WO2019015359A1 - Gold-film electrode, electrochemical biosensor electrode, sensor, and manufacturing method therefor - Google Patents

Abstract

A gold-film electrode, an electrochemical biosensor electrode, a sensor, and a manufacturing method therefor. The gold-film electrode comprises: a substrate; and a gold film on the substrate, the thickness of the gold film being 200-400 nm, the surface roughness (Ra) of the surface of the side of the gold film facing away from the substrate being 5-10 nm and the pore depth of the surface being 20-40 nm. The surface roughness (Ra) and the surface pore depth are produced by means of a roughening treatment, the roughening treatment comprising a chemical etching treatment. The electrochemical biosensor electrode comprises: the gold-film electrode and a finishing layer formed on the gold film of the gold-film electrode, the finishing layer comprising Prussian blue, the thickness thereof being 20-130 nm, and the Prussian blue existing at least partly in the form of spherical and/or cubic particles.

Description

Gold film electrode, electrochemical biosensor electrode, sensor and preparation method thereof Technical field

The invention belongs to the field of nano biomaterials and electrochemical biosensors, and relates to a surface roughening gold film electrode and a hydrogen peroxide sensor based thereon, and a preparation and detection method thereof.

Background technique

Many biological and chemical reaction processes involve the participation or production of hydrogen peroxide (H ₂ O ₂ ). Especially in living organisms, many reaction by-products of oxide enzymes contain H ₂ O ₂ . The quantitative detection mode for many hormones and metabolites in the organism, such as blood sugar, lactic acid, cholesterol, alcohol, etc., is usually based on the oxidation-reduction reaction of the enzyme-catalyzed substrate with H ₂ O ₂ to generate light and electric signals, and then these signals are detected. And finished. Therefore, in the fields of food processing, textile industry, paper bleaching, pharmaceutical, clinical medicine and disinfectant manufacturing, it has important application value for the accurate and quantitative detection of hydrogen peroxide.

At present, the analysis methods for H ₂ O ₂ mainly include fluorescence spectroscopy, chemiluminescence, spectrophotometry, and electrochemical detection. Among them, the electrochemical method is considered to be the best detection method in the preparation of biosensors because of its simple operation, low equipment cost, accurate measurement results, and especially low detectable substance concentration. The direct H ₂ O ₂ electrochemical detection method usually applies a constant potential to the surface of a chemically/electrochemically modified electrode containing platinum, gold, glassy carbon, etc., which reacts with hydrogen peroxide, and simultaneously detects the redox reaction current. achieve. Horseradish peroxidase (HRP) and Prussian blue (PB) are widely used hydrogen peroxide enzymes in recent years. Since PB's electrocatalytic reduction of H ₂ O ₂ is carried out at a low potential, many electroactive substances such as uric acid, acetaminophen and ascorbic acid can be excluded, so the PB modified electrode has a very high detection of H ₂ O ₂ . Strong selectivity.

For the substrate of the modified electrode, the magnetron sputtering gold film is chemically stable, and can be fixed in various forms of the PB functional layer, such as electrochemical deposition, screen printing, and chemical synthesis coating. At the same time, since the thickness of the gold film is small, it has the ability to be flexibly prepared, and the comfort of the sensor electrode for the living body can be improved. However, the surface roughness of a conventional magnetron-sputtered gold film is extremely low, which is disadvantageous for the fixation of an electrochemically active functional layer. By performing surface treatment, such as de-alloying to obtain a surface porous structure, etc., the stability of the sensor functional layer can be improved and the work performance can be improved. However, the nanoporous gold film obtained by dealloying has many surface cracks and is easy to be broken, and is easily separated from the substrate during the preparation process, and is not suitable for subsequent processing. Therefore, the surface treatment process of the magnetron sputtering gold film is improved, the working performance of the sensor functional layer can be improved, the brittleness of the electrode surface can be avoided, and the subsequent processing can be performed, thereby obtaining a more stable, sensitive and wider detection range. Chemical sensor.

Citation 1, which discloses a method for preparing a hydrogen peroxide electrochemical sensor, which uses a surface of a glassy carbon electrode sheet to plate a multilayer modified film, the first layer of which is naphthol green. The detection limit of the sensor is 0.9 μM, and the corresponding linear range is 8 × 10 ^-6 to 1.8 × 10 ^-4 M.

Citation 2 discloses an electrochemical sensor which electrodeposits gold nanoparticles and magnetic ferroferric oxide on the surface of a glassy carbon electrode and discloses a detection limit of 50 nM.

Citation 3 discloses a hydrogen peroxide non-enzyme electrochemical sensor and a preparation method thereof, which are modified with noble metal nanoparticles such as gold. It uses ion beam deposition to deposit precious metals, and controls the deposition amount within 5 layers. The linear range of detection of hydrogen peroxide is 4 to 44M.

Citation 4 discloses a gold microsphere-titanium nitride nanotube array composite material and a preparation method thereof, and also provides an application of the composite material in preparing an enzyme-free hydrogen peroxide electrochemical sensor. It demineralizes the titanium nitride nano-array tube on the titanium nitride substrate and forms gold microspheres on the top of the array tube.

Citation 5, which uses cyclic voltammetry to electrochemically deposit a Prussian blue film on a platinum electrode in two different electrolytes, and measures the cyclic voltammetric behavior of the modified film in a potassium chloride solution. Electrochemical impedance spectroscopy of the seed film. The electrochemical impedance spectroscopy measurements of the modified Prussian blue film platinum electrode show that the deposition conditions and the thickness of the deposited film have an effect on the electron transport process.

Therefore, it can be seen that there is still room for improvement in the prior art for obtaining high detection sensitivity, wide detection linear range, low detection limit, good use durability, and a relatively convenient preparation method.

List of cited documents

Patent literature

Citation 1: CN102043002A

Citation 2: CN101986147A

Citation 3: CN103792271A

Citation 4: CN103952763A

Non-patent literature

Citation 5: "Electrodeposition of Prussian Blue Film and Its Electrochemical Impedance Spectroscopy", Zhang Fenfen, et al., Chemical Research and Application, Vol. 15, No. 2, April 2003.

