CN110618179A - Glucose electrochemical microelectrode sensor based on nano porous metal film - Google Patents

Abstract

The invention discloses a preparation method of a nanoporous metal film, and the porous film is loaded with glucose oxidase, which is used as a microelectrode sensor for blood sugar monitoring, and relates to the processing technology and sensor technology of the porous metal film. On the polyimide substrate, magnetron co-sputtering of metals and metal oxides in different proportions and thicknesses, treated with deionized water or weak acid solution, removes metal oxides, leaving a three-dimensional porous network structure and interconnected metals Layer, as the support layer and conductive layer for loading glucose oxidase (GOD), the other side of the insulating substrate is coated with Ag/AgCl as a reference-counter electrode to form an electrochemical two-electrode system, and then coated with a polymer as The diffusion-restricting layer and the anti-interference hydrophilic layer form a glucose sensing microelectrode. The nanoporous gold membrane has a large specific surface area, which effectively increases the active area of the electrode and the GOD loading capacity. It has good permeability and high conductivity, and can also protect the microelectrode enzyme membrane sensing layer from physical damage to a certain extent. The microelectrode sensor has a wide linear range and good long-term stability.

Description

A Glucose Electrochemical Microelectrode Sensor Based on Nanoporous Metal Membrane

technical field

The invention belongs to the field of electrochemical biosensors, and relates to a processing technology of a metal film with a porous structure, a microelectrode and an electrochemical glucose sensor based on the porous metal film, and their preparation methods.

Background technique

Diabetes is one of the common chronic diseases that seriously threaten human health. Real-time monitoring of blood glucose is helpful for patients and doctors to evaluate the condition, formulate reasonable and scientific treatment plans or adjust existing plans. Therefore, glucose sensors are also the most researched and commercial products in the field of biosensors. the earliest biosensors. The core component of the glucose sensor is the enzyme electrode composed of glucose oxidase (GOD) effectively immobilized on the bioactive interface. In practical applications or existing marketed blood glucose monitoring sensors generally use needle-type microelectrodes, which are less traumatic and can reduce the physiological immune response of the human body. If Abbott’s Freestyle Navigator has been approved by the US Food and Drug Administration (FDA), the sensor probe has a diameter of less than 0.4 mm and is inserted 5 mm below the skin without collecting fingertip blood , to obtain blood glucose indicators by monitoring tissue fluid glucose levels. However, due to electrode miniaturization and size limitations, the loadable area of GOD is greatly reduced, and the amount of enzyme loaded is insufficient, which reduces the linear range and sensitivity of the sensor, and even fails to work normally. According to clinical requirements, the upper limit of the linear area of the glucose sensor Must reach 30mM. New materials with excellent properties researched in the laboratory are often unable to be applied due to lack of spatial fixation.

In order to solve the problem of loading GOD enzyme without increasing the size of the microelectrode, there are mainly two ways to solve it. One is to increase the number of microelectrodes, such as the needle-shaped glucose sensor for subcutaneous tissue implantable real-time monitoring mentioned in domestic patent CN200910097842.2 (authorized announcement number CN 101530327B) and CN200410101080.6, using one or two needle-shaped working An electrode and a reference electrode form a two-electrode system. During implantation, 2-3 electrodes need to be implanted for measurement. The domestic patent CN201510783056.3 (authorized announcement number CN 105266826 B) mentions a subcutaneous tissue implanted needle-like The glucose sensor has a split three-electrode system with a working electrode, a counter electrode and a reference electrode at the bottom of the base. These methods will increase the area of trauma. Another method is to prepare a three-dimensional network structure on the surface of the microelectrode to increase the specific surface area of the active surface of the electrode, such as doping zero-dimensional quantum dots, metal nanoparticles, one-dimensional nanotubes, or two-dimensional Nanosheets such as graphene and zinc oxide, these methods have good performance, but the preparation process is complicated and requires harsh processing conditions and techniques. The simplest way to obtain a three-dimensional network structure is to directly prepare an electrode with a three-dimensional structure surface film, such as a mesoporous carbon film electrode, a porous silicon film electrode, a porous metal film electrode, etc. Domestic patent CN201510750828.3 invented a porous active graphene microelectrode. After immobilizing glucose oxidase, it realized the direct electrochemistry of the enzyme and the rapid electrochemical determination of glucose; the domestic patent CN201810700292 used hollow tubular graphene fibers to fill glucose oxidation The enzyme, glucose oxidase, is coated inside the continuous graphene, which has an excellent enzyme protection effect, and the excellent conductivity of graphene enables rapid charge transfer and transmission; patent CN201710592096 provides a surface-roughened gold A film electrode is used, and an electrochemical hydrogen peroxide sensor is constructed based on the gold film electrode. The method uses magnetron sputtering to prepare a gold film layer, and then uses chemical etching to roughen the surface of the gold film. However, generally speaking, the three-dimensional structure of the electrode surface has a thickness limit. Increasing the thickness will increase the probability of coating peeling off and affect the durability and stability of the sensor electrode. Therefore, it is necessary to invent a method that can obtain a firm coating film that is not easy to fall off , a simple and reliable method for obtaining three-dimensional structured surface electrodes.

