JPH05203614A - Electrode-enclosure type detection element, and intermittent automatic analysis device and method - Google Patents

Abstract

PURPOSE:To provide a disposable detection element which can detect subject matter included in blood or urine by using a porous body and electrode carrying biological catalyst such a enzyme, and to provide an intermittent automatic analysis device and method using the detection element. CONSTITUTION:A detection element 10 includes a porous reaction body 4 carrying biological catalyst to detect change by reaction of subject matter in sample from a sample entrance 1 with catalyst by an electrode 6. An analysis device includes a means to let the sample stay after it is filled in a reaction chamber 5, a reaction change signal processing means, and a sample motion/stay control means. Need of a means to inject the sample into a detection system or carrier liquid for transfer can therefore be eliminated.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention utilizes a porous material carrying a biological catalyst such as an enzyme and an electrode to detect a target substance contained in blood, urine or the like and is disposable. Detection element, and an intermittent automatic analyzer and method using the detection element.

[0002]

2. Description of the Related Art As an apparatus for continuously analyzing a biological liquid sample such as blood or urine, for example, a flow injection apparatus having a reaction section in which glass beads or chitosan beads carrying an immobilized enzyme are packed in a tubular column. Is conventionally known. However, in this apparatus, beads are packed in a column having a narrow inner diameter of about 1 to 2 mm, so that the procedure for immobilizing the enzyme is difficult and complicated. In addition, since it is difficult to remove the air bubbles that have entered the column, it is necessary to always keep the inside of the column in a wet state in order to prevent air bubbles from being mixed, and thus conditioning is difficult. Furthermore, if the column is always kept in a wet state, the enzyme will be easily deactivated due to the influence of contaminants in the water and long-term storage will be difficult, and the contaminants such as proteins and bacteria in the test sample will cause clogging. It also had the drawback of being easy. The flow injection device also requires an injector for injecting the sample into the analysis system and a carrier liquid for transporting the sample to the reaction section.

As a similar device utilizing an immobilized enzyme, an enzyme sensor is known in which an immobilized enzyme membrane is mounted on a sensitive membrane of an electrode, a cellulose membrane is coated thereon, and these are immobilized by an O-ring. Has been. However, in this type of enzyme sensor, since the immobilized enzyme membrane is in direct contact with the electrode surface, it is susceptible to deterioration due to oxidation, is not stable and has a short life, and also mounts the immobilized enzyme membrane on the electrode surface. Also, it was troublesome to remove it. Furthermore, a dense film is required to allow only the inspection object to reach the electrode surface and not the inhibitor, but such a film is technically difficult to manufacture and mass production is difficult.

[0004]

SUMMARY OF THE INVENTION Therefore, the object of the present invention is that the enzyme immobilization operation is simple, the enzyme activity can be easily maintained for a long period of time, and the interference by the inhibitor can be effectively prevented. It is another object of the present invention to provide a means capable of performing measurement accurately, highly accurately, and in a short time. Another object of the present invention is to provide means for injecting a sample or standard solution into the detection system, or automatic analysis means that does not require the use of a carrier liquid for transferring the sample or standard solution into the detection system. ..

[0005]

According to the present invention, there are provided (1) a housing having a sample inlet and a sample outlet,
(2) Porosity filled in the housing by supporting a biological catalyst capable of reacting with a test object contained in the sample and causing a change detectable by the electrode, on the entire surface of the porous support. (3) an electrode that is enclosed in the housing and is in contact with the porous reactant, and can detect a change due to a reaction between a test object contained in a sample and a biological catalyst, And (4) a sensing element, characterized in that it comprises a terminal capable of sending a signal from said electrode to an external reader. The present invention also provides (1) the detection element according to claim 1, (2) means for allowing a predetermined amount of sample to be introduced into the reaction chamber of the detection element from the sample inlet of the detection element, (3)
Means capable of holding a predetermined amount of sample in a stagnant state in the reaction chamber, (4) a signal from an electrode which detects a change caused by a reaction between a test object contained in the sample and a biological catalyst, A device for processing, (5) means capable of removing an inspected sample from the reaction chamber via a sample outlet, and (6) a device for controlling the movement and stagnation of the sample, the intermittent automatic Regarding analytical equipment. Furthermore,
According to the present invention, a predetermined amount of sample is put into the reaction chamber of the detection element from the sample inlet of the detection element, and the sample is held in the reaction chamber in a stagnant state, and the test object and biological substance contained in the sample It is characterized in that a change due to reaction with a catalyst is generated, the change is detected by an electrode, a signal from the electrode is sent to a reader, and an inspected sample is removed from the reaction chamber via a sample outlet. , Also relates to an intermittent automatic analysis method.

