JPS6353502B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides three items that enable one analyzer to measure chemical components contained in body fluids such as blood, particularly urea nitrogen, creatinine (CRE), and glucose (GLU). It is related to analyzers.

Recently, the usefulness of clinical tests has been increasingly talked about. For example, for patients undergoing dialysis therapy using an artificial kidney, blood is used as a sample and three clinical tests are performed: blood urea nitrogen (BUN), CRE, and GLU. This is considered extremely important. Traditionally, analysis of these three items was done based on manual methods, but recently, the development of multi-item automatic analysis equipment has progressed,
Some have already been commercialized. However, the commercialized devices are not only expensive, but also
Since it uses reagents or solution enzymes, it has the disadvantage of high running costs.
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 was made with attention to these circumstances, and it is possible to perform measurements using whole blood as a test fluid.
Moreover, the aim is to provide an analyzer that can quickly measure the three items of BUN, CRE, and GLU at low running costs.

That is, the present invention is an analyzer that measures three items, urea nitrogen, creatinine, and glucose, using a body fluid as a sample. The part contains an immobilized enzyme membrane with immobilized creatinine amidohydrolase, creatine amidinohydrolase, and sarcosine oxidase and a hydrogen peroxide electrode, and the glucose measuring part has an immobilized enzyme membrane with immobilized glucose oxidase and a hydrogen peroxide electrode. are arranged respectively, and each measurement part is arranged in series, and these are connected by a line pipe, and a compensation flow path is formed in parallel with the line pipe that connects the measurement parts of the three items, and the compensation flow path is equipped with an ammonia measuring section equipped with an ammonia electrode corresponding to the urea nitrogen measuring section, and an immobilized enzyme membrane on which creatinamidinohydrolase and sarcosine oxidase are immobilized and hydrogen peroxide corresponding to the creatinine measuring section. The gist is that a creatine measuring section with electrodes is provided, and a correction calculation section for each measured value is also provided. In addition, as an immobilized enzyme membrane,
Those described in JP-A-52-55691 and JP-A-54-102193 can be used.

To enzymatically measure urea, urease is usually used to measure ammonia, which is a decomposition product, but in the present invention, an immobilized enzyme membrane comprising urease supported on a suitable film or sheet is used. The structure is such that the reaction between urease and substrate can be carried out using this method. In other words, since the enzyme is retained in a solid state, it has the advantage that it can 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. Various decomposition products are generated.
This decomposition product may be measured by any method selected from physicochemical methods, chemical methods, and biochemical methods. However, for ammonia, which is a decomposition product of BUN by urease, it is difficult to measure it using a urease-immobilized enzyme membrane. The recommended method is to use an ammonia electrode equipped with an ammonium ion, or to measure the amount of ammonium ions generated using conductivity. The ammonia electrode is a part of the enzyme electrode in the so-called ammonium electrode method, in which a substrate is brought into contact with the enzyme thin film covering the surface of the ion electrode in a stationary or flowing state, and the concentration of the generated ions is measured. Find the concentration of BUN). Contact at rest, e.g. 0.5
Repeat the measurement several times for 10 seconds every second.
A method to calculate the concentration by calculating linear regression coefficients based on the method of least squares is called the rate method.On the other hand, a method to determine the concentration by bringing the enzyme into contact in a fluid state to carry out an enzyme reaction and continuously measuring the results. This method is called the Frösl method, but which of these methods to adopt is at the discretion of the person implementing the present invention. In measurements using an ammonia electrode, if alkaline conditions with a pH of 11 or higher are formed, most of the ammonia becomes gaseous and easily passes through the gas permeable membrane of the electrode, but under high pH conditions there is a risk of deactivation of urease. Since it is strong, it is best to perform measurements under conditions that are slightly closer to the alkaline side.