Nanoporous metal film is a metal material with a nanoscale pore structure, which contains a large number of three-dimensional interconnected nanoscale pores and skeletons. It not only has the characteristics of metal conductivity and ductility, but also has small size effect, The unique properties of nanomaterials such as surface effect, quantum size effect and quantum tunneling effect. In addition, its porous structure also makes it have the characteristics of high specific surface area, low density, good permeability and high conductivity. At present, it has been widely used in catalysis, sensing, separation, filtration, biomaterials, fuel cells and other fields. The high specific surface area of the porous metal film can increase the active area of the electrode and the loading of electroactive substances. The nanoscale pore structure and metal The conductivity makes it highly catalytic to certain substances, and the nanoporous gold film also shows good stability and regeneration, and is easy to recycle and reuse.

The preparation methods of nanoporous metals mainly include dealloying method, template method and electrochemical method. The template method is to load the metal on a pre-established template through a certain technology, and then remove the template by physical and chemical methods to obtain a porous target metal. Commonly used templates include ionic solution polymers, biological templates, and porous alumina. Wait. The template method can better control the shape and size of the obtained nanoporous metal, but due to the constraints of the template, the adjustment of the structure or size of the porous metal can only be achieved by readjusting the template, and an appropriate method must be used to remove the template, so The operation is more complicated and cumbersome. Electrochemical method, using pure metal as the substrate, anodizes the metal by continuously applying voltage in a certain solution, so that part of the metal on the surface gradually dissolves into the electrolyte, thereby obtaining a nanoporous metal film. Although this method is widely used, as described in the above-mentioned patent CN201710592096, what is obtained is often only a roughened surface, and the pores cannot reach deep inside, and the pore diameter of the pores is not easy to adjust. The speed is difficult to control, and the obtained pore size is not ideal. When the pore size is too small (<5 nanometers), GOD and other proteins cannot enter the pores. When the pore size is too large (>200nm), the fixed GOD is easy to fall off. On the other hand, metals that are corroded and dissolved into the electrolyte are difficult to recycle, which also causes waste of metals and environmental pollution. The dealloying method uses the difference in activity between different metals to selectively dissolve one or more metals in the alloy in a corrosive solution, thereby obtaining a single-component nanoporous metal. The alloying method has been studied more in the Au-Ag alloy system. Gold and silver can form a single-phase infinite solid solution. The two metal components have a large enough standard electrode potential difference. The silver atoms can be corroded and dissolved, and the remaining gold atoms diffuse on the interface. And gather to form the skeleton of nanoporous gold. The dealloying method is generally simple and convenient to operate, and the obtained nanostructure has a high specific surface area and a uniform structure, and the pore size can be obtained through dynamic adjustment and control of the corrosion process, which is suitable for large-scale industrial production. However, when using this method, an alloy with a specific composition and the same crystal phase needs to be prepared in advance. It is difficult to find a suitable alloy system for most metals, and the scope of application is narrow, and the formation of the gold film requires re-condensation of the gold component, and the original rigidity is destroyed. , the adhesion of the film decreases, and it is easy to deform and fall off.

Contents of the invention

Aiming at the problems of low sensitivity and narrow linear range of the microelectrode glucose oxidase sensor due to size limitation and small amount of loaded enzyme in the prior art, the present invention provides a method for preparing a microelectrode based on a three-dimensional porous metal film and glucose oxidase sensor. Sensor preparation method. The preparation of the porous metal film in the present invention does not require a template, the pore diameter is easy to adjust, the bonding force with the substrate is strong, and it is not easy to fall off. The method has a wide range of applications, and a series of metals including gold and platinum can be obtained by this method. Porous structure, used for A large amount of glucose oxidase is loaded to improve the sensitivity of the sensor and increase the linear range.

The present invention is achieved through the following technical solutions:

1. The present invention first provides a kind of porous metal film, and it comprises:

polyimide substrate;

a porous metal film on said substrate;

The thickness of the porous metal film is 50-200nm;

The pores of the porous metal film are 10-100nm;

Applicable metals have good electrical conductivity, stable chemical properties, not easy to oxidize at room temperature, and good biocompatibility. They can be gold, platinum, iridium, rhenium, tungsten, tantalum, hafnium, silver, palladium, rhodium, ruthenium, and titanium. Any one, or an alloy composed of them;

The polyimide substrate thickness of the supporting substrate of the porous metal film can be selected from 0.05-1mm, preferably 0.5mm;

2. The invention provides a kind of preparation method of porous metal membrane:

On the polyimide substrate, use double-target magnetron co-sputtering to deposit metal and metal oxide (such as MgO, CaO) mixture films with different thicknesses and different proportions, and soak in deionized water or weak acid solution to make the metal The oxide is removed, leaving a three-dimensional porous metal layer.