A representative embodiment of the sensing element according to the invention is shown in FIG.
Shown in. The detection element 10 comprises a housing 3 having a sample inlet 1 and a sample outlet 2 at the front and back. The housing 3 is
It is preferably made of a non-breathable and water resistant material. Inside the housing 3 is a reaction chamber 5 filled with a porous reactant 4, and an electrode 6 provided on the inner wall surface of the housing 3.
The porous reactant 4 comprises a porous support and a biological catalyst supported on the entire surface of the porous support. Electrode 6
Are in electrical communication with terminals 7 provided on the outer surface of the housing. If necessary, a mesh plate 8 can be provided between the sample inlet 1 and the sample outlet 2 and the reaction chamber 5.

The material of the porous support that forms the porous reaction body 4 is not particularly limited, but a fiber aggregate (for example, non-woven fabric, felt, woven fabric, knitted fabric) or foam or a composite thereof is used. It is particularly preferable that the non-woven fabric has a three-dimensional structure. At least a part of the electrode 6 and the porous reactant 4 may be in contact with each other. The electrode 6 may be provided on the inner wall surface of the housing by etching or plating a metal foil, or a metal plate, a metal rod, or the like may be wrapped with the porous reaction body 4.

The detection element of the present invention can be used for detecting and quantifying various biological test objects contained in an arbitrary sample, and in particular, a biological liquid sample such as blood, serum or plasma. Detection of lipids (eg cholesterol, phospholipids, neutral fats) contained in etc., sugars or nitrogen compounds (eg urea, uric acid, creatinine), nitrogen compounds contained in urine (eg uric acid, urea nitrogen) And used for quantification. Before carrying out the inspection with the detection element of the invention,
Optionally, the biological fluid sample can be diluted with saline or the like, or treated with a drug such as an anticoagulant.

A biological catalyst is supported on the entire surface of the porous support constituting the porous reactant contained in the detection element of the present invention. The biological catalyst may be any as long as it can react with an object to be tested contained in a sample to cause a change detectable by an electrode, for example, an enzyme,
Organelles, microorganisms, animal and plant cells can be used. In the present invention, a biological catalyst is selected according to the type of test object. It is preferred to select a combination that will eventually produce hydrogen peroxide or ammonia by the reaction of the test object with one or more biological catalysts (especially enzymes). Although many examples are already known as combinations thereof, typical examples are glucose-glucose oxidase (hydrogen peroxide), pyruvate-pyruvate oxidase (hydrogen peroxide), and urate-uricase (peroxidation). Hydrogen), urea-urease (ammonia), neutral fat-lipase and glycerol oxidase (hydrogen peroxide), cholesterol-cholesterol oxidase (hydrogen peroxide), L amino acid-L amino acid oxidase (hydrogen peroxide), ethanol-alcohol oxidase (Hydrogen peroxide) etc. Further, the electrode is selected according to the type of the reaction product of the test object and the biological catalyst.
For example, with respect to the hydrogen peroxide production reaction, the reaction can be detected by amperometry using a platinum electrode or a carbon electrode. Also, for the ammonia production reaction, an ammonia electrode or an ion selective field effect transistor (FET) is used.
An electrode or the like can be preferably used.

Immobilizing the biological catalyst on a porous support,
As a method for supporting, any immobilization method can be used, but for example, a carrier binding method in which a biological catalyst is directly or indirectly bound to the surface of a porous support by a covalent bond or an ionic bond ( For example, the method described in JP-A-60-224618) or a comprehensive method for entrapping and immobilizing a biological catalyst in a lattice of a polymer gel such as silk fibroin or in a resin film (JP-A-2-245189). Alternatively, the methods described in JP-A-60-120988, etc.) can be used. In particular,
According to the method described in JP-A-2-245189, a silk fibroin aqueous solution containing a biological catalyst is impregnated into a fiber assembly (particularly one mainly composed of hydrophilic fibers), dried, and insolubilized with alcohol, The biological catalyst is encapsulated and fixed in the silk fibroin gel layer, and the silk fibroin gel layer does not pass large molecules such as enzyme proteins but small molecules such as the test object that is the reaction substrate. Therefore, it is desirable as a means for immobilizing a biological catalyst.

Next, a method of using the detection element 10 according to the present invention, and an automatic analyzer and an automatic analysis method of the present invention will be described with reference to the embodiment shown in FIG. First, the flow path switching valve 11 is connected to the cleaning liquid tank 12, and the cleaning liquid (for example, physiological saline) in the cleaning liquid tank 12 is sent to the detection element 10 to fill the reaction chamber 5. Next, the flow path switching valve 11 is connected to the standard liquid tank 13. The standard solution stored in the standard solution tank 13 is a standard reagent having a known concentration, which serves as a control for the test object that may be contained in the sample in the sample tank 14.
The pump 15 is operated to remove the cleaning liquid in the reaction chamber 5, and the reaction chamber 5 is filled with the standard liquid to pump the pump 15.
To stop. The reaction between the standard test analyte of known concentration in the standard solution and the biological catalyst proceeds, causing a detectable change (eg, increased reaction products or decreased reaction consumption). These changes are detected by the electrode 6 and sent to the data processing controller 16 via the terminal 7. After the reaction between the standard test object and the biological catalyst is completed or after reacting for a predetermined time, the flow path switching valve 11 is connected to the cleaning liquid tank 12, and the standard liquid is removed by the pump 15. The cleaning liquid is sent to the reaction chamber 5 and filled therein.

