KR20120138575A - An analytical method of a biochemical analyte using a chemiluminescent analytical chip placed in a spectrofluorometer - Google Patents

Abstract

PURPOSE: A method for analyzing biochemical substances using a chemiluminescence analysis chip installed in a spectrophotofluorometer is provided to analyze the biochemical substances by installing the chemiluminescence analysis chip in a dark room chamber of the common spectrophotofluorometer, thereby rapidly analyzing a small amount of the biochemical substances. CONSTITUTION: A method for analyzing biochemical substances using a chemiluminescence analysis chip installed in a spectrophotofluorometer comprises next steps. A chemiluminescence analysis chip is installed in the inside of the spectrophotofluorometer. A biochemical specimen, a spectroscopic analysis reagent, and an oxidizing agent are injected by using a syringe pump connected to outside of the spectrophotofluorometer. The intensity of the chemiluminescence is measured by using the spectrophotofluorometer.

Description

Analytical method of a biochemical analyte using a chemiluminescent analytical chip placed in a spectrofluorometer

The present invention relates to a method for analyzing biochemicals using a chemiluminescence analysis chip mounted on a fluorescence spectrophotometer, and more specifically, a chemiluminescence analysis chip is mounted inside a commercial fluorescence spectrometer and biochemically mounted on the chemiluminescence assay chip from the outside. The present invention relates to a method for rapidly and accurately analyzing trace amounts of biochemicals by injecting a substance sample, a spectroscopic reagent, and an oxidant to induce a chemiluminescence reaction and then measuring the chemiluminescence intensity emitted to the outside with a fluorescence spectrophotometer.

As a method for analyzing biochemicals, electrochemical methods for performing analysis through measurement of electrical signals and spectroscopic methods for performing analysis by measuring light such as fluorescence or chemiluminescence are generally used. Among them, the spectral analysis method using light is an analysis method that is widely used for the analysis of trace amounts of biochemicals because of its excellent sensitivity. In order to perform analysis using fluorescence, light is irradiated to the analytical sample solution from the outside, and the analysis is performed by detecting fluorescence at an angle perpendicular to the irradiation path of the light. However, in the case of analysis using chemiluminescence, the concentration of the sample to be analyzed is analyzed by detecting the intensity of light emitted by self-emission generated by a chemical reaction inside the sample solution without irradiating light to the sample solution from the outside. As such, the analysis using chemiluminescence has a relatively low noise signal compared to the analysis using fluorescence, and thus a relatively high signal strength is obtained, which is particularly useful for analyzing trace amounts of biochemicals.

The term lumonescence was first coined by the German scientist Eilhardt Wiedemann in 1888 and refers to cold light, which is generally opposed to incandescent light from hot objects. It is divided into bioluminescence, chemiluminescence, electroluminescence, fluorescence, phosphorescence, radioluminescence, thermoluminescence, etc. Among them, fluorescence refers to a phenomenon of emitting electromagnetic radiation by being exposed to radiation having the same or greater energy. Over the last few decades, fluorescence spectroscopy using this fluorescence has been used for the determination of metal ions and organic compounds. In general, materials that can be measured by fluorescence spectroscopy can also be measured by absorption spectroscopy, but fluorescence spectroscopy has higher sensitivity and selectivity than absorption spectroscopy.

Chemiluminescence refers to luminescence generated from molecules reaching an excited state by a chemical reaction. Chemiluminescent reagents include luminol and luminol derivatives, dioxetane, lophine, lucigenin, acridinium salts, indole derivatives, Schiff base , Oxalate, diphenoylperoxide, ruthenium compound (Ru (bpy) ₃ ²⁺ ), and the like. Recently, research groups on the analysis of amino acids using the ruthenium compound (Ru (bpy) ₃ ²⁺ ) and the chemiluminescence reaction of amino acids have been conducted by various research groups. The chemiluminescence method has a relatively simple measuring device, and the analysis sensitivity is superior to the absorption spectroscopy and the fluorescence method.