Preferably, the magnetron sputtering metal and metal oxide targets have a standard size of 2 inches and a purity of 99.999%;

Preferably, the heating temperature of the magnetron sputtering substrate is 200°C;

Preferably, metal and metal oxide use radio frequency sputtering power supply;

The magnetron sputtering air pressure is 3-10 millitorr, preferably 5 millitorr, and the substrate speed is 10-30 rpm, preferably 20 rpm;

The different proportions of metals and metal oxides, wherein the proportion of metal oxides can be: 15%, 30%, 45%, 60%, 70%, 80%, preferably 70%, the greater the proportion of metal oxides , the larger the pore diameter of the prepared metal film, the pore diameter can be adjusted from 10-100nm.

The thickness of the metal and metal oxide mixture film is different, optional 50-200nm, preferably 100nm;

The thickness of the metal and metal oxide mixture film is different, optional 50-200nm, preferably 100nm;

The cleaning duration of the solution is 10-60 minutes, preferably 30 minutes, repeated three times;

3. based on porous metal film, the present invention has constructed a kind of microelectrode glucose sensor, and it comprises:

polyimide substrate;

Porous metal films on substrates;

The modified layer on the porous metal film and the modified layer on the substrate;

Described substrate divides A and B two sides, and A side prepares porous metal film according to claim 4 and 5, makes enzyme electrode after filling and fixing glucose oxidase in metal film pore and is sensor working electrode; B side is coated with silver/chloride Silver is the counter electrode-reference electrode, forming a two-electrode electrochemical glucose sensor.

The porous metal surface A on which the glucose oxidase is immobilized is a working electrode, the silver/silver chloride-coated surface B is a reference electrode, and the platinum wire is a counter electrode, forming a three-electrode electrochemical glucose sensing system.

The modified layer of the porous metal membrane working electrode is an enzyme membrane sensing layer, a diffusion limiting layer, an anti-interference hydrophilic layer, and an optional catalytic layer from the inside to the outside.

The enzyme membrane sensing layer includes: glucose oxidase, glutaraldehyde crosslinking agent, bovine serum albumin, nano-gold, nano-platinum, carbon nanotubes and other nanoparticle components, which are fixed by drip coating or soaking or spraying GOD is in the pores of the gold film.

The catalytic layer is arranged between the enzyme film sensing layer and gold, and can be prepared by electrochemical deposition methods such as nano-platinum and other precious metal nanoparticles, platinum black, ferrocene and Prussian blue as representatives of electronic mediators, etc. .

The diffusion limiting layer can be any one of polyurethane, polyvinyl alcohol, polycarbonate or polyvinyl chloride, and is prepared by soaking or drip coating.

The anti-interference hydrophilic layer can be any one of polyvinyl alcohol, chitosan or modified chitosan, polyethylene glycol or oligosaccharides.

The counter-reference electrode on the B side of the substrate includes silver metal, silver chloride, a diffusion-limiting layer and an anti-interference hydrophilic layer. Silver/silver chloride is plated with a layer of silver by magnetron sputtering, which is obtained after chlorination or directly coated with Ag/AgCl slurry.

4. The present invention provides a kind of preparation method and the step of the microelectrode glucose sensor based on porous metal film, mainly comprising:

(1) Clean the substrate and dry it: use ethanol and isopropanol to ultrasonically clean it for 10 minutes, and blow it dry with nitrogen; optional oxygen plasma treatment can increase the hydrophilicity of the substrate surface, and the coating thickness is more uniform and the roughness is smaller ;

(2) Preparation of porous metal film: On a clean substrate, magnetron co-sputtering of metal and magnesium oxide mixed films of different ratios and thicknesses, and cleaning in deionized water to form a porous metal film;

(3) To prepare a silver/silver chloride electrode, first, on the opposite side of the substrate porous metal film, use evaporation or magnetron sputtering to sputter a layer of 50-200nm silver metal, and use silver paste or welding technology to lead out the metal surfaces on both sides , using one of the following methods to make a silver/silver chloride electrode:

Constant current electroplating technology, the silver metal film is used as the anode, the porous metal film is used as the cathode, the current is 0.1-0.3mA, immersed in 0.1mol/L hydrochloric acid solution, chlorinated for 2-6 hours, and cleaned with deionized water;

Constant voltage electroplating is adopted, the silver metal film is used as the anode, the porous metal film is used as the cathode, a 5V DC voltage is applied on both sides of the electrodes, the electrolyte is 0.1mol/L hydrochloric acid solution, lasts for 10-30 minutes, and is cleaned with deionized water;

(4) Porous metal film modified catalytic layer: the catalytic layer between the enzyme film layer and the inner wall of the metal hole, optional platinum nanoparticles, Prussian blue, etc., prepared by any of the following methods:

