Glucose sensor with gel-immobilized glucose oxidase and gluconolactonase

A glucose sensor in which a gel is in contact with a pH sensing element. The gel contains immobilized glucose oxidase and gluconolactonase (EC 3.1.1.17). Gluconolactonase is present in an amount effective to accelerate the hydrolysis of D-glucono-.delta.-lactone.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates to a glucose sensor, more particularly a 
glucose sensor comprising a gel in which glucose oxidase is immobilized 
and a pH-sensing element. 
2. Description of the Related Art 
A variety of types of glucose sensors have been proposed to determine the 
concentration of glucose in a solution. The conventional techniques of 
immobilized enzyme electrode probes are shown in "Solid Phase 
Biochemistry" published by John Wiley & Sons, New York, 1983, page 479 to 
505. Although many models of glucose sensors have been proposed, their 
common principle for measuring the concentration of glucose is based on 
the oxidation reaction of glucose which is catalyzed by glucose oxidase 
(abbreviated as GOD hereinafter). 
In the glucose sensor, oxygen (O.sub.2) consumed by the oxidation reaction 
of glucose, and hydrogen peroxide (H.sub.2 O.sub.2) and gluconic acid 
which are produced by the oxidation reaction of glucose are sensed or 
measured by means of electrochemical elements to determine the 
concentration of glucose. The electrochemical elements may consist of a pH 
glass electrode or a hydrogen-ion-sensitive field effect transistor 
(abbreviated as pH-ISFET, hereinafter) which detects pH-change or 
pH-variation caused by the production of gluconic acid. 
An example of the pH glass electrode is disclosed in "Biochimica et 
Biophysica Acta," Nilsson et al., 1973, vol 320, page 529, while a variety 
of models of the glucose sensors using the pH-ISFET have been proposed 
recently, for example "IEEE Transactions on Electron Devices," 1986, vol. 
ED-33, No. 1, page 47. In these conventional glucose sensors, a gel 
containing a GOD, which is available on the market, is held or deposited 
on a pH-sensing region of the pH-ISFET. 
In the pH-change sensing type of enzymatic glucose probes above-mentioned, 
protons dissociated from gluconic acid produced by hydrolysis of 
D-glucono-.delta.-lactone, which is a direct product of the oxidation 
reaction of glucose are detected. 
Gluconic acid possesses a dissociation constant (pKa) lower than 4 and 
hence is dissociated almost completely into a proton and a conjugate base 
at a pH range higher than 6. On the other hand, the 
D-glucono-.delta.-lactone which is produced by the oxidation reaction of 
glucose is almost completely hydrolyzed at a pH range higher than 5, since 
equilibrium in the hydrolysis reaction of the D-glucono-.delta.-lactone at 
the pH range higher than 5 is shifted excessively to the side of gluconic 
acid formation. 
Therefore, if the pH is higher than 6, the D-glucono-.delta.-lactone which 
is a direct product of the oxidation reaction of glucose is hydrolyzed 
almost completely into gluconic acid which in turn is dissociated almost 
completely into a proton and a conjugate base, so that it can be 
considered that there is a relationship of 1:1 between the amount of 
glucose which is consumed by the oxidation reaction of glucose and the 
amount of protons which are detected. In the pH-change sensing type of 
enzymatic glucose probes, since the pH-change created on the pH sensing 
region is detected through the above-mentioned reaction scheme it is 
preferable that the hydrolysis reaction of D-glucono-.delta.-lactone 
proceed as fast as possible and also that the consumption rate of glucose 
and the formation rate of gluconic acid are balanced. 
However, the rate constant of reaction in the hydrolysis of 
D-glucono-.delta.-lactone is in the order of 10.sup.-3 sec.sup.-1 (Y. 
Pocker et al., "Journal of American Chemical Society," 1973, vol 95, page 
113) in the case of spontaneous hydrolysis reaction. This means that it 
takes more than 10 minutes to hydrolyze half of the amount of 
D-glucono-.delta.-lactone at ambient temperature and at neutral pH. 
Therefore, in the pH-change sensing type of enzymatic glucose probes in 
which the oxidation reaction of glucose proceeds in the gel containing the 
reference substance GOD, if the velocity of the hydrolysis of 
D-glucono-.delta.-lactone is slow, the greater portion of the 
D-glucono-.delta.-lactone which is produced by the oxidation reaction of 
glucose disappears or is lost out of the gel before it is converted to 
gluconic acid, so that the portion which disappears or is lost does not 
contribute to pH-change on the pH-sensing region of the glass pH-electrode 
or of the pH-ISFET. 
