CN110618179A - Glucose electrochemical microelectrode sensor based on nano porous metal film - Google Patents
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Abstract
The invention discloses a preparation method of a nano porous metal film, glucose oxidase is loaded on the porous film, a microelectrode sensor for monitoring blood sugar is used, and the invention relates to a processing technology of the porous metal film and a sensor technology. The method comprises the following steps of carrying out magnetron co-sputtering on metals and metal oxides with different proportions and thicknesses on a polyimide substrate, treating with deionized water or a weak acid solution, removing the metal oxides, leaving metal layers with three-dimensional porous network structures and connected with each other, serving as a supporting layer and a conducting layer for loading Glucose Oxidase (GOD), coating Ag/AgCl on the other surface of an insulating substrate as a reference-counter electrode to form an electrochemical two-electrode system, coating a high polymer as a diffusion limiting layer, and forming the glucose sensing microelectrode after an anti-interference hydrophilic layer is coated. The specific surface area of the nano porous gold film is large, the active area of the electrode and the GOD loading capacity are effectively increased, the permeability is good, the conductivity is high, and the physical damage of a microelectrode enzyme film sensing layer can be protected to a certain extent. The microelectrode sensor has wide linear range and good long-term stability.
Description
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 preparation methods thereof.
Background
Diabetes is one of common chronic diseases seriously threatening human health, and the real-time monitoring of blood sugar is beneficial to patients and doctors to evaluate the state of illness, and make a reasonable and scientific treatment scheme or adjust the existing scheme, so that the glucose sensor is also the most researched and most commercialized biosensor in the field of biosensors. The most central component of a glucose sensor is Glucose Oxidase (GOD), which is effectively immobilized on an enzyme electrode consisting of a bioactive interface. In practical application or the blood sugar monitoring sensor sold in the existing market generally adopts needle-type microelectrodes, which have small wound and can reduce the immune response of human physiology. For example, the blood glucose index can be obtained by monitoring the glucose level in tissue fluid without collecting fingertip blood, by obtaining a free transient glucose monitoring system sensor (Freestyle Navigator) from Abbott, Authority Food and Drug Administration (FDA), a diameter of a sensor probe is less than 0.4 mm, and the sensor probe is inserted 5mm below the skin. However, due to the miniaturization and size limitation of the electrode, the loading area of GOD is greatly reduced, the amount of loaded enzyme is insufficient, the linear range, sensitivity and other performances of the sensor are reduced, even the sensor cannot work normally, and the upper limit of the linear region of the glucose sensor must reach 30mM according to clinical requirements. New materials with excellent performance researched in laboratories are often not applicable due to no spatial fixation.
Under the requirement of not increasing the size of the microelectrode, two approaches are mainly used for solving the problem of loading GOD enzyme. One is to increase the number of microelectrodes, for example, the needle glucose sensor for subcutaneous tissue implantation real-time monitoring proposed in domestic patent CN200910097842.2 (publication No. CN 101530327B) and CN200410101080.6 uses one or two needle working electrodes and a reference electrode to form a two-electrode system, 2-3 electrodes are required to be implanted for measurement when implanted, the domestic patent CN201510783056.3 (publication No. CN 105266826B) proposes a needle glucose sensor for subcutaneous tissue implantation, and the bottom of the base is provided with a split three-electrode system of a working electrode, a counter electrode and a reference electrode. These methods increase the area of the wound. The other 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 nano sheets of graphene, zinc oxide and the like in a modifier on the surface of the electrode. The simplest three-dimensional network structure is obtained by directly preparing electrodes with surface films with three-dimensional structures, such as mesoporous carbon film electrodes, porous silicon film electrodes, porous metal film electrodes and the like. The domestic patent CN201510750828.3 discloses a porous active graphene microelectrode which realizes direct electrochemistry of enzyme and rapid electrochemical determination of glucose after glucose oxidase is fixed; in the domestic patent CN201810700292, hollow tubular graphene fibers are filled with glucose oxidase, the glucose oxidase is coated inside continuous graphene, so that an excellent enzyme protection effect is achieved, and the charge is rapidly transferred and transmitted due to excellent graphene conductivity; patent CN201710592096 provides a gold film electrode with roughened surface, and an electrochemical hydrogen peroxide sensor is constructed based on the gold film electrode. However, generally speaking, the three-dimensional structure of the electrode surface has thickness limitation, and increasing the thickness increases the probability of coating film falling off, which affects the durability and stability of the sensor electrode, so that a method for obtaining a coating film with firm and difficult falling off, and a simple and reliable method for obtaining a three-dimensional structure surface electrode are needed to be invented.
