JPH09159645A - Urine sugar biosensor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To enlarge the working range of urine sugar biosensor by forming a glutaraldehyde cross-linked bovine serum albumin film and a photocross-linked resin film sequentially on a working electrode thereby enhancing the selectivity of sensor. SOLUTION: Distilled water and glutaraldehyde aqueous solution are added sequentially to bovine serum albumin to prepare a mixed liquid which is then dripped onto a working electrode (anode) to progress cross-linking of glutaraldehyde aqueous solution under saturated vapor thus forming a glutaraldehyde cross-linked bovine serum albumin film. On the other hand, glucose oxidase(GOD) and distilled water are then added sequentially to photocross-liked polyvinyl alcohol to produce a dope liquid which is then applied onto the film thus formed by means of a spinner. It is then dried and irradiated with UV-rays thus fixing the GOD inclusively in the photocross-liked polyvinyl alcohol. The inventive urine sugar biosensor is excellent in sensor characteristics, especially selectivity, and the working range thereof can be enlarged.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a urine sugar biosensor. More specifically, the present invention relates to a urine sugar biosensor having excellent sensor characteristics, particularly selectivity.

[0002]

2. Description of the Related Art Japanese Patent Publication No. 2-35933 describes an immobilized enzyme electrode. This immobilized enzyme electrode is prepared by applying an albumin solution containing a polyfunctional aldehyde onto a conductive substrate, After forming the crosslinked albumin layer at ~ 10 ° C,
It is produced by applying a solution containing a polyfunctional aldehyde and an H ₂ O ₂ -forming oxidase to form an H ₂ O ₂ -forming oxidase-containing layer on the crosslinked albumin layer.

However, the immobilized enzyme electrode thus manufactured has the following drawbacks. (1) When the H ₂ O ₂ forming oxidase-containing layer is formed, the polyfunctional aldehyde used therein penetrates into the crosslinked albumin layer, and the structure of the crosslinked albumin layer (crosslink density, structure of bovine serum albumin itself, etc.) Changes, and there is a drawback that the sensor characteristics using this as an electrode, particularly the selectivity, tend to change. (2) The cross-linked albumin layer and the H ₂ O ₂ -forming oxidase-containing layer do not have sufficient adhesiveness and have a drawback that they are easily peeled off. (3) When the H ₂ O ₂ forming oxidase-containing layer is formed, heat of about 40 ° C. is applied in order to cross-link glucose oxidase and the polyfunctional aldehyde in a short time of about 30 minutes, Heating is not preferable from the viewpoint of maintaining enzyme activity.

[0004]

An object of the present invention is to provide a urinary sugar biosensor in which a glutaraldehyde cross-linked bovine serum albumin film and a glucose oxidase-containing film are sequentially formed on the working electrode. It is about providing what is excellent in terms.

[0005]

SUMMARY OF THE INVENTION The object of the present invention is as follows.
This is achieved by a urine sugar biosensor in which a glutaraldehyde-crosslinked bovine serum albumin film and a glucose oxidase-containing photocrosslinked resin film are sequentially formed on the working electrode.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION The base sensor of the urinary sugar biosensor according to the present invention is an H ₂ O ₂ sensor, which is structurally a two-pole structure of a working electrode and a counter electrode, or a working electrode, a counter electrode and a reference electrode. Any of the structures can be taken.

As the working electrode (anode) material, platinum, gold,
Carbon, etc., as the counter electrode (cathode) material, platinum, silver,
Carbon or the like is used, and silver / silver chloride or the like is used as the reference electrode material. The working electrode and the counter electrode are formed on a substrate made of ceramics, glass, plastic, paper or the like by a vapor deposition method, a sputtering method, a screen printing method or the like, and a reference electrode is generally formed by a screen printing method.

First, a glutaraldehyde-crosslinked bovine serum albumin film is formed on the working electrode by the following method. A mixed solution is prepared by adding 1 ml of distilled water to about 5 to 50 μg of bovine serum albumin, and then adding an aqueous solution of glutaraldehyde in an amount such that the final concentration of glutaraldehyde is about 0.1 to 0.4%. An appropriate amount (about 0.1 to 10 μl) of the prepared mixed solution is dropped on the working electrode, and then the mixture is left to stand at about 2 to 10 ° C. for about 0.5 to 5 hours under saturated vapor of an aqueous glutaraldehyde solution. After that, add about 10 more in a constant temperature bath of about 30-45 ℃.
Let stand for ~ 60 minutes to complete the cross-linking reaction between bovine serum albumin and glutaraldehyde.