Summary of the invention

Problems to be solved by the invention

In view of the above problems, the present invention has been made in an effort to provide a surface roughening treatment of a gold film electrode, an electrochemical biosensor electrode based on the gold film electrode, and an electrochemical biosensor based on the electrodes, particularly for detecting the same At the time of the hydrogen peroxide concentration, it is possible to obtain characteristics of detection detection sensitivity, detection linear range, and detection limit and use durability.

In addition, the present invention also provides a simple and effective preparation method for preparing the above gold film electrode, electrochemical biosensor electrode, and electrochemical biosensor based on these electrodes.

Solution to solve the problem

The present invention first provides a gold film electrode comprising:

Substrate

a gold film on the substrate,

The thickness of the gold film is 200 to 400 nm.

The surface of the gold film opposite to the substrate has a surface roughness Ra of 5 to 10 nm and a surface having a pore depth of 20 to 40 nm.

According to the gold film electrode described above, the surface roughness Ra and the surface pore depth are obtained by roughening treatment, and the roughening treatment preferably includes a chemical etching treatment.

According to the gold film electrode described above, the gold thin film is formed using a sputtering deposition method.

According to the gold film electrode described above, a metal transition layer is present between the substrate and the gold thin film, and the metal transition layer contains at least one selected from the group consisting of Cr, Ti, and alloys thereof.

According to the gold film electrode described above, the metal transition layer is formed by a sputtering deposition method and has a thickness of 10 to 40 nm.

In another aspect, the present invention provides an electrochemical biosensor electrode, the electrode comprising: the gold film electrode described above, and a finishing layer formed on the gold film of the gold film electrode,

The modified layer includes Prussian blue and has a thickness of 20 to 130 nm.

At least a portion of the Prussian blue is present in the form of spherical and/or cubic particles.

According to the sensor electrode described above, the modified layer is formed by an electrochemical deposition method.

In another aspect, the present invention provides an electrochemical biosensor which is based on the gold film electrode of any of the above or the sensor electrode of any of the above.

In another aspect, the present invention provides an electrochemical biosensor for detecting hydrogen peroxide, wherein the sensor is based on the sensor described above, and the sensitivity of detecting the hydrogen peroxide by the electrochemical biosensor for detecting hydrogen peroxide is 270 to 430 mA/Mcm ² , the detection limit is 0.27 μM or more, and the linear range is 1 to X μM, wherein X is 1000 or more and 4500 or less.

In addition, the present invention provides a method of preparing a gold film electrode, the method comprising:

a step of depositing a gold thin film having a thickness of 200 to 400 nm on a substrate by a sputtering method, and

The surface of the gold thin film is roughened so that the surface roughness Ra is 5 to 10 nm and the pore depth of the surface is 20 to 40 nm.

According to the method described above, the roughening includes performing a chemical etching process.

A method according to the above, comprising the step of pre-depositing a metal transition layer comprising at least one selected from the group consisting of Cr, Ti, and alloys thereof before depositing a gold thin film on the substrate.

In addition, the present invention provides a method of preparing an electrochemical biosensor electrode, the method comprising:

a step of forming a gold film electrode as a base electrode by the method for producing a gold film electrode as described above, and a step of forming a modified layer on the base electrode,

The step of forming a modified layer on the substrate electrode is to form a modified layer by an electrochemical deposition method, the modified layer including Prussian blue,

The thickness of the gold film is 200-400 nm, and the thickness of the modified layer is 20-130 nm.

At least a portion of the Prussian blue is present in the form of spherical and/or cubic particles.

Further, the present invention provides a method for preparing an electrochemical biosensor for detecting hydrogen peroxide, comprising the method according to any one of the above, wherein the detection sensitivity of the electrochemical biosensor for detecting hydrogen peroxide is 270 ～ 430 mA/Mcm ² , the detection limit is 0.27 μM or more, and the linear range is 1 to X μM, wherein X is 1000 or more and 4500 or less.

Effect of the invention

The invention uses a magnetron sputtering gold film which is surface roughened by a chemical etching process, and deposits a film including Prussian blue by using an electrochemical deposition method, thereby obtaining a wide linear detection range based on the Prussian blue film modification. High sensitivity, low detection limit, and excellent durability, as well as a highly selective electrochemical biosensor, especially suitable for the detection of hydrogen peroxide.

In addition, the gold film electrode, the electrochemical biosensor electrode and the electrochemical biosensor for detecting hydrogen peroxide provided by the invention have simple and effective processes, are more suitable for industrial mass production, and reduce manufacturing costs.

DRAWINGS

Fig. 1 is a view showing the surface structure of a magnetron-sputtered gold film and a roughened gold film prepared in Example 1. Figure (a) shows the surface structure of the magnetron-sputtered gold film measured by atomic force microscopy (AFM), Figure (b) shows the height information curve of the line segment shown in Figure (a), and Figure (c) shows the atomic force microscope (AFM). The surface structure of the roughened gold film measured, and (d) is the height information curve of the line segment shown in (c).

Fig. 2 is a view showing the surface structure of a hydrogen peroxide sensor having a thickness of 25 nm prepared in Example 2. Figure (a) is a scanning electron microscope (SEM) image, Figure (b) is an atomic force microscope (AFM) measured PB film and gold film interface, and Figure (c) is the height of the line segment shown in Figure (b) information.

Fig. 3 is a view showing the surface structure of a hydrogen peroxide sensor having a thickness of 70 nm prepared in Example 3. Figure (a) is a scanning electron microscope (SEM) image, Figure (b) is a PB film and gold film interface measured by atomic force microscopy, and Figure (c) is the height information of the line segment shown in Figure (b).

Fig. 4 is a view showing the surface structure of a 100 nm-thickness hydrogen peroxide sensor prepared in Example 4. Figure (a) is a scanning electron microscope (SEM) image, Figure (b) is a PB film and gold film interface measured by atomic force microscopy, and Figure (c) is the height information of the line segment shown in Figure (b).

Fig. 5 is a view showing the surface structure of a 120 nm-thickness hydrogen peroxide sensor prepared in Example 5. Figure (a) is a scanning electron microscope (SEM) image, Figure (b) is a PB film and gold film interface measured by atomic force microscopy, and Figure (c) is the height information of the line segment shown in Figure (b).

