬酸的缓冲溶液(溶液的pH为6.8，PBS)，电解质溶液中吡咯的浓度为0.2mol/L和柠檬酸的浓度为0.1mol/L，采用计时电位法控制电化学反应，反应电流为1.5mA/cm 2，反应时间为20秒，氯掺杂聚吡咯上沉积有纳米锥结构的聚吡咯/柠檬酸复合物(纳米锥结构柠檬酸掺杂的聚吡咯沉积在被激光刻蚀出的凹槽中)，得到即纳米锥结构的聚吡咯/柠檬酸和纳米针结构的二氧化钛微区相间排列的的工作电极(未接枝葡糖氧化酶的葡萄糖传感器电极)。
本实施例制备的复合材料结构与实施例1相似，检测性能也与实施例1相似。
实施例3
(1)钛表面碱热处理：钛片(0.5mm×10mm×10mm)依次在丙酮、乙醇和去离子水中超声清洗10min。然后采用氢氟酸(0.54mol/L)和硝酸(0.25mol/L)混合酸清洗钛片表面的氧化层，清洗时间为7min；酸处理后的材料在去离子水中超声清洗10min，干燥待用，得到预处理的钛片；将预处理的钛片浸入装有70mL浓度为2mol/L的NaOH溶液的水热反应釜中，在100℃恒温条件下反应24h；反应结束后，取出，用去离子水浸泡冲洗，在真空干燥箱中 干燥待用，钛片表面形成纳米针结构的二氧化钛层；
(2)在室温条件下，使用红外光纤激光打印机对表面形成纳米针结构的二氧化钛层的钛片进行刻蚀，软件设置平行光路(光路与光路之间的间隔是100μm，光斑的宽度是仪器固定的30μm)，设置激光器的功率为5W，扫描速度为100mm/s；部分纳米针结构的二氧化钛层被激光刻蚀掉，形成多个凹槽或沟槽，凹槽的深度与纳米针结构的二氧化钛层的厚度相同；所述纳米针结构的二氧化钛层被凹槽分割成多条平行长方形区域，所述凹槽的横截面也为长方形；凹槽与被刻蚀后的纳米针结构的二氧化钛层交替排布；获得微图案的纳米针结构的二氧化钛层；
(3)通过电化学方法在凹槽中电沉积氯掺杂的聚吡咯：
选用三电极模式，表面有微图案的纳米针结构的二氧化钛层的钛片为工作电极，铜片为对电极，饱和甘汞电极为参比电极，电解质溶液中吡咯的浓度为0.2mol/L，盐酸的浓度为0.25mol/L，采用计时电位法控制电化学反应，反应电流30mA/cm 2，反应时间为20秒，凹槽图案中(被激光刻蚀部分)沉积一层氯掺杂的聚吡咯，反应完后，浸泡在去离子水中以除去表面没有反应的吡咯和盐酸，得到沉积有氯掺杂聚吡咯的钛电极；
(4)纳米锥结构的聚吡咯构建：
选用三电极模式，沉积有氯掺杂的聚吡咯的钛电极为工作电极，铜片为对电极，饱和甘汞电极为参比电极，电解质溶液为吡咯和柠檬酸的缓冲溶液(溶液的pH为6.8，PBS)，电解质溶液中吡咯的浓度为0.2mol/L和柠檬酸的浓度为0.1mol/L，采用计时电位法控制电化学反应，反应电流为1.5mA/cm 2，反应时间为20秒，氯掺杂聚吡咯上沉积有纳米锥结构的聚吡咯/柠檬酸复合物(纳米锥结构柠檬酸掺杂的聚吡咯沉积在被激光刻蚀出的凹槽中)，得到即纳米锥结构的聚吡咯/柠檬酸和纳米针结构的二氧化钛微区相间排列的的工作电极(未接枝葡糖氧化酶的葡萄糖传感器电极)。
本实施例制备的复合材料结构与实施例1相似，检测性能也与实施例1相似。
实施例4
(1)钛表面碱热处理：钛片(0.5mm×10mm×10mm)依次在丙酮、乙醇 和去离子水中超声清洗10min；然后采用氢氟酸(0.54mol/L)和硝酸(0.25mol/L)混合酸清洗钛片表面的氧化层，清洗时间为7min；酸处理后的材料在去离子水中超声清洗10min，干燥待用，得到预处理的钛片；将预处理的钛片浸入装有70mL浓度为2mol/L的NaOH溶液的水热反应釜中，在100℃恒温条件下反应24h，反应结束后，取出样品用去离子水浸泡冲洗，在真空干燥箱中干燥待用，钛片表面形成纳米针结构的二氧化钛层；
(2)在室温条件下，使用红外光纤激光打印机对表面形成纳米针结构的二氧化钛层的钛片进行刻蚀，软件设置平行光路(光路与光路之间的间隔是100μm，光斑的宽度是仪器固定的30μm)，设置激光器的功率为2.8W，扫描速度为100mm/s；部分纳米针结构的二氧化钛层被激光刻蚀掉，形成多个凹槽或沟槽，凹槽的深度与纳米针结构的二氧化钛层的厚度相同；所述纳米针结构的二氧化钛层被凹槽分割成多条平行长方形区域，所述凹槽的横截面也为长方形；凹槽与被刻蚀后的纳米针结构的二氧化钛层交替排布；获得微图案的纳米针结构的二氧化钛层；
(3)通过电化学方法在凹槽中电沉积氯掺杂的聚吡咯：
选用三电极模式，表面有微图案的纳米针结构的二氧化钛层的钛片为工作电极，铜片为对电极，饱和甘汞电极为参比电极，电解质溶液中吡咯的浓度为0.2mol/L，盐酸的浓度为0.25mol/L，采用计时电位法控制电化学反应，反应电流20mA/cm 2，反应时间为20秒，凹槽图案中(被激光刻蚀部分)沉积一层氯掺杂的聚吡咯，反应完后，浸泡在去离子水中以除去表面没有反应的吡咯和盐酸，得到沉积有氯掺杂聚吡咯的钛电极；
(4)纳米锥结构的聚吡咯构建：
选用三电极模式，沉积有氯掺杂的聚吡咯的钛电极为工作电极，铜片为对电极，饱和甘汞电极为参比电极，电解质溶液为吡咯和柠檬酸的缓冲溶液(溶液的pH为6.8，PBS)，电解质溶液中吡咯的浓度为0.2mol/L和柠檬酸的浓度为0.2mol/L，采用计时电位法控制电化学反应，反应电流为0.9mA/cm 2，反应时间为20秒，氯掺杂聚吡咯上沉积有纳米锥结构的聚吡咯/柠檬酸复合物(纳米锥结构柠檬酸掺杂的聚吡咯沉积在被激光刻蚀出的凹槽中)，得到即纳米锥结构的聚吡咯/柠檬酸和纳米针结构的二氧化钛微区相间排列的的工作电极(未 接枝葡糖氧化酶的葡萄糖传感器电极)。
本实施例制备的复合材料结构与实施例1相似，检测性能也与实施例1相似。
实施例5
将实施例1制备的葡萄糖传感器电极(未接枝葡萄糖氧化酶)浸泡在含有牛血清蛋白(50mg/mL)和葡萄糖氧化酶(25mg/mL)的PBS中，在4℃条件下反应30min，用PBS浸泡清洗，保存在4℃冰箱中备用。
采用电化学工作站进行葡萄糖检测试验。选用三电极模式，微图案葡萄糖传感器电极(接枝葡萄糖氧化酶)为工作电极，铜片为对电极，饱和甘汞电极为参比电极，含有不同浓度的葡萄糖的PBS溶液作为电解质溶液，用于模拟真实血糖检测。测试在37℃条件下进行，对工作电极施加0.3V的电压，待电流曲线稳定之后，记录电流值。葡萄糖的浓度范围是0.1-4.0mmol/L。
图4为实施例5中葡萄糖传感器电极检测的电流响应与葡萄糖的浓度关系图。如图4所示，电流值随着葡萄糖浓度变化的趋势图，对曲线进行线性拟合，得到电流值(y)与葡萄糖浓度(x)的线性方程：y＝4.4872x+13.652，线性相关系数R 2＝0.9964。因此，制备的葡萄糖传感器电极对葡萄糖的检测具有较高的可靠性。
实施例2～4制备的电极进行接枝葡萄糖氧化酶，对葡萄糖的检测也具有较高的可靠性。

Claims (10)

  1. 一种葡萄糖传感器电极，其特征在于：包括钛电极，钛电极的表面由纳米针二氧化钛形成的区域与由纳米锥结构聚吡咯形成的区域组成，每一个由纳米锥结构聚吡咯形成的区域与由纳米针二氧化钛形成的区域相邻，每一由纳米锥结构聚吡咯形成的区域与区域之间为由纳米针二氧化钛形成的区域；纳米锥结构聚吡咯形成的区域接枝有能够检测葡萄糖的酶；由纳米针二氧化钛形成的区域和由纳米锥结构聚吡咯形成的区域其中至少有一种区域为微区。
  2. 根据权利要求1所述葡萄糖传感器电极，其特征在于：所述由纳米针二氧化钛形成的区域和由纳米锥结构聚吡咯形成的区域其中至少有一种区域为多个独立区域；每一个独立区域为微区；
    所述能够检测葡萄糖的酶为葡萄糖氧化酶；所述纳米锥结构聚吡咯为掺杂或未掺杂的纳米锥结构聚吡咯；所述由纳米锥结构聚吡咯形成的区域与由纳米针二氧化钛形成的区域形成电势差。
