JPS6211593B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to an enzyme-based analyzer, and particularly relates to an enzyme-based analyzer that is suitable for expanding the measurement range and obtaining highly accurate measurement values in an analyzer using an oxidoreductase that uses oxygen as an acceptor, such as glucose oxidase. . Conventional analyzers that use oxidoreductases that use oxygen as receptors are often used in the field of clinical testing, as exemplified by Japanese Patent Publication No. 1983-8318. This is based on the fact that enzymes have so-called high reaction specificity, catalyzing only specific substances, so even small analyzers with simple mechanisms can be expected to have excellent properties. One widely used medical analyzer is a glucose analyzer that measures glucose in biological fluids. In this case, by utilizing the oxidizing action of an enzyme called β-D-glucose:oxydiene oxidoreductase (commonly known as glucose oxidase), dissolved oxygen that is quantitatively consumed by the reaction of equation (1) below, or The amount of hydrogen peroxide produced is detected and the glucose concentration is calculated.

[Table] This reaction is similar to enzymes that oxidize substrates other than glucose, such as cholesterol and uric acid, and oxygen in the solution is directly involved in the reaction. Therefore, in an analyzer that uses such an oxidoreductase, it is important whether the amount of oxygen in the test solution is sufficient to oxidize the substrate in the same test solution. Therefore,
In glucose analyzers and the like, a test liquid such as blood is diluted with a buffer solution and analyzed to avoid a shortage of dissolved oxygen. BACKGROUND ART Recently, analyzers such as blood multi-item testing devices that can analyze items such as electrolytes, blood gases, and glucose in a small amount of sample in a short time have been needed in emergencies and in the pediatric field. In this case, it is desirable to have a small and lightweight device in order to be compatible with other test items and to bring it to the bedside, and it is desirable to have a device that is simple and can quickly output results without requiring analysis operations such as dilution. There must be. However, the amount of oxygen in serum is only about 2.2 x 10 ^-3 mol/dl, which is equivalent to 40 mg/dl in terms of glucose, and when glucose is analyzed using conventional methods without dilution, there is often a shortage of dissolved oxygen. , reaction cells immobilized with various oxidoreductases are connected in series, and uric acid, glucose, cholesterol, etc.
Even if diluted when analyzing with a single device,
Since dissolved oxygen is consumed in the reaction cells upstream of the flow, the reaction cells downstream of the flow are always starved of dissolved oxygen. Therefore, there was a drawback that the measurement range of the substrate was significantly narrowed. In addition, if the dilution ratio is increased for the purpose of replenishing dissolved oxygen, errors associated with this and accuracy problems due to a decrease in the concentration of the substrate will occur. Devices such as enzyme-based multi-item analyzers have not been possible using conventional methods. An object of the present invention is to provide an enzyme-based analyzer with a wide quantitative range and high accuracy. The present invention is as follows. In other words, one of the reasons why the quantitative range of an enzyme-based analyzer becomes narrow and the precision decreases is that the dissolved oxygen in the sample differs for each sample, and that there is not enough oxygen in the sample to match the amount of substrate. It depends. Therefore,
In order to disassemble this, the substrate concentration in the sample can be adjusted according to the dissolved oxygen concentration using methods such as dilution, but as mentioned above, there are problems with accuracy, the equipment configuration is complex, and the size is large. This makes it expensive and unsuitable for inspection equipment used at the bedside. Therefore, it was discovered that since the dissolution rate of oxygen from the gas phase to the aqueous phase is sufficiently fast, the above problem can be solved by replenishing oxygen in the gas phase by shortening the diffusion distance to the enzyme reaction area. In other words, an insoluble carrier immobilized with an oxidoreductase that acts as an oxygen acceptor and beads made of a substance with high oxygen transfer ability such as fluorocarbon are uniformly packed into the same column, and this is used as an enzyme reaction column. do. In this way, by installing beads that replenish oxygen very close to the enzymatic reaction area that consumes oxygen, the measurement range can be expanded without affecting other measurement items or the detection system, resulting in high precision. Enzyme-based analyzers and multi-item analyzers can be realized with a small size, simple configuration, and a blind price. Examples of the present invention will be described below. FIG. 1 shows an embodiment of the invention. In the figure, reference numeral 1 denotes a sample injection port through which a sample, which is an object to be measured, is injected. The sample injected through the sample injection port 1 is configured to be sent to the enzyme reaction section 2 by a carrier solution 7. 3 is a detector, which detects the result of the reaction by the enzyme reaction section 2; The result of this detector 3 is amplified by an amplifier 4 and recorded on a recorder 6.
A pump 5 is used to send an appropriate amount of the sample together with the carrier solution 7 to the enzyme reaction section 2. Since it is constructed in this way, the carrier solution 7 is a buffer solution with a pH close to the optimum pH for the enzyme reaction.
The sample is discharged by the pump 5 through the sample injection port 1, the enzyme reaction section 2, and the detector 3. A specimen such as serum is injected into the carrier solution 7 through the sample injection port 1 and transported to the enzyme reaction section 2. The enzyme reaction section 2 is a crumb uniformly filled with beads on which an enzyme suitable for the substrate to be measured is immobilized and beads made of a substance with high oxygen transfer ability.For example, when measuring glucose concentration, According to equation (1), glucose is oxidized and dissolved oxygen in the portion corresponding to the sample band is consumed. At this time, hydrogen peroxide is generated in an equimolar amount to the oxidized glucose, which is detected by the detector 3 to obtain a signal related to the glucose concentration. FIG. 2 is a side sectional view of a main part showing an embodiment of the enzyme reaction section according to the present invention. The interior of the column body 2 is uniformly filled with enzyme-immobilized beads 8 and fluorocarbon beads 9 as a substance with high oxygen transfer ability. In addition to fluorocarbons, inorganic adsorbents such as certain zeolites are also known as substances with high oxygen transfer ability, but these have problems with contamination by blood components and blood coagulation. However, organic adsorbents such as fluorocarbons do not have the above-mentioned problems and are therefore effective in analyzing biological fluids, particularly blood. The shapes of the enzyme-immobilized carrier and the fluorocarbon are not particularly limited in order to fully exhibit the effects of the present invention, but spherical shapes are preferable to minimize the spread of the sample band and the diffusion distance of oxygen. desirable. In addition, in order to make the filling state uniform and close-packed, the sizes of these two beads are set to R, where R is the diameter of the enzyme-immobilized beads and r is the diameter of the fluorocarbon beads.
It is desirable that the relationship is >r, and more preferably that R/r is in the range of 3 to 5. According to this example, even if the concentration of the substrate in the sample band is high compared to dissolved oxygen, if the oxygen partial pressure in the sample band decreases during the reaction, the enzyme is released from the fluorocarbon, and this oxygen To promote the enzyme reaction, the highest response value is about 10 compared to the conventional example.
It has the effect of becoming more than twice as expensive. FIG. 3 is a flow path diagram of a multi-item analyzer in which two enzyme reaction sections according to the present invention are connected in series. 2a, 2
b is a reaction column filled with beads immobilized with different types of oxidases and fluorocarbon beads, respectively. A sample such as serum is injected through the sample injection port 1 and selected by the pump 5 into each enzyme reaction section. First, a substrate suitable for this enzyme is oxidized in the first enzyme reaction section 2a, and oxidized according to the amount. Since hydrogen peroxide is generated, this is detected by a detector 3a such as a hydrogen peroxide electrode. this,
Hydrogen peroxide remaining undecomposed by the detector 3a is decomposed while passing through a column 10 on which a hydrogen peroxide decomposing enzyme such as catalase is immobilized. Hereinafter, the same thing will happen in the second enzyme reaction section 2b and the detector 3b. Signals from the detectors 3a and 3b are taken into an amplification and arithmetic circuit 11, and output to a display device 12 and a digital printer 13. According to this embodiment, it is possible to immobilize various oxidoreductases and combine them with a hydrogen peroxide electrode, which has the effect of allowing multiple tests to be performed almost simultaneously using a small amount of sample. In the embodiment shown in FIG. 3, the number of combinations of enzyme reaction sections and detectors is two, but this number is not particularly limited and can be increased or decreased as necessary. FIG. 4 is a calibration curve when glucose in serum was measured according to the example shown in FIG. Using the buffer liquid shown in Table 1 as the carrier solution, the flow rate was
This is when the liquid was pumped at 1.0ml/min and 10μ of serum was injected.

