JPH0630763A - Quantitative analyzer - Google Patents

Abstract

PURPOSE:To enable determination of a very small amount of sample, reduction of consumption of a reagent, miniaturization of an analyzer and reduction of preparing cost. CONSTITUTION:The analyzer for determining a substance contained in a liquid from chemiluminescing amount utilizing an enzymatic reaction and chemiluminescing reaction is obtained by forming an enzymatic reaction part 3, a mixing part 4 and detection part 5 on one sheet of silicon substrate using an anisotropic etching. Consequently, dead space necessary for connecting each part can be made small and simultaneously total volume of the analyzer can be made small. Thereby, the very small amount of sample is not spread by the dead space and a consumption amount of reagent can be kept to several ten mul in one time measurement. Since light source is unnecessary, miniaturization of the analyzer is made possible and since many analyzers are collectively prepared, the preparing cost can also reduced.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a device for quantitative analysis utilizing enzyme reaction in the fields of medicine, pharmacy, chemical analysis and food industry.

[0002]

2. Description of the Related Art Conventionally, an enzyme flow injection analyzer is generally used as a device for quantitative analysis by an enzyme reaction. As shown in FIG. 5, a pump 7 for creating a flow of a carrier 9, a sample introducer 8 for introducing a certain amount of sample into the flow of the carrier, a packing material on which an enzyme is immobilized are filled. It comprises an enzyme reaction part 3 for carrying out an enzymatic reaction with the substance to be measured contained in the sample, and a detection part 5 for detecting the change in the amount of the enzymatic reaction product or the amount of the substance in the carrier due to the enzymatic reaction. For the detection unit, an absorbance detector that quantifies the amount of light of a substance, an electrochemical detector that electrochemically redox a substance with an electrode, and quantifies from a redox current flowing at that time is used.

Further, as a detector of the enzyme flow injection analyzer, a detector for quantifying using a chemiluminescence reaction has been devised, and a chemiluminescence detector having a constitution as shown in FIG. 6 is commercially available. . This is a pump 7 for introducing the chemiluminescent reagent 2 after the enzyme reaction part 3 in the enzyme flow injection analyzer, a mixer 19 for mixing the enzyme reaction product and the chemiluminescent reagent, a spiral flow cell 20 for detecting chemiluminescence, and The photomultiplier tube 21 that converts the amount of chemiluminescence into an electric signal is installed.

In these enzyme flow injection analyzers, each unit is a separate part, and when used, they are appropriately combined and connected by piping or wiring.

[0005]

However, in the conventional enzyme flow injection analyzer, each unit is connected by pipes with separate parts, especially the enzyme reaction column and the detector are separate parts. Since the enzyme column and the detection part are made by machining and there is a limit to miniaturization, the substances generated in the enzyme reaction column diffuse while flowing to the detection part, and it is possible to quantify a small amount of sample. It was difficult to measure in the low efficiency region.

Further, when the detector is an absorbance detector, a light source is required, which makes it difficult to downsize the device, and an electrochemical detector is susceptible to interference by the electrode active substance contained in the sample. Had. Further, since the total volume of the device becomes large to some extent, the flow rate of the carrier must be around 1 ml / min at the time of measurement, and there is a problem that the amount of reagent consumed becomes large when continuous measurement is performed.

Similarly, in the conventional method utilizing chemiluminescence detection, the substance produced in the enzyme reaction column diffuses while flowing into the mixer, or the chemiluminescence generated in the mixer is detected. There is a problem that it is difficult to quantify a small amount of sample and measure in a low concentration region because it is attenuated while moving to the spiral flow cell, which is a part of the flow cell, and there is a problem that the consumption of the carrier and the chemiluminescent reagent increases. It was

[0008] In addition to this, mechanical processing is involved in the production of the enzyme reaction column, the mixer and the spiral flow cell, and there is a problem that there is a limit to miniaturization and a cost is required because precise processing is required. was there. Then, the objective of this invention is to integrate the enzyme reaction part, the mixing part, and the detection part on one board | substrate, and to obtain the quantitative analysis device which solved such a conventional subject.

[0009]

In order to solve the above problems, the quantitative analysis device of the present invention utilizes a chemiluminescence reaction as a detection means. As a result, a light source for detection is not required, the device can be easily downsized, and the influence of interfering substances in the sample can be reduced.

At the same time, the enzyme reaction section, the mixing section, and the detection section are formed on the same substrate, and the distance from the enzyme reaction section to the detection section is minimized to minimize substance diffusion and luminescence attenuation. Hold down. At the same time, the total volume of the device is reduced to reduce the consumption of carrier and luminescent reagent. Furthermore, by using anisotropic etching of a silicon substrate instead of machining to manufacture these, a large number of them can be processed at the same time so that they can be manufactured at low cost.

[0011]

In the quantitative analysis device configured as described above, the substance to be measured contained in the sample is consumed as a substrate by the enzyme reaction in the enzyme reaction part, and another substance is produced. Further, in the mixing section, the enzymatic reaction product is mixed with the chemiluminescent reagent to generate light having a specific wavelength. afterwards,
While flowing through the detector, the intensity of light is converted into the intensity of an electric signal (current value, voltage value) by a photodiode or a photomultiplier tube. The intensity of this electric signal reflects the chemiluminescence intensity, which depends on the amount of the enzyme reaction product.

[0012] Naturally, the amount of the enzymatic reaction product depends on the amount of the substrate (measurement target substance) originally contained in the sample, so that the strength of the measured electric signal is ultimately dependent on the measurement target substance. It reflects the concentration. In addition, by configuring the enzyme reaction part, mixing part, and detection part on the same substrate and minimizing the distance from the enzyme reaction part to the detection part, it is possible to minimize substance diffusion and luminescence attenuation. Became.

[0013]

Embodiments of the present invention will be described below with reference to the drawings. (Example 1) FIG. 1 is a schematic view of a quantitative analysis device of the present invention.

In FIG. 1, a sample 1 measured in a fixed amount by a sample introduction device 8 is introduced into a carrier 9 and sent to an enzyme reaction section 3 by a pump 7. In the enzyme reaction part 3, a specific substance contained in the sample is consumed by the enzyme reaction, and another substance is produced. This enzymatic reaction product is sent to the mixing section 4 and mixed with the chemiluminescent reagent 2 sent by the pump 7 to cause a chemiluminescent reaction. Then, the mixed liquid moves to the detection unit 5. While flowing through the detection unit 5, the amount of chemiluminescence is converted into an electric signal by the photodiode 6 installed outside. Since the intensity of the electric signal reflects the concentration of the substance to be measured contained in the sample 1, it can be quantified.

FIG. 2 is a diagram showing an example of a concrete structure of the quantitative analysis device of the present invention. The quantitative analysis device of the present invention has a structure in which a silicon substrate 18 and a glass substrate 17 are joined. A carrier inlet 10, a chemiluminescent reagent inlet 11, an enzyme reaction unit 12, a mixing unit 13, and a flow cell unit 14 are provided on the silicon substrate. Enzyme reaction part 12
An enzyme-immobilized filler 15 having an enzyme immobilized on the surface is filled therein. A photodiode 16 for converting chemiluminescence into an electric signal is installed right above the flow cell part. At the time of measurement, a carrier containing the sample is introduced from the carrier introduction port by a pump. In the enzyme reaction part, an enzyme reaction product is produced by the enzyme reaction depending on the amount of the measurement object in the sample, and is mixed with the chemiluminescent reagent introduced from the chemiluminescent reagent introducing port in the mixing part.

The chemiluminescence generated there is measured by a photodiode installed outside while flowing through the flow cell section. Here, the photodiode is used to measure the amount of light emission, but the same measurement can be performed by using a photomultiplier tube instead of the photodiode.

(Example 2) In this example, the quantitative analysis device of the present invention was used to fill the enzyme reaction part with a packing material having glucose oxidase immobilized thereon, and to quantify the glucose contained in the sample. Will be described.

In FIG. 1, glass beads having a diameter of 100 μm, on the surface of which glucose oxidase has been immobilized by 3-aminopropyltriethoxysilane and glutaraldehyde by a Schiff bond, are filled in the enzyme reaction part 3 and the carrier 9 of pH 7 is filled. The phosphoric acid buffer solution was added to the chemiluminescent reagent 2 at 20 μl / min by the pump 7 at 0.47 mmo.
Using a solution containing 1 / l of luminol and 6.7 mmol / l of potassium ferricyanide, 60 by pump 7
It was supplied at μl / min. 0.2 μl in sample introducer 8
Was weighed and introduced into the carrier. FIG. 3 shows a calibration curve of the quantitative analysis device of the present invention in which response values for various glucose concentrations are graphed. 10 mg / dl ~
A linear relationship was established between the glucose concentration and the response value in the concentration range of 300 mg / dl.

