KR20100059577A - Glucose biosensor based on porous conducting polymer nanorods on an electrode and method for preparing the same - Google Patents

Abstract

PURPOSE: A high-sensitivity porous glucose biosensor including a porous conductive polymer nano-rod structure and a preparing method thereof are provided to prevent the reduction of electrode area due to the miniaturization of device. CONSTITUTION: A high-sensitivity porous glucose biosensor comprises a reference electrode, a relative electrode and a working electrode. The working electrode comprises conductive polymer nano-rods and a porous silicon material. A Au layer is laminated on the top of the silicon material and a Ti layer is laminated between the silicon material and the Au layer. The conductive polymer nano-rods are formed on the top of the Au layer.

Description

Glucose biosensor based on porous conducting polymer nanorods on an electrode and method for preparing the same

The present invention relates to a highly sensitive porous glucose biosensor and a method of manufacturing the same, and more particularly to a glucose biosensor comprising a reference electrode, a counter electrode, and a working electrode, wherein the working electrode includes nanorods of a conductive polymer. In addition, it is characterized in that the enzyme is fixed, thereby maximizing the area of the working electrode to solve the problem of reduction of the electrode area according to the miniaturization of the device, high sensitivity, high sensitivity glucose bio-capable of detecting low concentration glucose A sensor can be provided.

More than 90% of the domestic biosensors are medical biosensors, and most of them are blood glucose measurement.The domestic market of these blood glucose biosensors reaches about 40 billion won, and the market size is expected to continue to expand as the number of diabetics increases every year. It is becoming.

Biosensor is a system that immobilizes a substance that can react with a specific substance on the surface of an electrode and converts it into an electrical signal to quantitatively analyze its components. In such biosensors, the amount of reaction-related substances immobilized on the electrode surface is one of the important factors affecting the sensitivity of the biosensor. However, in recent years, as the biosensor becomes smaller in size, the area for fixing the materials that can react with a specific substance is also reduced, and serious limitations of the development of a highly sensitive miniaturized sensor are appearing.

To overcome this limitation, sensors are being developed that incorporate nanotechnology, one of which uses nanorods. However, the existing nanorods are mostly made of semiconductors and must be synthesized by using expensive equipment under high temperature and high pressure. Most of these nanorods are easily synthesized vertically on a substrate without using a porous membrane. There was a downside. On the other hand, conventional biosensors using conductive polymers had many thin film structures, which had problems of orientation due to immobilization of biomaterials using adsorption. In addition, compared to semiconductor nanorods, nanorod biosensors using them have a problem in that the synthesis is easy but the sensitivity is not good.

Korean Patent Publication No. 2000-8880 discloses a glucose sensor using a protein complex conjugated with a polymer anion (AMPS) and glucose oxidase (GOD) as a dopant of a conductive polymer. In order to solve the problem of the protein immobilization method used in the conventional glucose sensor, the sensor implemented a sensor system of a type doped with a polymer thin film structure on the thin film electrode.

In addition, Korean Patent Publication No. 2005-104171 discloses a method for quantitatively measuring glucose by using an Ag / AgCl electrode, and Korean Patent Publication No. 2004-26323 discloses a biosensor containing medium-porous platinum and a glucose concentration using the same. A measuring method is disclosed.

However, the preceding inventions have a problem in that the current flow is not smooth because the electrode area for fixing the enzyme to the biosensor is small and it is difficult to miniaturize and the orientation of the enzymes is not good.

On the other hand, the inventor disclosed a high-sensitivity biosensor and a composite biosensor including the same in Patent Registration No. 10-729147 (registration date June 11, 2007). The present invention relates to a biosensor including a reference electrode, a counter electrode, and a working electrode, and including an enzyme fixing part on an upper part of the working electrode as in the present invention. Is characteristic. As a follow-up study, the inventors continued to study biosensors and developed a method for maximizing the effective working area by introducing a nanorod structure to the working electrode part through the present invention.

Therefore, the first problem to be solved by the present invention is to solve the problem of reducing the electrode area according to the miniaturization of the device by maximizing the area of the working electrode, high response speed, high sensitivity glucose biosensor that can detect even low concentration of glucose To provide.

The second object of the present invention is to provide a method for manufacturing the glucose biosensor.

