JP3477511B2 - Biosensor using gold platinum electrode - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION Many biosensors have so far focused on measuring metabolism-related substances such as glucose, cholesterol and lactic acid. However, there are cases where physiological phenomena can be estimated by measuring multiple items at the same time. Depressive dehydration in the elderly and infants is a typical example. Although the direct indicator of dehydration is osmotic pressure, there has been no means for directly measuring osmotic pressure using a micro blood sample. The present invention relates to a sensor fusion method and a device therefor capable of estimating a physiological state by fusing a urea sensor, a sodium sensor, and a glucose sensor produced by immobilizing three kinds of enzymes on a gold black and platinum black composite electrode.

[0002]

[Approximate measurement formula of blood osmotic pressure] If the osmotic pressure is determined by the molecular concentration, it can be approximated by Π = RTΣCi from the concentration (Ci) of the main chemical species in blood. By utilizing this, the osmotic pressure can be measured by biosensing. The table below shows the major components in blood, Since an electrically neutral condition is satisfied as a component, an approximate value can be obtained by Na ⁺ . As main chemical species, Na ^+, glucose, and urea are selected, and Π ≈ 1.86 × Na ⁺ (mEq / L) + 1/18 × glucose (mg / dL) + 1 / 2.8 × urea nitrogen (mg /
can be approximated by dL). Clinically, potassium may or may not be present. By utilizing this, an osmotic pressure sensor can be constructed by constructing a multi-sensor capable of measuring Na +, glucose, and urea.

Such a multi-sensor can estimate dehydration by measuring the concentration of a bioactive substance. That is, a device capable of estimating a physiological phenomenon by sensor fusion can be manufactured.

Potentiometric sodium sensors and amperometric glucose sensors have been produced for Na ⁺ and glucose, respectively, but no sensor capable of measuring urea with high sensitivity has been produced. Conventionally, urea detection has been attempted by a method in which ammonia produced from urea by urease is measured by potentiometry. But,
In this method, sufficient output cannot be generated for a sample having a buffering action such as serum.

Therefore, it can be expected that urea can be measured amperometrically by using a complex enzyme system comprising a three enzyme system of urease, glutamate dehydrogenase, and glutamate oxidase. This method is not affected by the buffering effect of the sample. This enzyme system ultimately converts urea into peroxide and attempts to detect oxidation.

It is desirable to firmly fix the enzyme by such an electrode for a complex enzyme system and a gold-mercaptide bond, and to highly sensitively detect the product peroxide with platinum or the like at a platinum black electrode.

By the way, in the conventional enzyme sensor, the oxidation detection by amperometry is effective when the electrode active material is produced, but the electrode method cannot be adopted when the product cannot be detected by the electrode. Therefore, it has been attempted to adopt a so-called complex enzyme system, which is a method of further converting an intermediate product using another enzyme to obtain an electrode active material as a product. For example, there is a method of immobilizing a plurality of enzymes in a film shape and attaching this to an electrode.

[0008]

In view of the biosensors of the prior art as described above, the problem to be solved by the invention is to make urea of the above-mentioned enzymes (1), (2) and (3) extremely porous. It is intended to covalently bond and immobilize mainly to a rich composite metal electrode (for example, composed of gold and silver) by a gold-mercaptide bond and to detect with high sensitivity by platinum.

A biosensor capable of measuring urea in body fluid can be provided by the method using such three kinds of enzymes.
The blood osmotic pressure can be estimated by using the trienzyme sensor, the sodium sensor, and the glucose sensor.

[0010]

It has been found that the above problems can be solved by fusing a sensor for measuring each concentration.

That is, in the first approach, the present invention provides a porous electrode material such as composite gold black / platinum black having a porous surface, a group of enzymes such as transferase, synthase, and electrode activity such as oxidoreductase. It consists of enzymes that produce substances. Here, gold black serves as a stable immobilization of an enzyme by a gold mercaptide bond, and platinum black serves as a highly active catalyst electrode.

In the present invention, the porous electrode is a metal compound such as chloroauric acid or chloroplatinic acid which is reductively (electrochemically applied a negative potential) deposited on the surface of the electrode and has a surface area of It is about 100 to several thousand times that of a smooth electrode.

In the present invention, a complex enzyme system is constructed for the detection of urea, which is the most difficult to detect. Here, the complex enzyme system is composed of three kinds of enzymes. In the complex enzyme system, when immersed in a measurement sample such as a body fluid, an electrode active substance is produced from an electrode inactive substrate A (measurement target) by the three types of enzymes as described below.

In the present invention, immobilization of an enzyme (or an antibody) means that a sensitive group is introduced into the surface of a gold / platinum black composite metal electrode by a gold-mercaptide bonding method, and a plurality of enzymes are bound to the sensitive group. That is. Furthermore, the platinum black electrode and the carbon electrode are to directly immobilize the enzyme.

