KR20120113085A - An electrode for enzyme biofuel cell and a method thereof - Google Patents

Abstract

PURPOSE: An electrode for enzymatic fuel cell is provided to maintain activity of fixed support for several months, to have high stability because separation of fixed enzymes is prevented, and to maintain a constant density of power generated in the electrode. CONSTITUTION: An electrode for enzymatic fuel cell comprises enzymes, and an electron conductor and an enzyme immobilizer fixing the enzymes. The enzyme immobilizer is that an ethylenediamine-based compound represented by chemical formula 1: NH2-(CH2)n-NH2 modified from polyacrylonitrile. The enzyme immobilizer has a nanofiber membrane shape of polyacrylonitrile. An enzymatic biofuel cell comprises the electrode. The enzymatic biofuel uses one or more selected from organic compounds comprising sugar, carbohydrate, organic acid, alcohol, fatty acid, hydrocarbon, ketone, aldehyde, amino acid, protein and nucleic acid as fuel.

Description

Electrode for Enzyme Fuel Cell and Manufacturing Method Thereof {AN ELECTRODE FOR ENZYME BIOFUEL CELL AND A METHOD THEREOF}

The present invention relates to an electrode for an enzyme fuel cell and a method of manufacturing the same. In detail, the present invention relates to an electrode for an enzyme fuel cell and a method for preparing the same, which use a polymer nanofiber membrane modified as an enzyme-fixing support to maintain the activity of an enzyme and increase its stability to prevent desorption of the enzyme. .

Enzyme fuel cells have received a lot of attention because of the potential for use as a power source for human implantable medical devices, but there is a problem in that the amount of power generated therefrom is low. In particular, in order to use it as a human implantable power source, the size of the enzyme fuel cell should be reduced to several micrometers or less. In this case, the amount of generated power is further reduced, which makes it difficult to supply sufficient power to the human implantable medical device.

To improve the performance of the enzyme fuel cell, it is necessary to improve the performance of the enzyme electrode, a key element thereof. The enzyme electrode is prepared by immobilizing the enzyme on a well-electrode electrode, and the immobilization step of the enzyme is very important. In immobilizing the enzyme on the electrode material, there are several considerations to improve performance, such as compatibility between the enzyme and the support or the environment in which the support is attached and the shape of the support.

In an electrode for an enzyme fuel cell, in order to fix an enzyme to a support, the support is generally activated to have a highly reactive functional group and then covalently bonded to a functional group of an enzyme protein, that is, an amino group, a carboxyl group, a thiol group, an aromatic hydroxy group, or the like. The method is used.

This method of enzymatic fixation by covalent bonding is inexpensive because it can be used by modifying various kinds of scaffolds, and it is possible to arbitrarily adjust the surface of the scaffold, and to stably bind enzymes compared to enzyme fixation by other physical bonds. have. Many methods have been reported in the literature on immobilization of enzymes, but nevertheless new methods for useful methods of immobilizing enzymes while maintaining or enhancing the relevant properties such as their properties, activity, stability and selectivity Is still needed.

An object of the present invention is to provide an electrode for an enzyme fuel cell for continuously maintaining the activity of a fixed enzyme and increasing the stability of the enzyme. In addition, the present invention provides an electrode for an enzyme fuel cell comprising the electrode.

In order to solve the above problems, the present invention includes an enzyme, an enzyme fixing support to which the enzyme is immobilized, and an electron conductor, and the enzyme support comprises polyacrylonitrile (PAN) according to Equation 1 below. It provides an electrode for an enzyme fuel cell, characterized in that modified to.

[Formula 1]

NH _2- (CH ₂ ) _n -NH ₂

Where n is 3 to 20.

The enzyme-fixed support is preferably in the form of a nanofiber membrane of polyacrylonitrile. In addition, the electrode may be used as an anode. In addition, the electrode may be used as a reduction electrode.

Enzymes included in the anode are glucose oxidase, glucose dehydrogenase, alcohol redox enzyme, aldehyde dehydrogenase, CH-CH redox enzyme, amino acid redox enzyme, CH-NH redox enzyme, lactate dehydrogenase, lactose One or more oxidoreductases selected from the group consisting of dehydrogenase, pyruvate dehydrogenase, formate dehydrogenase and formaldehyde dehydrogenase.

The enzyme included in the cathode may be at least one redox enzyme selected from the group consisting of laccase, bilirubin oxidase, superoxide dismutase and peroxidase.