In fact, it was confirmed by the present inventor that no response to 
glucose was observed in a case where GOD of high purity (GI obtained from 
ORIENTAL KOBO Co., Ltd. and Buhlinger Mannheim) was used in an pH-ISFET 
glucose sensor which was constructed according to a method disclosed in 
Japanese Patent Application No. 59-209165 (Laid-Open No. 61-88135, 
published May 6, 1986), even if the buffer capacity of a solution to be 
measured was lowered to the order of 0.002. This means that it is 
necessary to accelerate hydrolysis of D-glucono-.delta.-lactone in the 
pH-change sensing type of enzymatic glucose probes. 
In the course of a study which was conducted to solve such problems, it was 
found that some of the GOD available on the market exhibited 
gluconolactonase (EC 3.1.1.17) activity which accelerates the hydrolysis 
of D-glucono-.delta.-lactone and that the out-put of the pH-ISFET glucose 
sensors containing such gluconolactonase were as high as the same level of 
those that had been reported. 
Although it was already reported in "Biotechnology and Bioengineering," 
1977, Vol. 19, page 185, that gluconolactonase is contained in 
commercially available GOD, this paper relates to reactions for preparing 
gluconic acid from maltose but mentions nothing about utilization of 
gluconolactonase in the pH-change sensing type of enzymatic glucose 
sensors. 
The present inventor found that the gluconolactonase can be used as an 
accelerator of the hydrolysis of D-glucono-.delta.-lactone and completed 
present invention. 
Therefore, an object of the present invention is to overcome the problem of 
the prior arts above, mentioned by accelerating the hydrolysis of 
D-glucono-.delta.-lactone and to provide an improved glucose sensor whose 
out-put signals are sufficiently high and hence is applicable for 
practical uses even at low concentrations of glucose. 
SUMMARY OF THE INVENTION 
The present invention provides a glucose sensor comprising a gel in which 
glucose oxidase is immobilized and a pH sensing element, characterized in 
that the gel contains gluconolactonase (EC 3.1.1.17). 
The proportion of the gluconolactonase to be added to the glucose 
oxidase-containing gel is such an amount as is necessary and satisfactory 
to hydrolyze immediately the D-glucono-.delta.-lactone which is produced 
by the oxidation reaction of glucose. Good results are achieved when 500 
units of gluconolactonase exist in 1 cm.sup.3 of the gel in which the 
enzyme is immobilized in order to keep the consumption rate of glucose 
caused by the oxidation reaction of glucose in equilibrium with the 
formation rate of gluconic acid. The amount of the enzyme is determined by 
the method of SHIMIZU ("Analysis Method of Enzymes," Khodansha, 1977, page 
22), assuming that Michaelis' constant is approximately 10 mM (G. D. 
Bailey, "Archives of Biochemistry and Biophysics," Vol 192, No. 2, 1979, 
page 482). In actual operation, more than 500 units of enzyme are 
preferably added to the gel in order to compensate deactivation of the 
enzyme during the preparation stage of the enzyme as well as physical 
hindrance cause by the matrix of the gel. 
As mentioned above, there is a possibility that the commercially available 
GOD may contain gluconolactonase as a contaminant. However, the proportion 
of the gluconolactonase in the GOD is too small to guarantee the 
above-mentioned active amount in the gel, therefore the amount of GOD 
itself must be adjusted to assure the above-mentioned activity. It often 
happens that too much of the GOD, which make preparation of the gel 
difficult, is required when the amount of gluconolactonase is too small. 
Moreover, the out-put of the pH-change sensing type of enzymatic glucose 
probes which are constructed by the commercially available GOD available 
on the market such as disclosed in the document mentioned above, is 
relatively weaker than the other type of enzyme sensors such as penicillin 
sensors (S. D. Caras et al., "Anal. Chem.," 1985, vol. 57, page 1920) or 
urea sensors (IEEE Transaction on Electron Devices, 1986, vol. ED-33, No. 
1, page 47). 
Therefore, the amount of gluconolactonase contained as a contaminant in the 
GOD available on the market is insufficient to obtain practical activity. 
This means that even if the GOD available on the market contains 
gluconolactonase as a contaminant, it is still necessary to add a 
predetermined amount of gluconolactonase freshly. 
The pH-sensors used in the present invention may be a pH-ISFET, a glass 
sensor or the like. 
According to the present invention, gluconolactonase, which catalyzes the 
hydrolysis reaction of D-glucono-.delta.-lactone, is added to the 
GOD-containing gel in order to promote the hydrolysis of 
D-glucono-.delta.-lactone and to balance the rate of glucose consumed in 
the gel with the formation rate of gluconic acid which is produced by the 
hydrolysis of D-glucono-.delta.-lactone, so that the sensitivity or 
out-put of the pH-sensor is improved. 