The nano porous metal film is a metal material with a nano pore structure, a large number of three-dimensionally communicated nano-scale pores and frameworks are arranged inside the nano porous metal film, and the nano porous metal film not only has the characteristics of conductivity, ductility and the like of metal, but also has the special properties of nano materials such as small-size effect, surface effect, quantum size effect, quantum tunnel effect and the like. In addition, the porous structure of the composite material also has the characteristics of high specific surface area, low density, good permeability, high conductivity and the like. The nano-porous gold membrane is widely applied to the fields of catalysis, sensing, separation, filtration, biological materials, fuel cells and the like at present, the high specific surface area of the porous metal membrane can increase the active area of an electrode and the load capacity of electroactive substances, the nano-porous structure and the metal conductivity enable the nano-porous gold membrane to have high catalytic activity on certain substances, and the nano-porous gold membrane also shows good stability and reproducibility and is easy to recycle.
The preparation method of the nano-porous metal mainly comprises an alloy removing method, a template method and an electrochemical method. The template method is to load a metal on a pre-established template through a certain technology, and then remove the template through physical, chemical and other methods, thereby obtaining a porous target metal, wherein the commonly used templates include ionic solution polymers, biological templates, porous alumina and the like. The template method can control the shape and size of the obtained nano-porous metal well, but due to the constraint of the template, the adjustment of the structure or size of the porous metal can be realized only by readjusting the template, and meanwhile, the template is removed by adopting a proper method, so the operation is complex and tedious. An electrochemical method, in which a pure metal is used as a substrate, and a voltage is continuously applied in a certain solution to anodize the metal, so that a part of the metal on the surface is gradually dissolved in an electrolyte, thereby obtaining a nanoporous metal film. Although the method is generally applied, as described in the above-mentioned patent CN201710592096, the obtained surface is often only roughened, the pores cannot reach the deep inside, the pore diameter of the pores is not easy to adjust, the transverse and radial speeds during corrosion are difficult to control, the obtained pore diameter is not ideal, when the pore diameter is too small (< 5 nm), proteins such as GOD cannot enter the pores, and when the pore diameter is too large (> 200nm), the fixed GOD is easy to fall off. On the other hand, metals dissolved in the electrolyte by corrosion are difficult to recycle, and waste of metals and environmental pollution are caused. The dealloying method is to selectively dissolve one or more metals in the alloy in a corrosive solution by utilizing the activity difference between different metals, so as to obtain the nano-porous metal with a single component. The dealloying method is widely researched in an Au-Ag alloy system, gold and silver can form a single-phase infinite solid solution, two metal components have a large enough standard electrode potential difference, silver atoms can be corroded and dissolved, and residual gold atoms are diffused and aggregated on an interface to form a nano-porous gold skeleton. The dealloying method is simple and convenient to operate on the whole, the prepared nano structure is high in specific surface area and uniform in structure, the aperture can be obtained by dynamic adjustment and control of the corrosion process, and the dealloying method is suitable for large-scale industrial production. However, when the method is used, the alloy with the specific composition and the same crystal phase needs to be prepared in advance, most metals are difficult to find an applicable alloy system, the application range is narrow, the gold component needs to be condensed again in the gold film forming process, the original rigidity is damaged, the adhesive force of the film is reduced, and the film is easy to deform and fall off.
Disclosure of Invention
Aiming at the problems of low sensor sensitivity, narrow linear range and the like caused by the limitation of size and small enzyme loading quantity of a microelectrode glucose oxidase sensor in the prior art, the invention provides a microelectrode preparation method based on a three-dimensional porous metal film and a glucose sensor preparation method. The preparation of the porous metal film in the invention does not need a template, the pore diameter of pores is easy to adjust, the bonding force with a substrate is strong, the porous metal film is not easy to fall off, the method has wide application range, a series of metals including gold, platinum and the like can obtain a porous structure by the method, and the porous metal film is used for loading a large amount of glucose oxidase, improving the sensitivity of the sensor and increasing the linear range.
The invention is realized by the following technical scheme:
1. the present invention first provides a porous metal film, which comprises:
a polyimide substrate;
a porous metal film on the substrate;
the thickness of the porous metal film is 50-200 nm;
the gap of the porous metal film is 10-100 nm;
the applicable metal has the characteristics of good conductivity, stable chemical property, difficult oxidation at room temperature, good biocompatibility and the like, and can be any one of gold, platinum, iridium, rhenium, tungsten, tantalum, hafnium, silver, palladium, rhodium, ruthenium and titanium, or an alloy consisting of the gold, the platinum, the iridium, the rhenium, the tungsten, the tantalum, the hafnium, the silver, the palladium, the rhodium, the ruthenium and the titanium;
the thickness of the polyimide substrate of the support substrate of the porous metal film can be selected from 0.05-1mm, and is preferably 0.5 mm;
2. the invention provides a preparation method of a porous metal film, which comprises the following steps:
on the polyimide substrate, double-target magnetron co-sputtering is adopted, mixed films of metal and metal oxide (such as MgO and CaO) with different thicknesses and different proportions are deposited, and the mixed films are soaked in deionized water or weak acid solution to remove the metal oxide, so that the three-dimensional porous metal layer is left.
