JPH0243477B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for quantifying biological components using immobilized enzymes.

Conventionally, when quantifying biological components such as bile acids that exist in trace amounts in serum, it is difficult to linearly detect the components to be measured such as bile acids using a detector such as a fluorometer. utilizes the catalytic action of an enzyme to transfer a sample solution to which a reactant that reacts in the presence of the enzyme to be measured is added to a column filled with an enzyme immobilized on a carrier. (referred to as an enzyme column), where the component to be measured reacts with the reactant to produce a reduced coenzyme, which is detected by a fluorescence detector and the amount of the component to be measured contained in the sample solution is calculated. is being carried out.

For example, in quantifying electromagnetic waves, nicotinamide adenine dinucleotide (NAD+
The sample solution to which the enzyme 3α-hydroxysteroid dehydrogenase (hereinafter referred to as 3α-HSD) has been added is
) is passed through an immobilized enzyme column packed with a carrier on which bile acids are immobilized, and the bile acids and NAD+ are reacted there. As a result, the fluorescent substance NADH is generated in an equimolar amount with the bile acids, and the NADH is It can be detected with a fluorometer, or the NADH generated above is oxidized to NAD by the action of the enzyme diaphorase in the presence of resazurin, and at the same time resazurin is reduced to produce resorufin, a fluorescent substance. The fluorescence of resorufin has been measured.

However, the amount of the component to be measured cannot be accurately quantified unless the blank value, which is the measured value shown when the biological sample solution does not pass through the immobilized enzyme column, is measured. A measurement device without an immobilized enzyme column was prepared separately. However, in this case, in order to measure the blank value and obtain the detected value of the biological sample liquid that has passed through the immobilized enzyme column, it is necessary to collect the biological sample twice, and the detectors are separate. The drawbacks were that weighing errors were likely to occur and the measurement time was long.

The purpose of the present invention is to eliminate such drawbacks, and its gist is to immobilize a biological sample solution containing a component to be measured between a branch flow path to which an immobilized enzyme column is connected. The biological sample liquid flowing through the branch channel to which the immobilized enzyme column is connected is diverted to a branch channel to which the enzyme column is not connected, and is brought into contact with the immobilized enzyme within the column to cause a reaction with the analyte component. The biological sample liquids that have passed through each of the branch channels are merged, and the biological sample liquids that have passed through each of the branch channels are made to reach the same detector with a time difference, thereby obtaining the blank value and the biological sample liquid. A method for quantifying a biological component using an immobilized enzyme, which is characterized by sequentially detecting detected values of a biological sample solution containing reaction products and quantifying the amount of the component to be measured based on these detected values. .

Next, a method for quantifying biological components using the immobilized enzyme of the present invention will be described in more detail with reference to the drawings.

Reference numeral 1 denotes a buffer tank, and the buffer in the tank contains a component capable of reacting with the component to be measured by the action of an enzyme, such as NAD+ in the case of bile acid analysis.
Reference numeral 2 denotes a constant flow pump, which is provided to feed the buffer solution at a constant flow rate.

Reference numeral 3 denotes a syringe for a biological sample containing a component to be measured, and the biological sample and the buffer solution are combined in the syringe 3. The biological sample and the buffer are uniformly mixed to form a biological sample liquid. After the biological sample liquid exits the injector 3, it is branched into branch channels 4 and 5. An immobilized enzyme column 7 is connected to the branch flow path 4 via a coiled flow pipe 6, and a coiled flow pipe 8 having the same diameter as the flow pipe 6 is connected to the branch flow path 5. It is formed. The branch channels 4 and 5 are both provided in parallel and have approximately the same length.

The biological sample liquid diverted to the branch channel 4 is introduced into the immobilized enzyme column 7 through the coiled flow channel 6, and when it comes into contact with the immobilized enzyme within the column 7, it is subjected to its catalytic action, and the sample to be measured is The components react with the reactive components in the liquid to produce reaction products. The biological sample liquid branched into the branch flow path 5 flows through a coiled flow conduit 8 . The coiled flow pipes 6 and 8 are, for example, wound stainless steel tubes with an inner diameter of 0.2 mm and a length of 10 m, and the pressure loss caused by this is significantly larger than that caused by the immobilized enzyme column 7 and detector 9. , the ratio of flow rates in the branch channels 4 and 5 is approximately 1:1.

The biological sample liquids that have passed through each of the branch channels 4 and 5 are combined. A detector 9 is provided in the confluence channel 10, and the biological sample liquid that has passed through the branch channel 5 first reaches the detector 9, and then, after a slight time lag, the biological sample liquid that has passed through the branch channel 4 reaches the detector 9. The sample liquid reaches the detector 9.

The biological sample liquid that has passed through the branch channel 5 first reaches the detector 9 to measure the blank value of the biological sample liquid, and then, after a slight time lag, the biological sample solution that has passed through the branch channel 4 is detected. By reaching the vessel 9, detection values of the biological sample liquid containing reaction products are sequentially obtained.

