JPH0236178B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a device capable of measuring creatinine (CRE) among chemical components contained in body fluids such as blood.

Recently, the usefulness of clinical tests has been increasingly talked about, and for example, for patients undergoing dialysis therapy using an artificial kidney, it is extremely important to measure the amount of CRE in the blood. Conventionally, CRE measurements have been carried out using manual methods, but the development of automatic analytical instruments has progressed recently, and some have already been commercialized. However, the commercialized devices have the disadvantage that not only the devices themselves are expensive, but also the running costs are high because they use reagents or solution enzymes. In addition, when monitoring during dialysis therapy using an artificial kidney or performing measurements from the bedside or immediately after blood collection, whole blood measurements are required, but conventional colorimetric methods cannot be used for whole blood measurements, so immobilized enzyme beads are attached to the column. Sometimes it is filled and measured. However, it has been pointed out that this method has the disadvantage that it is prone to clogging due to the use of whole blood.

The present invention has been made with attention to these circumstances, and it is possible to perform measurements using whole blood as a test liquid.
The purpose of this invention is to provide a measuring device that can quickly measure the amount of CRE at low running costs.

That is, in the present invention, a main line and a compensation line are formed in parallel through which test liquids are to flow, and a creatinine measuring section with an immobilized enzyme membrane is installed in the main line.
Moreover, the gist lies in that each compensation line is provided with a creatine measuring section with an immobilized enzyme membrane, and each measuring section is connected to a correction calculation section. As the immobilized enzyme membrane, those described in JP-A-52-55691 and JP-A-54-102193 can be used.

In measuring CRE, creatinine amidohydrolase is used as the enzyme.The generated creatine is then decomposed into sarcosine and urea by the action of creatine amidinohydrolase, and the sarcosine is further decomposed into glycine, formaldehyde and urea by the action of sarcosine oxidase. Decomposes into hydrogen peroxide. This hydrogen peroxide is then measured using an amperometric hydrogen peroxide electrode.

In the present invention, the reaction between the enzyme and the substrate is carried out using an immobilized enzyme membrane in which creatinine amidohydrolase, creatine amidinohydrolase, and sarcosine oxidase (hereinafter referred to as enzymes) are each supported on a suitable film or sheet. Various configurations are adopted. In other words, since the enzyme is retained in a solid state, it has the advantage of being able to be used continuously as long as it does not become inactivated, and when the substrate comes into contact with the above-mentioned immobilized enzyme membrane, an enzyme reaction specific to the substrate occurs. However, various decomposition products are generally generated. This decomposition product may be measured by any method selected from physicochemical methods, chemical methods, and biochemical methods, but hydrogen peroxide, which is a decomposition product of CRE by creatinine amide hydrolase, etc., can be measured as described above. A method using an amperometric hydrogen peroxide electrode is recommended.

In the amperometric type hydrogen peroxide electrode according to the present invention, a substrate is brought into contact with an enzyme thin film covering the surface of the electrode in a static state or a flowing state, and the concentration of the generated hydrogen peroxide is measured. In this case CRE)
Find the concentration of Contact in a stationary state, e.g.
The method of repeating measurements for 10 seconds every 0.5 seconds several times and calculating the linear regression coefficient based on the least squares method to determine the concentration is called the rate method. A method for determining the concentration by continuously measuring the results is called a flow-through method, and the person implementing the present invention is free to choose which of these methods to adopt.

Next, the configuration and effects of the analyzer will be explained according to a representative example of the present invention.

Figures 1 and 2 show sample quantification 6 used in the present invention.
An explanatory diagram of the operation of the direction valves _V1 and _V2 , and FIG. 3 is a conceptual diagram of the entire device. In FIG. 3, the upper line _L1 connected to the six-way valve _V1 is the main line, and the lower line _L2 is the compensation line.The reason for providing these lines is as follows. In other words, there is a non-negligible amount of creatine in the blood.
When quantifying hydrogen peroxide in the measurement of CRE, the above-mentioned creatine is also detected. Therefore, only creatine is measured on the compensation line _L2 , and this value is subtracted from the measured value on the main line _L1 to determine the correct CRE.

<Washing step> According to FIG. 2, _a washing solution, preferably a buffer solution, is introduced along the arrow A' ₁ , and is caused to flow through the six-way valves V ₁ and V ₂ in the order of -, respectively. Let it release. Note that this flow is performed by suction from pump _P3 . On the other hand, pumps P ₁ and P ₂ are operated to suck the buffer solution in the storage tank 9, and the arrow B' ₁ →B' ₂ in FIG.
and flow along C′ ₁ →C′ ₂ , the main line of the measuring device
A buffer solution is passed through _L1 and compensation line _L2 .
The flow of the buffer solution in the six-way valves _V1 and _V2 is →→→.