Subsequently, the flow path switching valve 11 is connected to the sample tank 14
The cleaning liquid is removed by the pump 15 and the liquid sample is sent to the reaction chamber 5, and the pump 15 is stopped when the reaction chamber 5 is filled with the liquid sample. When the liquid sample contains test substances, they come into contact with the biological catalyst immobilized on the porous support, and the reaction between the two proceeds to cause a detectable change. The sample is kept in the reaction chamber 5 until the above reaction is completed or until it is reacted for a predetermined time, and the change during that time is detected by the electrode 6 in the same manner as described above, and is sent to the data processing control device 16 via the terminal 7. send. The detected value processed by the data processing control device 16 is recorded by the recorder 17 and displayed in an appropriate format. The various liquids removed from the reaction chamber 5 are sent to the waste liquid tank 18.

When the inspection of one sample is completed, the flow path switching valve 11 is again brought into contact with the cleaning liquid tank 12 to clean the reaction chamber 5 with the cleaning liquid, and the above operation can be repeated.
Alternatively, the inspected detection element 10 may be taken out and replaced with a new detection element 10. The operation of the flow path switching valve 11 and the pump 15 can be controlled by the data processing control device 16. If the sample tank 14 is replaced with a new one one after another, continuous automatic analysis can be performed. By simultaneously installing a plurality of detection elements 10 and a plurality of standard solution tanks 13, automatic analysis of a plurality of test objects can be performed. Can be done.

[0014]

The porous reactant filled in the reaction chamber of the detection element according to the present invention has a simple procedure for immobilizing the enzyme,
The enzyme activity can be easily maintained for a long period of time, the interference by the inhibitory substance can be effectively prevented, and further, the measurement can be performed accurately, highly accurately, and in a short time. Further, according to the intermittent analyzer and method according to the present invention, unlike the conventional flow-type automatic analysis method, a means (for example, a microsyringe) for injecting a sample or standard solution into an analysis system, or a sample or standard solution is analyzed. The carrier liquid transferred into the system becomes unnecessary. Further, since the reaction time can be freely set in the reaction chamber, it can be used for various automatic inspections and is also suitable for the continuous method.

[Brief description of drawings]

1 is a cross-sectional view of one embodiment of the sensing element of the present invention.

FIG. 2 is an explanatory diagram of one mode of the analyzer of the present invention.

[Explanation of symbols]

1 ... Sample inlet; 2 ... Sample outlet; 3 ... Housing;
4 ... Porous reactant; 5 ... Reaction chamber; 6 ... Electrode; 7 ...
・ Terminal; 8 ・ ・ Mesh plate; 10 ・ ・ Detection element; 11 ・
・ Flow switching valve; 12 ・ ・ Washing liquid tank; 13 ・ ・ Standard liquid tank; 14 ・ ・ Sample tank; 15 Pumps; 16 ・ ・ Data processing control device; 17 ・ ・ Recorder; 18 ・ ・ Waste liquid tank

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification number Office reference number FI technical display location G01N 35/08 A 8310-2J 7235-2J G01N 27/46 336 B 7235-2J 336 Z

Claims (3)

[Claims] 1. (1) A housing having a sample inlet and a sample outlet, (2) a porous biological catalyst capable of reacting with a test object contained in the sample and causing a change detectable by an electrode. A porous reaction material which is carried on the entire surface of the support and is filled in the housing, and (3) an inspection target which is enclosed in the housing and is in contact with the porous reaction material, and which is contained in the sample. An electrode capable of detecting a change due to a reaction with a biological catalyst, and (4)
A detection element comprising a terminal capable of sending a signal from the electrode to an external reader. 2. The detecting element according to claim 1, (2)
Means capable of introducing a predetermined amount of sample into the reaction chamber of the detection element from the sample inlet of the detection element, (3) Means capable of retaining and retaining a predetermined amount of sample in the reaction chamber, (4) Sample A device for processing a signal from an electrode which detects a change generated by a reaction between a test object contained therein and a biological catalyst, (5) removing an inspected sample from the reaction chamber through a sample outlet. And (6) a sample movement and stagnation control device, an intermittent automatic analyzer. 3. An object to be inspected contained in the sample, wherein a predetermined amount of sample is put into the reaction chamber of the detection element from the sample inlet of the detection element according to claim 1, and the sample is held in the reaction chamber while being held therein. Change due to the reaction of the biological catalyst with the biological catalyst, the change is detected by the electrode, the signal from the electrode is sent to the data processing controller, and the inspected sample is removed from the reaction chamber via the sample outlet. An intermittent automatic analysis method characterized by: