JP4785505B2 - Enzyme immobilized biosensor - Google Patents

Description

  The present invention relates to an enzyme-immobilized biosensor for detecting and measuring aldehyde compounds or alcohol compounds.

  Studies on biosensors with immobilized enzymes have been conducted in the past. For example, biosensors (EEE type sensors) with immobilized enzymes on platinum black surfaces measure the blood glucose level of diabetic patients. It has been put into practical use as a sensor (Patent Document 1).

  However, in the case of a conventional enzyme-immobilized biosensor, the types of enzymes that can actually be used are limited, such as glucose oxidase, cholesterol oxidase, urease, and the like.

  On the other hand, in recent years, interest in formaldehyde, which is a causative substance of “chemical sensitivity”, has increased, and tap water has been disinfected for formaldehyde widely used in adhesives, chemical materials, wood preservatives, etc. In addition to the deepening of knowledge that ozone used for pretreatment to remove iron and manganese components that cause unpleasant flavors, ozonize some organic substances to generate formaldehyde, it is carcinogenic and metamorphogenic. From the viewpoint, it is desired to realize a method for detecting and measuring the gas phase or the liquid phase by a simple method.

  As methods for analyzing and measuring formaldehyde, DNPH (2,4-dinitrophenylhydrazine) derivatized solid-phase adsorption / solvent extraction / high performance liquid chromatography and gas chromatography have been used. However, the measurement takes time, and the apparatus for analysis is complicated and expensive. For this reason, the actual condition of the simple measuring method has been demanded.

  From such a background, it is conceivable to detect and measure aldehyde compounds such as formaldehyde with an EEE-type biosense using oxygen fixation. So far, detection and measurement of aldehyde compounds can be performed easily and accurately. An enzyme-immobilized biosensor that makes it possible has not been realized.

In addition, from the medical field, there has been a demand for a method for easily detecting and measuring alcohol compounds together with aldehyde compounds.
JP-A-10-267888

  The present invention eliminates the conventional problems from the circumstances as described above, and easily detects and measures aldehyde compounds such as formaldehyde in the gas phase or liquid phase, and further alcohol compounds. It is an issue to provide a new enzyme-immobilized biosensor that can be used.

  The enzyme-immobilized biosensor of the present invention is characterized by the following as a solution to the above problems.

First: Alcohol oxidase (AOX) is immobilized on the surface of the hydrogen peroxide electrode, and a cross-linked film of polyallyl compound or polyacryl compound is disposed on the outermost surface as a polymer coating to oxidize hydrogen peroxide. The presence of an aldehyde compound or an alcohol compound can be detected and measured from the current value or a change thereof.

  Second: At least a part of the hydrogen peroxide electrode is constituted by a platinum electrode and platinum black deposited on the surface thereof, and alcohol oxidase (AOX) is immobilized on the platinum black surface.

  According to the enzyme-immobilized biosensor of the present invention having the features as described above, the presence of an aldehyde compound or an alcohol compound can be easily detected and measured in a short time without the need for a complicated and expensive apparatus as in the prior art. can do. The aldehyde compound and the alcohol compound can be detected and measured even in the gas phase or in the liquid phase.

  In addition, according to the present invention, manufacture as a sensor is simple and easy.

  The present invention has the features as described above, and an embodiment thereof will be described below.

  In the enzyme-immobilized biosensor of the present invention, it is an essential requirement that alcohol oxidase (AOX) is immobilized on the surface of the hydrogen peroxide electrode. The basic principle is to detect this.

  In the case of formaldehyde, the reaction of the enzyme AOX and the electrode reaction will be described as follows.

  In this reaction, the amount of change in current when a constant potential is applied is measured. That is, the concentration of formaldehyde can be indirectly measured from the oxidation current of hydrogen peroxide generated by the AOX reaction.

  On the other hand, the enzyme AOX reacts with the alcohol compound in the same manner, and for example, hydrogen peroxide is generated as follows.

  As described above, according to the electrode reaction which is the principle of the present invention, detection and measurement of these compounds can be performed by detecting an oxidation current by an oxidation reaction in the case of aldehyde compounds including formaldehyde and alcohol compounds. become. Therefore, in the present invention, a sensor for detecting and measuring an aldehyde compound and a sensor for detecting and measuring an alcohol compound are realized.

  However, as understood from the above, it is assumed that the detected current value includes a response derived from an aldehyde compound and a response derived from alcohol. For this reason, when detecting and measuring an aldehyde compound such as formaldehyde, it may be necessary to suppress a response due to the alcohol compound.

  Therefore, in the enzyme-immobilized biosensor of the present invention, the selectivity of the response of the aldehyde compound or alcohol compound can be enhanced by disposing a polymer coating as the outermost surface.

  The polymer film as the outermost coating layer in the present invention enhances the stability of immobilization of alcohol oxidase (AOX) immobilized on the surface of the hydrogen peroxide electrode, and improves durability in the gas phase or liquid phase. Although it is indispensable for realizing, the polymer film can be adsorbed by using an alcohol compound-adsorbing material, an alcohol compound-excluding material, or an aldehyde compound-adsorbing material. It is also possible to improve the response selectivity. In general, the selectivity of the terdehydride compound and alcohol compound depends on the hydrophilicity and hydrophobicity of the polymer membrane, or the presence or absence of steric exclusion, but the solubility of the test substance in the polymer membrane And can be controlled based on the difference in permeability of the polymer membrane.

  For example, in order to control the response by an alcohol compound, it is considered to use PAAM (polyallylamine) as a polymer coating.

  As the hydrogen peroxide electrode constituting the enzyme-immobilized sensor of the present invention, various types may be used as long as the oxidation current of the peroxide water as described above can be accurately detected. Among them, a representative one that is constituted by a platinum electrode and platinum black deposited on the platinum electrode, or one that at least makes this constitution essential is preferably considered. As for the electrode on which platinum black is deposited and a method for producing the electrode, known means as a conventional EEE type sensor may be appropriately employed. Moreover, it is considered to use not only platinum black but also a porous metal such as gold black.

  Alcohol oxidase (AOX) immobilized on a hydrogen peroxide electrode such as platinum black can be immobilized by various methods and means similar to those of other conventional enzymes. For example, immobilization with glutaraldehyde (GA) is considered suitable. In the case of the sensor of the present invention, the immobilized amount of alcohol oxidase (AOX) depends on the combination of the type and characteristics of the polymer film on the outermost surface of the sensor, It can be determined as appropriate in consideration of the desired detection sensitivity.

  After the AOX oxygen immobilization, the outermost surface layer is coated with a polymer membrane. In this case, the polymer membrane is protected by alcohol oxidase (AOX), hydrophilicity or hydrophobicity related to the response selectivity of the test substance. It has desired properties and characteristics such as degree of property, adsorption or permeability. Preferred examples of the polymer for forming such a polymer film include polyallyl compounds such as polyallylamine (PAAM), polyacryl compounds such as polyacrylamide, and the like. It is also effective that these polymer membranes are crosslinked. Examples of means for crosslinking include dialdehyde compounds such as GA and water-soluble carbodiimide compounds.

  The film thickness of the polymer coating in the enzyme-immobilized biosensor of the present invention can be determined appropriately as described above.

  The selective response of the aldehyde compound as described above can be made possible depending on the type of the polymer film constituting the polymer coating.

  The electric circuit for applying a constant potential to the electrode and detecting the oxidation current in the enzyme-immobilized biosensor of the present invention can have various configurations as in the prior art. For example, a three-electrode system of a working electrode, a reference electrode, and a counter electrode, or a bipolar system can be used, and the electrolyte can be various.

  Then, when performing detection measurement of an actual aldehyde compound or alcohol compound, quantitative measurement is possible by creating a calibration curve between the detected current value and concentration. Further, qualitatively, detection is possible by a change in the detected current value.

  Therefore, an example will be shown below and will be described in more detail. Of course, the invention is not limited by the following examples.

<1> Preparation of enzyme-immobilized sensor electrode The platinum exposed surface at the end of a 0.1 mm diameter platinum electrode sealed in a glass tube is polished and polished by applying a constant potential (−80 mV) in a chloroplatinic acid solution. Platinum black was electrodeposited on the surface. Next, anodization was performed by applying a constant potential (1200 mV) in phosphate buffer.