Fig. 6 is a graph showing the performance of hydrogen peroxide detected by the hydrogen peroxide sensors prepared in Examples 2 to 5. Wherein a, b, c, and d are the hydrogen peroxide chronograph current curves of the hydrogen peroxide sensor described in Examples 2, 3, 4, and 5 under a constant pressure of -0.1 V (relative to Ag/AgCl).

Fig. 7 is a graph showing the relationship between the current density and the hydrogen peroxide concentration of the hydrogen peroxide sensors prepared in Examples 2 to 5. Among them, a to d are the relationship between the current density and the hydrogen peroxide concentration of the hydrogen peroxide sensor prepared in Examples 2 to 5, respectively, which are calculated and fitted by FIG.

Figure 8: Hydrogen peroxide sensors prepared in Examples 2 to 5 were tested for durability by cyclic voltammetry. In the graphs a to d, the hydrogen peroxide sensors prepared in the examples 2 to 5 were subjected to 1000 cycles of voltammetry, and the CV curve images obtained at intervals of 100 cycles were used.

Figure 9: Durability curves of the hydrogen peroxide sensors prepared in Examples 2 to 5 calculated by Figure 8, the points in the curve are divided by the first cycle volt-ampere curve by the area of the cyclic voltammogram per 100 intervals The percentage of area obtained is calculated from Figure 8.

Detailed ways

The detailed description of the various embodiments of the present invention is set forth in the claims and claims For example, in the present invention, "M" is used to mean 1 mol/liter or 1 mol/L for the sake of brevity.

<First embodiment>

A first embodiment of the present invention provides a gold film electrode comprising: a substrate, a gold thin film on a substrate, the gold thin film formed by a sputtering deposition method, having a thickness of 200 to 400 nm, and The surface of the gold film opposite to the substrate is roughened.

Base

The substrate of the present invention is not particularly limited as long as it can achieve the above effects of the present invention. As the substrate conventional in the art can be used as long as it has the required conductivity and support.

The substrate of the present invention may be selected from the group consisting of carbon, glassy carbon substrates, and semiconductor-based substrates or conductive polymer-based substrates.

Examples of the carbon-based substrate include carbon substrates such as graphite, carbon nanotubes, graphene, diamond-like carbon, and boron-doped diamond. Examples of the semiconductors include a semiconductor transparent conductive film such as silicon or ITO (indium tin oxide), IZO (indium tin oxide), AZO (aluminum-doped zinc oxide), or FTO (fluorine-doped tin oxide). Further, the conductive polymer film is obtained by processing a film or a sheet material formed of a conductive polymer. The conductive polymer is a type of polymer material in which a polymer having a conjugated π-bond is chemically or electrochemically "doped" to convert it from an insulator to a conductor. Polymer conductive materials are generally divided into two types: composite type and structural type: 1 composite type polymer conductive material, which is made of general-purpose polymer materials and various conductive materials by filling compounding, surface compounding or lamination. 2; structural polymer conductive materials. It refers to the polymer structure itself or a polymer material that has a conductive function after being doped. According to the conductivity, it can be divided into polymer semiconductors, polymer metals, and the like.

Further, the substrate of the present invention is preferably a silicon wafer from the viewpoint of obtaining an electrode having a high detection range, high sensitivity, and low detection limit.

In addition, the substrate can be subjected to the necessary cleaning and/or activation prior to use of the substrate of the present invention. The specific washing and activating means are not particularly limited, and can be carried out by a method common to the art as long as the final effect of the present invention is not impaired.

Gold film and metal transition layer

In the present invention, a method of sputter deposition is used to deposit a gold film on the substrate, and the gold film is subjected to surface roughening treatment.

Specifically, a method of magnetron sputtering deposition is used in the present invention. Sputter deposition is a technique in which a surface of a material is bombarded with a charge ion in a vacuum environment to deposit bombarded particles on the surface of the substrate. Magnetron sputtering deposition methods are generally classified into balanced magnetron sputtering and unbalanced magnetron sputtering. For the present invention, balanced magnetron sputtering is preferably used from the viewpoint of substrate size and process simplicity.

Balanced magnetron sputtering is commonly referred to as conventional magnetron sputtering. The effective control of the secondary electrons by the magnetic field, so that the disadvantage of the two-pole sputtering is its own advantages. The principle of balanced magnetron sputtering is to make the secondary electrons in the electromagnetic field perpendicular to each other, and are bound to the surface of the target to make a circular motion along the magnetic field line of the "racetrack", which improves the ionization rate of the gas even if the working pressure Decreasing to the order of 10 ^-1 ~ 10 ^-2 Pa can still increase the plasma density, which can increase the incident ion density, which is beneficial to reduce the sputtering voltage and increase the deposition rate. The secondary electrons can only be separated after the energy is exhausted. The surface of the target falls on the anode, so the substrate avoids the bombardment of secondary electrons, and the temperature rise of the substrate is low and there is no damage. Balanced magnetron sputtering can be effectively applied to the surface modification of temperature-critical substrate materials.

In the present invention, the thickness of the gold thin film is controlled to be 200 to 400 nm, preferably 200 to 350 nm, and more preferably 250 to 330 nm. If the thickness of the gold film is too small, there is a fear that the surface defects cannot be eliminated, and the detection performance of the sensor electrode is noisy or reproducible. However, if the thickness is too large, the performance of the sensor electrode cannot be significantly improved on the one hand, and the manufacturing cost is increased by the other invention.

The surface roughening treatment of the above gold film can be performed by chemical etching. Specifically, as an example, the gold film may be subjected to chemical etching by immersing it in a potassium iodide (KI) solution (also referred to as an I ₂ -KI solution) to perform surface roughening. The soaking time can be set as needed.

The surface roughened gold film has a rougher surface structure than the magnetron sputtered gold film, and the pore depth between the particles is deeper, has a larger specific surface area and reactivity, and the fixing efficiency of the PB functional layer is improved. The hydrogen peroxide electrochemical biosensor prepared by using the gold film electrode has lower detection limit and higher sensitivity. The surface roughened gold film has, for example, a surface roughness Ra of 5 to 10 nm and a surface having a pore depth of 20 to 40 nm, preferably, a surface roughness Ra of 6 to 7.5 nm, and a pore depth of the surface is 28 to 35 nm.

Further, in a preferred embodiment of the present invention, a metal transition layer is previously deposited on the substrate prior to performing the above-described sputter deposition of the gold film.

