JPS63144248A - Biosensor - Google Patents

Abstract

PURPOSE:To speedily and easily determine a specific component in an organic body sample with high accuracy by uniting electrode systems and a porous body. CONSTITUTION:Conductive carbon paste is printed on an insulating substrate 1, and heated and dried to form an electrode system of a counter electrode 2, a measurement electrode 3, and a reference electrode 4. Insulating paste is printed so that the electrode system is covered partially and electrodes 2'-4' which operate electrochemically are left; and a heat treatment is carried out to form an insulating layer 5. Further, both-side adhesive tapes 6 as cohesive structure bodies are adhered to the insulating layer 5 at an interval across the electrode systems 2'-4'. At this time, one end of a rayon cut piece 7 as a liquid holding layer is placed on one tape 6 and the other end is sandwiched between the other tape 6 and insulating layer 5. Then, a filter layer 9 for removing solid components from sample liquid is fixed in a holding frame 8 and a porous body 10 is further held in the holding frame 8. The holding frame 8 is adhered to the tapes 6 and the one end of the rayon cut piece 7 is sandwiched between the tape 6 and filter layer 9. Further, a resin-made cover 11 is adhered to unit the whole body.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a biosensor that can quickly and easily quantify specific components in various minute amounts of biological samples without diluting the sample liquid.

Conventional technology In the past, a biosensor as shown in Figure 6 has been proposed as a method for quantifying specific components in biological samples such as blood with high precision without performing operations such as diluting or stirring the sample solution. (For example, Japanese Patent Application Laid-Open No. 59-188852)
. This biosensor has leads 15.1 on an insulating substrate 12.
A measuring electrode 13 and a counter electrode 14 each made of platinum or the like having 6 are embedded, and the exposed portions of these electrode systems are covered with a porous body 17 carrying an oxidoreductase and an electron acceptor. When the sample liquid is dropped onto the porous body, the oxidoreductase and electron acceptor in the porous body are dissolved in the sample liquid, and an enzymatic reaction proceeds with the substrate in the sample liquid to reduce the electron acceptor. After the enzymatic reaction is completed, the reduced electron acceptor is electrochemically oxidized, and the substrate concentration in the sample solution is determined from the oxidation current value obtained at this time.

Problems to be Solved by the Invention In such a conventional configuration, the porous body can be easily used for measurement by replacing it for each measurement, but the electrode system requires operations such as cleaning. On the other hand, if the electrode system, including the electrode system, could be disposed of after each measurement, the measurement operation would be extremely simple, but it would be extremely expensive due to the electrode materials such as platinum and the structure. do not have.

As a result of various studies on these points, the present invention has developed an inexpensive disposable type biotechnology that can extremely easily quantify specific components in biological samples quickly and with high precision by integrating an electrode system and a porous body. It provides a sensor.

Means for Solving the Problems In order to solve the above problems, the present invention provides an electrode system consisting of at least a measurement electrode and a counter electrode on an insulating substrate, reacts an enzyme, an electron acceptor, and a sample solution, and performs the reaction. In a biosensor that electrochemically detects a change in substance concentration with the electrode system and measures the substrate concentration in a sample liquid, through a space formed using an adhesive structure on the electrode system,
A porous body carrying an oxidoreductase and an electron acceptor is installed and integrated with the electrode system and the substrate.

Further, as a liquid retaining layer for reliably supplying the sample liquid onto the electrode system, one end of the hydrophilic porous body is connected to the adhesive structure and a filtration layer provided to remove solid components from the sample liquid. The other end was placed between the adhesive structure and the insulating substrate, and the adhesive structure was placed in the space and on the electrode system. By installing the liquid-retaining layer in this way, the sample liquid is directionally guided from one end to the other end sandwiched between the adhesive structure and the filtration layer. Air can be effectively replaced with the sample liquid. This can prevent the formation of bubbles and the resulting decrease in measured values.

Effects According to the present invention, a disposable type biosensor including an electrode system can be constructed, and the substrate concentration can be extremely easily measured by adding a sample liquid to a porous body.

Moreover, one end of the liquid retaining layer is sandwiched between the adhesive structure and the filtration layer, and the other end is sandwiched between the adhesive structure and the insulating substrate.
Since it is installed in the space and above the electrode system, it is possible to reliably supply the sample liquid onto the electrode system, and there is no drop in the measured value due to air bubbles on the measurement tip, making it possible to perform highly accurate measurements.

Example An embodiment of the present invention will be described below.