For example, in the case of measuring the amount of urea as an example of measuring a metabolite in a body fluid, the above (1) to
Since the enzymatic reaction of (3) and the electrochemical reaction of (4) occur at the same time, urea can be measured by measuring the produced hydrogen peroxide.

In the present invention, in order to measure the osmotic pressure in the measurement sample, the following three-electrode system is adopted as an example. In a three-electrode system, a predetermined potential is set with respect to the reference electrode, and the current amount (or charge amount) when hydrogen peroxide is oxidized
Is measured and the amount and concentration of the substrate are determined based on the measured value.

The fusion type composite electrode of the present invention is
It can be used as a working electrode (measuring electrode) in an electrochemical measuring method, and is widely used in an electrochemical measuring method using the electrode (biosensor) of the present invention as a working electrode. A two-electrode system composed of a counter electrode which also functions as a reference electrode and a three-electrode system in which the reference electrode and the counter electrode are separated from each other are constructed, and the change caused by the reaction is measured by the amount of current and the potential difference. A predetermined potential is applied to the working electrode, but elements other than the biosensor of the present invention are well known and need not be described further.

In the present invention, the electrode active substance includes a substance that can be easily oxidized or reduced electrochemically, and at this time, a substance that can be easily oxidized and reduced by using an electron mediator. Further, sodium ions are used as Na ⁺ electrodes using a glass electrode or an ion selective membrane.

In the present invention, the complex enzyme system for urea measurement adopted employs at least one kind of enzyme to convert a biological substance which is not an electrode active substance into another kind of electrode inactive substance, and at least one enzyme finally becomes an electrode. It is composed of what is converted into an active substance. A gold-black / platinum-black composite electrode composed of such a composite enzyme system is schematically shown in FIG. FIG. 1A shows a state in which three enzymes are immobilized inside the porous holes of complex gold and platinum black, and FIG. 1B shows three types of enzymes covalently bonded by a gold mercaptide bond. The state is schematically shown.

[0020]

[Example 1] (Production method of composite gold black and platinum black) -0.10 V was applied to an electrode in which chloroplatinic acid and chloroauric acid were mixed at a ratio of 1: 1.
By applying the potential of, an electrode made of gold black and platinum black can be manufactured. FIG. 2 shows the result of elemental analysis by XMA of the composite electrode. Since Kα rays of platinum and gold overlap and cannot be clearly discriminated, attention was paid to Lα rays. The Lα rays of platinum and gold were observed when both compounds were detected at a ratio of 1: 1.

[0021]

Example 2 (Preparation of Sodium Ion Sensitive Membrane and Preparation and Evaluation of Sodium Electrode) PVC (Polyvinyl Chloride), Bis-12-crown-4, NPOE, TF
PB was dissolved in THF and developed on a petri dish to form a sodium ion sensitive membrane. This film was attached to a silver-silver oxide electrode to form a liquid film type electrode. The electrodes were evaluated by measuring the potential. The potential response was between 10 mM and 1 M, and a potential response close to the slope of the Nernst equation was obtained. (Fig. 3) Also, in the phosphate buffer (pH = 7), the slope became gentle and 10
Almost no change in potential was observed in the concentration range of mM or less.

(Preparation and Evaluation of Glucose Sensor) While the platinum electrode has a strong catalytic activity for oxidizing hydrogen peroxide, it is strongly affected by contaminants in blood such as ascorbic acid and uric acid. On the other hand, by increasing the glucose response of the sensor, we thought that the non-specific response could be made relatively small enough to be ignored. In this patent application, we tried to immobilize a large amount of enzyme on the electrode. Glucose oxidase (GOx) was immobilized on a platinum black electrode and cross-linked with glutaraldehyde (GA). Sensor evaluation was performed by applying a constant potential of +500 mV. A constant potential of +500 mV is applied also to a platinum black / gold black composite electrode. G
The sensor in which Ox was immobilized only once significantly suppressed the response in serum. When the number of enzyme immobilization treatments was increased, glucose concentration-dependent current was observed in serum. The result is shown in FIG.

(Preparation and Evaluation of Urea Sensor) Since the electric potential change detection urea sensor which has been applied for conventionally has a limit in the measurement principle in a buffer solution, a current detection type urea sensor is manufactured. Therefore, glutamate oxidase (GL
Ox), glutamate dehydrogenase (GLDH),
A urea sensor with a three-enzyme system consisting of urease was prepared (Fig. 5). A gold-platinum black composite electrode GLOx, GLDH, or urease was immobilized on a platinum black electrode, and GA crosslinking treatment was performed. Sensor evaluation was performed by applying a constant potential of +500 mV. The electrode with only GLox fixed is 1
There was a linear response to glutamate between 0 μM and 1 mM. The electrode (ammonia sensor) on which GLDH is immobilized via GA is NADPH and α-.
A response current was seen between 1 and 4 mM for ammonia in the presence of KG (Fig. 6). An electrode (urea sensor) was prepared by further immobilizing urease on the electrode on which these two enzymes were immobilized. This sensor is 10-80
The response current was about 0.1 to 1.6 nA up to mM, and sufficient sensitivity and output could not be obtained. It is considered that this is because the activity of the enzyme was decreased because the GA was crosslinked in the enzyme molecule by repeated GA treatments during the production of the sensor.
Therefore, when a sensor in which three kinds of enzymes were immobilized at one time was prepared, the sensitivity was significantly improved and 1 mM urea could be sufficiently measured.