Direct potentiostatic electrodeposition of Pt nanoparticles in chloroplatinic acid solution: use a porous gold film electrode as the working electrode, a platinum wire as the counter electrode, and Ag/AgCl as the reference electrode, connect to the electrochemical workstation, and immerse the electrode in H ₂ In the PtCl ₆ solution, at the potential of -0.25V, the platinum nanoparticles were deposited by the constant potential method, and the deposition time was 120s. After washing with deionized water, they were scanned in a 0.5mol/L H ₂ SO ₄ solution until they were stable;

Electrodeposition of Pt nanoparticles by cyclic voltammetry: In 0.5-5.0mmol/L HPtCl ₄ solution, in the range of 1.5-0.3V, scan 5-50 cycles at a scan rate of 50-150mV/s cyclic voltammetry, one-step deposition Pt nanoparticles;

Two-step cyclic voltammetry electrodeposition of Pt nanoparticles: the porous gold film electrode was placed in K ₂ SO ₄ solution for cyclic voltammetry scanning, and the surface was activated. The activated electrode was placed in a mixed solution of 2M K ₂ PtCl ₄ and 0.1M K ₂ SO ₄ for cyclic voltammetry scanning, and the obtained electrode was placed in H ₂ SO ₄ solution for cyclic voltammetry scanning, so that the coordination of platinum converted into platinum nanoparticles.

First prepare platinum nano-sol and then absorb by soaking: Dissolve 129.4 mg of chloroplatinic acid in 91.5 mL of water, dissolve 5 mg of polyvinylpyrrolidone (PVP) in 5 mL of water, mix the two solutions, and slowly add 1 mL of 0.1 mol/ 1. Sodium borohydride, the resulting mixed solution was left to stand at room temperature for 24 hours; the gold film electrode was immersed in 0.1wt% octadecyltrimethylammonium chloride (STAC) solution, and after standing for 5 seconds, it was taken out to dry, and then Place in platinum nano-sol for 30 minutes, electrostatically adsorb a layer of platinum nano-particle catalytic layer, take it out, rinse with deionized water, remove unimmobilized platinum nanoparticles on the surface, and dry;

Electrodeposition of Prussian blue: one side _of the porous metal film is used as the working electrode, the _Ag / _AgCl side is used as the reference electrode, and the Pt wire is used as the counter electrode. Electrochemical deposition was carried out in the electrolyte, using a constant voltage of 0.4V, and the deposition time was 10-30s to obtain a porous metal film electrode modified by Prussian blue.

(5) Immobilizing glucose oxidase on the working electrode: adopting the classic chemical cross-linking method, using glutaraldehyde (GA) as the cross-linking agent, preparing glucose oxidase (GOD) and bovine serum albumin (BSA) Oxidase mixed solution, mixed fully and evenly, drip-coated on the surface of the electrode, dried at 4°C, rinsed with deionized water to fix the oxidase, repeated drip-coating and rinsing, repeated 1-4 times, dried for use;

(6) The working electrode and reference electrode of the microelectrode are coated with a diffusion-limiting layer and an anti-interference hydrophilic layer: the polymer of the diffusion-limiting layer can be any one of Nafion, polyurethane, polyvinyl alcohol, polycarbonate or polyvinyl chloride One, prepared by soaking or dripping method. The anti-interference hydrophilic layer can be any one of Nafion, polyvinyl alcohol, chitosan or modified chitosan, polyethylene glycol or oligosaccharides, and is prepared by soaking or drip coating.

(7) Laser cutting is used to batch cut the coated substrate into the required size of the micro-electrode.

Compared with the prior art, the present invention has the following beneficial effects:

(1) The porous three-dimensional metal film provides an active interface with a high specific surface area, which can be used as a supporting substrate for further modification, and the GOD loading capacity is greatly increased, so that the prepared glucose has a wider response range (0.025mmol/L- 25.5mmol/L) and high sensitivity (8.20μA/mM);

(2) The porous metal film is not only the supporting layer of the modified layer, but also the conductive layer of the sensor. The good electrical conductivity of the metal can make the charge transfer and transmission fast;

(3) GOD goes deep into the pores of the porous metal film and has a certain protective effect on the enzyme. The nano-sized metal frame has a certain catalytic effect on the detected intermediate product hydrogen peroxide, which can amplify the current signal;

(4) The preparation of the porous metal film does not require a template, and the method has a wide range of applications. The metals or alloys of gold, platinum, iridium, rhenium, tungsten, tantalum, hafnium, silver, palladium, rhodium, ruthenium, titanium listed in this patent are all Can;

(5) Porous metal membranes can be prepared by adjusting the ratio of co-sputtering MgO or CaO to adjust the pore size and density. In addition to loading GOD, it can also load modifiers such as nanoparticles of different sizes;

(6) The working electrode and reference electrode-counter electrode of the sensor are integrated on the two surfaces of a single microelectrode, which can reduce trauma when implanted;

(7) The substrate selected by the present invention has good rigidity and toughness, and is not easy to break when implanted, and does not need additional guide pins;

(8) The manufacturing method of the microelectrode glucose electrochemical sensor of the present invention is simple and easy to coat, has good repeatability, can be prepared in large scale and batches, and has great application value and prospect.