A glucose sensor will be described as an example of a biosensor. FIG. 1 shows an embodiment of a glucose sensor, and is an exploded view of the constituent parts. A conductive carbon paste (t) is printed on an insulating substrate 1 made of polyethylene terephthalate by screen printing and dried by heating to form a counter electrode 2. Measuring pole 3. An electrode system consisting of a reference electrode 4 is formed. 2' then partially covers the electrode system and becomes the electrochemically active part of each electrode.

Insulating paste is printed in the same manner as described above so as to leave 3' and 4' (each 1-), and heat-treated to form an insulating layer 50. Furthermore, the electrode system (2' l 3' l '') ”
A double-sided adhesive tape 6 as an adhesive structure is adhered to the insulating layer 6 in parallel with an interval of 2 mm in a sandwiching manner.

At this time, one end of a rayon piece 7 (4×4+a++ size) is placed on top of the double-sided adhesive tape 6 as a liquid retaining layer, and the other end is sandwiched between the double-sided adhesive tape 6 and the insulating layer 6. FIG. 2 is a plan view of the electrode system section showing the positional relationship between the electrode system and the liquid retaining layer.

Next, polycarbonate is fixed as a filtration layer 9 for removing solid components 1 such as red blood cells from the sample liquid in a resin holding frame 8 with holes, and enzymes,
And the porous body 10 is held as a carrier for an electron acceptor. When the holding frame 8 thus constructed is adhered to the double-sided adhesive tape 6, one end of the rayon piece 7 will be sandwiched between the double-sided adhesive tape 6 and the filter layer 9. Furthermore, a resin cover 11 having an opening having a diameter smaller than that of the porous body 1o is adhered to integrate the whole body.

A cross-sectional view along the measurement electrode 3 of this integrated biosensor is shown in FIG. The porous body 1o used above was prepared by impregnating a nylon nonwoven fabric with a solution prepared by dissolving 100 da of glucose oxidase as an oxidoreductase and 160 q of potassium ferricyanide as an electron acceptor in a phosphate buffer solution with a pH of 6.e, and then drying it under reduced pressure. It was made by

Glucose standard solution was dropped as a sample solution into the porous body 1o of the glucose sensor configured as described above, and after 2 minutes of dropping, the sample solution was dropped 7 minutes in the anode direction with respect to the measurement electrode with reference to the reference electrode.
Apply a pulse voltage of 00 mV for 10 seconds, and after a certain period of time,
For example, the oxidation current value measured after 10 seconds is 0. In this case, the added glucose reacts with potassium ferricyanide by the action of glucose oxidase supported on the porous body 1o to produce potassium ferrocyanide. Therefore, an oxidation current based on the concentration of potassium ferrocyanide generated by applying a pulse voltage in the anode direction as described above is obtained, and this current value corresponds to the concentration of glucose, which is the substrate.

Human blood mixed with sodium fluoride as a glycolytic inhibitor and left at room temperature for 1 hour (hereinafter referred to as sample blood) was dropped onto the above glucose sensor, and after 2 minutes 700
! IIv, a 10-second pulse was applied, and the current value was measured 10 seconds after the pulse was generated. The results are shown in FIG. The average value of the measured current with 10 samples is 3.03μ
The coefficient of variation was 2.97%. FIG. 4B shows the results of a similar experiment conducted using the same sample blood as used in the above experiment, but on a glucose sensor with the above configuration except that the rayon section 7 was removed. The average value of the measured current with 10 samples is 2.00 μm, and the coefficient of variation is e O,
3%, and the variation in measured current values is wider than in the former case. When the glucose sensor that showed a particularly low value was disassembled, it was found that sample blood was not being supplied onto the electrode system.

In addition, as a liquid retaining layer, a rayon piece 7' having a size of 2 x 8 m+ was placed on the long side of the double-sided adhesive tape 6, and the long side was not sandwiched between the double-sided adhesive tape 6 and the insulating layer 6. The glucose sensor was installed along a double-sided adhesive tape, and the other configuration was the same as that of this example, and the results were measured using 10 samples using the same sample blood used in the above experiment. Shown in C of the figure. FIG. 6 is a plan view of the electrode system of this glucose sensor.

The average value of the measured current rI'i is 2.35 μm, and the coefficient of variation is 3.
It was 0.6%. For those that showed particularly low values,
As a result of the decomposition, it was confirmed that although the sample blood was supplied onto the electrode system, bubbles were formed on the measurement electrode 3'.