Preferably, the magnetron sputtering metal and metal oxide target material has a standard size of 2 inches and a purity of 99.999 percent;
preferably, the magnetron sputtering substrate is heated to 200 ℃;
preferably, the metals and metal oxides are sputtered using a radio frequency power supply;
magnetron sputtering under pressure of 3-10 mTorr, preferably 5 mTorr, at substrate rotation speed of 10-30 rpm, preferably 20 rpm;
the metals and the metal oxides with different proportions can be as follows: 15%, 30%, 45%, 60%, 70%, 80%, preferably 70%, the larger the metal oxide content is, the larger the pore diameter of the prepared metal membrane is, and the pore diameter can be adjusted from 10-100 nm.
The thickness of the metal and metal oxide mixture film is different, and can be selected from 50-200nm, preferably 100 nm;
the thickness of the metal and metal oxide mixture film is different, and can be selected from 50-200nm, preferably 100 nm;
the solution washing lasts for 10-60 minutes, preferably 30 minutes, and is repeated for three times;
3. based on a porous metal film, the invention constructs a microelectrode glucose sensor, which comprises:
a polyimide substrate;
a porous metal film on the substrate;
a modification layer on the porous metal film and a modification layer on the substrate;
the substrate is divided into two surfaces A and B, wherein the surface A is provided with a porous metal film according to claims 4 and 5, and an enzyme electrode is prepared as a sensor working electrode after glucose oxidase is filled and fixed in pores of the metal film; and coating silver/silver chloride on the surface B as a counter electrode-reference electrode to form the two-electrode electrochemical glucose sensor.
The surface A of the porous metal surface for fixing the glucose oxidase is a working electrode, the surface B coated with silver/silver chloride is a reference electrode, and a platinum wire is a counter electrode, so that the three-electrode electrochemical glucose sensing system is formed.
The porous metal film working electrode modification layer is an enzyme film sensing layer, a limiting diffusion layer, an anti-interference hydrophilic layer and an optional catalysis layer from inside to outside.
The enzyme membrane sensing layer comprises: glucose oxidase, glutaraldehyde cross-linking agent, bovine serum albumin, and optionally nano-gold, nano-platinum, carbon nano-tube and other nano-particle components, and the GOD is fixed in the pores of the gold film by drop coating or soaking or spraying.
The catalytic layer is arranged between the enzyme membrane sensing layer and gold, can be selected from noble metal nanoparticles such as nano platinum and the like, electronic mediators represented by platinum black, ferrocene, Prussian blue and the like, and is prepared by an electrochemical deposition method.
The diffusion limiting layer can be made of any one of polyurethane, polyvinyl alcohol, polycarbonate or polyvinyl chloride by a soaking or drop coating method.
The anti-interference hydrophilic layer can be any one of polyvinyl alcohol, chitosan or modified chitosan, polyethylene glycol or oligosaccharide.
And the counter electrode-reference electrode on the B surface of the substrate comprises silver metal, silver chloride, a diffusion limiting layer and an anti-interference hydrophilic layer. The silver/silver chloride is obtained by plating a layer of silver by a magnetron sputtering method and chloridizing or directly coating Ag/AgCl slurry.
4. The invention provides a preparation method and steps of a microelectrode glucose sensor based on a porous metal film, which mainly comprise the following steps:
(1) cleaning and drying the substrate: ultrasonically cleaning the mixture for 10 minutes by using ethanol and isopropanol respectively, and drying the mixture by using nitrogen; oxygen plasma treatment can be selected, so that the hydrophilicity of the surface of the substrate is increased, the coating thickness is more uniform, and the roughness is smaller;
(2) preparing a porous metal film: carrying out magnetron co-sputtering on metal and magnesium oxide mixed films with different proportions and different thicknesses on a clean substrate, and cleaning in deionized water to form a porous metal film;
(3) preparing a silver/silver chloride electrode, namely firstly utilizing evaporation or magnetron sputtering of a layer of 50-200nm silver metal on the opposite surface of a porous metal film of a substrate, respectively leading out leads on metal surfaces on two sides by utilizing silver paste or welding technology, and preparing the silver/silver chloride electrode by utilizing one of the following methods:
in the constant current electroplating technology, a silver metal film is taken as an anode, a porous metal film is taken as a cathode, the current is 0.1-0.3mA, the silver metal film is immersed in 0.1mol/L hydrochloric acid solution, chlorinated for 2-6 hours, and then washed by deionized water;
electroplating by adopting a constant voltage method, wherein a silver metal film is taken as an anode, a porous metal film is taken as a cathode, 5V direct current voltage is applied to two sides of the electrode, an electrolyte is 0.1mol/L hydrochloric acid solution, the duration is 10-30 minutes, and the silver metal film is cleaned by deionized water;
(4) and (3) modifying the catalytic layer by using the porous metal film: the catalytic layer between the enzyme membrane layer and the inner wall of the metal hole can be selected from platinum nanoparticles, Prussian blue and the like, and is prepared by any one of the following methods:
direct potentiostatic electrodeposition of Pt nanoparticles in chloroplatinic acid solution: connecting porous gold membrane electrode as working electrode, platinum wire as counter electrode, Ag/AgCl as reference electrode to electrochemical workstation, and immersing the electrode in H2PtCl6In the solution, platinum nanoparticles are deposited by a potentiostatic method under a potential of-0.25V for 120s, and the solution is washed by deionized water and then is subjected to a reaction at a concentration of 0.5mol/LH2SO4Scanning in the solution until the solution is stable;
cyclic voltammetry electrodeposition of Pt nanoparticles: 0.5-5.0mmol/L HPtCl4In the solution, in the range of 1.5-0.3V, scanning for 5-50 circles with cyclic voltammetry at the scanning speed of 50-150mV/s, and depositing Pt nano particles by a one-step method;
two-step cyclic voltammetry electrodeposition of Pt nanoparticles: placing a porous gold film electrode at K2SO4And (3) performing cyclic voltammetry scanning in the solution to activate the surface. Placing the activated electrode at 2M K2PtCl4And 0.1M K2SO4The obtained electrode is placed in H2SO4And (3) performing cyclic voltammetry scanning in the solution to convert the platinum complex into platinum nanoparticles.