Detailed ways

In order to facilitate understanding of the present invention, the present invention enumerates the following examples. It should be clear to those skilled in the art that the embodiments are only for helping to understand the present invention, and should not be regarded as specific limitations on the present invention.

Example 1

A method for preparing a porous gold film microelectrode, using polyimide as a substrate, magnetron co-sputtering gold and magnesium oxide, washing with acetic acid aqueous solution to remove MgO, and obtaining a porous gold film surface, the specific operation steps are as follows:

(1) Take a commercial polyimide substrate whose length, width, and thickness are 8 mm, 2 mm, and 0.5 mm, respectively, and treat it with oxygen plasma for 30 minutes, then clean it with water, ethanol, and isopropanol, and dry it with nitrogen. use;

(2) Magnetron co-sputtering of gold and MgO, the purity of gold and magnesium oxide targets is 99.999%, and the size is 2 inches. During magnetron sputtering, the pressure is 5 millitorr, the substrate speed is 20 rpm, and the substrate is heated to 200 ℃.

(3) Magnesium oxide uses 150W, and gold uses different powers to obtain different proportions of co-sputtering films:

(4) The obtained coating film and the substrate are placed in deionized water, after soaking for 30 minutes, replace the deionized water, soak again, carry out this process three times, take out nitrogen and blow dry for later use;

(5) On the back of the substrate, magnetron sputtering is used to coat a metal silver film with a thickness of 100nm. During the sputtering process, the substrate is heated to 200°C to make the silver metal film adhere to the substrate more firmly;

(6) Using gold wire ball bonding technology, lead two gold wire electrodes on the surface of porous gold and silver metal, 1 mm away from the end of the substrate;

(7) With the silver metal film as the anode and the gold film as the cathode, a direct current of 0.25mA is applied to both ends, and the electrolyte is 0.1mol/L hydrochloric acid. After chlorination for 5 hours, wash 3 times with deionized water;

(8) The obtained one side is a microelectrode with a porous gold surface and the other side is a silver/silver chloride surface.

Example 2

The second embodiment of the present invention provides a glucose electrochemical biosensor based on the preparation of the porous gold film microelectrode in the first embodiment, and the specific operation steps are as follows:

(1) choose sputtering 70% magnesia ratio, the sample that total thickness is 100nm carries out this embodiment, under this condition, pore aperture is in the scope of 20-100nm;

(2) modify a layer of Prussian blue in the pores of the porous gold film, the method is:

Immerse the microelectrode in the concentrated HNO ₃ :HCl:H ₂ O solution with a volume ratio of 1:3:4 for 3 minutes, wash the surface of the electrode thoroughly with deionized water, wash it repeatedly with ethanol and deionized water, and dry it with nitrogen gas ;

One side of the porous gold film is used as the working electrode, the _Ag / _AgCl side is used as the reference electrode, and the Pt wire is used as the counter electrode. Electrochemical deposition was carried out in a mixed electrolyte of 2.5mmol/L FeCl ₃ and HCl, 0.1mol/L HCl was used to adjust the pH of the electrolyte to 1.0-2.5, a constant voltage of 0.4V was used, and the deposition time was 10s to obtain Prussian blue modified Porous gold film electrode, Prussian blue into a spherical shape, with a thickness of about 15nm;

The obtained Prussian blue modified electrode was activated in a mixed solution of 0.1mol/L HCl and 0.1mol/L KCl, using a voltage of -50-350mV for cyclic voltammetry scanning, with a scan rate of 50mV/s, and 50 cycles; /L KCl in 0.1mol/L pH 6.8 phosphate buffer solution, use a constant potential polarization of -50mV for 100s, wash with deionized water, dry with nitrogen, and bake in an oven at 90°C for 1 hour to make the modification slightly The electrode is completely dry and ready for use;

(3) Using the chemical cross-linking method to immobilize glucose oxidase on the Prussian blue modified porous gold film electrode, the method steps are:

Use 0.1mol/L phosphate buffer (pH 7.0) to prepare 4mg/mL GOD enzyme solution and 4mg/mL bovine serum albumin solution, take equal volumes of the two solutions and mix them, ultrasonically disperse for 10 minutes, take 2-4μL of the resulting mixture solution carefully Drop-coat on the surface of the Prussian blue modified porous gold membrane electrode, after drying, drop-coat 1.5-4% (v/v) glutaraldehyde solution on the surface of the enzyme membrane for cross-linking and fixing for 30 minutes, the cross-linking temperature is 35 ℃ water bath . Rinse the surface of the electrode repeatedly with 0.1 mol/L phosphate buffer solution, repeat the steps of drop-coating GOD and glutaraldehyde 2-4 times, and dry it at 4°C to obtain a porous gold film working electrode with immobilized glucose oxidase.