In order to solve the first problem, the present invention is a glucose biosensor comprising a reference electrode, a counter electrode and a working electrode, the working electrode comprises a nanorod of a conductive polymer, the enzyme is fixed It provides a glucose biosensor characterized in that.

According to one embodiment of the invention, the working electrode preferably comprises a porous silicon substrate, the depth and diameter of the pores of the porous silicon substrate is not limited in size, but is generally about 1 μm to 10 μm.

According to another embodiment of the present invention, a gold (Au) layer may be stacked on the silicon substrate, and a titanium (Ti) layer may be stacked between the silicon substrate and the gold (Au) layer.

According to another embodiment of the present invention, the nanorod of the conductive polymer is preferably formed on the gold (Au) layer, the conductive polymer is, for example, polyacetylene (polyacetylene), polyaniline (polyaniline), It may be selected from the group consisting of polypyrrole, polythiophene, poly sulfur nitride.

According to another embodiment of the present invention, the enzyme may be glucose oxidase, and the enzyme is fixed through a covalent bond with the conductive polymer.

According to another embodiment of the present invention, platinum (Pt) nanoparticles may be inserted and dispersed in the nanorod of the conductive polymer, the size of the platinum (Pt) nanoparticles is preferably within 50nm. When the platinum nanoparticles are dispersed in the biosensor, the enzyme is immobilized on the surface of the platinum nanoparticles.

In order to solve the second problem, the present invention provides a method of manufacturing a glucose biosensor comprising a reference electrode, a counter electrode and a working electrode, forming a porous silicon layer on the working electrode; Forming nanorods of a conductive polymer on the porous silicon layer; And it provides a high sensitivity porous glucose biosensor manufacturing method comprising the step of fixing by binding the enzyme and the rod of the conductive polymer.

The present invention also provides a method of manufacturing a glucose biosensor comprising a reference electrode, a counter electrode and a working electrode, the method comprising: forming a porous silicon layer on the working electrode; Forming nanorods of a conductive polymer on the silicon layer; Dispersing platinum (Pt) nanoparticles between the nanorods of the conductive polymer; And it provides a high sensitivity porous glucose biosensor manufacturing method comprising the step of directly bonding and fixing the platinum (Pt) nanoparticles and enzyme.

According to an embodiment of the present invention, the method may further include depositing a gold (Au) layer on the porous silicon layer, and further comprising depositing a titanium (Ti) layer on the gold (Au) layer. It may include.

According to the high-sensitivity porous glucose biosensor according to the present invention, the nanorod structure of the conductive polymer is introduced on the work electrode of the porous silicon substrate to maximize the work electrode area, thereby reducing the electrode area reduction caused by the miniaturization of the device. In addition, by dispersing platinum nanoparticles in the nanorod structure, the sensitivity of the electron transport medium may be greatly increased. Accordingly, the present invention provides a high sensitivity electrochemical biosensor capable of detecting even a low concentration of glucose and having a fast response time.

The present invention relates to a highly sensitive electrochemical biosensor having a fast response time and capable of detecting even low concentrations of glucose and a method of manufacturing the same. The present invention is characterized by combining nanotechnology and biotechnology. Specifically, a porous structure was introduced using silicon substrates based on MEMS technology to increase the area of the working electrode part due to the miniaturization of the sensor. The area was maximized.

In addition, the present invention is to disperse the platinum nanoparticles on the conductive polymer to serve as a mediator of electron transfer generated by the chemical reaction. By using the covalent bonding method on the electrode thus prepared, the enzyme, which is a biomaterial, is immobilized to realize a high sensitivity biosensor capable of detecting fast response time and low concentration.

1 is a structural diagram of a biosensor according to the present invention. In the center of the figure is a working electrode, after depositing a gold thin film on a porous silicon substrate to form a conductive polymer nanorod, and dispersed the platinum nanoparticles thereon. The SEM photographs of the figure show the deposition of a gold thin film on the porous silicon structure of the working electrode, the conductive polymer nanorods, and the platinum nanoparticles dispersed thereon. Meanwhile, the left electrode of the working electrode is a reference electrode of Ag / AgCl and the right electrode represents a platinum (Pt) counter electrode.