Firstly, preparing platinum nano sol and then adsorbing by soaking: dissolving 129.4 mg of chloroplatinic acid in 91.5mL of water, dissolving 5mg of polyvinylpyrrolidone (PVP) in 5mL of water, mixing the two solutions, slowly adding 1mL of 0.1mol/L sodium borohydride while stirring, and standing the obtained mixed solution at room temperature for 24 hours; immersing the gold film electrode into 0.1 wt% octadecyl trimethyl ammonium chloride (STAC) solution, standing for 5 seconds, taking out and airing, then placing in platinum nano sol for 30 minutes, electrostatically adsorbing a platinum nano particle catalyst layer, taking out and washing with deionized water, removing platinum nano particles with non-immobilized surfaces, and airing;
electrodeposition of prussian blue: one surface of a porous metal film is taken as a working electrode, an Ag/AgCl surface is taken as a reference electrode, a Pt wire is taken as a counter electrode, and KCl and K are contained3[Fe(CN)6]、FeCl3Carrying out electrochemical deposition in mixed electrolyte of HCl, using constant voltage of 0.4V and deposition time of 10-30s to obtain Prussian blue modified porous metal membrane electrode,
(5) immobilization of glucose oxidase on the working electrode: adopting a classical chemical crosslinking method, taking Glutaraldehyde (GA) as a crosslinking agent, preparing oxidase mixed solution from Glucose Oxidase (GOD) and Bovine Serum Albumin (BSA), fully and uniformly mixing, dripping the mixed solution on the surface of an electrode, drying the electrode at 4 ℃, washing the electrode with deionized water to obtain immobilized oxidase, repeatedly dripping and washing the immobilized oxidase, repeatedly repeating the dripping and washing for 1 to 4 times, and drying the immobilized oxidase for later use;
(6) coating a diffusion limiting layer and an anti-interference hydrophilic layer on a working electrode and a reference electrode of the microelectrode: the diffusion-limiting layer polymer can be any one of Nafion, polyurethane, polyvinyl alcohol, polycarbonate or polyvinyl chloride, and is prepared by a soaking or drop coating method. The anti-interference hydrophilic layer can be prepared by any one of Nafion, polyvinyl alcohol, chitosan or modified chitosan, polyethylene glycol or oligosaccharide through a soaking or drop coating method.
(7) And cutting the coated substrate into the size required by the microelectrode in batches by adopting laser cutting.
Compared with the prior art, the invention has the following beneficial effects:
(1) the porous three-dimensional metal film provides an active interface with high specific surface area, can be used as a supporting substrate for further modification, and greatly increases the GOD loading, so that the prepared glucose has a wider response range (0.025mmol/L-25.5mmol/L) and high sensitivity (8.20 muA/mM);
(2) the porous metal film is used as a supporting layer of the modification layer and is also used as a conductive layer of the sensor, and the metal has good conductive property, so that charges can be rapidly transferred and transmitted;
(3) the GOD extends into pores of the porous metal film, has a certain protection effect on enzyme, and the nano-sized metal frame has a certain catalytic effect on the detected intermediate product hydrogen peroxide and can amplify current signals;
(4) the preparation of the porous metal film does not need a template, and the method has wide application range, and the metal of gold, platinum, iridium, rhenium, tungsten, tantalum, hafnium, silver, palladium, rhodium, ruthenium and titanium or the alloy thereof listed in the patent can be used;
(5) the preparation of the porous metal film can adjust the pore diameter and the density of the pores of the porous metal film by adjusting the proportion of co-sputtered MgO or CaO, and besides loading GOD, the porous metal film can also load modifiers such as nano-particles with different sizes and the like;
(6) the working electrode and the reference electrode-counter electrode of the sensor are integrated on two surfaces of a single microelectrode, so that the wound can be reduced when the microelectrode is implanted;
(7) the substrate selected by the invention has good rigidity and toughness, is not easy to break when implanted, and does not need to be additionally provided with a guide pin;
(8) the manufacturing method of the microelectrode glucose electrochemical sensor is simple and feasible in film coating, good in repeatability, capable of being prepared in large scale and in batch, and has great application value and prospect.