(4) Nafion is rich in a large amount of hydrophilic sulfonic acid groups, which has good water solubility, and the Nafion membrane also has good cation selectivity and biocompatibility. In this embodiment, the microelectrode is selected The surface of the working electrode and the reference electrode is modified with a layer of Nafion film to increase the hydrophilicity and anti-interference ability of the sensor. Mix 0.1mol/L phosphate buffer and 5% Nafion ethanol solution in a ratio of 10:1 to prepare a diluted Nafion solution; take 2 - 4 μL of the above Nafion solution was drip-coated on both sides of the electrode, and dried in the air for 30 minutes.

(5) All electrodes were placed in 0.1mol/L PBS (pH 7.0) before the test and scanned at a voltage of 0-1.0V until a stable voltammogram was obtained. After the test, they were placed in 0.1mol/L PBS (pH 7.0), Under the voltage of 0-1.0V, cyclic voltammetry scans 20 cycles (scanning rate 50mV/s) for reconstruction. When the electrode is idle, keep it under constant humidity conditions at 4°C.

(6) Response of porous gold film glucose electrochemical microelectrode sensor to different concentrations of glucose.

A conventional three-electrode test system was adopted, with the constructed porous gold film microelectrode as the working electrode, Ag/AgCl as the reference electrode, platinum wire (diameter 1 mm) as the counter electrode, and 20 mL of PBS (pH 7.0) buffer as the electrolyte , The response current of the enzyme electrode to glucose was measured at the electrode potential +0.6V vs. Ag/AgCl, and the enzyme electrode was scanned by cyclic voltammetry at a voltage of 0-+0.6V. During the current measurement, a magnetic stirrer was used to stir slowly to make the glucose molecules diffuse evenly in the buffer. All tests were performed at room temperature.

Glucose solution was prepared with deionized water, adding a few drops of 0.1mol/L HCl to adjust the pH to acidic to prevent microbial growth, and the newly prepared glucose solution was allowed to stand overnight to fully convert α-glucose into β-glucose, the substrate of GOD.

The porous gold film glucose electrochemical microelectrode sensor produced a certain current response to the gradually increasing glucose concentration. , 1.0,1.2,1.4,1.65,1.9,2.2,2.65,3.2,3.8,4.2,4.6,5.2,6.2,7.2,8.2,9.2,10.4,12.9,15.5,20.5,25.5mmol/L.

The modified porous gold film electrode has a fast and sensitive response to glucose, and the time to reach 95% of the maximum response current is _≤8s , indicating that H2O2 is easy to diffuse in the modified film, and the glucose concentration is between 0.025 mM and _2.15 mM It conforms to the linear equation: i(μA)=0.79525+8.19829C(mmol/L), the correlation coefficient is 0.9983 (RSD=3.2～4.7%, n=5), and the sensitivity of the sensor obtained from the formula is 8.20 μA/mM, The detection limit was 8.2 mol/L (S/N=3).

(7) Anti-interference performance test of porous gold film glucose electrochemical microelectrode sensor.

Add physiological concentration of interferents, Prussian blue modified porous gold glucose biosensor to 1mmol/L glucose, 0.1mmol/L ascorbic acid (AA), 0.5mmol/L uric acid (UA) and 0.1mmol/L acetaminophen (AP) The time-current response shows that the electrode has good anti-interference ability, and the current generated by the interfering substance has basically no effect on the detection of glucose.

(8) Stability test of porous gold film glucose electrochemical microelectrode sensor.

Continuously measure the response current of the porous gold film glucose electrochemical microelectrode to 1mmol/L glucose in 45 days, and the current decreases slowly with time. After 45 days, the response current of the glucose biosensor has only decreased by 15.7% relative to the maximum response current, indicating that The gold film and the gold film modification have good hydrophilicity and biocompatibility, which protect the enzyme and are conducive to the maintenance of the enzyme activity, so that the electrode has good stability.

Description of drawings

Fig. 1 is the schematic diagram of the preparation of porous metal membrane and the preparation of glucose sensor by immobilizing glucose oxidase;

Figure 2 is a scanning electron microscope picture of a porous gold membrane electrode after modifying Prussian blue, GOD enzyme layer and Nafion membrane;

Figure 3 is the chronoamperometry curve of the Prussian blue modified porous gold GOD electrode in response to different glucose concentrations, tested in 20mL pH 7.0 phosphate buffer solution, test potential +0.6V, electromagnetic stirring, room temperature, arrows indicate glucose after a single injection total concentration.

Fig. 4 is the working curve of the Prussian blue modified porous gold glucose biosensor (n=5, RSD=3.2-4.7%). The inset is the linear fit of the working curve: linear range: 0.025–2.15 mmol/L.

Figure 5 shows the time-current response of the Prussian blue modified porous gold glucose biosensor to 1mmol/L glucose, 0.1mmol/L ascorbic acid (AA), 0.5mmol/L uric acid (UA) and 0.1mmol/L acetaminophen (AP). (+0.6V vs. Ag/AgCl; 0.1M PBS, pH 7.0)

Fig. 6 is the response current (n=5) of the Prussian blue modified porous gold glucose biosensor to 1 mmol/glucose within 45 days, and the error bars represent the standard deviation. Tested in 20 mL pH 7.0 phosphate buffer, +0.6 V vs. Ag/AgCl; magnetic stirring, room temperature.