The porous electrode of the conductive polymer nanorods in which the metal nanoparticles are used in the present invention has an electrode area of 4.31 times larger than the conductive polymer flat electrode of the thin film structure, thereby maximizing the area for immobilizing the biomaterial. Dispersion of the metal nanoparticles on the conductive polymer increased the electron transfer effect generated in the chemical reaction, resulting in a 22.1-fold increase in sensitivity. In addition, immobilization of biomaterials using covalent bonds improves orientation, has a fast response speed within 10 seconds, and enables detection up to a low concentration range. Therefore, the present invention is expected to be the basis technology for the development of high sensitivity miniaturization sensor.

Hereinafter, the present invention will be described in more detail with reference to preferred examples, but the present invention is not limited thereto.

Example 1 Fabrication of Silicon Electrode Containing Nanorods of Conductive Polymer

First, porous silicon to be used as a substrate was prepared. Specifically, the porous silicon layer on the working electrode through a 40-minute anode etching process using a HF: C ₂ H ₅ OH: H ₂ O solution (1: 3: 2 mixture) at a current density of -6 mA / cm ² Formed. The depth and diameter of the pores were about 5 μm and 2 μm, respectively.

Next, the manufacturing steps of the electrode including the nanorods of the conductive polymer are as follows. First, prior to the gold thin film deposition, 200 Å of titanium (Ti) layer was deposited on the porous silicon and planar silicon substrates using RF sputtering. Then, a gold (Au) layer was deposited on the electrode on which the titanium layer was laminated, and the flat gold electrode and the porous gold electrode were respectively displayed. In general, titanium is used as the underlayer to enhance the adhesion strength between the gold thin film and the silicon substrate.

Next, aniline polymerization was carried out using electrochemical deposition under constant voltage. Polyaniline nanorods were electrochemically deposited on planar silicon and porous silicon-based gold electrodes for 720 seconds at 0.75 V in an electrolyte solution containing 0.2 MH ₂ SO ₄ and 0.1 M aniline. It was then washed with distilled water and dried in an oven at 40 ° C. for one day.

Example 2: Platinum Nanoparticle Dispersion Step

Each electrode modified with the planar gold / polyaniline nanorods and porous gold / polyaniline nanorods obtained in Example 1 was subjected to multi-cyclic voltammetry (CV) in 0.5 MH ₂ SO ₄ and 2.0 mM K ₂ PtCl ₆ . Pt nanoclusters were inserted into the polyaniline nanorods by treating at 0.40 to -0.25 V voltage conditions at a rate of 50 mV s ⁻¹ using the Nt. Accordingly, planar silicon electrodes and porous silicon electrodes including gold / polyaniline nanorods / platinum nanoparticles were formed, respectively. The optimized cycle count was about 30.

2 is a schematic diagram of a porous electrode including gold / polyaniline nanorods / platinum nanoparticles. The upper right figure in the figure shows the structure of the working electrode modified according to the present invention, and the lower right picture is a surface SEM image on a polyaniline nanorod with platinum nanoparticles (scale bar: 100 nm). The size of the platinum nanoparticles measured in this example was 30 to 40 nm.

Example 3: Enzyme Immobilization Step

The enzymes were immobilized on gold electrodes modified with polyaniline nanorods. In the following scheme, a scheme giving a fixation process of glucose oxidase is shown.

According to Scheme 1 below, in the absence of platinum (Pt) particles, the gold / polyaniline nanorod electrode was treated with glutaaldehyde (GA) to combine enzymes and polyaniline and then rinsed with distilled water and dried under nitrogen gas purge. I was. In the next step, an amide bond was formed between the N-terminus of the enzyme molecule and the glutahydroactivated electrode by immersing the electrode in a 50 mM phosphate buffer solution containing pH 7.0 for 24 hours.

Scheme 1

On the other hand, in the case of an electrode in which platinum (Pt) nanoparticles are dispersed, the enzyme is directly fixed on the surface of the platinum (Pt) particles by adsorption without modifying glutaaldehyde, as shown in Scheme 2 below.

Scheme 2

After the enzyme layer was formed, it was rinsed with a flowing phosphate buffer solution to remove residual monomer or weakly bound enzyme molecules.