Detailed Description
For the purpose of facilitating an understanding of the present invention, the present invention will now be described by way of examples. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.
Example 1
A method for preparing a porous gold film microelectrode takes polyimide as a substrate, gold and magnesium oxide are subjected to magnetron co-sputtering, and an acetic acid aqueous solution is used for cleaning and removing MgO, so that the surface of a porous gold film is obtained, and the method comprises the following specific operation steps:
(1) taking a commercial polyimide substrate with the length, width and thickness of 8mm, 2mm and 0.5mm respectively, treating the substrate with oxygen plasma for 30min, cleaning the substrate with water, ethanol and isopropanol respectively, and drying the substrate with nitrogen for later use;
(2) the gold and MgO are co-sputtered by magnetron sputtering, the purity of the gold and magnesium oxide target material is 99.999 percent, the size of the target material is 2 inches, the pressure is 5 millitorr, the rotating speed of the substrate is 20 r/min, and the substrate is heated to 200 ℃.
(3) The magnesium oxide uses 150W, and the gold uses different powers to obtain co-sputtered films with different proportions:
(4) placing the obtained coating film and the substrate in deionized water, soaking for 30 minutes, replacing the deionized water, soaking again, carrying out the process for three times, taking out nitrogen and drying for later use;
(5) plating a silver film on the back of the substrate by magnetron sputtering, wherein the thickness of the silver film is 100nm, and the substrate is heated to 200 ℃ in the sputtering process so that the silver film is more firmly attached to the substrate;
(6) adopting gold wire ball bonding technology, leading out two gold wire electrodes on the surfaces of the porous gold and silver metal, wherein the distance between the two gold wire electrodes and the tail end of the substrate is 1 mm;
(7) taking a silver metal film as an anode and a gold film as a cathode, applying 0.25mA direct current to two ends of the silver metal film and the gold film, chloridizing for 5 hours by using 0.1mol/L hydrochloric acid as electrolyte, and washing for 3 times by using deionized water;
(8) the obtained microelectrode with one surface being a porous gold surface and the other surface being a silver/silver chloride surface.
Example 2
The second embodiment of the invention provides a glucose electrochemical biosensor based on the preparation of the porous gold film microelectrode of the first embodiment, which comprises the following specific operation steps:
(1) selecting a sample sputtered with 70% of magnesium oxide proportion and with the total thickness of 100nm to carry out the embodiment, wherein under the condition, the pore diameter of the pore is in the range of 20-100 nm;
(2) modifying a layer of Prussian blue in pores of the porous gold film, wherein the method comprises the following steps:
immersing microelectrode in concentrated HNO with volume ratio of 1:3:43:HCl:H2O solution for 3 min, deionized water to clean the electrode surface, and ethanolRepeatedly cleaning with deionized water, and blow-drying with nitrogen;
one surface of the porous gold film is taken as a working electrode, an Ag/AgCl surface is taken as a reference electrode, a Pt wire is taken as a counter electrode, and the counter electrode is connected with an electrochemical workstation and contains 0.1mol/LKCl and 2.5mmol/LK3[Fe(CN)6]、2.5mmol/LFeCl3Carrying out electrochemical deposition in mixed electrolyte of HCl and 0.1mol/L HCl for adjusting the pH value of the electrolyte to 1.0-2.5, using constant voltage of 0.4V and deposition time of 10s to obtain a Prussian blue modified porous gold film electrode, wherein the Prussian blue is spherical and has the thickness of about 15 nm;
activating the obtained Prussian blue modified electrode in a mixed solution of 0.1mol/L HCl and 0.1mol/L KCl, and performing cyclic voltammetry scanning by using a voltage of-50-350 mV at a sweep rate of 50mV/s for 50 times; then, in 0.1mol/LpH 6.8.8 phosphoric acid buffer solution containing 0.1mol/L KCl, polarizing for 100s by using a constant potential of-50 mV, washing by using deionized water, drying by using nitrogen, placing in a drying oven at 90 ℃, and baking for 1 hour to completely dry the modified microelectrode for later use;
(3) fixing glucose oxidase on a Prussian blue modified porous gold membrane electrode by adopting a chemical crosslinking method, wherein the method comprises the following steps:
preparing 4mg/mL GOD enzyme solution and 4mg/mL bovine serum albumin solution by using 0.1mol/L phosphate buffer solution (pH 7.0), mixing the two solutions with equal volumes, performing ultrasonic dispersion for 10 minutes, carefully dripping 2-4 mu L of the obtained mixed solution on the surface of a Prussian blue modified porous gold membrane electrode, drying in the air, dripping 1.5-4% (v/v) glutaraldehyde solution on the surface of the enzyme membrane, and performing crosslinking and fixing for 30 minutes in a water bath at the crosslinking temperature of 35 ℃. Repeatedly washing the surface of the electrode by 0.1mol/L phosphate buffer solution, repeatedly performing the step of dripping GOD and glutaraldehyde for 2-4 times, and drying in the air at 4 ℃ to obtain the porous gold membrane working electrode for fixing glucose oxidase.