A glucose oxidase (GOD) -containing photocrosslinking resin film is formed on the glutaraldehyde-crosslinked bovine serum albumin film thus formed. Such GOD
The formation of the photo-crosslinking resin film containing a stilbazolium group, a photo-crosslinkable group such as styrylpyridinium group about 0.5 ~ 3.5 mol%
Polyvinyl alcohol content (saponification value approx. 70 to 10
0, the degree of polymerization of about 500-4000) to an aqueous solution of a photo-crosslinkable resin represented by about 5 to 50% by weight, preferably about 10 to 20% by weight, GOD (e.g., 11.1% photocrosslinking). 165,800 Units / g of solid PVA aqueous solution 3.6g
(0 mg) and distilled water are added to prepare a dope solution having a total amount of about 5 to 10 g, and an appropriate amount of this solution is dripped with a spinner or the like so as to cover the glutaraldehyde cross-linked bovine serum albumin film, and then about 10 to 30. Dry for about 0.5 to 2 hours at a temperature of ° C., then photocrosslinking by irradiating with ultraviolet rays for a predetermined time (about 0.5 to 10 minutes), wash with water and develop as necessary,
A photocrosslinking film is formed.

It is preferable to further form a GOD-free photocrosslinking resin film on these laminated films. In that case,
G from the dope used to form the GOD-containing photocrosslinking resin film
The film is formed by the same operation method using the one without OD.

[0011]

[Action] and

EFFECTS OF THE INVENTION Glucose in urine sugar is oxidized by glucose oxidase immobilized by a photocrosslinking resin film and converted into gluconolactone, and at the same time, H ₂ O ₂ is generated. Since the amount of H ₂ O ₂ generated is proportional to the glucose concentration, the amount of glucose can be quantified by measuring the amount with a base sensor (H ₂ O ₂ sensor).

In the urinary sugar biosensor based on the above operation principle, the sensor characteristics, especially the selectivity can be remarkably improved by adopting a constitution in which glucose oxidase is comprehensively immobilized on the photocrosslinking resin film. By further forming a glucose oxidase-free photocrosslinking resin film on such a glucose oxidase-containing photocrosslinking resin film, the calibration range can be remarkably expanded without impairing its excellent selectivity.

[0013]

Next, the present invention will be described by way of examples.

Example 1 (Production of Electrode) An alumina substrate (Kyocera product A-493) was ultrasonically cleaned, and then a platinum film was formed on the entire surface of the substrate using a platinum vapor deposition apparatus. Then, a positive type resist (OFPR800 manufactured by Tokyo Ohka Kogyo Co., Ltd.) was used to form an electrode pattern using a spin coater 1H-DX manufactured by Mikasa and a mask aligner M-2L. After that, plasma etching equipment (made by ULVAC
RBH-3030) was used for plasma etching to confirm continuity, and then cut into a predetermined size with a dicing saw (DAD-2H / 6T manufactured by DISCO).

Of the three electrodes formed on the substrate, two were used as the working electrode and the counter electrode, and one was used as the lead electrode for the reference electrode. A silver / silver chloride electrode was formed on this platinum electrode for reference electrode. When forming the silver / silver chloride electrode, first use a silver paste (Asahi Chemical Laboratory product LS-504J) to form a silver electrode by coating and baking by screen printing, and then using a current in 0.1M hydrochloric acid electrolyte. Density 0.6mA / c
Perform constant current electrolysis for 20 minutes at m ² (HA-501 / HB-1 manufactured by Hokuto Denko).
04 used), and formed as a reference electrode on the substrate.

(Formation of Laminated Film on Working Electrode) 1 ml of distilled water was added to 20 μg of bovine serum albumin (product A-6003 of Sigma),
Further, 10 μl of a 20 wt% glutaraldehyde aqueous solution was added to prepare a mixed solution. Add 2.5 μl of this mixture to the working electrode (area
0.16 cm ² ), and the mixture was allowed to stand for 2 hours at 4 ° C under saturated steam of an aqueous solution of glutaraldehyde, and then crosslinked at 40 ° C for 30 minutes.