Depending on factors such as the conditions of sputter deposition control, when the gold film is directly deposited on the substrate, there is a lattice mismatch in the gold/substrate interface, which in some cases will lead to the initial formation of the gold film. The length of time also causes surface defects and unevenness to form easily when depositing a gold film. In other cases, it may also result in the adhesion of the gold film deposited on the substrate to the substrate being affected, thereby causing undesirable sensor aging in subsequent electrochemical cycles.

Therefore, pre-forming a metal transition layer between the gold film and the substrate is advantageous in avoiding the above problems. That is, in a preferred embodiment of the present invention, the metal transition layer can well solve the lattice mismatch existing between the substrate and the gold film and increase the bonding of the gold film to the substrate.

The metal transition layer may be deposited by magnetron sputtering deposition as described above, and deposited to a thickness of 10 to 40 nm, preferably 20 to 30 nm. For the thickness of the metal transition layer, if it is too small, it will not function as a lattice match, and if it is too large, the economy will be deteriorated.

The material of the metal transition layer is not particularly limited as long as it can satisfy the improvement of the interface bonding property of the base/gold film. For example, it may be selected from at least any of Cr, Ti, and alloys thereof. Preferably, in the present invention, the material of the metal transition layer is different from gold, and particularly preferably, in the present invention, the metal transition layer is formed using Cr.

<Second Embodiment>

A second embodiment of the present invention provides an electrochemical biosensor electrode, a gold film electrode, and a modified layer formed on the gold film of the gold film electrode, wherein the modified layer includes Prussian blue, and The thickness is from 20 to 130 nm, and at least a portion of the Prussian blue is present in the form of spherical and/or cubic particles.

Finishing layer

The modifying layer of the present invention is an electrochemically active material layer having a specific selectivity. Among the modified layers of the present invention, Prussian Blue (PB) is included.

In some embodiments of the invention, a layer of Prussian blue is deposited electrochemically on top of the gold film. Typically, when depositing, a gold film can be used as the working electrode, Ag/AgCl as the reference electrode, Pt wire as the counter electrode, and Fe ³⁺ and Fe(CN) ₆ ⁴⁺ , K ⁺ and HCl. The mixed solution was electrochemically deposited as an electrolytic solution, and electrochemically deposited at a constant deposition voltage of 0.4 V (relative to Ag/AgCl electrode) to obtain a PB modified electrode.

By controlling the deposition time, the deposition thickness of the PB layer can be controlled. In the present invention, the thickness of the PB layer is 20 to 130 nm. The deposition time may be from 10 to 240 s. In some cases, the deposition time may be from 5 to 260 s, and the deposition time may be appropriately adjusted as long as the desired deposition thickness is obtained.

In the present embodiment, by controlling and adjusting the thickness of the PB deposition, it is possible to obtain an electrochemical hydrogen peroxide biosensor electrode having a high detection linear range, high sensitivity, low detection limit, good durability, and strong selectivity.

In some existing studies, it has been proposed to reduce the deposition thickness of the PB layer to improve the sensitivity of the detection. However, the present inventors have found that if the thickness of the PB deposited layer is too low, the PB deposited layer may not be uniformly covered, and some defects occurring in the deposited layer during the PB deposition process may not be obtained and effectively repaired. Therefore, from this point of view, the thickness of the PB layer in the present application should be 20 nm or more and further 25 nm or more. On the other hand, the present inventors have found that when the deposition thickness of the PB layer reaches a certain level, for example, 70 nm or more, the sensitivity tends to increase as the thickness of the deposition increases. The reason is presumed as follows: With the increase of the deposition thickness of the PB layer, the microscopic morphology of PB is mainly from the (amorphous) spherical transition to the predominantly cubic (crystalline) granular shape, and the cubic (crystalline) granular shape is more favorable. The charge conduction in the PB layer increases the response current and improves the sensitivity.

Furthermore, as the thickness of the PB deposit increases, the linear range of detection is significantly broadened. The reason for speculation may be that as the thickness of the PB deposit increases, the defects on the surface of the PB are improved, and therefore, at a constant voltage, a wide linear range can be exhibited, and the linear correlation coefficient is greater than 0.99.

In addition, with the increase of the deposition thickness of PB layer, the microscopic morphology of PB transitions from mainly (amorphous) spherical to mainly cubic (crystalline) granular, and with the change of the morphology of PB layer, it affects Durability of the use of the electrode. In some cases, since the amorphous spherical particles are mainly in the initially formed PB layer, the durability increases as the thickness of the PB increases. However, after further deposition of the PB layer, cubic PB particles are gradually formed in the PB layer, and the durability hardly changes during the (amorphous) spherical transition to the predominantly cubic (crystalline) granular shape. . When the PB deposition is continued after the formation of the cubic crystal form of the PB particles, the cubic (crystalline form) granular form gradually grows, resulting in a further increase in durability. However, when the cubic PB particles in the PB layer grow to a certain extent, cracks may occur on the surface of the PB layer due to the action of the grain boundaries.

Therefore, it can be seen that for the deposition thickness of the PB layer on the gold thin film, it is necessary to consider not only excellent sensitivity but also a wide detection linear range, durability of use, and the like. Therefore, from this point of view, the PB deposition thickness of the present invention is from 20 to 130 nm, preferably from 25 to 130 nm, more preferably from 70 to 120 nm.

Other active substances

In the present embodiment, in addition to the electrochemical deposition of PB on the gold film as the active material layer, without limitation, other activities may be deposited in the same layer or in a separate layer other than the PB layer according to actual needs. substance.

The active substance may be various enzymes. There may be mentioned one or more of glucose oxidase, lactate oxidase, horseradish catalase, cholesterol oxidase, xanthine oxidase, acetylcholinesterase, and organophosphine hydrolase.

The above active materials can be deposited by conventional deposition methods in the art. Further, one or more layers of deposition may be performed as needed, provided that the technical effects of the present invention are not impaired.

<Third embodiment>

In a third embodiment of the invention, an electrochemical biosensor is provided. The sensor is based on a gold film electrode as described or defined in <First Embodiment>, or based on a sensor electrode as described or defined in <Second Embodiment>.

The above sensors can be used directly as direct sensors to detect small molecules sensitive to PB, such as hydrogen peroxide.

