JPS6333667B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method and apparatus for measuring urea that indirectly measures the concentration of urea contained in a sample such as blood from the amount of change in conductivity of a predetermined absorption liquid. It is.

In recent years, advances in biomedical measurements have enabled continuous telemetry of physiological variables such as body temperature, gastrointestinal pressure, blood pressure, rate of respiration, and biological potential, as well as in vivo pO ₂ , Measurement methods, methods, and devices for continuously measuring pCo ₂ , blood PH and electrolytes, and gastric PH have become of interest. As one such biomedical measurement method, urease oxygen, which is a urea-degrading enzyme, is applied to urea in the sample to cause the enzymatic reaction of formula (1) below, and the generated NH ₃ is converted into NH ₃ gas. There is a method of indirectly measuring urea in a subject by detecting it with an electrode or an NH ₄ ⁺ cation electrode.

However, in the above urea measurement method, the relationship between the output signal from the electrode and the NH ₃ concentration is an exponential relationship according to the Nernst equation as shown in equation (2) below, and the liquid junction potential difference of the reference electrode A slight change in
It had a drawback that it was said to be a major source of error when measuring NH ₃ concentration.

E=E ₀ +2.303RT/F(log[NH ₃ ]+log a) ...(2) [log[NH ₃ ]+log a=log([NH ₃ )xa)=log
[OH ^- ] [OH ^- ] / [NH ₃ ] = a E: equilibrium electrode potential, Eo: standard electrode potential, R: gas constant, T: absolute temperature, F: Faraday constant, [NH ₃ ]: NH ₃ concentration, [OH- ^] : OH ^- concentration, a: constant] The present invention was made in view of these drawbacks, and its purpose is to quickly remove urea contained in specimens such as whole blood. The object of the present invention is to provide a method and device for measuring urea that can be used to measure urea.

Hereinafter, the present invention will be explained in detail using figures. FIG. 1 is an explanatory diagram of the configuration of an embodiment of the present invention,
In the figure, 1a to 1c are containers, 2a is an absorption liquid such as _HNO3 solution, 2b is a carrier solution, 2c is a carrier waste liquid, 3a is an absorption liquid inlet, 3b is a carrier solution inlet, and 3c is a carrier solution outlet. ,3d
4 is an absorption liquid outlet; 4 is a double peristaltic pump that sends the absorption liquid and carrier solution to their respective channels;
5 is a sample injector for injecting a predetermined amount of the analyte into a carrier solution; 6 is an immobilized enzyme on which is immobilized a urease enzyme that acts on urea contained in the analyte to cause an enzyme reaction; and 7a is an immobilized enzyme; For example, a tube made of a cation exchange membrane such as Nafion (trade name); 7b is installed in the tube 7a, and a cathode made of iron wire or platinum wire; 7c is installed, for example, wound in a spiral shape on the outside of the tube 7a; 7d is a power source that applies a predetermined voltage between the cathode and anodes 7b and 7c, and 7e is a sodium salt such as Na ₂ CO ₃ or NaOH that fills the outside of the tube 7a. 7f is an electrolytic cell 8a containing the tube, saturated solution 7e, etc.
For example, a crystalline material that does not allow ions to pass through but allows gas to pass through.
A thin film made of a polymeric substance such as PTFE, 8b is a second chamber forming a part of the channel through which the absorption liquid 2a flows, 8c is a second chamber forming a part of the channel through which the carrier solution 2b flows, The second chamber 8b is adjacent to the first chambers 8d and 8 through the thin film 8a.
e is an inlet and an outlet for the absorption liquid in the second chamber 8b, 8f and 8g are an inlet and an outlet for the carrier solution, etc. in the first chamber 8c, and 8h is composed of the first chamber 8c, the second chamber 8b, etc. For example, the flow cell 9a detects the conductivity of the absorption liquid.
A micro flow cell type electrode made of SUS316, 9b is a conductivity meter that is connected to the electrode 9a and displays the measured value after processing the measurement signal output from the electrode 9a, 10a is the peristaltic pump 4 from the absorption liquid inlet 3a, Inlet port 8d, second chamber 8d, outlet port 8
e, the first flow path leading to the absorption liquid discharge 3d via the electrode 9a, and 10b, the carrier solution inlet 3b, the peristaltic pump 4, the sample injector 5,
This is a second flow path leading to the carrier solution outlet 3c via the immobilized enzyme 6, the cation exchange membrane tube 7a, the inlet 8f, the first chamber 8c, and the outlet 8g. Note that the carrier solution 2b is, for example, KH ₂ EDTA,
It consists of a mixed solution of benzoic acid, NaCl, etc., and the pH is adjusted to the optimum pH for the enzyme reaction so that the enzyme reaction can be carried out efficiently in the immobilized enzyme 6. Further, FIG. 2 is a principle explanatory diagram illustrating the phenomenon of cation exchange within the electrolytic cell 7e,
In the figure, the same symbols as in FIG. 1 are used with the same meaning, and the explanation here will be omitted.

The operation of the embodiment of the present invention will be described below with reference to FIGS. 1 and 2. In FIG. 1, when the peristaltic pump 4 is driven, the absorption liquid 2a flows through the first channel 10a and is collected into the container 1a, and the carrier solution 2b flows through the second channel 10b and is collected into the container 1c. will be disposed of.
In this state, a predetermined amount (for example, 20μ) of the analyte is transferred from the sample injector 5 to the second flow path 10b.
When the analyte is injected into the carrier solution, it reaches the immobilized enzyme 6, and the urea in the analyte undergoes an enzymatic reaction as described in (3) below.