The chemiluminescence reaction of luminol (luminol, 5-amino-2,3-dihydrophthal-azine-1,4-dione) and hydrogen peroxide (H ₂ O ₂ ) proceeds as follows when a catalyst is present.

These species can be quantified if the concentration of the species catalyzing or inhibiting the reaction has a linear relationship with the chemiluminescence intensity generated in the reaction. The oxidation of luminol proceeds under basic whereas peroxyoxalate can undergo chemiluminescence at neutral. The reaction between peroxyoxalate and hydrogen peroxide (H ₂ O ₂ ) proceeds as follows to show chemiluminescence.

Various types of commercial fluorescence spectrometers capable of performing analysis of biochemicals using chemiluminescence are commercially available and used for analysis of trace amounts of daily biochemicals. Generally, a square analytical sample cell having a side length of 1 cm is mounted in a dark chamber of a commercial fluorescence spectrophotometer, and an analyte sample, a spectroscopic reagent, and an oxidizing agent are injected into the dark chamber chamber lid. The concentration of the sample to be analyzed is quantified by detecting light by operating a commercial fluorescence spectrophotometer.

Recently, the development of analytical technology using miniaturized analytical chips such as lab-on-a-chip and microfluidic chips as a means to analyze small quantities of biochemicals quickly and accurately Research and development. In the analysis of the biochemicals based on the lab-on-a-chip or the microfluidic chip, there is a difficulty in performing an analysis on a very small amount of sample solution in the microchannel or microchamber of the analysis chip. Therefore, it is necessary to use a high-sensitivity detector that can analyze the trace amount of sample solution inside the analysis chip.

In order to efficiently analyze trace amounts of biochemicals, it is produced by manufacturing a chemiluminescence analysis chip which is mounted on a commercial fluorescence spectrophotometer to perform analysis and injecting biochemicals, spectroscopic reagents and oxidants to be analyzed into the chemiluminescence analysis chip. By detecting the intensity of chemiluminescence using a high sensitivity detector of a commercial fluorescence spectrophotometer, it is necessary to develop a technology for analyzing a trace amount of biochemicals quickly and accurately.

In view of the above, the inventors of the present invention installed a chemiluminescence analysis chip inside a commercial fluorescence spectrophotometer, and injected a chemiluminescence sample, a spectrometry reagent, and an oxidant into the chemiluminescence analysis chip from the outside to chemiluminescence reaction. The present invention was completed by confirming that a trace amount of biochemicals could be analyzed quickly and accurately by measuring the chemiluminescence intensity emitted to the outside after induction of fluorescence spectrophotometer.

An object of the present invention is to mount a chemiluminescence assay chip on a commercial fluorescence spectrophotometer to perform analysis of biochemicals, the advantages of the chemiluminescence assay chip that can quickly analyze a small amount of biochemicals and high sensitivity analysis of a commercial fluorescence spectrophotometer Combining the advantages, it provides a biochemical analysis method that can analyze a small amount of biochemicals more quickly and accurately more efficiently.

In order to solve the above problems, the present invention provides a method for analyzing a biochemical material using a chemiluminescence analysis chip mounted on a fluorescence spectrophotometer comprising the following steps.

1) mounting the chemiluminescence analysis chip inside the fluorescence spectrophotometer;

2) injecting a biochemical sample, a spectroscopic reagent and an oxidizing agent into the chemiluminescent assay chip using a syringe pump connected to the outside of the fluorescence spectrophotometer; And

3) measuring the chemiluminescence intensity emitted to the outside with a fluorescence spectrophotometer.

Step 1 is a step of mounting the chemiluminescence analysis chip inside the fluorescence spectrometer, in order to detect the intensity of chemiluminescence emitted by the chemical reaction of the spectroscopic analysis reagent and the oxidant, the chemiluminescence analysis chip first inside the fluorescence spectrophotometer Step in mounting.