Further, as described above, a sensor including other active material layers can be used as an indirect sensor. The molecules or components such as glucose, protein, and the like can be detected by using the enzymes mentioned above in combination.

Further, the above-described sensor may be subjected to necessary encapsulation or any other modification as long as the effects of the present invention are not impaired. Through the necessary packaging, it can be applied to various occasions, and it is beneficial to improve the safety of use and ensure the effectiveness of detection.

Further, in the embodiment of the present invention, an electrochemical biosensor including the gold film electrode in <First Embodiment> or the sensor electrode in <Second Embodiment> is provided at the same time, and based on this, is prepared as Working electrode. In addition, the sensor further includes a reference electrode and a counter electrode. The reference electrode can be a conventional reference electrode in the art, such as a calomel electrode, a silver/silver chloride electrode, and the like. For the counter electrode, a Pt wire or a plate or the like can be used.

Typically, the product of the present embodiment is an electrochemical biosensor that can directly detect the concentration of hydrogen peroxide. Specifically, in the same electrolytic cell, the present embodiment adopts a three-electrode system, a working electrode, a reference electrode, and a counter electrode, wherein the working electrode is a surface-roughened magnetron sputtering gold film. Thereafter, a certain amount of deposition liquid is added to the electrolytic cell, and a constant voltage is applied to the working electrode, and a Prussian blue film is deposited on the surface of the gold film after a certain time. The obtained Prussian blue modified electrode was subjected to a certain chemical and physical process, and then placed in the same electrolytic cell, and the Prussian blue modified electrode was used as a working electrode to measure the hydrogen peroxide concentration. During the measurement, a constant voltage is still applied to the working electrode, a certain amount of phosphate buffer solution (PBS) is added to the electrolytic cell, and different concentrations of hydrogen peroxide solution are dropped under stirring, and the current intensity response is detected.

<Fourth embodiment>

In this embodiment, the present invention provides a method of preparing a gold film electrode, preparing an electrochemical biosensor electrode, and preparing an electrochemical biosensor for detecting hydrogen peroxide.

The method for preparing a gold film electrode includes:

a step of depositing a gold thin film having a thickness of 200 to 400 nm on a substrate by a sputtering method, and

The surface of the gold film is roughened to have a surface roughness Ra of 5 to 10 nm and a surface depth of 20 to 40 nm.

According to the above method, the roughening is performed by a chemical etching treatment.

According to the above method, in the step of forming the substrate electrode, further comprising the step of pre-depositing a metal transition layer comprising Cr, Ti, and an alloy thereof before depositing the gold thin film on the substrate At least either.

In addition, the method for preparing an electrochemical biosensor electrode of the present embodiment includes the above steps, and a step of depositing a modifying layer on the substrate electrode, wherein the step of forming a modifying layer on the substrate electrode is to form a modification by an electrochemical deposition method. a layer comprising Prussian blue, the gold film having a thickness of 200 to 400 nm, the modified layer having a thickness of 20 to 130 nm, and at least a portion of the Prussian blue being present in the form of spherical and/or cubic particles.

In addition, the method for preparing an electrochemical biosensor for detecting hydrogen peroxide according to the present embodiment includes the above steps, and optionally, may further include a step of forming or preparing another active material layer, preparing a reference electrode, a counter electrode, and optionally The necessary packaging steps.

In these steps, the apparatus or equipment to be used is not particularly limited as long as it satisfies the effects sufficient to achieve the present invention.

More specifically, the technical solution for preparing the surface roughened gold film electrode in the present embodiment is as follows:

(1) A 300 μm-thick silicon wafer is placed in a magnetron sputtering apparatus with ultra-pure chromium (chromium content ≥99.99 wt.%) as a target, first depositing a layer of 20 nm thick chromium metal; A gold content of ≥99.99 wt.% is used as a target, and a gold film having a thickness of 200 to 400 nm is sputter deposited.

(2) The obtained gold film was formed into a square gold film electrode having a size of 10 mm × 10 mm by a cutting process, and ultrasonically washed and blown dry in dilute hydrochloric acid, dilute sodium hydroxide solution, deionized water, acetone and alcohol.

(3) The gold film after pretreatment is immersed in a solution containing potassium iodide (KI) for a certain period of time, for example, 5 to 20 s, preferably 10 s. Then, it is sequentially washed with dilute sodium hydroxide solution, deionized water, acetone and alcohol, and dried.

Further, the technical solution for preparing the electrochemical biosensor electrode in the present embodiment is as follows:

(4) The surface roughened gold film electrode obtained in the above (3) is used as a working electrode, Ag/AgCl is used as a reference electrode, and Pt wire is used as a counter electrode to contain Fe ³⁺ and Fe(CN) _{6 .} A mixed solution of ⁴⁺ , K ⁺ and HCl was electrochemically deposited for the electrolyte, the deposition time was 10 to 240 s, and the deposition voltage was 0.4 V (relative to the Ag/AgCl electrode) to obtain a PB modified electrode.

(5) The obtained PB modified electrode is subjected to cyclic voltammetry activation in an electrode activation solution containing K ⁺ and HCl, the voltage range is -0.05 V to 0.35 V, the voltage scanning rate is 50 mV/s, and the number of cycles is 25 to 40. , preferably 35 times. Then, the potentiostatic polarization is carried out in a phosphate buffer, the constant potential is -0.1 to 0.1 V (relative to the Ag/Cl electrode), preferably -0.05 V (relative to the Ag/Cl electrode), and the stable polarization time is 120 to 600 s, preferably 600s. The PB modified electrode was then washed with deionized water and dried at 100 ° C for 1 h to obtain the electrochemical biosensor electrode.

Further, the deposition solution in the step (4) is 2.5 mM FeCl ₃ + 2.5 mM K ₃ Fe(CN) ₆ + 0.1 M KCl + 0.12 M HCl solution, which is prepared from the above inorganic salt and acid solution and 100 mL of deionized water.

Further, the deposition time in the step (4) was 10 s, the deposition voltage was 0.4 V (relative to the Ag/AgCl electrode), and the thickness of the PB film was 25 nm.

Further, the deposition time in the step (4) is 40 s, the deposition voltage is 0.4 V (relative to the Ag/AgCl electrode), and the thickness of the PB film is 70 nm.

