JP2502656B2 - Biosensor manufacturing method - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing a biosensor capable of quantifying a specific component in various trace amounts of a biological sample quickly and simply without diluting a sample solution. .

2. Description of the Related Art Conventionally, a biosensor as shown in FIG. 5 has been proposed as a method for highly accurately quantifying a specific component in a biological sample such as blood without performing operations such as dilution and stirring of a sample solution. ing. (For example, JP-A-59-166852). This biosensor has leads 12, 13 on an insulating substrate 9.
Measuring electrode 10 and counter electrode made of platinum, etc.
11 is buried and the exposed parts of these electrode systems are covered with a porous body 14 carrying an oxidoreductase and an electron acceptor. When the sample solution is dropped onto the porous body, the oxidoreductase and the electron acceptor in the porous body are dissolved in the sample solution, and the enzyme reaction proceeds with the substrate in the sample solution to reduce the electron acceptor. After completion of the enzymatic reaction, the reduced electron acceptor is electrochemically oxidized, and the concentration of the substrate in the sample solution is determined from the oxidation current value obtained at this time.

Problems to be Solved by the Invention With such a conventional configuration, the porous body can be easily measured by replacing it with each measurement, but the electrode system requires an operation such as cleaning. On the other hand, if it is possible to dispose of each measurement including the electrode system, it will be very simple in terms of measurement operation, but it will have to be very expensive in terms of the electrode material and configuration such as platinum. Absent. Also,
In the conventional structure, it was observed that the liquid dropped onto the electrode and the wetting on the electrode surface became non-uniform, and an unstable response was exhibited due to bubbles remaining on the electrode surface.

Means for Solving the Problems In order to solve the above problems, the present invention provides, on an insulating substrate, an electrode system including at least a measurement electrode and a counter electrode by printing or coating of a carbon paste, and then the surface of the electrode. After polishing and heat treatment, a hydrophilic polymer layer is formed on the electrode surface, and the substrate is integrated with oxidoreductase and electron acceptor.

Effects According to the present invention, it is possible to construct a disposable type biosensor that can measure the substrate concentration extremely easily and has excellent storage stability.

Example One example of the present invention will be described below.

Example 1 A glucose sensor will be described as an example of a biosensor. FIG. 1 shows a glucose sensor manufactured as an example of a method for manufacturing a biosensor of the present invention, and is an exploded view of constituent parts. An electrically conductive carbon paste containing a resin binder is printed in parallel strips by screen printing on an insulating substrate 1 made of polyethylene terephthalate, and heated and dried to form an electrode system composed of a counter electrode 2, a measurement electrode 3, and a reference electrode 4. To form. Next, an insulating paste mainly composed of polyester is applied so as to partially cover the electrode system and leave 2 ', 3', 4 '(each 1) which becomes an electrochemically acting portion of each electrode. Similarly, printing is performed and heat treatment is performed to form the insulating layer 5. next,
After polishing the exposed parts of 2 ', 3', 4 ', in air
It heat-processed at 100 degreeC for 4 hours. Next, a 0.5 wt% aqueous solution of carboxymethyl cellulose (hereinafter abbreviated as CMC) as a hydrophilic polymer was spread on the electrode and dried. After that, the resin-made holding frame 6 with holes is adhered to the insulating layer 5. Next, a solution in which glucose oxidase as an enzyme is dissolved in a phosphate buffer solution is developed so as to cover the electrode systems 2 ', 3', 4'and dried to carry the enzyme. Then,
The porous body 7 carrying the electron acceptor is held in the hole of the holding frame. Further, a resin cover 8 having an opening having a diameter smaller than the outer diameter of the porous body is adhered to integrate the whole body. The attachment of the holding frame and the formation of the enzyme layer and the hydrophilic polymer layer are not particularly limited to the above order.

Measuring electrode 3 for the integrated biosensor
A cross-sectional view along the line is shown in FIG. The porous material used above is made of nylon non-woven fabric, potassium ferricyanide 400 mg as an electron acceptor, PH5.6 phosphate buffer 1 mL containing 0.025 wt% concentration of surfactant (polyethylene glycol alkyl phenyl ether). It was prepared by impregnating the base material with the liquid dissolved in, crystallizing it by immersing it in ethanol containing a surfactant with a concentration of 0.025 wt%, and then drying it under reduced pressure.

The glucose standard solution was dropped as a sample solution into the porous body of the glucose sensor configured as described above, and two minutes after the dropping, a pulse voltage of 700 mV was applied with reference to the reference electrode to polarize the measurement electrode in the anode direction. .

The added sample solution rapidly spreads on the electrode surface while dissolving the enzyme, electron acceptor and CMC into a viscous liquid, and no residual air bubbles were observed. It is considered that this is because the wettability of the electrode surface was improved by the hydrophilic polymer layer previously formed on the electrode.

On the other hand, glucose in the added sample liquid reacts with potassium ferricyanide carried on the porous body 7 by the action of glucose oxidase carried on the electrode to produce potassium ferrocyanide. Therefore, by applying the pulse voltage in the direction of the anode, an oxidation current proportional to the concentration of potassium ferrocyanide produced is obtained, and this current value corresponds to the concentration of glucose as a substrate.

