JP2000338085A - Method for detecting chemiluminescence and microchip electrophoretic apparatus using the method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a small-sized microchip electrophoretic apparatus of a low cost by using a chemical reaction luminescence method as a detector. SOLUTION: A migration liquid is filled in a sample introducing channel 20 and a separate channel 21, a sample liquid is filled in a reservoir R1, and a luminescent reagent is filled in a reservoir R4. After a sample is introduced into the channel 20, the sample existing in an intersection of both the channels is allowed to migrate electrophoretically in a direction of the reservoir R4 through the channel 21. Components separated while migrating are sequentially reacted with the reagent in sequence arriving at the reservoir R4 and light emitted. The emitted light is detected by a photomultiplier tube 22 arranged directly under the reservoir R4.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a chemiluminescence detection method for separating a liquid sample component using, for example, an electrophoresis chip and detecting the separated components, and a microchip electrophoresis apparatus using the method. About.

[0002]

2. Description of the Related Art Capillary electrophoresis (CE) is used for analyzing biological components such as peptides, proteins, nucleic acids, and saccharides, as well as for separating components having similar structures such as optical resolution and isotope separation. This method is widely used for clinical medicine, pharmaceuticals, monitoring of environmental substances, and the like.

[0003] As a detector of a capillary electrophoresis apparatus, a detector such as an ultraviolet-visible spectrophotometer has been mainly used so far. However, in recent years, chemiluminescence detection has been considered as a simpler and more sensitive detector. Vessels have been developed. Since a chemiluminescence detector measures luminescence generated by a chemical reaction between a reagent and a sample component, a special light source is not required, the cost is low, and the operation is simple. Also,
Since there are few noise sources, highly sensitive detection can be achieved.

As a specific example of the chemiluminescence detection method, the principle of a peroxalate-based chemiluminescence reaction will be described. In the chemiluminescence of peroxalate, oxalic acid derivatives (for example, bis (2,2,
4,6-Trichlorophenyl: TCPO), bis (oxalate)
4-dinitrophenyl: DNPO)) to form 1,2-dixetanedione or substituted 1,2-dixetanedione, which are active intermediates with high energy. An energy transition occurs from this intermediate to excite a coexisting fluorescent substance, and when this returns to the ground state, it emits light consistent with its fluorescence spectrum. This fluorescent substance (fluorophore) is again excited by the active intermediate to emit light, and by repeating this, strong light is emitted. In this way, strong luminescence can be obtained with one molecule of fluorophore.

[0005] When the substance to be analyzed itself has fluorescence, it is used as it is, and when it does not, it is derivatized to a fluorescent substance, so that quantification with higher sensitivity than fluorescence analysis is possible. is there. As described above, when using a peroxalate-hydrogen peroxide chemiluminescence reaction,
Fluorescent dyes acting as sensitizers can be detected,
Analysis of amino acids, peptides, proteins, and saccharides labeled with various fluorescent dyes is possible. In addition, when a luminol-hydrogen peroxide chemiluminescence reaction is used, various substances that serve as reaction catalysts such as metal ions, metal complexes, and heme proteins can be detected with high sensitivity.

[0006] However, there are several problems in connecting such a chemiluminescence detector with electrophoresis. That is, this luminescent reagent is poorly water-soluble, requires an organic solvent as a solvent, and the luminescent reaction may also proceed under non-aqueous conditions. Further, it is easily hydrolyzed and is very unstable in an aqueous system, so that the reaction activity tends to decrease immediately.
For this reason, a method of previously mixing with an aqueous solution to have conductivity necessary for electrophoresis cannot be adopted from the viewpoint of the solubility and stability of the reagent.

Therefore, as a configuration for combining an electrophoresis apparatus using a capillary tube with a chemiluminescence detector,
Conventionally, the configuration shown in FIGS. 8 and 9 is used. FIG. 8 is an overall configuration diagram of a capillary electrophoresis apparatus,
FIG. 9 is an enlarged view of a portion A in FIG.