Further, the deposition time in the step (4) is 120 s, the deposition voltage is 0.4 V (relative to the Ag/AgCl electrode), and the thickness of the PB film is 100 nm.

Further, the deposition time in the step (4) is 240 s, the deposition voltage is 0.4 V (relative to the Ag/AgCl electrode), and the thickness of the PB film is 120 nm.

Further, the electrode activation solution in the step (5) is 0.12 M HCl + 0.1 M KCl solution, and is prepared from the above inorganic salt and concentrated hydrochloric acid solution and 100 mL of deionized water.

Further, the phosphate buffer in the step (5) is a 0.05 M KH ₂ PO ₄ /K ₂ HPO ₄ +0.1 M KCl solution, which is prepared from the above inorganic salt powder and 100 mL of deionized water.

In the present embodiment, the above electrode can be used as a direct sensor for the detection of hydrogen peroxide. In another embodiment, with other active substance enzymes, as described above, detection of substances such as glucose, protein, etc. (ie, indirect) can be achieved. sensor).

The functional film (PB film) of the hydrogen peroxide sensor electrode of the present invention has a thickness of 20 to 130 nm, preferably 25 to 130 nm, more preferably 70 to 120 nm, and a detection limit of hydrogen peroxide of 0.27 μM or more; and a detection sensitivity of 270. ～430 mA/Mcm ² , preferably 330 to 430 mA/Mcm ² , more preferably 380 to 430 mA/Mcm ² ; linear range is 1 to X μM, wherein X is 1000 or more and 4500 or less, preferably X is 2,000 or more and 4500 or less. Preferably, X is 3,500 or more and 4500 or less.

Compared with the prior art, the beneficial effects of the present invention are mainly as follows: the present invention uses hydrogen peroxide formed by modifying a PB functional layer by using a surface roughened gold film electrode as a substrate electrode and electrochemical deposition method. The sensor has high sensitivity, wide linear range, low detection limit and excellent durability. The preparation and fixing of the functional layer is completed in one time. Compared with other sensors, the invention adopts a magnetron sputtering gold film, which has the advantages of low cost, simple operation, and comfortable living environment for flexible preparation and application.

Example

The invention is further described below in conjunction with specific embodiments, but the scope of protection of the invention is not limited thereto.

The sensitivity and durability test method for detecting hydrogen peroxide by the hydrogen peroxide sensor of the present invention is as follows:

(1) Sensitivity and detection range test:

The hydrogen peroxide sensor (Au/PB electrode) of the present invention is used as a working electrode, Ag/Cl is a reference electrode, and the Pt sheet is a counter electrode. In the 100 mL phosphate buffer (pH=6.2) described in the step (5), under a slight stirring condition (rotor rotation speed of 200-250 RMP), the chronoamperometry method is used, and the quantitative concentration of the hydrogen peroxide solution is continuously added dropwise for sensitivity. And linear range test, a constant potential is applied to the working electrode during the experiment, and the voltage range is -0.15 to 0.15 V (relative to the Ag/Cl electrode), preferably -0.1 V.

(2) Durability test:

The hydrogen peroxide sensor (Au/PB electrode) of the present invention is used as a working electrode, Ag/Cl is a reference electrode, and the Pt sheet is a counter electrode. The durability of the sensor was tested by cyclic voltammetry in 100 mL of phosphate buffer (pH = 6.2) as described in step (5). The cyclic voltammetry voltage range is -0.05 to 0.35 V (relative to Ag/AgCl electrode), the scanning rate is 50 mV/s, and the number of cycles is 600 to 1200 times, preferably 1000 times, by calculating the cyclic voltammogram area of each 100 times. Durability test.

Example 1

(1) Magnetron sputtering gold film treatment

A silicon wafer with a thickness of 200-400 μm is placed in a magnetron sputtering apparatus, and ultra-pure chromium (chromium content ≥99.99 wt.%) is used as a target. First, a layer of chrome metal having a thickness of 20 nm is deposited; then gold (gold) A content of ≥99.99 wt.% is used as a target, and a gold film having a thickness of 200 to 400 nm is sputter deposited. After that, the obtained magnetron sputtered gold film was cut into a 10 mm×10 mm square gold film electrode, and ultrasonically cleaned in dilute hydrochloric acid, dilute sodium hydroxide solution, deionized water, acetone and alcohol for 30 min, and then a nitrogen spray gun was used. Blow dry and obtain the pretreated gold film for use. The surface structure of the gold film is shown in Figures 1(a) and (b).

(2) Surface roughening treatment of magnetron sputtering gold film

The gold film obtained in the step (1) was immersed in a 30 vol.% potassium iodide (KI) solution and allowed to stand for 10 s. Then, it is sequentially washed with dilute sodium hydroxide solution, deionized water, acetone and alcohol, and dried. The surface structure of the roughened gold film is shown in Figures 1(c) and (d).

Example 2

(1) Preparation of Prussian blue modified film

Using a three-electrode system, the roughened gold film electrode treated in the step (2) of Example 1 was used as a working electrode, Ag/AgCl was used as a reference electrode, and Pt wire was used as a counter electrode to a final concentration of 2.5 mM FeCl _{3 .} , with a final concentration of 2.5 mM K ₃ Fe(CN) ₆ , a final concentration of 0.1 M KCl and a final concentration of 0.12 M HCl solution for electrochemical deposition of the electrolyte, deposition time of 10 s, deposition voltage of 0.4 V (relative A PB modified electrode having a thickness of 25 nm was obtained at an Ag/AgCl electrode.

(2) The obtained PB modified electrode was subjected to cyclic voltammetry activation in a mixed solution of HCl having a final concentration of 0.12 M and a final concentration of 0.1 M in KCl, the voltage range was -0.05 V to 0.35 V, and the voltage scanning rate was 50 mV/s. The number of cycles is 25. Then, in a 100 mL phosphate buffer (pH=6.2) containing 0.05 M KH ₂ PO ₄ /K ₂ HPO ₄ +0.1 M KCl, constant potential stable polarization, constant potential -0.05 V (relative to Ag/Cl electrode), stable pole The time is 120s. Thereafter, the PB modified electrode was washed with deionized water, dried with a nitrogen gas gun, and dried at 100 ° C for 1 h to obtain a hydrogen peroxide biosensor. The surface structure and functional layer thickness of the hydrogen peroxide biosensor are shown in FIG.