The electrode for an enzyme fuel cell includes HQS (8-hydroxyquinoline-5-sulfonic acid hydrate), quinone compound, vitamin K3 (2-methyl-1,4-naphthoquinone), biorogen compound, nicotinamide compound , A riboflavin compound, ABTS (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonate), metal complexes of Os, Ru, Fe and Co It may further comprise an electron transfer medium.

In addition, the present invention provides an enzyme fuel cell comprising the electrode for the enzyme fuel cell. The enzyme fuel cell may use at least one of organic compounds including sugars, carbohydrates, organic acids, alcohols, fatty acids, hydrocarbons, ketones, aldehydes, amino acids, proteins and nucleic acids as fuel.

In the electrode for an enzyme fuel cell according to the present invention, the activity of the immobilized enzyme lasts for more than 75% for several months, and the desorption of the immobilized enzyme is prevented, so that the stability is high and the density of power generated from the electrode is kept constant.

1 schematically illustrates the process of treating a polyacrylonitrile support with hexamethylenediamine.
2 schematically illustrates a process of immobilizing an enzyme on a support to which hexamethylenediamine is attached.
3 is a graph showing a cyclic voltammogram of an electrode of Example 1. FIG.
4 is a graph showing a cyclic voltammogram of an electrode of Example 2. FIG.
FIG. 5 is a SEM photograph of the PAN nanofiber membrane of Example 1. FIG.
6 is a SEM photograph of the PAN nanofibrous membrane before (a) and after (b) modification.
7 is a SEM photograph showing the enzyme immobilized on the PAN nanofibrous membrane.
FIG. 8 shows FI-IR graphs of respective stages of electrode fabrication.

An electrode for an enzyme fuel cell according to the present invention includes an enzyme, an enzyme fixing support to which the enzyme is fixed, and an electron conductor. The enzyme support may be used by modifying a polymer compound with an ethylenediamine-based compound represented by the following formula (1).

[Formula 1]

NH _2- (CH ₂ ) _n -NH ₂

In Formula 1 n is 3 or more. Preferably it is 3-20, More preferably, it is 3-10. In Formula 1,-(CH ₂ ) _n -may be linear or branched. However, when n is 6 or less, it is preferable that it is linear.

The ethylenediamine-based compound serves as a crosslinking covalent bond between the polymer compound and the enzyme used as a support. The ethylenediamine-based compound includes two amine groups as shown in Chemical Formula 1. One of the two amine groups binds to the polymer compound used as the enzyme fixation support, and the other binds to the enzyme and the peptide, so that the enzyme may be immobilized on the support. The-(CH ₂ ) _n -portion of the ethylenediamine-based compound has an effect of adding elasticity and flexibility to the bond between the immobilized enzyme and the support. This is due to the characteristics of the hydrocarbon in which the carbon-to-carbon single bond rotates. The elasticity and flexibility have the effect of increasing the enzyme-substrate binding and preventing the enzyme from detaching from the support. In general, when an enzyme is immobilized to a support by covalent bond, the activity of the enzyme is lost during the covalent reaction.

The electrode for an enzyme fuel cell according to the present invention can overcome the above disadvantages by increasing the elasticity and flexibility between the enzyme and the support. In Formula 1, n is preferably 3 to 10. If n is less than 3, the effect of elasticity and flexibility does not appear, which is not preferable. If n is greater than 20, the distance between the enzyme and the support becomes far, which may cause an obstacle in the movement of electrons between the electron conductors attached to the enzyme and the support. Preferably n is 3 to 10. The-(CH ₂ ) n-moiety in the ethylenediamine compound may be linear or branched.

However, when n is 6 or less, a linear diameter is preferable. When n is 6 or less and branched, the elasticity and flexibility effects are reduced. In one specific embodiment of the present invention, the ethylenediamine-based compound is hexamethylenediamine.

In the electrode for an enzyme fuel cell according to the present invention, the enzyme fixation support may be used by modifying a polymer compound with the ethylenediamine compound. According to a specific embodiment of the present invention, the support is modified to have an amine group which is highly reactive by modifying the support so that the support can be immobilized by covalently binding to the carboxy group (-COOH), one of the functional groups of the enzyme protein. The advantage of the enzyme-support covalent bond is that it is possible to be stable and can be reused for a long time, and it is inexpensive because it can be used by modifying various kinds of support, and the support surface can be arbitrarily adjusted.

The polymer compound that can be used as the enzyme immobilization support can be used as long as it can be modified to have a carboxyl group to bind with the ethylenediamine-based compound. According to one specific embodiment of the present invention, the polymer compound is a polyacrylonitrile (PAN) -based compound, but is not limited thereto. The PAN can be modified by treatment with NaOH.