In the present invention, the chemiluminescence analysis chip is preferably mounted vertically inside the fluorescence spectrophotometer.

As used herein, the term "chemiluminescent assay chip" refers to an assay chip that detects an analyte by inducing a chemiluminescence reaction between a spectroscopic reagent and an oxidant.

The chemiluminescence analysis chip comprises an analysis chip body; A fluid injector including a sample inlet for injecting an analysis target sample formed in front of the upper part of the analysis chip, a spectroscopic reagent inlet for injecting a spectroscopic reagent, and an oxidant inlet for injecting an oxidant; A fluid outlet formed at the rear of the analysis chip main body; A microchannel formed in the analysis chip body in communication with the fluid inlet and the fluid outlet; And a microchamber disposed as a part of the microchannel between the fluid inlet and the fluid outlet.

The chemiluminescent assay chip is a sample to be analyzed by injecting a sample to be analyzed together with a spectroscopic reagent and an oxidant through a fluid injection unit and measuring the intensity of light absorption or emission generated through the reaction. Can be used to analyze the presence and / or amount of

The chemiluminescence assay chip body is a main body of an analysis chip that analyzes by measuring the intensity of light absorption or emission does not affect the absorption or emission of light by the biochemicals and spectroscopic reagents to be analyzed. As made of the material, it may be made of a material which does not absorb or absorbs light in the near ultraviolet region or the visible wavelength region is very small.

Specifically, the chemiluminescence analysis chip main body may be manufactured using materials such as glass-based, quartz, polycarbonate polymers, silicone-based polymers such as polydimethylsiloxane, and olefin-based polymers such as polypropylene. have.

Preferably, the oxidant inlet is located behind the sample inlet and the spectroscopic reagent inlet. This means that the analyte sample and the spectroscopic reagent injected through the sample inlet for the sample injection and the spectroscopic reagent inlet for the spectroscopic reagent injection reach the oxidant inlet for the oxidant injection disposed behind the microchannel. This is because the efficiency of the analysis can be increased by reaching the oxidant inlet and introducing the oxidant after being sufficiently mixed in the microchannel beforehand.

The microchannel may have a circular or polygonal cross section, and a circular diameter or a length of one side of the polygon may be in the range of 30 to 800 micrometers in size.

The microchamber is formed in a circular or polygonal cross section, and the length of one side of the circular diameter or polygon may be in the range of 30 to 800 micrometers in size.

The chemiluminescence assay chip may further include a substrate disposed on a rear surface of the assay chip body.

The substrate is made of a material such as glass, polymer, metal, and the like, and is preferably flat, and may be made of a material having a larger width than the analysis chip main body and easily bonding to the analysis chip main body.

In the present invention, a general commercial fluorescence spectrophotometer may be used.

As used herein, the term "commercial fluorescence spectrophotometer" refers to a commercial fluorescence spectrophotometer commercial product that is manufactured and sold for the purpose of measuring the intensity of light to measure the presence and concentration of analyte. You can therefore use a commercial product that has the function of measuring or measuring over time.

Preferably, in the present invention, the chemiluminescence assay chip may be mounted vertically in a dark chamber inside the fluorescence spectrophotometer.

Step 2 is a step of injecting a biochemical sample, a spectroscopic reagent and an oxidant into the chemiluminescent assay chip using a syringe pump connected to the outside of the fluorescence spectrophotometer, the sample, spectroscopic reagent and oxidant to the chemiluminescent assay chip Injecting the chemiluminescence reaction is a step.

In the present invention, the biochemical material is an analyte of analytical process using a chemiluminescence assay chip and increases or decreases the intensity of chemiluminescence generated by the reaction of a spectroscopic reagent and an oxidant. By detecting the degree, the presence and concentration of the biochemical can be analyzed.

By additionally adding metal ions as a catalyst to the spectroscopic reagent, the chemiluminescence intensity can be further increased to improve the accuracy and sensitivity of the analysis. Copper ions, cobalt ions or iron ions may be used as the metal ions. Specifically, the metal ion may be used Cu ^{2 +} , Co ^{2 +} , Fe ^{3 +} or Fe ^{2 +} , but is not limited thereto.