(4) Hydrogen peroxide current response test:

A hydrogen peroxide biosensor with a PB functional layer thickness of 30 nm was placed in 0.05 M phosphate buffer (pH=6.2), and under a slight agitation condition (rotor rotation speed of 200 RMP), a chronoamperometry method was used to continuously add a quantitative concentration. The hydrogen peroxide solution was tested for sensitivity and linear range, and a 0.1 V (relative to Ag/Cl electrode) constant potential was applied to the working electrode during the experiment. The results are shown in the curve a in Fig. 6 and the curve a in Fig. 7, and the detection limit of hydrogen peroxide is 0.85 μM (signal-to-noise ratio is 3), the linear range is 1 μM to 1000 μM, and the detection sensitivity is 338 mA/Mcm ² .

(5) Durability test of hydrogen peroxide biosensor

The hydrogen peroxide sensor (Au/PB electrode) of the present invention is used as a working electrode, Ag/Cl is a reference electrode, and the Pt sheet is a counter electrode. The durability of the sensor was tested by cyclic voltammetry in phosphate buffer (pH = 6.2). Cyclic voltammetry has a voltage range of -0.05 to 0.35 V (relative to Ag/AgCl electrode), a scan rate of 0.05 V/s, and a number of cycles of 1000 cycles, every 100 cycles of the volt-ampere curve and the area of the first cyclic voltammetry curve. The proportional calculation yields the cyclic voltammogram shown in a of Fig. 8 and the a curve in Fig. 9. Experimental data shows that the capacitor has a capacitance decay rate of about 35% after 1000 cycles of volt-ampere scanning.

Example 3

Using a three-electrode system, the roughened gold film electrode treated in the step (2) of Example 1 was used as a working electrode, Ag/AgCl was used as a reference electrode, and Pt wire was used as a counter electrode, and the electrolyte in Example 2 was used. Electrochemical deposition, deposition time of 40 s, deposition voltage of 0.4 V (relative to Ag / AgCl electrode) to obtain a PB modified electrode with a thickness of 70 nm. The obtained PB modified electrode was subjected to cyclic voltammetry activation in the activation solution of Example 1, with a voltage range of -0.05 V to 0.35 V, a voltage scanning rate of 50 mV/s, and a number of cycles of 35. Thereafter, the potentiostatic polarization was carried out in the phosphate buffer (pH = 6.2) in Example 2, the constant potential was -0.05 V (relative to the Ag/Cl electrode), and the stable polarization time was 240 s. Thereafter, the PB modified electrode was washed with deionized water, dried with a nitrogen gas gun, and dried at 100 ° C for 1 h to obtain a hydrogen peroxide biosensor. The surface structure and functional layer thickness of the hydrogen peroxide biosensor are shown in FIG. The detection sensitivity and linear range of the sensor for hydrogen peroxide (detection method is the same as in the second embodiment) are shown in the curve b of Fig. 6 and the curve of b in Fig. 7, and the detection limit of hydrogen peroxide is 0.27 μM (the signal-to-noise ratio is 3) The linear range is from 1 μM to 2000 μM, and the detection sensitivity is 272 mA/Mcm ² . The durability test of the electrode (test method is the same as in Example 2) is shown in Figure 8 b and Figure 9 b curve, the capacitance decay rate after 1000 cycles of voltammetric scanning is about 22.5%.

Example 4

Using a three-electrode system, the roughened gold film electrode treated in the step (2) of Example 1 was used as a working electrode, Ag/AgCl was used as a reference electrode, and Pt wire was used as a counter electrode, and the electrolyte in Example 2 was used. Electrochemical deposition, deposition time of 120 s, deposition voltage of 0.4 V (relative to Ag / AgCl electrode) to obtain a PB modified electrode with a thickness of 100 nm. The obtained PB modified electrode was subjected to cyclic voltammetry activation in the activation solution of Example 2, with a voltage range of -0.05 V to 0.35 V, a voltage scanning rate of 50 mV/s, and a number of cycles of 30. Thereafter, the potentiostatic polarization was carried out in the phosphate buffer (pH = 6.2) in Example 2, the constant potential was -0.05 V (relative to the Ag/Cl electrode), and the stable polarization time was 600 s. Thereafter, the PB modified electrode was washed with deionized water, dried with a nitrogen gas gun, and dried at 100 ° C for 1 h to obtain a hydrogen peroxide biosensor. The surface structure and functional layer thickness of the hydrogen peroxide biosensor are shown in FIG. The sensitivity and linear range of the sensor for hydrogen peroxide (see Example 2 for the detection method) are shown in the c curve in Figure 6 and the c curve in Figure 7. The detection limit of hydrogen peroxide is 0.35 μM (the signal-to-noise ratio is 3), the linear range is 5 μM to 3500 μM, and the detection sensitivity is 383 mA/Mcm ² . The durability test of the electrode (test method is the same as in Example 2) is shown in Figure 8 c and the curve c in Figure 9, and the capacitance decay rate after 1000 cycles of voltammetry is 25%.

Example 5

Using a three-electrode system, the roughened gold film electrode treated in the step (2) of Example 1 was used as a working electrode, Ag/AgCl was used as a reference electrode, and Pt wire was used as a counter electrode, and the electrolyte in Example 2 was used. Electrochemical deposition, deposition time of 240 s, deposition voltage of 0.4 V (relative to Ag / AgCl electrode) to obtain a PB modified electrode with a thickness of 120 nm. The obtained PB modified electrode was subjected to cyclic voltammetry activation in the activation solution of Example 2, the voltage range was -0.05 V to 0.35 V, the voltage scanning rate was 50 mV/s, and the number of cycles was 35. Thereafter, the potentiostatic polarization was carried out in the phosphate buffer (pH = 6.2) in Example 2, the constant potential was -0.05 V (relative to the Ag/Cl electrode), and the stable polarization time was 600 s. Thereafter, the PB modified electrode was washed with deionized water, dried with a nitrogen gas gun, and dried at 100 ° C for 1 h to obtain a hydrogen peroxide biosensor. The surface structure and functional layer thickness of the hydrogen peroxide biosensor are shown in FIG. The detection sensitivity and linear range of the sensor for hydrogen peroxide (see Example 2 for the detection method) are shown in the d curve of Fig. 6 and the d curve of Fig. 7, and the detection limit of hydrogen peroxide is 0.28 μM (the signal-to-noise ratio is 3) The linear range is from 1 μM to 4500 μM, and the detection sensitivity is 424 mA/Mcm ² . The durability test of the electrode (test method is the same as in Example 2) is shown in Figure 8 d and the d curve in Figure 9, and the capacitance decay rate after 1000 cycles of voltammetry scanning is about 15%.