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. Note that if urea produced during these decomposition processes is mixed into the sample at the BUN measurement stage, the measured BUN value will appear higher, reducing measurement accuracy. Therefore, when performing measurements by assembling a serial pipeline as in the present invention, it is recommended to complete the BUN measurement first and then arrange the CRE measurement.

GLU is measured using glucose oxidase as the enzyme, focusing on the latter of the produced gluconic acid and hydrogen peroxide, and measuring using a hydrogen peroxide electrode as in the case of CRE. Furthermore, GLU is
Since it originally contains considerably more than BUN and CRE, it is not considered to be a particularly serious problem even if the sample is diluted during these measurements or if impurities, especially hydrogen peroxide, are mixed in. do not have. Therefore, there is no problem in incorporating the method in such a way that the measurement is performed at the final stage. Note that hydrogen peroxide can also be corrected by subtracting the value at the time of CRE measurement.

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.
FIG. ₃ is a conceptual diagram of _the entire apparatus, and FIG. 4 is an explanatory diagram showing the measurement time schedule. In addition, in Fig. 3, the 6-way valve V ₁
The upper line L ₁ connected to is a measurement line, and the lower line L ₂ is a compensation line. The reason for providing these lines is as follows. In other words, NH ₄ ⁺ exists in the blood, albeit in a small amount, and when quantifying ammonia in BUN measurement, the above method is used.
NH ₄ ⁺ is also detected together. Therefore, only NH ₄ ⁺ is measured in the compensation line L ₂ , and the measurement line
Subtract this value from the measured value at L ₁ to determine the correct BUN. Also, since there is a non-negligible amount of creatine in the blood, when measuring CRE, this contaminated creatine is also detected.
The measured value of CRE appears high. Therefore, only creatine is measured on the compensation line _L2 , and this value is subtracted from the measured value on the measurement line _L1 to obtain the correct CRE. Regarding GLU, there are no impurities that require correction as mentioned above, so
There is no need to make any special measurements on the compensation line _L2 . Furthermore, by forming the compensation line _L2 parallel to the measurement line _L1 , BUN and CRE can be determined with high accuracy without being affected by dilution due to diffusion of the sample flowing within the measurement line _L1 . .

<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, so that it flows along the arrow A' ₂ . 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 C′ ₁ →C′ ₂ , and the measuring line of the analyzer
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. Incidentally, all of 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 maintain a suitable pH, etc., and the sample and buffer solution are sent to mixing coils M ₁ and M ₂ . Note that the area within the chain line area in FIG. 3 is a temperature adjustment area, which is adjusted by the temperature indicator controller 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 rates of the sample flowing through the measurement line L ₁ and the compensation line L ₂ are set to be the same and arbitrary by the microcomputer MC and drive interface MI. is adjusted so that the speed is given.

<Measurement of BUN> The tip of the mixture of sample and buffer solution (hereinafter referred to as 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 BUN measurement.
That is, the BUN value is measured by the flow-through method at the electrode 1, and the NH ₄ ⁺ is measured at the electrode 4, and the results are input to an analog-to-digital converter system (hereinafter referred to as ADC system) 7. The calculation gives the correct BUN value. and 90% to the maximum value of BUN
The stage at which the following values reappear is determined to be the end point of BUN measurement. In addition, the response speed of BUN is generally slow, so before a value of 90% or less is obtained, the following photosensor
The end of BUN measurement can also be determined by Ps3 and Ps4.

<CRE measurement> Next, the tip of the test liquid is placed on the photosensor Ps3 and
Detects Ps4. and the flow condition of the test liquid,
The pumps P 1 and P ₂ are stopped at the timing when the highest concentration portion estimated from the responsiveness of the BUN measurement electrode ₁ reaches the CRE measurement electrode 2 and the creatine measurement electrode 5. Here, the rate method is used, and the CRE concentration is measured every 0.5 seconds for 10 seconds, and the linear regression coefficients are calculated according to the least squares method. Furthermore, creatine was measured using electrode 5.
Inputting it into the ADC system 7 to obtain a correct CRE measurement value is the same as in the case of BUN.