The applicant declares that although the present invention has described the process equipment and process flow of the present invention in detail through the above-mentioned embodiments, the present invention is not limited to the above-mentioned detailed process equipment and process flow, that is, it does not mean that the present invention must rely on the above-mentioned detailed process equipment and process flow can be implemented, the above description should not be considered as limiting the present invention. Any improvements, modifications and substitutions of the present invention will be apparent to those skilled in the art after reading the above disclosure. Therefore, the protection scope of the present invention should be defined by the appended claims.

Claims (6)

1. A porous metal membrane, characterized in that, comprising: Polyimide substrate; a nanoporous metal film on said substrate; The thickness of the porous metal film is 50-200nm; The pores of the porous metal film are 10-100nm. 2. The porous metal membrane as claimed in claim 1, characterized in that the metal of the porous membrane has good electrical conductivity, stable chemical properties, not easily oxidized at room temperature, and good biocompatibility, and can be gold, platinum, iridium, Any one of rhenium, tungsten, tantalum, hafnium, silver, palladium, rhodium, ruthenium, titanium, or an alloy composed of them. 3. The porous metal membrane supporting substrate according to claim 1, characterized in that the material is polyimide, preferably with a thickness of 0.5 mm. 4. A method for preparing a porous metal membrane, characterized in that: On the polyimide substrate, use double-target magnetron co-sputtering to deposit different thicknesses and different proportions of metal and metal oxide (such as MgO, CaO) mixture films, and wash with deionized water or weak acid solution to dissolve the metal oxide, leaving a three-dimensional porous metal layer. Preferably, the magnetron sputtering metal and metal oxide targets have a standard size of 2 inches and a purity of 99.999%; Preferably, the heating temperature of the magnetron sputtering substrate is 200°C; Preferably, metal and metal oxide use radio frequency sputtering power supply; Magnetron sputtering air pressure is 3-10 millitorr, preferably 5 millitorr, substrate speed 10-30 rpm, preferably 20 rpm; The different proportions of metals and metal oxides, wherein the proportion of metal oxides can be: 15%, 30%, 45%, 60%, 70%, 80%, preferably 70%; The thickness of the metal and metal oxide mixture film is different, optional 50-200nm, preferably 100nm; The solution cleaning lasts for 10-60 minutes, preferably 30 minutes, and is repeated three times. 5. A microelectrode glucose sensor based on porous metal membrane, characterized in that, comprising: A substrate as claimed in claims 1-5; The porous metal film on the substrate described in claims 1-5; The modified layer on the porous metal film and the modified layer on the substrate; Described substrate divides A and B two sides, and A side prepares porous metal film according to claim 4 and 5, makes enzyme electrode after filling and fixing glucose oxidase in metal film pore and is sensor working electrode; B side is coated with silver/chloride Silver is the counter electrode-reference electrode, forming a two-electrode electrochemical glucose sensor. The porous metal surface A on which the glucose oxidase is immobilized is a working electrode, the silver/silver chloride-coated surface B is a reference electrode, and the platinum wire is a counter electrode, forming a three-electrode electrochemical glucose sensing system. The modified layer of the porous metal membrane working electrode is an enzyme membrane sensing layer, a diffusion limiting layer, an anti-interference hydrophilic layer, and an optional catalytic layer from the inside to the outside. The enzyme membrane sensing layer includes: glucose oxidase, glutaraldehyde crosslinking agent, bovine serum albumin, nano-gold, nano-platinum, carbon nanotubes and other nanoparticle components, which are fixed by drip coating or soaking or spraying GOD is in the pores of the gold film. The catalytic layer is arranged between the enzyme film sensing layer and gold, and can be prepared by electrochemical deposition methods such as nano-platinum and other precious metal nanoparticles, platinum black, ferrocene and Prussian blue as representatives of electronic mediators, etc. . The diffusion limiting layer can be any one of polyurethane, polyvinyl alcohol, polycarbonate or polyvinyl chloride, and is prepared by soaking or drip coating. The anti-interference hydrophilic layer can be any one of polyvinyl alcohol, chitosan or modified chitosan, polyethylene glycol or oligosaccharides. The counter-reference electrode on the B side of the substrate includes silver metal, silver chloride, a diffusion-limiting layer and an anti-interference hydrophilic layer. Silver/silver chloride is plated with a layer of silver by magnetron sputtering, which is obtained after chlorination or directly coated with Ag/AgCl slurry. 