The spectroscopic reagent is a reagent that emits light by a chemical reaction in the chemiluminescence assay chip, luminol (luminol), luminol derivatives, dioxetane (dioxetane), lophine (lucigenin), Acridinium salts, indole derivatives, oxalate, ruthenium compounds (Ru (bpy) ₃ ²⁺ ) and the like can be used. The spectroscopic reagent may be dissolved in an appropriate concentration in a buffer solution of various pH ranges.

The oxidizing agent may be hydrogen peroxide (H ₂ O ₂ ) or iron cyanide (Fe (CN) ₆ ) and the like as a substance causing chemiluminescence by the oxidation reaction of the spectroscopic reagent. The oxidant may be prepared by using a specific concentration in a buffer of a specific pH.

Step 3 is a step of measuring the chemiluminescence intensity emitted to the outside with a fluorescence spectrophotometer, the chemiluminescence intensity emitted to the outside by the oxidation reaction of the chemiluminescent reagent to analyze the presence and / or concentration of biochemicals Is measured with a fluorescence spectrophotometer.

Biochemical analysis method using a chemiluminescence analysis chip mounted on a fluorescence spectrophotometer according to the present invention is to mount a chemiluminescence analysis chip on a fluorescence spectrophotometer to analyze the biochemicals, and the spectroscopic analysis reagent in the chemiluminescence analysis chip By detecting the intensity of chemiluminescence emitted by the chemical reaction of the oxidizing agent using the fluorescence spectrophotometer, the presence or concentration of the biochemical can be analyzed.

The biochemical substance to be analyzed increases the intensity of light emission by promoting the light emitting reaction of the spectroscopic reagent, or decreases the emission intensity of light by interrupting the light emitting reaction of the spectroscopic reagent. It can be used to analyze the presence and concentration of the biochemicals to be analyzed.

In the present invention, biochemicals that can be analyzed using the chemiluminescence analysis chip and fluorescence spectrophotometer include antioxidants, amino acids or sugars, but are not limited thereto.

The antioxidants are antioxidant enzymes such as superoxide dismutase and vitamins such as vitamin C and E, which remove free radicals generated in vivo or inhibit oxidation reaction in solution. Phytochemicals such as flavonoids and the like.

Examples of the sugars include glucose or sucrose.

Hereinafter, the present invention will be described in detail with reference to the accompanying drawings.

1 is a schematic diagram showing an experimental setup for a biochemical analysis using a chemiluminescence analysis chip 20 mounted on a commercial fluorescence spectrophotometer 10 of the present invention.

In Figure 1 (a) is a structural diagram of the experimental device connected to the chemiluminescence analysis chip 20 mounted in the dark chamber chamber 11 of the commercial fluorescence spectrophotometer (b) (b) the structure of the chemiluminescence analysis chip 20 Indicates.

In FIG. 1A, the commercial fluorescence spectrophotometer 10 includes a dark chamber chamber 11, a light detection slit 12, a monochromator 13, a photomultiplier tube 14, and the like. The optical signal obtained from the commercial fluorescence spectrophotometer 10 is transmitted to the computer main body 50 to display the analysis result in the form of a spectroscopic spectrum 60 on the monitor 51.

The chemiluminescence analysis chip 20 may be mounted inside the dark chamber chamber 11, and the sample and spectroscopic analysis of the chemiluminescence analysis chip 20 are analyzed using a syringe pump 30 installed outside. Reagent and oxidant solutions may be injected. At this time, the chemiluminescence analysis chip 20 is preferably mounted vertically in the dark chamber chamber (11). By mounting and analyzing the chemiluminescence analysis chip 20 vertically as described above, the chemiluminescence analysis chip 20 is not only easily mounted in the dark chamber of the commercial fluorescence spectrophotometer, but also the position and the chemiluminescence analysis chip of the light detection slit 12 of the commercial fluorescence spectrophotometer. The position of the microchannel or the microchamber of 20) is well matched, and as a result, a high spectroscopic spectrum 60 can be easily obtained.