In this reaction, the nitrile group (-C≡N) is substituted with a carboxy group (-COOH), and the carboxy group (-COOH) of the modified PAN is reacted with hexamethylenediamine (HMDA) again to form an amine group. The enzyme is fixed to the support by peptide bonding with the carboxyl group of the enzyme protein to be immobilized (see FIGS. 1 and 2).

The enzyme fixation support is preferably in the form of a nanofibrous membrane (nanofibrous membrane). The support in the form of the nanofiber membrane can increase the number of enzymes fixed per unit area of the support, and has a porous property is not limited to the movement of electrons in the electrode.

In addition, there is an advantageous aspect in forming an electron conductor-support composite in which the conductive material is combined with or mixed with the conductive material which is an electron conductor. According to a specific embodiment according to the present invention, the support in the form of the nanofiber membrane may be obtained by, for example, applying an electrospinning technique.

Electron conductors are substances that conduct electrons. Electron conductors can be organic or inorganic in nature as long as they can (1) conduct electrons across the material, (2) have a high surface area, and (3) disperse into small particulate matter. The electron conductor can be a carbon-based material, a metal conductor, a semiconductor, a metal oxide or a strain conductor. According to one specific embodiment of the present invention, the preferred electron conductor is a carbon-based material. The carbonaceous material may be, for example, carbon black (Vulcan XC-72, E-tek), carbon nanotubes in the form of multiwall or single wall, carbon powder, carbon fiber, diamond-coated conductor, graphite, non-compression Graphite worm, delaminated refined flaky graphite, high performance graphite and carbon powder, platinum carbon or gold-coated carbon.

In one specific embodiment of the present invention, the electrode of the present invention is a form in which a support in the form of the nanofiber membrane is coated on a carbon material which is the electron conductor. In another specific embodiment of the present invention, the carbon material, which is the electron conductor, may be dispersed in the polymer compound, which is the support, to be used in the form of a polymer-carbon material composite. Since the form of the complex is similar to the form in which the enzyme is directly immobilized on the electrode, the electrode and the enzyme are electrically connected to each other, thereby providing an effect such as an increase in power density. The carbon material may preferably be selected from carbon fiber, carbon black or carbon nanotubes.

The electrode may be used as an oxidation electrode or a reduction electrode. In the present invention, the anode means an anode comprising a redox enzyme that catalyzes the oxidation of the substrate. Through oxidation of the substrate, the oxidizing electrode supplies electrons to the electrical circuit. On the other hand, the reduction electrode means a reduction electrode containing a redox enzyme for reducing the substrate. The reduction electrode receives electrons and serves to reduce the substrate.

Oxidoreductase refers to a reducing agent, ie, an enzyme that catalyzes the transfer of electrons from a hydrogen or electron donor to an oxidant, ie hydrogen or an electron acceptor. The oxidoreductase is preferably named a donor: acceptor oxireductase, but is commonly referred to as a donor dehydrogenase or an acceptor reductase. In particular, when oxygen is a receptor, oxidoreductases are sometimes referred to as donor oxidases.

In one specific embodiment of the present invention, the redox enzyme included in the anode can be used as long as it is an oxidoreductase capable of supplying electrons by oxidizing a substrate. Although not limited thereto, the redox enzymes included in the anode include, for example, glucose oxidase, glucose dehydrogenase, alcohol oxidase, aldehyde dehydrogenase, CH-CH oxidase, amino acid oxidase, CH -NH oxidoreductase, lactate dehydrogenase, lactose dehydrogenase, pyruvate dehydrogenase, formate dehydrogenase, and formaldehyde dehydrogenase, and one or more oxidoreductases selected from the group.

The type of enzyme may vary depending on the type of substrate to be used as fuel of the enzyme fuel cell. Substrates of enzymes are organic compounds such as sugars, carbohydrates, organic acids, alcohols, fatty acids, hydrocarbons, ketones, aldehydes, amino acids, proteins and nucleic acids. For example, when glucose is used as a fuel of an enzyme fuel cell, the redox enzyme included in the anode may be a glucose oxidase. Glucose oxidase releases electrons by oxidizing glucose to glucolactone.