(4) In the embodiment, a layer of Nafion film is modified on the surfaces of a working electrode and a reference electrode of a microelectrode to increase the hydrophilicity and the anti-interference capability of the sensor, and 0.1mol/L phosphate buffer solution and 5% Nafion ethanol solution are mixed according to the proportion of 10:1 to prepare diluted Nafion solution; 2-4 μ L of the Nafion solution was applied dropwise to both sides of the electrode and air dried for 30 minutes.
(5) All electrodes are placed in 0.1mol/L PBS (pH 7.0) before testing and scanned under 0-1.0V voltage until a stable voltammogram is obtained, and are reconstructed under 0.1mol/L PBS (pH 7.0) and cyclic voltammogram scanning under 0-1.0V voltage for 20 circles (scanning speed is 50mV/s), when the electrodes are idle, the electrodes are kept under the condition of constant humidity at 4 ℃, the modified surface level is upward, and the electrodes are protected against dust by a beaker buckle cover.
(6) The response of the porous gold film glucose electrochemical microelectrode sensor to glucose with different concentrations.
A conventional three-electrode test system is adopted, a constructed porous gold film microelectrode is taken as a working electrode, Ag/AgCl is taken as a reference electrode, a platinum wire (diameter is 1mm) is taken as a counter electrode, 20mL of PBS (pH 7.0) buffer solution is taken as electrolyte, the response current of an enzyme electrode to glucose is measured under the electrode potential +0.6V vs. Ag/AgCl, and cyclic voltammetry scanning is carried out on the enzyme electrode under the voltage of 0 to + 0.6V. The current was measured by slowly stirring with a magnetic stirrer to allow the glucose molecules to diffuse uniformly in the buffer. All tests were performed at room temperature.
The glucose solution is prepared by deionized water, a few drops of 0.1mol/L HCl are added to adjust the pH value to acidity to prevent the growth of microorganisms, and the newly prepared glucose solution is kept stand overnight to fully convert alpha-glucose into GOD substrate beta-glucose.
The porous gold film glucose electrochemical microelectrode sensor generates a certain current response to the gradually increased glucose concentration, and the glucose concentration is 0.025, 0.05, 0.1, 0.15, 0.2, 0.25, 0.35, 0.45, 0.55, 0.65, 0.8, 0.9, 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 and 25.5mmol/L in sequence.
The modified porous gold membrane electrode has quick and sensitive response to glucose, and the time for reaching the maximum response current of 95 percent is less than or equal to 8s, which shows that H2O2Diffusion in the modified film is easy, and the glucose concentration between 0.025mM and 2.15mM conforms to the linear equation: i.e. iThe sensitivity of the sensor is 8.20 mua/mM and the detection limit is 8.2mol/L (S/N: 3), which is obtained from the formula (mua) ═ 0.79525+8.19829C (mmol/L), the correlation coefficient is 0.9983(RSD ═ 3.2-4.7%, and N ═ 5).
(7) And (3) testing the anti-interference performance of the porous gold film glucose electrochemical microelectrode sensor.
The Prussian blue modified porous gold glucose biosensor has good anti-interference capability on time current response display electrodes of 1mmol/L glucose, 0.1mmol/L Ascorbic Acid (AA), 0.5mmol/L Uric Acid (UA) and 0.1mmol/L Acetaminophen (AP) by adding an interferent with physiological concentration, and the current generated by the interferent basically has no influence on the detection of the glucose.
(8) And (3) testing the stability of the porous gold film glucose electrochemical microelectrode sensor.
The response current of the porous gold film glucose electrochemical microelectrode to 1mmol/L glucose is continuously measured within 45 days, the current is slowly reduced along with the change of time, and the response current of the glucose biosensor is only reduced by 15.7 percent relative to the maximum response current after 45 days, which shows that the gold film and the gold film modifier have good hydrophilicity and biocompatibility, play a role in protecting enzyme, are beneficial to the maintenance of enzyme activity, and ensure that the electrode has good stability.
Drawings
FIG. 1 is a schematic diagram of a porous metal membrane preparation and immobilization of glucose oxidase to prepare a glucose sensor;
FIG. 2 is an electron scanning microscope picture of a porous gold film electrode after modification of Prussian blue, a GOD enzyme layer and a Nafion film;
FIG. 3 is a plot of the chronoamperometric response of Prussian blue modified porous gold GOD electrodes to different glucose concentrations, tested in 20mL of pH7.0 phosphate buffer, test potential +0.6V, with electromagnetic stirring, room temperature, and the arrow marks the total glucose concentration after a single sample application.
Fig. 4 is a working curve of the prussian blue modified porous gold glucose biosensor (n is 5, RSD is 3.2-4.7%). The inset is a linear fit of the working curve: linear range: 0.025-2.15 mmol/L.