  3. 根据权利要求2所述葡萄糖传感器电极，其特征在于：所述纳米锥结构聚吡咯为掺杂的纳米锥结构聚吡咯，所述掺杂的纳米锥结构聚吡咯为柠檬酸掺杂的纳米锥结构聚吡咯。
  4. 根据权利要求2所述葡萄糖传感器电极，其特征在于：当由纳米针二氧化钛形成的区域和由纳米锥结构聚吡咯形成的区域都为多个独立区域时，由纳米针二氧化钛形成的区域和由纳米锥结构聚吡咯形成的区域相间排列或周期性相间排列；
    当由纳米针二氧化钛形成的区域为整体区域，由纳米锥结构聚吡咯形成的区域为多个独立区域时，纳米锥结构聚吡咯形成的区域周期性排列，每一纳米锥结构聚吡咯形成的区域的四周为由纳米针二氧化钛形成的区域。
  5. 根据权利要求1所述葡萄糖传感器电极，其特征在于：所述纳米锥结构聚吡咯设置在氯掺杂的聚吡咯上，即纳米锥结构的聚吡咯与钛电极之间沉积有氯掺杂的聚吡咯。
  6. 根据权利要求1～5任一项所述葡萄糖传感器电极的制备方法，其特征在于：包括以下步骤：
    (1)钛表面碱热处理
    去除钛电极表面的氧化膜，得到预处理的钛电极；将预处理的钛电极置于 装有NaOH溶液的水热反应釜中进行碱热反应，钛电极表面形成纳米针结构二氧化钛膜层；
    (2)微图案的纳米针二氧化钛膜层构建
    在纳米针结构二氧化钛膜层上形成凹槽，获得具有微图案的纳米针结构二氧化钛膜层；
    (3)电沉积氯掺杂的聚吡咯
    选用三电极模式，导电金属为对电极，步骤(2)中具有微图案的纳米针结构二氧化钛膜层的钛电极为工作电极，电解质溶液为包含吡咯和氯离子的水溶液，采用计时电位法控制电化学反应，氯掺杂的聚吡咯沉积在具有微图案的纳米针结构二氧化钛膜层的凹槽图案中；
    (4)纳米锥结构聚吡咯构建
    选用三电极模式，导电金属为对电极，步骤(3)中沉积有氯掺杂的聚吡咯的钛电极为工作电极，电解质为包含吡咯和柠檬酸的缓冲溶液，采用计时电位法控制电化学反应，氯掺杂的聚吡咯上形成纳米锥结构柠檬酸掺杂的聚吡咯即纳米锥结构柠檬酸掺杂的聚吡咯沉积在凹槽图案中，获得具有纳米针结构二氧化钛和纳米锥结构聚吡咯的钛电极；
    (5)接枝能够检测葡萄糖的酶：
    将步骤(4)制备的钛电极上接枝能够检测葡萄糖的酶，得到葡萄糖传感器电极。
  7. 根据权利要求6所述葡萄糖传感器电极的制备方法，其特征在于：步骤(1)中所述碱热反应的温度为70～110℃；步骤(1)所述氢氧化钠溶液浓度为1～4mol/L；
    步骤(1)所述碱热处理的时间为12～36h；
    步骤(3)中所述电化学反应的电流为10～30mA/cm 2；电化学反应的时间为10s～40s；
    步骤(4)中所述电化学反应的电流为0.5～2.0mA/cm 2；电化学反应的时间为10～50s。
  8. 根据权利要求6所述葡萄糖传感器电极的制备方法，其特征在于：步骤(3)中所述氯离子的浓度为0.1～0.4mol/L，吡咯的浓度为0.1～0.3mol/L；
    步骤(4)中所述缓冲溶液的pH为6.8～7.2；
    步骤(4)中所述吡咯的浓度为0.1～0.3mol/L，柠檬酸的浓度为0.05～0.3mol/L；
    步骤(5)中能够检测葡萄糖的酶为葡萄糖氧化酶；此时步骤(5)的具体步骤为：将步骤(4)制备的钛电极浸泡于含有牛血清蛋白和葡萄糖氧化酶的PBS溶液中进行反应，得到葡萄糖传感器电极。
  9. 根据权利要求8所述葡萄糖传感器电极的制备方法，其特征在于：步骤(5)中所述牛血清蛋白和葡萄糖氧化酶的浓度分别为30～80g/L和10～50g/mL；
    步骤(5)中所述反应的温度为4～8℃；所述反应的时间为20～50min。
  10. 根据权利要求1～5任一项所述葡萄糖传感器电极的应用，其特征在于：所述葡萄糖传感器电极用于葡萄糖浓度检测。
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