FIG. 3 shows, as an example of the response characteristics of the sensor having the above-described configuration, the relationship between the current value 10 seconds after the voltage application and the glucose concentration, and showed extremely good linearity.
In the method for manufacturing the glucose sensor shown above, the temperature of the heat treatment step after polishing the carbon electrode is 100 ° C., 70
Each of the sensors was constructed in exactly the same way as above, except that no heat treatment was performed at 30 ° C.
Then, the response change to the glucose standard solution was examined. FIG. 4 shows the change when the initial response current was set to 10% for the sensor using electrodes at each heat treatment temperature. As is clear from the figure, at a treatment temperature of 60 ° C. or higher, the response change with storage is small, but at 50 ° C. or without heat treatment, the change is large. It is presumed that this is because the activity of the exposed surface portion of the polished carbon printed electrode is not stable. In addition, when the electrode surface was not polished, only about 1/3 of the response current when polishing was obtained, but the difference in the response current depending on the presence or absence of polishing is that the resin contained as a binder in the paste. It is considered that this is because the components partially cover the carbon surface. However, it is considered that the removal of the resin binder on the surface of the carbon electrode and the smoothing of the electrode surface are promoted by polishing, and further heat treatment can stabilize the activity of the exposed portion of the electrode.

According to the study of the present inventors, by heat-treating at a temperature of 60 to 170 ° C. in an oxygen atmosphere for 1 to 4 hours or more, favorable results were obtained in which the change in response current after storage was extremely small. In the heat treatment, at 50 ° C. or lower, the preferable result was not obtained as described above, and conversely, in the heat treatment at a temperature higher than 170 ° C., the response sensitivity was rather lowered. It can be considered that this is due to deterioration of the surface of the carbon electrode including deterioration of the resin binder in the carbon paste.

Example 2 The steps up to forming the hydrophilic polymer layer were performed in exactly the same manner as in Example 1, then, without using a storage frame, a phosphate buffer solution of glucose oxidase was developed on the electrode surface and dried. Next, finely divided potassium ferricyanide was dispersed in an organic solvent, and this was spread on the above-mentioned enzyme-supporting surface, and then the organic solvent was evaporated.

With respect to the glucose sensor having the hydrophilic polymer, enzyme, and electron acceptor supported on the electrode surface obtained as described above, the response to glucose concentration was measured in the same manner as in Example 1, and the concentration and the current value were measured. A good linear relationship was obtained between.

Regarding the arrangement in the case of integrating the electron acceptor and the redox enzyme in the method for producing the biosensor of the present invention, as shown in Example 1, a porous body is used, and either one or both of them are formed into a porous body. When carried and integrated with the insulating substrate, the dropped sample solution is surely spread on the porous body,
In addition, the carried enzyme or electron acceptor is also rapidly dissolved, so that the measurement time can be shortened. Further, as shown in Example 2, if both are carried and integrated on the electrode together with the hydrophilic polymer, it becomes structurally simple, for example, a holding frame becomes unnecessary, and the manufacturing advantage is great.

Further, the integration method is not limited to the shapes and combinations of the frame body, the cover and the like shown in the embodiments. As the porous body to be used, in addition to nylon nonwoven fabric, a porous body made of cellulose, rayon, ceramics, polycarbonate or the like can be used alone or in combination. Furthermore, the combination of the oxidoreductase and the electron acceptor is not limited to those in the above examples, and any combination can be used as long as it meets the gist of the present invention. on the other hand,
In the above embodiments, the case of the three-electrode system was described as the electrode system, but the two-electrode system including the counter electrode and the measurement electrode could also be used for measurement. Further, in forming the electrode, when the conductivity of the carbon paste is low, the lead may be formed in advance with silver paste or the like, and the carbon electrode may be formed on the lead.

As the water-absorbent polymer, gelatin, methylcellulose, etc. can be used in addition to CMC, and starch-based, carboxymethylcellulose-based, gelatin-based, acrylate-based, vinyl alcohol-based, vinylpyrrolidone-based, and maleic anhydride-based polymers are preferable. A hydrophilic polymer layer having a required film thickness can be formed on the electrode by applying and drying a solution of these water-absorbing or water-soluble hydrophilic polymers in an appropriate concentration.

The method for producing the biosensor of the present invention is not limited to the glucose sensor shown in the above-mentioned examples, and can be used in systems involving oxidoreductase such as alcohol sensor and cholesterol sensor. Further, as the electron acceptor, potassium ferricyanide used in the above examples is suitable because it reacts stably, but quinone type and ferrocene type can also be used.

EFFECTS OF THE INVENTION As described above, the biosensor manufacturing method of the present invention can provide a biosensor excellent in responsiveness and storability by subjecting an electrode system mainly composed of carbon to polishing and heat treatment.

[Brief description of drawings]

FIG. 1 is an exploded perspective view of a biosensor according to an embodiment of the present invention, FIG. 2 is a longitudinal sectional view of the biosensor, FIG. 3 is a response characteristic diagram of the biosensor, and FIG. 4 is the biosensor. FIG. 5 is a longitudinal sectional view of a conventional biosensor.

(72) Inventor Takashi Iijima 1006 Kadoma, Kadoma City, Osaka Prefecture Matsushita Electric Industrial Co., Ltd. (56) References JP-A-63-3248 (JP, A) JP-A-63-58149 (JP) , A) JP-A-1-54345 (JP, A)

Claims (4)

(57) [Claims] 1. An electrode system comprising at least a measuring electrode and a counter electrode is provided on an insulating substrate by printing or coating a carbon paste, and then the surface of this electrode is polished and heat-treated, and then the electrode surface is highly hydrophilic. A method for producing a biosensor, comprising forming a molecular layer and integrating the substrate with an oxidoreductase and an electron acceptor. 2. The method for producing a biosensor according to claim 1, wherein the hydrophilic polymer layer formed on the surface of the electrode carries an oxidoreductase and an electron acceptor and is integrated with the substrate. 3. The method for producing a biosensor according to claim 1, wherein both or either one of the oxidoreductase and the electron acceptor is previously supported on the porous body and then integrated with the substrate. 4. The method for producing a biosensor according to claim 1, wherein the heat treatment is performed at a temperature of 60 ° C. or higher in an oxygen atmosphere.