The chemiluminescence analysis chip 20 has a fluid inlet consisting of a sample inlet 22, a spectroscopic reagent inlet 23, and an oxidant inlet 24, and a fluid outlet 29 in the analysis chip body 21. The sample inlet 22 and the spectroscopic reagent inlet 23 are located in front, the oxidant inlet 24 is located in the middle, and the fluid outlet 29 is located in the rear. Between the fluid inlet and the fluid outlet 29, the microchannel 25 formed inside the main body of the analysis chip communicates with the fluid inlet 24 between the oxidant inlet 24 and the fluid outlet 29. Some sections of the microchannel 25 have a micro chamber 27.

After mounting the chemiluminescence analysis chip 20 perpendicular to the dark chamber chamber 11 of the commercial fluorescence spectrophotometer 10, the lid of the dark chamber chamber 11 is closed and the syringe pump 30 installed outside Using the sample injection syringe 31 is injected into the sample inlet 22 of the chemiluminescence assay chip 20 at a constant rate through the sample injection tube (32). In addition, the spectroscopic analysis reagent injection hole of the chemiluminescent assay chip 20 through the spectroscopic analysis reagent injection tube 34 using the syringe pump 30 to the spectroscopic analysis reagent injection syringe 33 Inject at 23 constant speed. In addition, the syringe pump 30 is injected into the oxidant injection hole 24 of the chemiluminescence assay chip 20 through the oxidant injection tube 36 using the oxidant injection syringe 35. The sample solution injected from the microchannel 25 reacts with the spectroscopic reagent and the oxidant to emit chemiluminescence. The emitted light passes through the monochromator 13 through a light detection slit 12. The light intensity is amplified in the photomultiplier 14. This amplified light signal enters the computer main body 50 and is displayed on the monitor 51 in the form of a spectroscopic spectrum 60. The amount of the sample to be analyzed may be quantified through the spectroscopic spectrum 60 displayed by the above process.

Figure 2 is a picture of the (a) chemiluminescence analysis chip actually manufactured in the present invention, (b) a fluorescence spectrophotometer equipped with a chemiluminescence assay chip connected to the syringe pump, (c) the chemical inside the dark chamber of the fluorescence spectrophotometer (D) Picture showing the luminescence analysis chip.

As shown in FIG. 1 and FIG. 2, after mounting the chemiluminescence analysis chip of the present invention on a commercial fluorescence spectrophotometer, enoxacin is used as an analyte, luminol is used as a spectroscopic reagent, and hydrogen peroxide or 3 to 5 show the results of detecting and analyzing the intensity of chemiluminescence generated by injecting these into the chemiluminescence analysis chip using iron cyanide with a commercial fluorescence spectrophotometer having a high sensitivity detection function.

Biochemical analysis method using a chemiluminescence analysis chip mounted on a commercial fluorescence spectrophotometer according to the present invention described above by mounting the chemiluminescence analysis chip in a dark chamber chamber of a commercial fluorescence spectrophotometer to perform analysis of biochemicals, By combining the advantages of chemiluminescence analysis chips that can rapidly analyze biochemicals with the high sensitivity of commercial fluorescence spectrophotometers, small quantities of biochemicals can be analyzed quickly and accurately.

Biochemical analysis method using a chemiluminescence analysis chip mounted on a commercial fluorescence spectrophotometer according to the present invention is commercial fluorescence spectroscopy using a chemiluminescence analysis chip manufactured in various sizes and shapes according to the biochemical material to be analyzed Easily mounted in the darkroom chamber of the photometer to perform the analysis and to be easily detached after analysis.