The inlet end of the capillary 30 for component separation is immersed in a liquid tank 31 filled with the electrophoretic liquid, and the outlet end of the capillary 30 is immersed in the liquid tank 32 filled with the electrophoretic liquid via the joint 33 of the part A. ing. A predetermined voltage is applied to the electrophoresis liquid in the liquid tank 31 from a voltage source (HV) 34, while the electrophoresis liquid in the liquid tank 32 is at the ground potential. Due to this potential difference, the electrophoresis liquid moves in the capillary 30 from the liquid tank 31 to the liquid tank 32. The liquid sample mixed into the electrophoresis running liquid from the sample introduction part 35 on the inlet side of the capillary 30 is subjected to component separation during electrophoresis in the capillary 30. A buffer (a component is the same as the electrophoresis liquid) supplied by the pump P1 and a luminescent reagent (TCPO + H ₂ O ₂ ) supplied by the pump P2 are introduced into the joint 33.

As shown in detail in FIG.
Has a double-tube structure in which the tip of the small-diameter separation capillary 30a on the left is inserted inside the large-diameter reaction capillary 30b on the right, and through the gap between the separation capillary 30a and the reaction capillary 30b. As described above, the buffer and the luminescent reagent are introduced. As a result, the buffer solution and the luminescent reagent are mixed in the joint 33 before merging with the electrophoresis solution, and become a sheath laminar flow (sheath flow) from the gap between the capillaries 30a and 30b, and the solution flowing out of the separation capillary 30a. Mix with. Then, the fluorescent sample and the luminescent reagent chemically react to generate luminescence. A photomultiplier tube 36 is provided close to the mixing area, and a signal detected by the photomultiplier tube 36 is counted by, for example, a photon counter.

[0010]

As described above, in the conventional capillary electrophoresis apparatus, the interface portion must be a double tube, and a high-precision pump for sending a luminescent reagent or a buffer is required. This makes it difficult to reduce the size of the device. Further, a complicated operation such as inserting the separation capillary 30a into the reaction capillary 30b in the joint 33 was required. Further, the separation capillary 30
If the gap between the outer diameter of “a” and the inner diameter of the reaction capillary 30b is large, the band of the eluted components spreads in the mixed region, and the separation performance is significantly impaired. In addition, since the luminescent reagent must always flow at a stable flow rate, the cost of the reagent also increases.

SUMMARY OF THE INVENTION The present invention has been made to solve these problems, and a main object of the present invention is to provide a method for detecting chemiluminescence, which has a simple structure and can achieve miniaturization and cost reduction of an apparatus. To provide a microchip electrophoresis apparatus using the same.

[0012]

Means for Solving the Problems To solve the above problems, the present inventor has applied a chemiluminescence to a microchip type separation / analysis apparatus such as microchip electrophoresis formed by bonding two transparent flat plates. I came up with the introduction of a detector. In microchip electrophoresis, since a width of a separation channel for electrophoresis is small, a fluorescence detector using a laser capable of extremely narrowing a beam diameter as excitation light has conventionally been used. Therefore, even if the electrophoresis chip itself can be manufactured at low cost, the detector must be expensive. The configuration using a fluorescence detector for microchip electrophoresis is described in JP-A-8-327594 by the present applicant.

According to a first aspect of the present invention, there is provided a pair of flat plates having substantially the same shape, wherein at least one of the flat plates has a surface on which a groove through which liquid flows is formed, and the other flat plate has a groove. A through hole is formed at a position substantially corresponding to the end of the groove, and a separation and analysis microchip formed by forming a separation flow path corresponding to the groove by bonding both flat plates with the groove inside. Separate liquid sample components,
A chemiluminescence detection method for detecting each of the separated components, wherein a luminescent reagent is introduced into a through hole at an outlet end of a separation channel, and the luminescent reagent and a component eluted into the through hole are sequentially reacted,
This is characterized in that the emitted light is detected.