<Measurement of GLU> When GRE measurement is completed, pumps P ₁ and P ₂ are operated and the flow rate suitable for GLU measurement (normally
The test solution is flowed at a rate of approximately 1.6 ml/min), and the tip is detected by the photosensor Ps5. The photosensor Ps6 and the electrode 6 are used to match the flow path conditions of the compensation line _L2 to those of the measurement line _L1 , and there is no need for them to have a special function when measuring GLU, and other means may be used. You may change it to The value measured by the GLU measurement electrode 3 is input to the ADC system 7 and displayed as a GLU value.
The end point of the GLU measurement is the point at which 90% of the maximum value reappears, as in the case of BUN.

<Resuming cleaning> When the GLU measurement is completed, switch the six-way valves V ₁ and V ₂ to the state shown in Figure 1, increase the flow rate, send the buffer solution to the measurement line L ₁ and the compensation line L ₂ , and release the test solution. do. The absence of the test solution is detected by photosensors Ps7 and Ps8, and the end of the washing 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, BUN and GLU were used as a flow rate method, and CRE was used as a rate method, but of course it is also possible to perform various combinations of these methods. For example, when measuring all three items using the rate method, pumps P ₁ and P ₂ are stopped when the detection level of photosensors Ps1 to Ps8 reaches a certain level or higher, and the pumps are measured every 0.5 seconds for 10 seconds. are measured, and each coefficient is determined by calculation using the method of least squares.

Since the device of the present invention is configured as described above,
The three items BUL, CRE, and GLU are measured continuously or simultaneously in one device, and each measurement section uses an immobilized enzyme membrane, making the device easy to handle and reducing running costs. can be achieved.

The three-item analyzer of the present invention may include three items as necessary.
In addition to the above items, equipment for measuring Na, K, Cl, etc. using the electrode method may be added.

[Brief explanation of the drawing]

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 apparatus of the present invention, and FIG. 4 is an explanatory diagram showing a measurement time schedule. P...pump, Ps...photo sensor, V...
Sample quantification 6-way valve, L ₁ ...Measurement line, L ₂
...compensation line.

Claims (1)

[Scope of Claims] 1. An analyzer that measures three items, urea nitrogen, creatinine, and glucose, using a body fluid as a sample, which includes an immobilized enzyme membrane on which urease is immobilized and an ammonia electrode in the urea nitrogen measuring section, The creatinine measurement section includes an immobilized enzyme membrane with immobilized creatinine amidohydrolase, creatinamidinohydrolase, and sarcosine oxidase, and a hydrogen peroxide electrode, and the glucose measurement section includes an immobilized enzyme membrane with immobilized glucose oxidase and peroxide. Hydrogen electrodes are respectively arranged and each measurement section is arranged in series, and these are connected by a line pipe, and a compensation flow path is formed in parallel with the line pipe connecting the three measurement sections, and the compensation The flow channel is provided with an ammonia measuring section equipped with an ammonia electrode corresponding to the urea nitrogen measuring section, and an immobilized enzyme membrane on which creatinamidinohydrolase and sarcosine oxidase are immobilized and a superimmobilized enzyme membrane corresponding to the creatinine measuring section. A three-item analyzer characterized in that it is equipped with a creatine measuring section equipped with a hydrogen oxide electrode and a correction calculation section for each measured value. 2. The three-item analyzer according to claim 1, in which a urea nitrogen measuring section, a creatinine measuring section, and a glucose measuring section are arranged in this order. 3. The three-item analyzer according to claim 1 or 2, wherein at least the urea nitrogen measuring section and the glucose measuring section are measured using an enzyme electrode using a flow-through method. 4. The three-item analyzer according to any one of claims 1, 2, or 3, which is equipped with a flow rate adjustment device for a test liquid flowing in a line pipe.