Biochemical analysis method using a chemiluminescence assay chip mounted on a commercial fluorescence spectrophotometer according to the present invention is mounted on a commercial fluorescence spectrophotometer to perform analysis of biochemicals from the microchamber of the chemiluminescence assay chip The minute chemiluminescence intensity generated can be detected with high sensitivity, and it is useful for analyzing trace amount of biochemicals because it is easy to process the data of the detected result and it is easy to obtain reproducible results with high precision which is the advantage of commercial fluorescence spectrophotometer. .

Biochemical analysis method using a chemiluminescence assay chip mounted on a commercial fluorescence spectrophotometer according to the present invention is generated by the chemical reaction of the spectroscopic reagent and oxidant in the microchamber of the chemiluminescence assay chip and the action of biochemicals affecting it By detecting the intensity of the chemiluminescence light, which is self-luminescence, by using a commercial fluorescence spectrophotometer, the biochemicals are analyzed. Therefore, a simple and simple operation of the device can quickly obtain high sensitivity biochemical analysis results.

1 is a schematic diagram showing an experimental setup for a biochemical analysis using a chemiluminescence analysis chip mounted on a commercial fluorescence spectrophotometer of the present invention. (A) shows the structural diagram of the experimental device connected to the chemiluminescence analysis chip mounted in the dark chamber of the commercial fluorescence spectrophotometer, and (B) shows the structure of the chemiluminescence analysis chip.
Figure 2 is a photo of the (a) chemiluminescence analysis chip actually manufactured in the present invention, (b) a fluorescence spectrophotometer equipped with a chemiluminescence analysis chip connected to a syringe pump (syringe pump), (c) a dark room of the fluorescence spectrophotometer The photo shows the chemiluminescence analysis chip inside the chamber. (D) The photo shows the chemiluminescence analysis chip mounted in the dark chamber.
3 is a chemiluminescence intensity obtained by injecting a chemiluminescence analysis chip prepared in a commercial fluorescence spectrophotometer and injecting a chemiluminescence reagent luminol (luminol) and an oxidizing agent hydrogen peroxide (H ₂ O ₂ ) 1, luminol - if injected is water analysis in the copper ion catalyst system Enoch reaper (enoxacin) - chemiluminescence intensity (2), and luminol in the case of injection of a copper ion (Cu ^{2 +)} catalyst for hydrogen peroxide reaction of hydrogen peroxide Increased chemiluminescence intensity (3).
Figure 4 shows the chemiluminescence intensity (1) and luminol-iron cyanide by the reaction of luminol and iron cyanide (Fe (CN) ₆ ) injected into the chemiluminescence assay chip after mounting a chemiluminescence assay chip in a commercial fluorescence spectrophotometer Increased chemiluminescence intensity (2) when an anoxal, enoxacin, was injected into the
5 is a concentration of enoxacin when luminoxin (enoxacin) is injected into the luminol-iron cyanide injected into the chemiluminescence assay chip at various concentrations after the chemiluminescence assay chip of the present invention is mounted on a commercial fluorescence spectrophotometer. It shows the chemiluminescence intensity according to.

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for describing the present invention more specifically, and the scope of the present invention is not limited by these examples.

Example  1: chemiluminescence of the present invention according to the addition of biochemicals Analysis chip  And fluorescence spectrophotometer Luminol and  Changes in Chemiluminescence Intensity by Oxidation of Hydrogen Peroxide

As shown in FIG. 1, a chemiluminescence assay chip was mounted in a commercial fluorescence spectrophotometer and chemiluminescence was generated by oxidation of luminol and hydrogen peroxide using the chemiluminescence assay chip and fluorescence spectrophotometer according to the present invention. The intensity change was investigated. In the present embodiment, the biochemicals used as samples were enoxacin, which is a kind of antibacterial agent.

The chemiluminescence intensity was mounted in a commercial fluorescence spectrophotometer, and after injecting a sample of enoxacin, a chemiluminescent reagent, luminol and hydrogen peroxide (H ₂ O ₂ ) into the chemiluminescence analysis chip. Measured. Alternatively it was injected additional copper ions (Cu ^{+ 2)} catalyst for the chemiluminescent reagent.

At this time, the concentration of luminol reagent was dissolved in KCl-NaOH buffer and adjusted to 1 mM, hydrogen peroxide (H ₂ O ₂ ) concentration was 0.1 M, and enoxacin (ENX) concentration was 0.1 mM. The pH was 12.8, the copper ion concentration was 0.1 mM, and the flow rate of the fluid was 30 ml / h.

The analysis results are shown in FIG. 3.

And in Figure 3 (1), chemiluminescence intensity when only one injection luminol (luminol) and the oxidizing agent is hydrogen peroxide _{(H 2 O 2), (} 2) is a luminol-copper ion (Cu ^{2 +),} the catalyst is injected into the hydrogen peroxide reaction (3) shows the increased chemiluminescence intensity when enoxacin, a sample, is injected into the luminol-hydrogen peroxide-copper ion catalyst system.

3, it can be seen that increased chemiluminescence intensity (2) was obtained by catalyzing the reaction of luminol-hydrogen peroxide with copper ions. In addition, as shown in FIG. 3, it can be seen that the chemiluminescence intensity is further increased when enoxacin is injected. Through this, it can be confirmed that even a small amount of biochemicals can be analyzed more quickly and accurately when using a combination of a chemiluminescence analysis chip and a fluorescence spectrophotometer.

Example  2: chemiluminescence of the present invention according to the addition of biochemicals Analysis chip  And fluorescence spectrophotometer Luminol and Iron cyanide  Changes in Chemiluminescence Intensity by Oxidation

As shown in FIG. 1, a chemical luminescence assay chip is mounted in a commercial fluorescence spectrometer, and the chemistry by oxidation of luminol and iron cyanide using the chemiluminescence assay chip and fluorescence spectrophotometer according to the present invention according to the addition of a biochemical material as a sample The emission intensity change was investigated. In the present embodiment, the biochemicals used as samples were enoxacin, which is a kind of antibacterial agent.

The chemiluminescence intensity is mounted in a commercial fluorescence spectrometer, and the chemiluminescence analysis chip is injected with enoxacin as a sample, luminol as a chemiluminescent reagent, and iron cyanide (Fe (CN) ₆ ) as an oxidizer. It was measured after.

At this time, the concentration of luminol reagent was dissolved in KCl-NaOH buffer and adjusted to 1 mM, iron cyanide concentration was 0.1 mM, enoxacin (ENX) concentration was 0.1 mM, and pH was 12.8. The flow rate of the fluid was 30 ml / h.

The analysis results are shown in FIG. 4.

In Figure 4 (1) shows the chemiluminescence intensity by the reaction of luminol and iron cyanide (Fe (CN) ₆ ), (2) increased when injecting enoxacin (enoxacin) as a sample to the luminol-iron cyanide The chemiluminescence intensity is shown.

As shown in Figure 4, it can be seen that the chemiluminescence intensity increased more than twice when the luminol-iron cyanide was injected when enoxacin, an analyte, was injected.

Example  3: chemiluminescence of the present invention according to the concentration of biochemicals Analysis chip  And fluorescence spectrophotometer Luminol and Iron cyanide  Changes in Chemiluminescence Intensity by Oxidation

As shown in FIG. 1, a chemiluminescence assay chip was mounted in a commercial fluorescence spectrophotometer and chemiluminescence was generated by oxidation of luminol and iron cyanide using a chemiluminescence assay chip and a fluorescence spectrophotometer according to the concentration of a biochemical material as a sample. The intensity change was investigated. In the present embodiment, the biochemicals used as samples were enoxacin, which is a kind of antibacterial agent.

The chemiluminescence intensity is mounted in a commercial fluorescence spectrophotometer and injected into the chemiluminescence analysis chip at different concentrations, and the chemiluminescence reagent luminol and iron cyanide (Fe) (CN) ₆ ) was measured after injection.

At this time, the concentration of luminol reagent was dissolved in KCl-NaOH buffer and adjusted to 1 mM, the concentration of iron cyanide was 0.1 mM, the pH was 12.8, and the flow rate of the fluid was 30 ml / h. It was set as. Meanwhile, the concentrations of enoxacin are 0.1 × 10 ^-6 M, 0.5 × 10 ^-6 M, 1 × 10 ^-6 M, 5 × 10 ^-6 M, 10 × 10 ^-6 M, 50 × 10 ^-6 M and 100 Each of them was different with ^x10-6 M.

The analysis results are shown in FIG. 5.

In Figure 5, (1) is the case where the concentration of enoxacin is 0.1 × 10 ^-6 M, (2) is 0.5 × 10 ^-6 M, (3) is 1 × 10 ^-6 M, (4 ) Is 5 × 10 ^-6 M, (5) is 10 × 10 ^-6 M, (6) is 50 × 10 ^-6 M, and (7) is 100 × 10 ^-6 M .

As can be seen in the results of Figure 5, it was confirmed that the chemiluminescence intensity increases step by step as the concentration of the analysis material enoxacin increases. Therefore, it can be seen that the analyte can be quantified by detecting the chemiluminescence intensity that increases as the concentration of the analyte increases.

While preferred embodiments of the present invention have been described above, the present invention is not limited to the above specific embodiments. It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the appended claims. And equivalents should also be considered to be within the scope of the present invention.

Claims (11)

Mounting a chemiluminescence analysis chip inside a fluorescence spectrophotometer;
Injecting a biochemical sample, a spectroscopic reagent, and an oxidant into the chemiluminescent assay chip using a syringe pump connected to the outside of the fluorescence spectrophotometer; And
A method of analyzing a biochemical using a chemiluminescence analysis chip mounted on a fluorescence spectrometer, comprising measuring the chemiluminescence intensity emitted to the outside with a fluorescence spectrophotometer.
The method of claim 1, wherein the chemiluminescence analysis chip is mounted vertically inside the fluorescence spectrophotometer.
According to claim 1, wherein the chemiluminescence analysis chip comprises an analysis chip body; A fluid injector including a sample inlet for injecting an analysis target sample formed in front of the analysis chip main body, a spectroscopic reagent inlet for injecting spectroscopic reagent, and an oxidant inlet for injecting an oxidant; A fluid outlet formed at the rear of the analysis chip main body; A microchannel formed in the analysis chip body in communication with the fluid inlet and the fluid outlet; And a microchamber disposed between the fluid inlet and the fluid outlet as a part of the microchannel, wherein the biochemical material using the chemiluminescence analysis chip mounted on the fluorescence spectrophotometer.
4. The method of claim 3, wherein the oxidant inlet is located behind the sample inlet and the spectroscopic reagent inlet.
The method of claim 1, wherein a metal ion is further added to the spectroscopic reagent as a catalyst.
The method of claim 5, wherein the copper ions, cobalt ions or iron ions are biochemical substances using a chemiluminescence analysis chip mounted on a fluorescence spectrophotometer.
The method of claim 1, wherein the biochemical is an antioxidant, an amino acid, or a sugar.
The method of claim 7, wherein the antioxidant is a superoxide dismutase, vitamin C, vitamin E, or flavonoid.
The method according to claim 7, wherein the sugar is glucose or sucrose. The method for analyzing a biochemical using a chemiluminescence assay chip mounted on a fluorescence spectrophotometer.
The method of claim 1, wherein the spectroscopic reagent is luminol, luminol derivative, dioxetane, ropin, lucigenin, acridinium salt, indole derivative, oxalate or ruthenium compound, using a chemiluminescence analysis chip mounted on a fluorescence spectrophotometer Method of analysis of biochemicals.
The method of claim 1, wherein the oxidant is hydrogen peroxide or iron cyanide.

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