In one aspect of the present invention, any of redox enzymes included in the reduction electrode can be used as long as it can reduce the substrate. Although not limited thereto, the redox enzyme included in the reduction electrode may be, for example, one or more redox enzymes selected from the group consisting of laccase, bilirubin oxidase, superoxide dismutase and peroxidase. For example, when using a laccase as an oxidoreductase included in the reduction electrode, the laccase reduces oxygen to water using the transferred electrons. As another example, when bilirubin oxidase is used as an oxidoreductase included in the reduction electrode, bilirubin oxidase oxidizes bilirubin to biliverdine and simultaneously reduces oxygen to water.

Electron transfer mediators are used to provide high power density to enzyme fuel cells. In the present invention, any electron transfer medium may be used as long as it is a compound capable of accepting or donating electrons. However, the electron transfer medium may be selected in consideration of reaction affinity with the enzyme used in the enzyme fuel cell, electron exchange rate with the conductive polymer, structural stability, and the like. For example, in one specific embodiment of the present invention, in the case where the electrode is an anode, if glucose oxidase is used, the electron transport medium is preferably FADH (oxidized or reduced).

Advantages and features of the present invention and methods for achieving the same will be apparent with reference to the embodiments described below in detail. However, the present invention is not limited to the embodiments disclosed below, but may be implemented in various different forms, only the embodiments are to make the disclosure of the present invention complete, and the general knowledge in the technical field to which the present invention belongs. It is provided to fully convey the scope of the invention to those skilled in the art, and the present invention is defined only by the scope of the claims.

Preparation of the electrode

1. Preparation of Nanofiber Membrane

PAN nanofibrous membranes were prepared using electrospinning technology. PAN was dissolved in N, N-dimethylformamide (DMF) to make 9% and 10% solutions, respectively, which were mechanically stirred at room temperature for two hours. The polymer solution was supplied to a capillary using a syringe, and a high voltage of 20 kV was applied to the capillary. The fibers were obtained using copper foil fixed to a stainless steel drum rotating at a speed of 150 rpm. The distance between the syringe and the drum was about 15 cm. The resulting nanofiber membranes were dried at 70 ° C. in vacuo. Figure 5 shows a SEM picture of the PAN nanofiber membrane prepared above.

2. Modification of Nanofiber Membrane

The PAN nanofibrous membrane prepared above was treated with NaOH at 50 ° C. for 1 hour and then washed. HCl was treated to remove excess alkali, washed with distilled water and dried. Next, the nanofiber membrane was treated with hexamethylenediamine (HMDA) at room temperature for 1 hour to obtain a nanofiber membrane having NH ₂ groups formed thereon. The nanofiber membranes were washed with distilled water and dried. 6 is a SEM photograph of the PAN nanofibrous membrane before (a) and after (b) modification. It can be seen that the shape of the nanofibers is maintained even after modification.

3. Electrode Manufacturing

0.18 g of multi-walled carbon nanotubes (Hanhwa Chemical) and 0.20 g of PVdF were added to 100 ml of NMP (N-methyl-2-pyrrolidone) and dispersed in a ball mill grinder for 1 hour. The dispersion was attached to the modified nanofiber membrane by a casting method and then dried to prepare an electrode.

4. Fixation of Glucose Oxidase

The electrode prepared above was immersed in 1 ml (5 mg / ml; pH 5.8) of glucose oxidase in 0.1 M sodium phosphate buffer solution and stored at room temperature for 24 hours. After confirming that the glucose was fixed to the membrane and washed with distilled water and 0.1M sodium phosphate buffer solution (pH 5.8). 7 is a SEM photograph showing the enzyme immobilized on the PAN nanofibrous membrane.

8 shows FT-IR graphs for each step of the electrode fabrication. The top graph of FIG. 8 is a FT-IR graph of the PAN nanofiber membrane, showing peaks for CN binding. In addition, the second graph from the top is an FT-IR graph of the PAN nanofibrous membrane after NaOH treatment to the PAN nanofibrous membrane, and a C = O peak can be confirmed around 1,750 cm ⁻¹ . In addition, the third graph 3500cm by FT-IR chart of the modified state to the final ethylene diamine compound by treating the nano-HMDA in the PAN membrane from above ^- it was confirmed that the peptide bond peak appears in the vicinity of ^1. The bottom graph is FT-IR data after attaching the enzyme to the modified PAN nanofibrous membrane, and it was confirmed that a large peak appeared around 3,500 cm ^-1 .

After preparing the electrode according to Example 1, the electrode was placed in a continuous reactor capable of flow control for 7 days, and then the safety of the enzyme was observed. The continuous reactor allowed the sodium phosphate buffer solution of pH 6.0 to continuously circulate at a rate of 1 m / s.