NH ₄ ⁺ produced by the reaction of formula (3) above and
HCO ₃ ^- is again transported to the carrier solution and reaches the cation exchange membrane tube 7a. At this time, power supply 7
When a predetermined voltage (for example, 2.5V) is applied between the cathode 7b and the anode 7c from d, if the saturated sodium salt solution 7e is NaOH, the following formula
It undergoes an electrolytic reaction like (4).

That is, in FIG. 2, the cathode 7b and the anode 7
When a predetermined voltage is applied from the power supply 7d during the period c, only Na ⁺ passes through the cation exchange membrane tube 7c and reaches the carrier solution 2b, and is generated by the cathodic reaction of the above equation (4). The carrier solution 2b reacts with OH ^- to generate NaOH, increasing the pH value of the carrier solution 2b. In this way, the PH value of the carrier solution in the cation exchange membrane tube 7a changes depending on the amount of electricity supplied from the power source 7d between the cathode 7b and the anode 7c. Therefore, by keeping the amount of electricity at a predetermined value, the cation exchange membrane tube 7
The carrier solution in a becomes alkaline with a pH of 10.5 or more, and the NH ₄ ⁺ generated in the above formula (3) undergoes the reaction of the following formula (5) and is converted to NH ₃ .

NH ₄ ⁺ +OH ^- →NH ₃ +H ₂ O ...(5) NH ₃ generated in the reaction of the above formula (5) is again transported to the carrier solution and reaches the first chamber 8c in the flow cell 8h, and then , which passes through the thin film 8a and reaches the second chamber 8b, where it reacts with the absorption liquid supplied through the first flow path 10a into the second chamber 8b and is absorbed as shown in the following formula (6). Ru.

NH ₃ +HNO ₃ →NH ₄ NO ₃ ...(6) NH ₄ NO ₃ generated in the reaction of the above equation (6) is carried to the absorption liquid and reaches the electrode 9a, but compared to HNO ₃ NH ₄ Since NO ₃ has a low electrical conductivity, the electrical conductivity of the absorption liquid changes before and after the reaction of equation (6) above. The change is detected by the electrode 9a, and the detection signal is subjected to predetermined arithmetic processing taking into account the contents of equations (3) to (6) to calculate the urea concentration in the subject;
The urea concentration is displayed on the conductivity meter 9b or the like.

According to the embodiment of the present invention as described above in detail, the PH of the carrier solution is adjusted to a desired value by using electrolytic reaction in the electrolytic cell 7f. Compared to the method of adding an alkaline reagent for adjustment, this method has the advantage that it does not require pipes or the like for the alkaline reagent, resulting in a significant cost reduction. It also has the advantage that urea contained in a sample can be measured quickly and accurately without being affected by large error factors as seen in the conventional example. Furthermore, by turning off the power supply 7d in Figure 1 when not measuring, it is possible to easily avoid contamination of a strong alkaline solution into the carrier solution, thereby extending the life of the enzyme in the immobilized enzyme 6. It also has advantages.
Furthermore, the present invention is a method for quantifying urea using a simple measurement principle and a robust detection end, and is capable of achieving speed and high sensitivity required for a urea measurement method in clinical tests, as well as whole blood measurement. It is also a practical measurement method that satisfies all conditions.

The case where the substrate is urea and the enzyme is urease has been described in detail above, but the same applies when the substrate is L-amino acid, aspartic acid, or creatinine and the enzyme is L-amino acid oxidase, asparaginase, or creatinase, respectively. I can say that.

[Brief explanation of the drawing]

FIG. 1 is an explanatory diagram of the configuration of an embodiment of the present invention, and FIG. 2 is an explanatory diagram of the principle of cation exchange. 1a to 1c...container, 2a...absorption liquid, 2b...
...Carrier solution, 2c...Sayaria waste liquid, 3a,
3b, 8b, 8f...inlet, 3c, 3d...outlet, 8c, 8g...outlet, 4...peristaltic pump, 5...sample injector, 6...immobilized enzyme, 7a...cation Exchange membrane tube, 7
b, 7c, 9a... Electrode, 7d Electric power... Power supply, 7e
... Sodium salt saturated solution, 7f ... Electrolytic cell,
8a... Thin film, 8b, 8c... Chamber, 8h... Flow cell, 9b... Conductivity meter, 10a, 10b...
flow path.

Claims (1)

[Scope of Claims] 1. Means for injecting an analyte into a channel through which a carrier solution flows, and urea in the analyte by an enzymatic reaction.
A means of converting it to NH ⁺ ₄ and a means of converting it to NH + 4 and
means for alkalinizing the carrier solution with OH ^- to convert the NH ⁺ ₄ to NH ₃ ; and means for permeating the NH ₃ through an ion-impermeable, gas-permeable membrane to the absorption liquid; A method for measuring urea, characterized in that the amount of urea in the sample is determined by detecting the amount of change in conductivity of the absorption liquid. 2. a double peristaltic pump that sends the absorption liquid and the carrier solution to the first and second channels, respectively;
a sample injector disposed downstream of the peristaltic pump and injecting the analyte into the second channel; an immobilized enzyme disposed downstream of the sample injector and having immobilized urease; a cation exchange membrane tube forming part of the flow path of the tube, a sodium salt saturated solution contained outside the tube, and an insoluble electrode for electrolyzing the solution and disposed downstream of the immobilized enzyme. a first chamber disposed downstream of the electrolytic cell and forming a part of the second flow path through an ion-impermeable, gas-permeable thin film; a flow cell in which a second chamber forming a part of the flow cell is provided adjacent to the flow cell; and an electrode disposed downstream of the second chamber in the first flow path to detect a change in conductivity of the absorption liquid. and a urea measuring device for quantifying urea in the subject.