FIG. 5 is a time current response of a 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 of the prussian blue modified porous gold glucose biosensor to 1 mmol/glucose over 45 days (n-5), error bars represent standard deviations. Tested in 20mL pH7.0 phosphate buffer, +0.6V vs. Ag/AgCl; stirring by electromagnetic stirring at room temperature.
The applicant states that although the present invention has been described in detail in the above examples, the present invention is not limited to the above detailed process equipment and process flow, i.e. it is not meant to imply that the present invention must be implemented in the above detailed process equipment and process flow, and the above description should not be taken as limiting the present invention. From reading the above, it will be apparent to those skilled in the art that any improvements, modifications and alternatives to the present invention will be apparent. Accordingly, the scope of the invention should be determined from the following claims.
Claims (6)
1. A porous metal film, comprising:
a polyimide substrate;
a nanoporous metal film on the substrate;
the thickness of the porous metal film is 50-200 nm;
the gap of the porous metal film is 10-100 nm.
2. The porous metal film of claim 1, wherein the metal of the porous film is characterized by good conductivity, chemical stability, low room temperature oxidation resistance, and good biocompatibility, and may be any one of gold, platinum, iridium, rhenium, tungsten, tantalum, hafnium, silver, palladium, rhodium, ruthenium, and titanium, or an alloy thereof.
3. The porous metal membrane support substrate of claim 1, wherein the polyimide material is preferably 0.5mm thick.
4. A method for producing a porous metal film, characterized by:
on the polyimide substrate, double-target magnetron co-sputtering is adopted to deposit mixed films of metal and metal oxide (such as MgO and CaO) with different thicknesses and different proportions, and the mixed films are cleaned by deionized water or weak acid solution to dissolve the metal oxide, so that the three-dimensional porous metal layer is left.
Preferably, the magnetron sputtering metal and metal oxide target material has a standard size of 2 inches and a purity of 99.999 percent;
preferably, the magnetron sputtering substrate is heated to 200 ℃;
preferably, the metals and metal oxides are sputtered using a radio frequency power supply;
magnetron sputtering under pressure of 3-10 mTorr, preferably 5 mTorr, at substrate rotation speed of 10-30 rpm, preferably 20 rpm;
the metals and the metal oxides with different proportions can be as follows: 15%, 30%, 45%, 60%, 70%, 80%, preferably 70%;
the thickness of the metal and metal oxide mixture film is different, and can be selected from 50-200nm, preferably 100 nm;
the solution wash lasts 10-60 minutes, preferably 30 minutes, repeated three times.
5. A microelectrode glucose sensor based on a porous metal film, comprising:
a substrate as recited in claims 1-5;
a porous metal film on the substrate as set forth in claims 1 to 5;
a modification layer on the porous metal film and a modification layer on the substrate;
the substrate is divided into two surfaces A and B, wherein the surface A is provided with a porous metal film according to claims 4 and 5, and an enzyme electrode is prepared as a sensor working electrode after glucose oxidase is filled and fixed in pores of the metal film; and coating silver/silver chloride on the surface B as a counter electrode-reference electrode to form the two-electrode electrochemical glucose sensor.
The surface A of the porous metal surface for fixing the glucose oxidase is a working electrode, the surface B coated with silver/silver chloride is a reference electrode, and a platinum wire is a counter electrode, so that the three-electrode electrochemical glucose sensing system is formed.
The porous metal film working electrode modification layer is an enzyme film sensing layer, a limiting diffusion layer, an anti-interference hydrophilic layer and an optional catalysis layer from inside to outside.
The enzyme membrane sensing layer comprises: glucose oxidase, glutaraldehyde cross-linking agent, bovine serum albumin, and optionally nano-gold, nano-platinum, carbon nano-tube and other nano-particle components, and the GOD is fixed in the pores of the gold film by drop coating or soaking or spraying.
The catalytic layer is arranged between the enzyme membrane sensing layer and gold, can be selected from noble metal nanoparticles such as nano platinum and the like, electronic mediators represented by platinum black, ferrocene, Prussian blue and the like, and is prepared by an electrochemical deposition method.
The diffusion limiting layer can be made of any one of polyurethane, polyvinyl alcohol, polycarbonate or polyvinyl chloride by a soaking or drop coating method.
The anti-interference hydrophilic layer can be any one of polyvinyl alcohol, chitosan or modified chitosan, polyethylene glycol or oligosaccharide.
And the counter electrode-reference electrode on the B surface of the substrate comprises silver metal, silver chloride, a diffusion limiting layer and an anti-interference hydrophilic layer. The silver/silver chloride is obtained by plating a layer of silver by a magnetron sputtering method and chloridizing or directly coating Ag/AgCl slurry.