As described above, the glucose sensor according to the present invention 
has the enzyme immobilized gel containing gluconolactonase, so that an 
increase sensor out-put is obtained even for lower concentrations of 
glucose and hence the lower limit of detection in the pH-sensing type 
glucose sensors is expanded.

DESCRIPTION OF THE PREFERRED EMBODIMENT 
Now, the present invention will be described in more detail with reference 
to examples. 
Gluconolactonase used in the examples was extracted from beef liver and 
purified by the method of Bailey et al. described in "Archives of 
Biochemistry and Biophysics," vol. 192, No. 2, page 482, 1979. The 
specific activity of the purified gluconolactonase was 1,100 units/mg at 
25.degree. C. and pH=7.5 (one unit of activity is defined as the amount of 
D-glucono-.delta.-lactone required to hydrolyze 1.mu.mol of 
gluconolactonase for one minute). 
GOD used was obtained from ORIENTAL KOBO Co., Ltd. 
EXAMPLE 1 
Preparation of the gel 
A layer of enzyme immobilized gel was formed on the gate region of a 
pH-ISFET by a lift-off method described in the Japanese Patent Application 
No. 59-209165 (Laid-Open No. 61-88135, published May 6, 1986) Table 1 
shows the composition of a protein solution spin-coated. 
TABLE 1 
______________________________________ 
Solution Part by volume 
______________________________________ 
30 wt % aqueous solution of bovine serum 
3 
albumin 
30 wt % aqueous solution of GOD 
3 
10 wt % aqueous solution of gluconolactonase.sup.(1) 
2 
5 wt % aqueous solution of glutaraldehyde 
2 
______________________________________ 
Note: 
.sup.(1) gluconolactonase is dissolved in an aqueous solution of 10 mM of 
MgCl.sub.2. 
As a control, a comparative example of an enzyme immobilized gel was 
prepared by the same method as above except that the solution of 
gluconolactonase is replaced by a protein solution containing 10 mM of 
MgCl.sub.2. 
Each layer of the enzyme immobilized gels prepared has a thickness of 1 
.mu.m. 
EXAMPLE 2 
Preparation of the gel 
An enzyme immobilized gel was prepared on the gate region of a pH-ISFET by 
a method described in "Sensors and Actuators," vol. 7, page 233. Table 2 
shows the composition of a protein solution prepared. 
TABLE 2 
______________________________________ 
Solution Part by volume 
______________________________________ 
10 wt % aqueous solution of 
1 
2-hydroxy methylmethacrylic acid 
20 wt % aqueous solution of GOD 
1 
10 wt % aqueous solution of gluconolactonase.sup.(1) 
1 
1 wt % aqueous solution of riboflavin 
1 
1 wt % aqueous solution of potassium 
1 
persulfate 
______________________________________ 
Note: 
.sup.(1) gluconolactonase is dissolved in an aqueous solution of 10 mM of 
MgCl.sub.2. 
A mixture having the composition was dropped on the gate wells on a 
pH-ISFET. Then, the above described is exposed to ultraviolet radiation to 
polymerize 2-hydroxy methylmethacrylic acid to obtain an enzyme 
immobilized gel. 
As a control, a comparative example of an enzyme immobilized gel was 
prepared by the same method as above except that the solution of 
gluconolactonase is replaced by an aqueous solution containing 10 mM of 
MgCl.sub.2. 
Each layer of the enzyme immobilized gels prepared has a thickness of 200 
.mu.m. 
EVALUATION OF RESPONSE PROPERTIES 
The results of the response properties of the glucose sensors prepared are 
shown in FIG. 1 and FIG. 2. 
The response of the glucose sensor is determined in a 20 mM 
(N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-NaOH) buffer solution 
(pH=7.5) at 25.degree. C. 
Curve "A" in FIG. 1 is a response curve of the glucose sensor prepared by 
Example 1 according to the present invention, while curve "B" in FIG. 1 is 
a response curve of the glucose sensor prepared by Example 2 according to 
the present invention. 
FIG. 2 shows response curves of the glucose sensors prepared by the 
comparative examples containing no gluconolactonase. Curve "a" in FIG. 2 
corresponds to the curve "A" in FIG. 1, while curve "b" in FIG. 2 
corresponds to the curve "B" in FIG. 1. 
From the comparison of FIG. 1 and FIG. 2, it was revealed that the out-put 
of glucose sensor having the gluconolactonase-containing GOD immobilized 
gel according to the present invention increases remarkably from 0.01 mM 
of the concentration of glucose, so that the glucose sensors according to 
the present invention can be used in a wide range of concentrations of 
glucose from 0.01 to 10 mM. To the contrary, the conventional glucose 
sensors having the GOD immobilized gel containing no gluconolactonase 
shown in FIG. 2 do not produce satisfactory sensor out-put over about 0.1 
mM of the concentration of glucose.