However, in these experiments, blood cells were removed from the sample blood that reached the electrode system by the filtration layer 9.

On the other hand, according to the configuration of this example, the sample dish was reliably supplied onto the electrode system, and the formation of bubbles at the measurement level was not observed, and a response with good reproducibility was obtained.

Furthermore, regarding the double-sided adhesive tape 6, the one on which the rayon section 7 is placed is set 0.61 to 0.3 m higher than the other tape, and the other parts are the same as in this example. Using the same blood that was used,
As a result of carrying out the same measurement as above with 10 samples, the average value of the measured current was 3.01 μm, and the coefficient of variation was 2.18%, which was better than the measurement results in this example shown in Fig. 4. , which was even more stable.

The hydrophilic porous material used as the liquid retaining layer is not limited to rayon, and its shape is not limited to those in the above embodiments.

Screen printing as a method for forming electrode systems can produce disposable biosensors with uniform characteristics at low cost, and in particular, it is possible to fabricate electrodes using carbon, which is an inexpensive and stable electrode material. This is a convenient way to form.

The method of integration in the biosensor of the present invention is as follows:
The present invention is not limited to the shapes and combinations of frames, covers, etc. shown in the examples. In addition, the porous body used is
In addition to nylon nonwovens, porous bodies made of cellulose, rayon, ceramic, polycarbonate, etc. can be used alone or in combination. Further, the combination of oxidoreductase and electron acceptor is not limited to 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, a three-electrode blade type electrode system was described, but measurements can also be made with a two-electrode blade type consisting of a counter electrode and a measurement electrode.

Effects of the Invention The biosensor of the present invention includes an insulating substrate and an electrode system,
By integrating a porous material carrying an oxidoreductase and an electron acceptor through a space formed using an adhesive structure on the electrode system, it is possible to extremely easily measure the substrate concentration in a biological sample. can do.

Furthermore, one end of the hydrophilic porous material is sandwiched between the adhesive structure and the filtration layer as a liquid retaining layer, and the other end is sandwiched between the adhesive structure and the insulating substrate, so that By installing it on the electrode system, we were able to improve measurement reproducibility.

[Brief explanation of the drawing]

FIG. 1 is an exploded perspective view of a biosensor that is an embodiment of the present invention, FIG. 2 is a plan view of the electrode system, FIG. 3 is a longitudinal sectional view thereof, and FIG. 4 is a response characteristic diagram of the biosensor. Figure 6 is the 4th
C in the figure is a plan view of the electrode system part of the biosensor used for measurement, and FIG. 6 is a longitudinal sectional view of a conventional biosensor. 1... Insulating substrate, 2, 2'... Counter electrode, 3゜3'... Measurement electrode, 4, 4'...
・Counter electrode, 6...Insulating layer, 6...Double-sided adhesive tape, ? , 7'...Liquid retaining layer (rayon section), 8...Retaining frame, 9...Filtering layer, 1
0...Porous body, 11...Resin cover, 12...Insulating substrate, 13...Measurement electrode, 14...Counter electrode, 15.16...Reed, 17...Porous body. Name of agent: Patent attorney Toshio Nakao and 1 other person 2nd
Figure N ~ C Tsutsukasa illusion) (3rd @ Figure 4 / z34s6 γ θ 9 10 1 constant number of times (Concave! 5 Ward 7' Figure 6

Claims (5)

[Claims] (1) An insulating substrate is provided with an electrode system consisting of at least a measurement electrode and a counter electrode, and a change in substance concentration during a reaction between an enzyme, an electron acceptor, and a sample liquid is electrochemically detected by the electrode system; In a biosensor that measures the substrate concentration of a sample solution, the insulating substrate, the electrode system, the liquid retaining layer, and the filtration layer are oxidized through a space formed using an adhesive structure on the electrode system. It is integrated with a porous body carrying a reductase and an electron acceptor, one end of the liquid retaining layer is sandwiched between the adhesive structure and the filtration layer, and the other end is sandwiched between the adhesive structure and the insulating substrate. A biosensor, characterized in that the biosensor is installed on an electrode system in the space part in between. (2) The biosensor according to claim 1, wherein the electrode system includes a measurement electrode, a counter electrode, and a reference electrode. (3) The biosensor according to claim 1 or 2, wherein the electrode system is made of a carbon-based material formed by screen printing on an insulating substrate. (4) The biosensor according to claim 1, wherein the liquid retaining layer is made of rayon. (5) The biosensor according to claim 1, wherein the adhesive structure has a height difference.