6. The microelectrode glucose sensor based on the porous metal film as claimed in claim 6, wherein the preparation method comprises the following steps:
(1) cleaning and drying the substrate: ultrasonically cleaning the mixture for 10 minutes by using ethanol and isopropanol respectively, and drying the mixture by using nitrogen; oxygen plasma treatment can be selected, so that the hydrophilicity of the surface of the substrate is increased, the coating thickness is more uniform, and the roughness is smaller;
(2) preparing a porous metal film: carrying out magnetron co-sputtering on metal and magnesium oxide mixed films with different proportions and different thicknesses on a clean substrate, and cleaning in deionized water to form a porous metal film;
(3) preparing a silver/silver chloride electrode, namely firstly utilizing evaporation or magnetron sputtering of a layer of 50-200nm silver metal on the opposite surface of a porous metal film of a substrate, respectively leading out leads on metal surfaces on two sides by utilizing silver paste or welding technology, and preparing the silver/silver chloride electrode by utilizing one of the following methods:
in the constant current electroplating technology, a silver metal film is taken as an anode, a porous metal film is taken as a cathode, the current is 0.1-0.3mA, the silver metal film is immersed in 0.1mol/L hydrochloric acid solution, chlorinated for 2-6 hours, and then washed by deionized water;
electroplating by adopting a constant voltage method, wherein a silver metal film is taken as an anode, a porous metal film is taken as a cathode, 5V direct current voltage is applied to two sides of the electrode, an electrolyte is 0.1mol/L hydrochloric acid solution, the duration is 10-30 minutes, and the silver metal film is cleaned by deionized water;
(4) and (3) modifying the catalytic layer by using the porous metal film: the catalytic layer between the enzyme membrane layer and the inner wall of the metal hole can be selected from platinum nanoparticles, Prussian blue and the like, and is prepared by any one of the following methods:
direct potentiostatic electrodeposition of Pt nanoparticles in chloroplatinic acid solution: connecting porous gold membrane electrode as working electrode, platinum wire as counter electrode, Ag/AgCl as reference electrode to electrochemical workstation, and immersing the electrode in H2PtCl6In the solution, platinum nanoparticles are deposited by a potentiostatic method at a potential of-0.25V for 120s, and the solution is rinsed by deionized water and then is subjected to a condition of 0.5mol/L H2SO4Scanning in the solution until the solution is stable;
cyclic voltammetry electrodeposition of Pt nanoparticles: 0.5-5.0mmol/L HPtCl4In the solution, in the range of 1.5-0.3V, scanning for 5-50 circles with cyclic voltammetry at the scanning speed of 50-150mV/s, and depositing Pt nano particles by a one-step method;
two-step cyclic voltammetry electrodeposition of Pt nanoparticles: placing a porous gold film electrode at K2SO4And (3) performing cyclic voltammetry scanning in the solution to activate the surface. Placing the activated electrode at 2M K2PtCl4And 0.1M K2SO4The obtained electrode is placed in H2SO4And (3) performing cyclic voltammetry scanning in the solution to convert the platinum complex into platinum nanoparticles.
Firstly, preparing platinum nano sol and then adsorbing by soaking: dissolving 129.4 mg of chloroplatinic acid in 91.5mL of water, dissolving 5mg of polyvinylpyrrolidone (PVP) in 5mL of water, mixing the two solutions, slowly adding 1mL of 0.1mol/L sodium borohydride while stirring, and standing the obtained mixed solution at room temperature for 24 hours; immersing the gold film electrode into 0.1 wt% octadecyl trimethyl ammonium chloride (STAC) solution, standing for 5 seconds, taking out and airing, then placing in platinum nano sol for 30 minutes, electrostatically adsorbing a platinum nano particle catalyst layer, taking out and washing with deionized water, removing platinum nano particles with non-immobilized surfaces, and airing;
electrodeposition of prussian blue: one surface of a porous metal film is taken as a working electrode, an Ag/AgCl surface is taken as a reference electrode, a Pt wire is taken as a counter electrode, and KCl and K are contained3[Fe(CN)6]、FeCl3Carrying out electrochemical deposition in mixed electrolyte of HCl, using constant voltage of 0.4V and deposition time of 10-30s to obtain Prussian blue modified porous metal membrane electrode,
(5) immobilization of glucose oxidase on the working electrode: adopting a classical chemical crosslinking method, taking Glutaraldehyde (GA) as a crosslinking agent, preparing oxidase mixed solution from Glucose Oxidase (GOD) and Bovine Serum Albumin (BSA), fully and uniformly mixing, dripping the mixed solution on the surface of an electrode, drying the electrode at 4 ℃, washing the electrode with deionized water to obtain immobilized oxidase, repeatedly dripping and washing the immobilized oxidase, repeatedly repeating the dripping and washing for 1 to 4 times, and drying the immobilized oxidase for later use;
(6) coating a diffusion limiting layer and an anti-interference hydrophilic layer on a working electrode and a reference electrode of the microelectrode: the diffusion-limiting layer polymer can be any one of Nafion, polyurethane, polyvinyl alcohol, polycarbonate or polyvinyl chloride, and is prepared by a soaking or drop coating method. The anti-interference hydrophilic layer can be prepared by any one of Nafion, polyvinyl alcohol, chitosan or modified chitosan, polyethylene glycol or oligosaccharide through a soaking or drop coating method.
(7) And cutting the coated substrate into the size required by the microelectrode in batches by adopting laser cutting.
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