(Calibration by osmometer) Albumin,
A simulated solution consisting of four components of glucose, NaCl and urea was measured by an osmometer, and the theoretical formula was compared with the measured values by the present fusion system and osmometer. As a result, for glucose and urea, the change in osmotic pressure with respect to the concentration was almost equal in the calculated value and the measured value (measured value by an osmometer, sensor fusion). On the other hand, the change in osmotic pressure with respect to the change in sodium concentration differed between the measured value and the calculated value, but this can be dealt with by applying a correction coefficient. The measurement was also performed in the presence of albumin, which is the main component of plasma protein, but no strong effect on osmotic pressure was observed.

[0025]

EFFECTS OF THE INVENTION By utilizing the osmotic pressure measuring device by fusion of the biosensor and the ion sensor of the present invention and the manufacturing method thereof, the symptoms of dehydration can be promptly estimated by the electrochemical device. As a result, a measuring device for measuring the sample can be realized. Such an example can be applied not only to dehydration but also to other physiological phenomena. As described above, although an electrocardiograph and a sphygmomanometer have been put to practical use as ME devices, it has been found that a physiological phenomenon can be estimated only by the fusion of a chemical sensor and a biosensor, and its ripple effect is great.

[Brief description of drawings]

[Figure 1] Gold black / platinum black composite electrode. The complex enzyme system (enzyme A, enzyme B and enzyme C) is immobilized on the porous surface. It is a schematic diagram in which each enzyme is immobilized by a gold mercaptide bond. At this set potential, the final product, hydrogen peroxide, is oxidized on the platinum black surface.

FIG. 2 is data showing that porous electrodes prepared by electrolytic reduction by mixing equal amounts of chloroauric acid and chloroplatinic acid are made of respective metals. Since the Kα rays of gold and platinum overlap,
Attention was paid to the Lα ray. Only platinum is seen in the electrode obtained by mixing 9: 1 chloroplatinic acid and chloroauric acid, and conversely, only gold is seen in the electrode obtained by mixing 1: 9.

FIG. 3 is a diagram in which a manufactured Na ⁺ electrode follows a Nernst response.

FIG. 4 is a diagram showing that an electrode on which glucose oxidase is immobilized many times is less susceptible to the influence of serum.

FIG. 5 is a diagram of substance conversion when hydrogen peroxide is produced from urea when three kinds of enzymes are used for amperometric detection of urea. Oxidation of the final product, hydrogen peroxide, is detected.

FIG. 6 is a diagram showing a relationship between a urea electrode and a sensor output obtained by the above method.

[Explanation of symbols]

1. Gold black XMA (X-ray microanalysis) results 2. 2. XMA (X-ray microanalysis) results of platinum black 3. XM results of composite porous electrode consisting of gold black and platinum black 4. Response to phosphate buffer and serum when glucose oxidase is immobilized once 5. 5. Response to phosphate buffer and serum when glucose oxidase is immobilized 4 times Responses to phosphate buffer and serum after immobilization of glucose oxidase 6 times

─────────────────────────────────────────────────── ─── Continuation of the front page (56) Reference JP-A-64-10165 (JP, A) Acute alcohol poisoning and plasma osmotic pressure, emergency medicine, August 10, 1984, Vol. 8, No. 8, 1017-1022 CHEMICAL SENSORS, Japan, March 26, 1997, Vol. 13, SupplementA, 117-120 (58) Fields investigated (Int.Cl. ⁷ , DB name) G01N 27/327 JISST file (JOIS)

Claims (4)

(57) [Claims] 1. A gold-platinum electrode containing both gold and platinum, which has a porous surface by electrolytically reducing a compound containing gold and a compound containing platinum. 2. A sensing device characterized in that an enzyme is mainly chemically bonded to a porous surface by gold mercaptide bonding or surface treatment on the gold-platinum electrode according to claim 1. 3. A method for producing a gold-platinum electrode containing both gold and platinum to form a porous surface by electrolytically reducing a compound containing gold and a compound containing platinum. 4. A method for producing a sensing device, characterized in that the enzyme is chemically bonded to the porous surface by gold mercaptide bonding or surface treatment on the gold-platinum electrode according to claim 3.