Patent Application
20070292890
The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which:
Now, a chemiluminescence chip according to the present invention and a chemiluminescence detecting apparatus using the same will be described in detail with reference to the accompanying drawings.
As shown in the drawings, the chemiluminescence chip according to the present invention is constructed such that a filling unit 1, in which an immobilized enzyme 2 is filled, is mounted in a space having an inlet port 1a, through which a material to be measured is introduced, and an outlet port 1b, through which the material to be measured is discharged, on a supporting structure 4.
In the chemiluminescence chip according to the present invention shown in
In the chemiluminescence chip according to the present invention shown in
Although not shown in the drawings, the chemiluminescence chip according to the present invention may be constructed such that the filling unit 1 is a groove formed in a flat plate, and the flat board is pressed against the supporting structure 4 by the transparent plate 3.
In the case that the filling unit 1 is formed of the transparent tube, it is preferable to use a tube having high chemical resistance and high translucency, such as a Teflon tube, as the transparent tube.
In the case that the filling unit 1 is formed of the synthetic resin sheet, it is preferable to use a sheet having high chemical resistance, such as a Teflon sheet, as the synthetic resin sheet.
In the case that the filling unit 1 is formed of the flat plate, it is preferable to use a synthetic resin plate or a metal plate having high chemical resistance, such as a Teflon sheet, as the flat plate. Also, the groove is formed in the flat plate by a cutting process or an optical etching process.
It is also preferable to use a plate having high chemical resistance and high translucency, such as a Teflon plate or a glass plate, as the transparent plate 3.
In the chemiluminescence chip according to the present invention, hydrogen peroxide, a compound having an imidazole ring, or an oxidizing enzyme having hydrogen peroxide generated therein may be used as the material to be measured. However, the material to be measured is not restricted to the above-specified materials.
Also, in the chemiluminescence chip according to the present invention, peroxidase, alkaliphosphatase, or glucose oxidase may be used as the above-mentioned enzyme. However, the enzyme usable in the present invention is not restricted to the above-specified enzymes. In addition, the immobilized phase of the immobilized enzyme 2 may be gel having amino group, such as kitosan gel, glass beads, polystyrene gel, or acryl gel. However, the immobilized phase of the immobilized enzyme 2 is not restricted to the above-specified immobilized phases.
Furthermore, in the chemiluminescence chip according to the present invention, when the filling unit 1 is formed in the shape of a zigzag as shown in
As shown in
Furthermore, the chemiluminescence detecting apparatus according to the present invention includes a heater 13 mounted in the dark box 11 for controlling the interior temperature of the dark box 11, and a temperature sensor 14 mounted in the dark box 11 for detecting the interior temperature of the dark box 11. Also, a computer 15 for processing image data photographed by the cooled CCD camera 12, is disposed outside the dark box 11, and a monitor 16 for displaying the image data processed by the computer 15 is also disposed outside the dark box 11. The material to be measured is repeatedly injected into the inlet pipe Pa by means of a liquid-forwarding pump 17 and a loop injector 18.
Hereinafter, the detailed construction of the chemiluminescence chip according to the present invention and the chemiluminescence detecting apparatus using the same will be described in the case that the material to be measured is hydrogen peroxide.
First, the chemiluminescence chip according to the present invention is constructed such that the filling unit 1 is formed of a tube, and the immobilized enzyme 2, which is obtained by immobilizing horseradish peroxidase (hereinafter, referred to as “HRP”) as enzyme, is filled in the tube, the tube is pressed against the supporting structure by the transparent plate 3.
A Teflon tube was used as the tube of the filling unit 1. Kitopal beads (a product from Fuzibo corporation) and imperforate glass beads containing amino group (a product from Kemco corporation) were used as the immobilized enzyme 2.
In the chemiluminescence detecting apparatus according to the present invention, an imidazole solution, pH of which has been adjusted by an alkali buffer solution, is introduced into the inlet pipe Pa by means of the liquid-forwarding pump 17, and an oxygenated water, which has been prepared with various concentrations, is injected into the inlet pipe Pa by means of the loop injector 18. Subsequently, the mixture of the imidazole solution and the oxygenated water flows along the inlet pipe Pa, and reaches the immobilized enzyme 2 of the filling unit 1 through the inlet port 1a of the filling unit 1 of the chemiluminescence chip T. As a result, chemiluminescence is generated in the immobilized enzyme 2. This chemiluminescence is photographed by the cooled CCD camera 12. The photographed image data is processed by the computer 15, and the processed image data is displayed on the monitor 16.
A tricine buffer solution, a tris hydrochloric acid buffer solution, or a boric acid buffer solution may be used as the alkali buffer solution. However, the alkali buffer solution usable in the present invention is not restricted to the above-specified buffer solutions. In this example, tricine buffer solution was used, and the concentration of the tricine buffer solution was 50 mmol/L. In this case, the value of pH was 9.4.
The imidazole usable in the present invention may be imidazole, 2-methylimidazole, 4-methylimidazole, 4-methyl-5-hydroxymethyl imidazole, allantoin, ethylene urea, histidine, pyrazole. However, the imidazole usable in the present invention is not restricted to the above-specified imidazoles. In this example, imidazole was used, and the concentration of the imidazole was 100 mmol/L.
Three kinds of oxygenated water, the concentrations of which are 9.8 μmol/L, 98 μmol/L, and 980 μmol/L, were prepared. Also, the amount of the oxygenated water used was approximately 5 to 50 μL, which is a small amount. In this example, the amount of the oxygenated water used was 20 μL.
Also, it is preferable that the flow speed of the imidazole in the inlet pipe Pa be low. In this example, the flow speed of the imidazole was 100 μL/min. However, it is also possible that the flow speed of the imidazole is less than 100 μL/min.
Furthermore, chemiluminescence is generated by the reaction of the immobilized HRP as the immobilized enzyme 2, imidazole, and oxygenated water, and, after the chemiluminescence is generated, the active center of the HRP is protonized, and therefore, the HRP is inactivated. However, the proton of the active center of the inactivated HRP is removed by the alkali buffer solution and the imidazole. As a result, the HRP is changed into an activated HRP, and therefore, the chemiluminescence is generated again.
As apparent from the above description, in the chemiluminescence detecting apparatus according to the present invention, when the material to be measured is hydrogen peroxide, the chemiluminescence image having a strength corresponding to the concentration of oxygenated water is displayed on the monitor 16.
Consequently, it is possible to easily and conveniently detect and quantify hydrogen peroxide in body fluids, such as sweat, tears, blood, urine, spit, and lymph, by selecting the body fluids as the hydrogen peroxide measuring sample. The detection and quantification of the hydrogen peroxide in the body fluids are necessary to measure oxidative stress and the substrate of oxidase enzyme, and are very useful in a clinical testing field.
Although the preferred embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.