  Immersion treatment of 800 units / ml alcohol oxidase (AOX) (SIGMA-ALDRICH Co .; Alcohl Oxidase from Hanseula sp) for 10 minutes, 1% glutaraldehyde (GA) aqueous solution for 5 minutes twice for platinum black electrode Repeatedly, alcohol oxidase (AOX) was immobilized on the surface.

Then, membrane treatment was performed by alternately immersing in a polyallylamine (PAAM) solution (0.1 g / 20 ml) for 10 minutes, in a 1% glutaraldehyde (GA) aqueous solution for 5 minutes, and in a polyallylamine (PAAM) solution in order of 10 minutes. gave. This was designated as a PAAM film treatment (once). Further, an electrode immersed in an aqueous GA solution for 5 minutes and in a PAAM solution for 10 minutes was subjected to PAAM film treatment (twice). A polymer film was formed as the outermost layer.
<2> Sensor configuration and measurement conditions The sensor electrode (SP) produced above was used as a working electrode (W) to form a sensor as in the configuration of FIG. In the configuration example of the three-electrode system in FIG. 1, the reference electrode (R) is an Ag / AgCl electrode, and the counter electrode (C) is a platinum electrode. In addition, ALS601S in a figure has shown the electrochemical analyzer, and controls the voltage and the measurement of the amount of electric current and the amount of clones by operation | movement of a computer.

  The detection measurement conditions using this sensor were as shown in Table 1 below.

[Table 1]
Measurement method: Three-electrode system Applied potential: 600 mV
Temperature: 25 ° C
Cell capacity: 10 mL
(Under stirring)
<3> Measurement Results 1) In the measurement under the above conditions, the change in the current value after the formaldehyde was dropped on the sensor in the case of the treatment of the PAAM film (one time) was observed over time. An example of the result is shown in FIG. The sensor response is confirmed as a significant change in the current value after dropping (P).

  FIG. 3 shows the relationship between formaldehyde concentration (mM) and response current (nA). By using this as a calibration curve, quantitative detection and measurement of the formaldehyde concentration of the test sample becomes possible.

  For example, from the verification of formaldehyde detection measurement as described above, it was confirmed that a sensor capable of formaldehyde detection measurement having the sensor performance shown in Table 2 below was realized.

[Table 2]
Concentration range: 0.1-100 mM
Response time: 10-30s
Continuous use time: 8hr
Moreover, about the sensor in this case, it was also confirmed that there is no deterioration in sensor performance for one month by refrigerated storage at a temperature of 4 ° C. in a 0.1 mM phosphate buffer. It can be seen that the durability of the performance is excellent.

  2) On the other hand, when the responsiveness to methyl alcohol was also evaluated, the results shown in FIG. 4 were obtained depending on the presence or absence of the outermost PAAM film.

For responses to the methyl A alcohol is suppressed it has been confirmed by the presence of PAAM film. Regarding formaldehyde, no change in response was observed depending on the presence or absence of such a film, and it was confirmed that the presence or absence of the film affects the durability of maintaining the performance over time.

It is the schematic which showed the structural example of the sensor of this invention. It is the figure which illustrated the change of the electric current value after formaldehyde dropping as an example. It is the figure which illustrated the relationship between formaldehyde concentration and response current. It is the figure which illustrated the relationship between a methyl alcohol concentration and an electric current.

Claims (2)

Alcohol oxidase (AOX) is immobilized on the surface of the hydrogen peroxide electrode, and a cross-linked film of a polyallyl compound or a polyacryl compound is disposed on the outermost surface as a polymer coating , and the oxidation current value of hydrogen peroxide, Alternatively, an enzyme-immobilized biosensor that can detect and measure the presence of an aldehyde compound or an alcohol compound based on the change.   2. The hydrogen peroxide electrode comprises at least a part of a platinum electrode and platinum black deposited on the surface thereof, and an alcohol oxidase (AOX) is immobilized on the surface of the platinum black. The enzyme-immobilized biosensor described.