The detection result by the detector 9 is recorded on the recorder 11.

In this way, the detected value and blank value of the biological sample solution containing the reaction product are obtained as a fluorescence intensity-time curve, and the difference in area is calculated, and the amount of the component to be measured is determined from a separately measured calibration curve. can do. The biological sample liquid that has exited the detector 9 is discharged to the outside of the system.

According to the method of the present invention, both the detected value and the blank value of the biological sample liquid that has passed through the immobilized enzyme column can be measured with the same detector in the same device, so the biological sample collector only needs to measure it once. Moreover, weighing errors are less likely to occur, and the measurement time can be further shortened.

Example Cellulose fine particles with a particle size of about 80 microns were used as a carrier, and 5 ml of ion-exchanged water was added to 5 ml of the fine particles.
After adding 10 ml of a 2M aqueous sodium carbonate solution and stirring, 1 ml of acetonitrile in which 2 g of cyanide bromide had been dissolved in advance was added, and the mixture was allowed to react for 90 seconds while stirring vigorously. The activated cellulose particles were quickly dissolved in 0.1M carbonate buffer (PH).
9.5), washed with ion-exchanged water and 0.1M carbonate buffer (PH9.5) containing 0.5M sodium chloride, and then washed with 0.1M carbonate buffer (PH9.5) containing 0.5M sodium chloride in which 44 mg of 3α-HSD was dissolved. 5) 5 ml was added and stirred at room temperature for 2 hours to react.

Next, 0.05% -mercaptoethanol was added to block the active sites still present on the surface of the microparticles on which 3α-HSD was immobilized by the above treatment.
2 at 4°C in 0.1M Tris-HCl buffer (PH8.0).
Allowed time to react.

The enzyme-immobilized microparticles thus obtained were mixed with a 0.1M acetate buffer (PH5.0) containing 0.5M sodium chloride,
Contains ion-exchanged water and 0.5M sodium chloride
After repeated washing with 0.1M carbonate buffer (PH9.5), it was packed into a column with a length of 100 mm and an inner diameter of 4 mm to prepare an immobilized enzyme-packed column.

The column obtained as above was used as column 7 in Figure 1, and 1 contained 199 mg of NAD+.
Pour 0.1M pyrophosphate buffer (PH9.5) into buffer tank 1, then pump the liquid at a flow rate of 1.2 ml/min with constant flow pump 2. When the liquid feeding stabilizes, take Yasuto's serum.
0.01 ml was injected through syringe 3 and mixed with the buffer. The sample solution thus obtained is divided into two parts and distributed to branch channels 4 and 5, respectively, and the sample solution branched to branch channel 5 is distributed through a coiled distribution pipe 8 with an inner diameter of 2 mm and a length of 10 m. In addition, the sample liquid branched into the branch channel 4 flows from a coiled flow channel 6 with an inner diameter of 2 mm and a length of 10 m through an immobilized enzyme column 7, and then flows into a confluence channel 10.
reached. Next, the sample liquid that passed through the branch channel 5 reached the detector 9 20 seconds after being injected into the syringe 3, and the sample solution that passed through the branch channel 4 reached the detector 9. , reached the detector 9 after 2 minutes.
Detector 9 detects excitation wavelength 360nm and fluorescence wavelength
The fluorescence intensity-time curve shown in FIG. 2 was obtained by using 460 nm. From this curve, the value of the area of peak (a) minus the area of peak (b) was determined, and from a separately measured calibration curve, a value of 8 μmol/blood bile acid was obtained.

[Brief explanation of the drawing]

FIG. 1 is an explanatory diagram showing an embodiment of the method of the present invention,
FIG. 2 shows the fluorescence intensity-time curve obtained in the example. Explanation of symbols 1... Buffer tank, 2... Constant flow pump, 3... Syringe, 4, 5... Branch flow path, 6,
8... Coiled flow pipe, 7... Immobilized enzyme column, 9... Detector, 10... Merging channel.

Claims (1)

[Claims] 1. Separate the biological sample solution containing the component to be measured into the branch channel to which the immobilized enzyme column is connected and the branch channel to which the immobilized enzyme column is not connected, and separate the branch channel to which the immobilized enzyme column is connected. The flowing biological sample liquid was brought into contact with the immobilized enzyme in the column to cause a reaction with the component to be measured, and the biological sample liquids that had passed through each of the branch channels were combined, and the biological sample liquid that had passed through each of the branch channels was combined. The blank value of the biological sample liquid and the detected value of the biological sample liquid containing reaction products are sequentially detected by allowing the biological sample liquid to reach the same detector with a time difference, and the measured component is determined based on these detected values. A method for quantifying biological components using immobilized enzymes, which is characterized by quantifying the amount.