<Sample injection> When washing has been sufficiently performed, pump _P3 is stopped and six-way valves _V1 and _V2 are switched as shown in FIG. Pumps P ₁ and P ₂ continue to operate.
The buffer solution is allowed to flow from B ₁ to B ₂ and from C ₁ to C ₂ through a six-way valve. Then, start injecting the sample along arrow A ₁ , and the sample enters each 6-way valve →→→
Flow in this order. Since the sample is blood, it is detected by the photosensor Ps when its tip reaches the photosensor Ps position. At this stage, the inside of the circuit →→→ is filled with sample, and a fixed amount of sample is retained. When targeting body fluids other than blood, it is also possible to omit the photo sensor and use pulse motors for pumps P ₁ and P ₂ , and estimate the arrival of the sample by detecting pulses and reaching a certain number of pulses. good. Note that all the photosensors described below can be considered in the same way.

<Measurement start> Switch the six-way valves V ₁ and V ₂ to return to the state shown in Figure 2, and transfer the quantitative sample to the arrow B' ₂ using the buffer solution introduced along the arrows B' 1 and C _{' 1} . and C′ ₂
direction and stop pump _P3 . Pump P ₃ may be stopped at the same time as the detection by photo sensor Ps, but if a suitable timer is used to stop pump P 3 after a certain period of time after detection, the buffer in the circuit This is extremely advantageous in terms of stabilizing measurement accuracy, as it provides time for the liquid to be completely discharged and replaced by the sample.

<Mixing> For subsequent specific measurements, it is necessary to completely mix the sample and buffer solution to adjust the optimum pH, etc., and the sample and buffer solution are sent to mixing coils M ₁ and M ₂ . The area within the chain line area in FIG. 3 is the temperature adjustment area, which is adjusted by the temperature indicating regulator TIC to maintain a temperature suitable for the enzyme reaction. Therefore, the samples in the mixing coils M ₁ and M ₂ are diluted with the buffer solution and at the same time are heated to a certain temperature. The pumps P ₁ and P ₂ are linked as shown by the dashed line, and the flow rate of the sample flowing through the main line L ₁ and the compensation line L ₂ is determined by the microcomputer.
Adjustment is made so that the same and arbitrary speed is given by the MC and the drive interface MI.

<CRE measurement> The tip of the sample and buffer mixture (hereinafter referred to as the test solution) that exits the mixing coil is a photo sensor.
When detected by Ps1 and Ps2, pump _P1
and P ₂ are controlled and adjusted to the optimum test liquid velocity (usually around 1 ml/min) for CRE measurement.
Then, the pumps P ₁ and P ₂ are stopped at the timing when the highest concentration portion estimated from the flow state of the test liquid reaches the CRE measurement electrode 2 and the creatine measurement electrode 5. That is, the rate method is adopted here, and the CRE concentration is measured every 10 seconds, for example, every 0.5 seconds, and linear regression coefficients are calculated according to the least squares method. Note that creatine is measured using electrode 5. These results are then input to an analog-to-digital converter system (hereinafter referred to as ADC system) 7 to obtain correct CRE measurement values.

<Resuming cleaning> When the CRE measurement is completed, switch the 6-way valves V ₁ and V ₂ to the state shown in Figure 1, increase the flow rate, and send the buffer solution to the main line L ₁ and compensation line L ₂ to remove the test solution. discharge. The absence of the test liquid is detected by the photosensors Ps7 and Ps8, and the end of the cleaning process is determined. On the other hand, pump P ₃ is also restarted,
Clean the inside of the 6-way valves V ₁ and V ₂ to prepare for the next sample injection.

In the above measurement example, CRE was measured using the rate method, but of course it is also possible to use the flow-through method.

Since the device of the present invention is configured as described above,
Since CRE is measured continuously and an immobilized enzyme membrane is used in the measuring section, the device is easy to handle and running costs can be reduced. Note that the measuring device of the present invention may be provided with a device for measuring Na, K, Cl, etc. by an electrode method, if necessary.

[Brief explanation of drawings]

Figures 1 and 2 are explanatory diagrams of the operation of the 6-way quantification valve, and Figure 3
The figure is an overall conceptual diagram of the device of the present invention. P...pump, Ps...photo sensor, V...
Sample quantification 6-way valve, L ₁ ... Main line, L ₂ ...
…compensation line.

Claims (1)

[Scope of Claims] 1. A measuring device for measuring creatinine in a body fluid, in which a main line and a compensation line through which a test liquid flows are formed in parallel, and the main line contains creatinine amidohydrolase, creatinine amidine, A creatinine measurement part was arranged to measure the generated H ₂ O ₂ by placing an enzyme membrane with immobilized hydrolase and sarcosine oxidase, and an immobilized oxygen membrane with creatine amidinohydrolase and sarcosine oxidase was placed in the compensation line. _{H2O2 _}
What is claimed is: 1. A creatinine measuring device comprising: creatinine measuring sections for measuring creatinine, and each measuring section is connected to a correction calculation section. 2. The creatinine measuring device according to claim 1, which is equipped with a flow rate adjusting device for the test liquid supplied to the measuring section.