After preparing the electrode according to Example 1, the electrode was placed in a continuous reactor capable of flow control for 25 days, and then the safety of the enzyme was observed. The continuous reactor allowed the sodium phosphate buffer solution of pH 6.0 to continuously circulate at a rate of 1 m / s.

Enzyme Stability Confirmation Experiment

A standard curve was obtained by the standard protocol of UV analysis (562 nm) according to Pierce BCA Protein Assay, which was used to determine the enzyme residual on the electrodes according to Examples 1, 2 and 3. In the case of Example 1, the fixed amount of enzyme was found to be 230 µg / mg. On the other hand, the electrode of Example 2 soaked in a continuous reactor containing a buffer for 7 days was 137.6 µg / mg, and the electrode of Example 3 soaked in 25 days was 133.0 µg. / mg. According to the experimental results, about 40% of the enzyme was released from the initial enzyme fixation for the first 7 days, but the amount of enzyme fixation after 3 weeks was almost the same as the enzyme fixation after 7 days. It was confirmed that the enzyme fixation force of.

Experimental Comparison of Electrical Characteristics of Electrode

About 1 mg of the electrode of Examples 1 and 2 was attached to the cleaned copper electrode surface using silver / epoxy resin. The electrodes of Examples 1 and 2 were placed in a vacuum oven and dried at room temperature for 1 hour, and then the electrode surface was wrapped with a dialysis membrane and fixed with an O-ring. Potentiometer (Potentiostat, CH Instrument) was used to measure the amount of current generated while changing the voltage at room temperature. The electrode cell was filled with 6 mM of a solution containing 20 mM of KCl and 10 mM of glucose in 100 mM potassium phosphate buffer (pH 7.0), and the electrode was immersed. The electrodes of Examples 1 and 2 were used as the working electrode, the counter electrode was platinum wire, and Ag / AgCl was used as the reference electrode. The scan speed was 50 mV / s.

3 is a graph showing the cyclic voltammogram of Example 1, and FIG. The same experiment was repeated three times using each electrode, and it was confirmed that the three peaks of each of the three experiments of Examples 1 and 2 showed a generated current peak near 250 mV. Through this, it was confirmed that the desorption of the enzyme was small enough to have little effect on the electrode performance in the electrode of Example 2, it was confirmed that the enzyme fixation force of the electrode according to the present invention is excellent.

Claims (9)

An enzyme, an enzyme fixing support to which the enzyme is immobilized, and an electron conductor,
The enzyme support is characterized in that the polyacrylonitrile (polyacrylonitrile; PAN) is modified with an ethylenediamine-based compound according to Formula 1, the enzyme fuel cell electrode:
[Formula 1]
NH _2- (CH ₂ ) _n -NH ₂
Where n is 3 to 20.
The method of claim 1,
The enzyme fixing support is characterized in that the nanofiber membrane form of polyacrylonitrile, enzyme fuel cell electrode.
The method according to claim 1 or 2,
The electrode is to be used as an anode, enzyme fuel cell electrode.
The method according to claim 1 or 2,
The electrode is to be used as a reducing electrode, the electrode for an enzyme fuel cell.
The method of claim 3,
Enzymes included in the anode are glucose oxidase, glucose dehydrogenase, alcohol redox enzyme, aldehyde dehydrogenase, CH-CH redox enzyme, amino acid redox enzyme, CH-NH redox enzyme, lactate dehydrogenase, lactose An electrode for an enzyme fuel cell, which is at least one redox enzyme selected from the group consisting of dehydrogenase, pyruvate dehydrogenase, formate dehydrogenase and formaldehyde dehydrogenase.
The method of claim 4, wherein
The enzyme included in the cathode is at least one redox enzyme selected from the group consisting of laccase, bilirubin oxidase, superoxide dismutase and peroxidase, enzyme fuel cell electrode.
The method according to claim 1 or 2,
HQS (8-hydroxyquinoline-5-sulfonic acid hydrate), quinone compound, vitamin K3 (2-methyl-1,4-naphthoquinone), biorogen compound, nicotinamide compound, riboflavin compound, ABTS (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonate), additionally includes an electron transfer mediator selected from the group consisting of metal complexes of Os, Ru, Fe and Co An electrode for an enzyme fuel cell.
An enzyme fuel cell comprising the electrode of claim 1.
9. The method of claim 8,
The enzyme fuel cell is an enzyme fuel cell that uses at least one of organic compounds including sugars, carbohydrates, organic acids, alcohols, fatty acids, hydrocarbons, ketones, aldehydes, amino acids, proteins and nucleic acids as fuel.

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