[Table] Thus, it is clear that by using this example, the maximum response value can be expanded to about 600 mg/dl. Therefore, according to this example, the substantial reaction site between the substrate in a sample such as serum or a solution containing the sample and the residual reductase that uses oxygen as an acceptor does not affect other systems at all. This has the following effects: (1) expansion of the maximum response value, (2) improvement of analysis accuracy, and (3) realization of a compact and low-cost multi-item analyzer. As explained above, according to the present invention, the quantification range can be widened and the accuracy can be increased.

[Brief explanation of the drawing]

1 and 3 are block diagrams showing one embodiment of the present invention, FIG. 2 is a diagram showing an enzyme reaction section according to the present invention, and FIG. 4 is a calibration curve for glucose obtained in the embodiment shown in FIG. 1. It is. 1...Sample injection port, 2...Enzyme reaction section, 3...
Detector, 4... Magnifier, 5... Pump, 6... Recorder, 7... Carrier solution, 8... Enzyme immobilized beads, 9... Fluorocarbon beads, 10... Hydrogen peroxide degrading enzyme ram , 11... Width amplification/arithmetic circuit, 12, 13... Output device.

Claims (1)

[Scope of Claims] 1. Direct or indirect detection of components produced or increased or decreased by the enzyme reaction with an enzyme reaction part in which one or more types of oxidoreductases that act as oxygen receptors are immobilized on an insoluble carrier. In the enzyme-based analyzer, the enzyme reaction section is composed of a solid made of a substance with a high oxygen transfer ability and an insoluble carrier with an enzyme immobilized on the surface or in the pores of the same container. An enzyme-based analyzer featuring: 2 In the invention described in claim 1,
An enzyme-based analyzer characterized in that the substance with high oxygen transfer ability is an organic compound such as fluorocarbon.