Test Example 1 Analysis of Morphology of Electrode Structure

In the preparation of a porous electrode having a hierarchical structure according to the present invention, the surface structure in each electrochemical process was analyzed by SEM, and the measured images are shown in FIGS. 3 and 4. As shown in FIG. 3B, a porous silicon layer was formed at the working electrode, and the Ti / Au layer was sputtered on the porous silicon substrate. The thickness of the Ti layer and the gold (Au) thin film layer were 200 kPa and 2000 kPa, respectively (Scale bar: A 5 μm, B 1 μm).

FIG. 4 shows SEM images of polyaniline nanorods before and after electrochemical deposition of platinum particles (Scale bar 1 μm). FIG. 4A shows a large surface area with entangled polyaniline nanorods of about 200 nm in diameter formed on the surface. Shows a three-dimensional nanoporous structure having. The layered porous electrode consists of micropores of porous silicon, and the nanoporous network of polyaniline nanorods provides an ideal surface for enzymatic immobilization with many platinum nanoparticles, as well as analytes at the active site. Can transport more and access more easily. As can be seen in FIG. 4B, the platinum nanoparticles are well dispersed and partially intercalated on the polyaniline nanorod surface. This well dispersed platinum (Pt) nanocatalyst can increase the electrocatalytic activity for the electrooxidation of hydrogen peroxide produced by the enzymatic reaction.

Test Example 2: Effective electrode area comparison experiment

In the following experiments, all currents were measured with an auto-lab microAUTOBIII (Eco Chemie, The Netherlands) to which three electrode cells were connected. The three electrode system consists of a platinum (Pt) counter electrode, an SCE reference electrode and a gold working electrode based on planar silicon or porous silicon.

The effective working area of the strain electrode was calculated from the Randles' slope and the results are summarized in Table 1. The following results show that the effective surface of the nanorod structure and the porous substrate is wider than the surface of the thin film structure or the flat substrate. In addition, the metal nanoparticles of the conductive polymer further increased the effective surface area of the electrode. The results below show that the porous electrode having a hierarchical structure (porous gold / polyaniline nanorods / platinum nanoparticles) has an active area of 5.48 times wider than the same amount of planar gold electrode.

electrode Linearity
(R ² ) Randles' slope Effective electrode area (cm ² ) Flat Gold (Au) 0.99584 0.14536 0.6199 Flat Gold / Polyaniline Thin Film 0.99543 0.18464 0.7874 Flat Gold / Polyaniline Nanorods 0.99953 0.38255 1.6313 Porous Gold (Au) 0.99921 0.30519 1.3014 Porous Gold / Polyaniline Nanorods 0.99356 0.77831 3.3189 Porous Gold / Polyaniline Nanorods / Platinum Nanoparticles 0.99562 0.79611 3.3948

FIG. 5 shows calibration curves for four different types of electrodes, including porous electrodes (porous gold / polyaniline nanorods / platinum nanoparticles) having a platinum (Pt) dispersed hierarchy. In Figure 5 (A) is a planar gold (Au) electrode, (B) is a planar gold (Au) / polyaniline nanorod / platinum nanoparticle electrode, (C) is a porous gold (Au) / polyaniline nanorod nanoparticle electrode and (D) shows the calibration curve of the porous gold (Au) / polyaniline nanorods / platinum nanoparticle electrodes.

Test Example 3: Sensitivity Comparison Experiment

Table 2 below shows the results of determining the glucose concentration for each electrode. As shown in the table below, porous gold (Au) / polyaniline nanorods / platinum nanoparticle sensors are 22.1 times more sensitive than planar gold (Au) / polyaniline nanorod sensors.

electrode Concentration range
(mM) Sensitivity
(μA / mM) Correlation coefficient
(R ² ) Flat Gold / Polyaniline Nanorods 0.6 to 10 1.61 0.9992 Planar Gold / Polyaniline Nanorods / Platinum Nanoparticles 0.3 to 2.5 7.28 0.9986 Porous Gold / Polyaniline Nanorods 0.15 to 5 13.66 0.9983 Porous Gold / Polyaniline Nanorods / Platinum Nanoparticles 0.0092-5 35.64 0.9996

The signal of the sensor essentially depends on the amount of enzyme immobilized on the sensor surface. Considering the function of glucose oxidase enzymes with limited reactivity, the high sensitivity of the sensor according to the invention is a result indicating that more enzymes can be immobilized on the porous electrode than on the planar electrode due to the enlargement of the active surface. In addition, according to the present embodiment, it can be seen that the high-density platinum nanoparticles well dispersed in the electrode can provide an even and wide active surface, and also play an important role in the catalytic activity for the electro-oxidation of hydrogen peroxide produced by the enzymatic reaction. have. Nanostructured conductive polymers are also believed to serve as mattresses for electron transfer, dispersion of metal particles and fixation of glucose oxidase.