Example 6 (Reference example)

The experimental conditions of Example 4 were repeated except that the time for depositing the PB film was adjusted to 280 s to obtain a thicker PB deposited film.

Example 7 (Reference example)

In the fifth embodiment, a hydrogen peroxide biosensor for comparison was obtained in the same manner as in the fifth embodiment except that the surface roughening treatment of the magnetron sputtering gold film was not performed without performing the above step (2). The detection sensitivity and linear range of the hydrogen peroxide were measured by the same detection method as in Example 2. The detection limit of hydrogen peroxide was 2.38 μM (signal-to-noise ratio was 3), and the linear range was 5 to 4500 μM. It is 281 mA/Mcm ² .

As can be seen from the above Examples 2 to 5, as the deposition time increases, the deposition thickness of the PB film gradually increases.

Example 2 shows that the deposition thickness is 30 nm in the case of a deposition time of 10 s, and the sensitivity is higher than that of Example 3 (deposition time 40 s, thickness 70 nm), showing that the thickness of the active material layer formed by the prior literature is smaller. The rule of higher sensitivity. However, Examples 3 to 5 show that the sensitivity increases as the thickness of the PB layer increases, which is different from the aforementioned rule pointed out in the prior literature, and it is presumed that it may be attributed to the change in the shape of the PB particles and the crystal form thereof.

In Examples 2 to 5, it was revealed that as the thickness of the PB film increases, the detection linear range gradually becomes wider. Therefore, the thickness of the PB film can be controlled to an appropriate range according to the data of Examples 2 to 5, Good detection sensitivity can be obtained, and a wide detection range can be achieved.

For Example 6, the same Example 6 was repeated while actually operating, and it was found that cracks appeared on the surface of some PB films, resulting in dispersion and instability of the test data, which may be due to an increase in deposition thickness (especially After the thickness of the PB film is larger than 130 nm, the cubic crystal in the PB film layer continuously grows, and the separation between the grain boundary and the grain boundary is related.

According to the comparison between Embodiment 7 and Embodiment 5, by performing the surface roughening treatment on the magnetron sputtering gold film as the sensor substrate, the sensitivity can be greatly improved, and at the same time, a wider detection range and a lower detection limit can be obtained.

Industrial availability

The gold film electrode and electrochemical biosensor of the present invention can be industrially produced and can be applied to the detection of hydrogen peroxide in a living body.

Claims (14)

1. A gold film electrode comprising:

   Substrate

   a gold film on the substrate,

   The thickness of the gold film is 200 to 400 nm.

   The surface of the gold film opposite to the substrate has a surface roughness Ra of 5 to 10 nm and a surface having a pore depth of 20 to 40 nm.
2. The gold film electrode according to claim 1, wherein the surface roughness Ra and the surface pore depth are obtained by roughening treatment, and the roughening treatment preferably includes a chemical etching treatment.
3. The gold film electrode according to claim 1 or 2, wherein the gold film is formed using a sputtering deposition method.
4. The gold film electrode according to any one of claims 1 to 3, wherein a metal transition layer is present between the substrate and the gold thin film, the metal transition layer comprising at least one selected from the group consisting of Cr, Ti, and alloys thereof .
5. The gold film electrode according to claim 4, wherein the metal transition layer is formed by a sputtering deposition method and has a thickness of 10 to 40 nm.
6. An electrochemical biosensor electrode comprising: the gold film electrode according to any one of claims 1 to 5; and a modified layer formed on the gold film of the gold film electrode,

   The modified layer includes Prussian blue and has a thickness of 20 to 130 nm.

   At least a portion of the Prussian blue is present in the form of spherical and/or cubic particles.
7. The sensor electrode according to claim 6, wherein the modifying layer is formed by an electrochemical deposition method.
8. An electrochemical biosensor comprising the gold film electrode according to any one of claims 1 to 5 or the sensor electrode according to claim 6 or 7.
9. An electrochemical biosensor for detecting hydrogen peroxide, wherein the sensor is the sensor according to claim 8, wherein the sensitivity of detecting the hydrogen peroxide by the electrochemical biosensor for detecting hydrogen peroxide is 270 to 430 mA/Mcm ² . The detection limit is 0.27 μM or more, and the linear range is 1 to X μM, wherein X is 1000 or more and 4500 or less.
10. A method of preparing a gold film electrode, the method comprising:

    a step of depositing a gold thin film having a thickness of 200 to 400 nm on a substrate by a sputtering method, and

    The surface of the gold thin film is roughened so that the surface roughness Ra is 5 to 10 nm and the pore depth of the surface is 20 to 40 nm.
11. The method of claim 10 wherein said roughening comprises performing a chemical etching process.
12. The method according to claim 10 or 11, comprising the step of pre-depositing a metal transition layer comprising at least one selected from the group consisting of Cr, Ti, and alloys thereof before depositing a gold thin film on the substrate. By.
13. A method of preparing an electrochemical biosensor electrode, the method comprising:

    a step of forming a gold film electrode as a base electrode by the method for producing a gold film electrode according to any one of claims 10 to 12, and a step of forming a modified layer on the base electrode,

    The step of forming a modified layer on the substrate electrode is to form a modified layer by an electrochemical deposition method, the modified layer including Prussian blue,

    The thickness of the gold film is 200-400 nm, and the thickness of the modified layer is 20-130 nm.

    At least a portion of the Prussian blue is present in the form of spherical and/or cubic particles.
14. A method for preparing an electrochemical biosensor for detecting hydrogen peroxide, comprising the method for preparing a gold film electrode according to any one of claims 10 to 12 or the method for preparing an electrochemical biosensor electrode according to claim 13 The detection sensitivity of the electrochemical biosensor for detecting hydrogen peroxide is 270 to 430 mA/Mcm ² , the detection limit is 0.27 μM or more, and the linear range is 1 to X μM, wherein X is 1000 or more and 4500 or less.

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