Further, when glucose in serum was measured based on this result, it was possible to obtain a measured value having a good correlation with a commercially available glucose measuring kit for clinical tests. The time required for one measurement was about 1 minute, the amount of the carrier used at this time was about 20 μl, and the amount of the chemiluminescent reagent was about 60 μl.

(Embodiment 3) In this embodiment, the quantitative analysis device of the present invention was used to fill the packing material in which the lactate oxidase was immobilized in the enzyme reaction part, and the lactic acid contained in the sample was quantified. Will be described.

In FIG. 1, lactate oxidase is immobilized on the surface by a Schiff bond using 3-aminopropyltriethoxysilane and glutaraldehyde and has a diameter of 100.
The enzyme reaction part 3 was filled with μm glass beads, and a phosphate buffer solution of pH 7 was added to the carrier 9 by a pump 7 to make 20 μm
0.47 mmol / l in chemiluminescent reagent 2 at 1 / min
Of luminol and 6.7 mmol / l potassium ferricyanide were used, and 60 μl /
It was supplied at min. The sample introducer 8 weighed 0.2 μl of the sample and introduced it into the carrier.

FIG. 3 shows a calibration curve of the quantitative analyzer of the present invention in which response values for various lactic acid concentrations are graphed. 4.
A linear relationship was established between the lactic acid concentration and the response value in the concentration range of 5 mg / dl to 45 mg / dl. Furthermore, when lactic acid in serum was measured based on this result, it was possible to obtain a measurement value having a good correlation with a commercially available lactic acid measurement kit for clinical tests. Also, the time required for one measurement is about 1
The amount of carrier used at this time is about 20μ.
1, the amount of chemiluminescent reagent was about 60 μl.

[0023]

In the quantitative analyzer of the present invention, the enzyme reaction section, the mixing section, and the detection section are integrated and produced by etching. Therefore, the dead volume required for connecting each section is compared with that of the conventional quantitative analyzer. The total volume of the device can be reduced at the same time. As a result, a small amount of sample did not diffuse in dead volume, and the amount of reagent consumed could be several tens μl in one measurement. In addition, the light source is unnecessary compared with the case of using an absorbance detector, etc., so that the device can be downsized, and by making a large number of them in a batch by etching,
The manufacturing cost could also be reduced.

[Brief description of drawings]

FIG. 1 is a schematic diagram of a quantitative analysis device of the present invention.

FIG. 2 is a diagram specifically showing the structure of the quantitative analysis device of the present invention.

FIG. 3 is a diagram showing a calibration curve for glucose in the quantitative analyzer of the present invention.

FIG. 4 is a diagram showing a calibration curve for lactic acid in the quantitative analysis device of the present invention.

FIG. 5 is a schematic diagram of an enzyme flow injection analyzer.

FIG. 6 is a schematic diagram showing a structure of a conventional chemiluminescence detector.

[Explanation of reference numerals] 1 sample 2 chemiluminescent reagent 3 enzyme reaction part 4 mixing part 5 detection part 6 photodiode 7 pump 8 sample introduction device 9 carrier 10 carrier introduction port 11 chemiluminescence reagent introduction port 12 enzyme reaction part 13 mixing part 14 Flow cell part 15 Enzyme-immobilized packing material 16 Photodide 17 Glass substrate 18 Silicon substrate 19 Mixer 20 Spiral flow cell 21 Photomultiplier tube

Claims (4)

[Claims] 1. An analyzer for quantifying a substance contained in a liquid by an amount of chemiluminescence using an enzyme reaction and a chemiluminescence reaction, wherein an enzyme reaction part for performing an enzyme reaction, an enzyme reaction product and chemiluminescence. A quantitative analysis device comprising a mixing section for mixing a reagent to induce a chemiluminescence reaction and a detection section for detecting chemiluminescence, and these three elements are formed on the same substrate. 2. The quantitative analysis device according to claim 1, wherein the substrate is a substrate obtained by adhering a silicon single crystal plate and a glass plate. 3. The quantitative analysis device according to claim 2, wherein the enzyme reaction part and the mixing part are produced by anisotropic etching of the silicon single crystal substrate. 4. The quantitative analysis device according to claim 3, wherein the detection unit is composed of a flow cell formed on the silicon single crystal substrate by anisotropic etching, and a photodiode or a photomultiplier tube installed outside.