Test Example 4 Measurement of Stability of Enzyme Electrode

The stability of the enzyme electrode was tested at 0.6 V in 0.1 M phosphate buffer containing 1 mM glucose. The test results showed that the sensor's reactivity was maintained for 45 days up to 80% of the initial response. The excellent shelf life of the present invention suggests that the high density porous surface on which platinum (Pt) is dispersed is not only compatible with the immobilized enzyme, but also facilitates the maintenance of the biological activity of glucose.

1 is a structural diagram and a SEM image of a highly sensitive porous glucose biosensor according to the present invention.

2 is a cross-sectional view and SEM image of a porous electrode including gold / polyaniline nanorods and gold / polyaniline / platinum nanoparticles according to the present invention.

Figure 3 is an SEM image of the surface structure in the manufacturing process of the porous electrode having a hierarchical structure in accordance with the present invention.

Figure 4 shows an SEM image of the polyaniline nanorods before and after electrochemical deposition of platinum particles according to the present invention. (Scale bar: 1㎛)

FIG. 5 shows calibration curves for four different types of electrodes, including porous electrodes (porous gold / polyaniline nanorods / platinum nanoparticles) having a platinum (Pt) dispersed hierarchy. In Figure 5 (A) is a planar gold (Au) electrode, (B) is a planar gold (Au) / polyaniline nanorod / platinum nanoparticle electrode, (C) is a porous gold (Au) / polyaniline nanorod nanoparticle electrode and (D) shows the calibration curve of the porous gold (Au) / polyaniline nanorods / platinum nanoparticle electrodes.

Claims (15)

A glucose biosensor comprising a reference electrode, a counter electrode, and a working electrode, wherein the working electrode includes nanorods of a conductive polymer and has an enzyme fixed thereto. The glucose biosensor of claim 1, wherein the working electrode comprises a porous silicon substrate. The glucose biosensor of claim 2, wherein a gold layer is stacked on the silicon substrate. The glucose biosensor of claim 3, wherein a titanium layer is stacked between the silicon substrate and the gold layer. The glucose biosensor of claim 4, wherein the nanorods of the conductive polymer are formed on the gold layer. The method of claim 1, wherein the conductive polymer is selected from the group consisting of the polyacetylene, polyaniline, polypyrrole, polythiophene, poly sulfur nitride Glucose biosensor, characterized in that. The glucose biosensor of claim 1, wherein the enzyme is glucose oxidase. The glucose biosensor of claim 1, wherein the enzyme is immobilized through a covalent bond with the conductive polymer. The glucose biosensor according to claim 1, wherein platinum (Pt) nanoparticles are inserted and dispersed in the nanorods of the conductive polymer. The glucose biosensor of claim 10, wherein the platinum (Pt) nanoparticles have a size of 50 nm or less. The glucose biosensor of claim 10, wherein the enzyme is immobilized on the surface of the platinum nanoparticles. In the method of manufacturing a glucose biosensor comprising a reference electrode, a counter electrode and a working electrode, Forming a porous silicon layer on the working electrode; Forming nanorods of a conductive polymer on the porous silicon layer; And Method of manufacturing a high sensitivity porous glucose biosensor comprising the step of fixing by binding the enzyme and the rod of the conductive polymer. In the method of manufacturing a glucose biosensor comprising a reference electrode, a counter electrode and a working electrode, Forming a porous silicon layer on the working electrode; Forming nanorods of a conductive polymer on the silicon layer; Dispersing platinum (Pt) nanoparticles between the nanorods of the conductive polymer; And Method for producing a highly sensitive porous glucose biosensor comprising the step of directly bonding and fixing the platinum (Pt) nanoparticles and enzyme. The method of manufacturing a highly sensitive porous glucose biosensor according to claim 12 or 13, further comprising depositing a gold (Au) layer on the porous silicon layer. The method of claim 14, further comprising depositing a titanium (Ti) layer on the gold (Au) layer.

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