6. as claimed in claim 6, a kind of microelectrode glucose sensor based on porous metal membrane, is characterized in that, preparation method comprises the following steps: (1) Clean the substrate and dry it: use ethanol and isopropanol to ultrasonically clean it for 10 minutes, and blow it dry with nitrogen; optional oxygen plasma treatment can increase the hydrophilicity of the substrate surface, and the coating thickness is more uniform and the roughness is smaller ; (2) Preparation of porous metal film: On a clean substrate, magnetron co-sputtering of metal and magnesium oxide mixed films of different ratios and thicknesses, and cleaning in deionized water to form a porous metal film; (3) To prepare a silver/silver chloride electrode, first, on the opposite side of the substrate porous metal film, use evaporation or magnetron sputtering to sputter a layer of 50-200nm silver metal, and use silver paste or welding technology to lead out the metal surfaces on both sides , using one of the following methods to make a silver/silver chloride electrode: Constant current electroplating technology, the silver metal film is used as the anode, the porous metal film is used as the cathode, the current is 0.1-0.3mA, immersed in 0.1mol/L hydrochloric acid solution, chlorinated for 2-6 hours, and cleaned with deionized water; Constant voltage electroplating is adopted, the silver metal film is used as the anode, the porous metal film is used as the cathode, a 5V DC voltage is applied on both sides of the electrodes, the electrolyte is 0.1mol/L hydrochloric acid solution, lasts for 10-30 minutes, and is cleaned with deionized water; (4) Porous metal film modified catalytic layer: the catalytic layer between the enzyme film layer and the inner wall of the metal hole, optional platinum nanoparticles, Prussian blue, etc., prepared by any of the following methods: Direct potentiostatic electrodeposition of Pt nanoparticles in chloroplatinic acid solution: use a porous gold film electrode as the working electrode, a platinum wire as the counter electrode, and Ag/AgCl as the reference electrode, connect to the electrochemical workstation, and immerse the electrode in H ₂ In the PtCl ₆ solution, at the potential of -0.25V, the platinum nanoparticles were deposited by the constant potential method, and the deposition time was 120s. After washing with deionized water, they were scanned in a 0.5mol/L H ₂ SO ₄ solution until they were stable; Electrodeposition of Pt nanoparticles by cyclic voltammetry: in 0.5-5.0mmol/L HPtCl ₄ solution, in the range of 1.5-0.3V, scan 5-50 circles at a scan rate of 50-150mV/s cyclic voltammetry, one-step deposition Pt nanoparticles; Two-step cyclic voltammetry electrodeposition of Pt nanoparticles: the porous gold film electrode was placed in K ₂ SO ₄ solution for cyclic voltammetry scanning, and the surface was activated. The activated electrode was placed in a mixed solution of 2M K ₂ PtCl ₄ and 0.1M K ₂ SO ₄ for cyclic voltammetry scanning, and the obtained electrode was placed in H ₂ SO ₄ solution for cyclic voltammetry scanning, so that the coordination of platinum converted into platinum nanoparticles. First prepare the platinum nano-sol and then absorb it by immersion: Dissolve 129.4 mg of chloroplatinic acid in 91.5 mL of water, dissolve 5 mg of polyvinylpyrrolidone (PVP) in 5 mL of water, mix the two solutions, and slowly add 1 mL of 0.1 mol/ 1. Sodium borohydride, the resulting mixed solution was left to stand at room temperature for 24 hours; the gold film electrode was immersed in 0.1wt% octadecyltrimethylammonium chloride (STAC) solution, and after standing for 5 seconds, it was taken out to dry, and then Place in platinum nano-sol for 30 minutes, electrostatically adsorb a layer of platinum nano-particle catalytic layer, take it out, rinse with deionized water, remove unimmobilized platinum nanoparticles on the surface, and dry; Electrodeposition of Prussian blue: one side _of the porous metal film is used as the working electrode, the _Ag / _AgCl side is used as the reference electrode, and the Pt wire is used as the counter electrode. Electrochemical deposition was carried out in the electrolyte, using a constant voltage of 0.4V, and the deposition time was 10-30s to obtain a porous metal film electrode modified by Prussian blue. (5) Immobilizing glucose oxidase on the working electrode: adopting the classic chemical cross-linking method, using glutaraldehyde (GA) as the cross-linking agent, preparing glucose oxidase (GOD) and bovine serum albumin (BSA) Oxidase mixed solution, mixed fully and evenly, drip-coated on the surface of the electrode, dried at 4°C, rinsed with deionized water to fix the oxidase, repeated drip-coating and rinsing, repeated 1-4 times, dried for use; (6) The working electrode and reference electrode of the microelectrode are coated with a diffusion-limiting layer and an anti-interference hydrophilic layer: the polymer of the diffusion-limiting layer can be any one of Nafion, polyurethane, polyvinyl alcohol, polycarbonate or polyvinyl chloride One, prepared by soaking or dripping method. The anti-interference hydrophilic layer can be any one of Nafion, polyvinyl alcohol, chitosan or modified chitosan, polyethylene glycol or oligosaccharides, and is prepared by soaking or drip coating. (7) Laser cutting is used to batch cut the coated substrate into the required size of the micro-electrode.