On the glutaraldehyde-crosslinked bovine serum albumin film thus formed, 11.1 of photocrosslinkable polyvinyl alcohol (product of Toyo Gosei Co., Ltd .; stilbazolium group content 1.3 mol%, degree of polymerization 1700, saponification value 88) was used. % Aqueous solution
3.6g glucose oxidase (Sigma product; Type V
II-S, G7016, 165,800 units / g solids) 16.6 mg and distilled water 3.46 g were sequentially added to prepare a dope solution 2.5 μl, spinner applied (200 rpm, 15 seconds), and left at room temperature for 1 hour UV irradiation was performed for 6 minutes to immobilize glucose oxidase in the photocrosslinked polyvinyl alcohol membrane.

(Measurement) The measurement of the manufactured biosensor was carried out using a current detector (LC-4B manufactured by BAS Co.), a sample injector (Model 231 manufactured by Gilson Co.), a pump (DMX-2000 manufactured by Sanuki Kogyo; a double plunger). Type) and a Teflon tube (outer diameter 1/16 inch, inner diameter 1.0 mm) were used for flow injection analysis.

The measurement conditions are 30 ° C., pH 6.9 and applied voltage.
0.6V vs. Ag / AgCl, cell volume 30μl, sample loop 10
μl, distance between sample injector sensors 1 m, flow rate 1.
4 ml / min, the buffer solution is a neutral phosphate standard solution (0.025M KH ₂ PO ₄ , 0.025M Na ₂ HPO ₄ ) 1 specified in JIS K-0020.
In 500 ml, 0.5 g (8.2 mM) benzoic acid as preservative and KC
l The addition of 1.86 g (50 mM) was used.

Glucose has a final concentration of 50, 100, 2
L-ascorbic acid in an amount such as 50, 500, 800, 1000 or 2000 mg / dl and mixed with it is 100 mg / dl
Used in each amount. The selectivity for L-ascorbic acid at these concentrations averages 0.9% F
S (100m when glucose output value of 100mg / dl is 100%)
When the output of 100% g / dl glucose was 100 nA, it was 0.9 nA, but N = 3). In addition, the glucose concentration calibration range is 0 with or without L-ascorbic acid.
It was ~ 800 mg / dl.

Example 2 In Example 1, a dope solution was spinner-coated on a glutaraldehyde-crosslinked bovine serum albumin film, left at room temperature, and then 2.5 μl of dope solution containing no GOD was spinner-coated (200 rpm, 15 seconds). ) And left at room temperature for 1 hour,
Thereafter, irradiation with ultraviolet rays for 5 minutes, washing with water, and development were carried out for 1 minute.

When the obtained sensor was evaluated in the same manner as in Example 1, the selectivity was 0.8% FS (n = 3), and the calibration range was expanded to 0 to 2000 mg / dl.

The glucose concentration and the current output (unit:
The relationship with μA) is as follows, and a calibration curve for each of the above calibration ranges can be created from this relationship. Glucose concentration (mg / dl) Example 1 Example 2 50 0.22 0.10 100 0.45 0.20 250 1.15 0.35 500 2.30 0.80 800 3.30 1.30 1000 3.45 1.75 2000 3.90 3.15

Example 3 Using the biosensor prepared in Example 1, commercially available urine (control urine; abnormal type, glucose concentration 25)
0 mg / dl), 1.20 μA (n = 3)
Output, which showed good agreement with the calibration result of Example 1.

Comparative Example In Example 1, after forming a glutaraldehyde cross-linked bovine serum albumin film, it was prepared from GOD 16.6 mg, bovine serum albumin 3.4 mg, distilled water 1 ml and 2% glutaraldehyde aqueous solution 10 μl on this film. 2.5 .mu.l of the dope solution was added dropwise, cross-linked at 40.degree. C. for 30 minutes and then washed with water for 1 minute.

When the obtained sensor was evaluated in the same manner as in Example 1, the selectivity was 2.6 (n = 3).
The glucose concentration calibration range was 0 to 800 mg / dl with and without L-ascorbic acid.

[Brief description of the drawings]

FIG. 1 is a calibration curve graph of glucose concentration in Examples 1 and 2.

Claims (2)

[Claims] 1. A urinary sugar biosensor comprising a glutaraldehyde-crosslinked bovine serum albumin film and a glucose oxidase-containing photocrosslinked resin film formed on a working electrode in this order. 2. The urine sugar biosensor according to claim 1, wherein a glucose oxidase-free photocrosslinking resin film is further formed on the glucose oxidase-containing photocrosslinking resin film.