According to a second aspect of the present invention, there is provided a microchip electrophoresis apparatus using the above-described chemiluminescence detection method, wherein a voltage applying means for electrophoresis for giving a predetermined potential difference to both ends of the separation channel, A light-receiving means disposed substantially immediately below the through-hole to detect light generated by a chemical reaction between a component eluted in the through-hole at the outlet end and electrophoresed in the through-hole at the outlet end by the potential difference and the luminescent reagent; And characterized in that:

[0015]

According to the first aspect of the present invention, there is provided a method for detecting chemiluminescence according to the first aspect of the present invention, wherein a through-hole is provided at an exit end of a separation channel of a microchip for separation analysis formed by bonding two flat plates. A luminescent reagent is introduced in advance, and component separation is started immediately thereafter. As a method for component separation, in addition to electrophoresis, electrochromatography or so-called liquid chromatography in which a buffer is passed at a small flow rate by a differential pressure between both ends of a separation channel (or a pressure from an inlet end) may be used. While passing through the separation channel, the sample component is separated, and when it reaches the outlet end with a time lag, a chemical reaction occurs with the luminescent reagent in the order of arrival to emit light. Quantitative analysis of each component can be performed by detecting this emitted light and measuring the emission intensity of each component.

Therefore, according to the chemiluminescence detection method of the present invention, the light source required in the conventional laser fluorescence detector or the like is not required, and the capillary separation unit and the detection unit are integrated. Since a special interface is not required, the entire device can be extremely miniaturized. Thereby, a portable analyzer can be used, and handling becomes very easy. Further, according to this chemical detection method, detection is sequentially performed after the components are completely separated in the separation channel, so that the detection itself does not impair the separation,
High separation performance can be maintained. Further, since there is no need to constantly flow the luminescent reagent, the cost of the luminescent reagent can be reduced.

In the invention of claim 2, electrophoresis is used as a method of separating components. That is, a potential difference is applied to both ends of the separation channel by the voltage applying means, and the sample is caused to migrate in the electrophoresis liquid filled in the separation channel.
As the voltage applying means, electrodes provided so as to be in contact with the electrophoresis liquid in the through holes at both ends of the separation channel,
And a voltage source for applying a voltage to the electrode from the outside. Since the electrode can be a conductive thin film formed on a flat plate surface by, for example, vapor deposition,
The chip itself can be reduced in size.

Further, in such an electrophoresis chip, it is preferable to provide a sample introduction channel having through holes at both ends so as to have a common part with the separation channel. At the time of sample injection, a liquid sample is injected into a through hole at one end of the sample introduction channel, and the sample is replaced by the entire sample introduction channel by electroosmotic flow or differential pressure (pressing). Next, a potential difference is applied to both ends of the separation channel as described above. Then, the sample in the common part of the sample introduction channel and the separation channel is injected into the separation channel, migrates toward the outlet end, and the components are separated between them. By controlling the voltage so that the sample does not diffuse from the sample introduction channel to the separation channel, a fixed amount of sample corresponding to the volume of the common part between the sample introduction channel and the separation channel is injected with good reproducibility. , Accurate quantitative analysis can be performed.

In such a chemiluminescence method, the wavelength detected by the light receiving means is the same as the fluorescence wavelength, or about 515 nm even in the case of a luminol-hydrogen peroxide system. Can be molded. Therefore, it is also useful for disposable chips.

[0020]

DESCRIPTION OF THE PREFERRED EMBODIMENTS A microchip electrophoresis apparatus according to one embodiment of the present invention will be described with reference to the drawings. FIG. 1 is an external perspective view of a microchip electrophoresis apparatus according to this embodiment, and FIG. 3 is a plan external view of an electrophoresis chip according to this embodiment.

First, the configuration of the microchip electrophoresis apparatus according to the present embodiment will be described with reference to FIGS. The electrophoresis chip 10 includes a pair of transparent flat plates 11 and 15 made of a glass plate, a quartz plate, or the like. Lower transparent plate 1
1 has a sample extension groove 12 and a migration groove 13 respectively.
Are formed so as to intersect with each other (this is referred to as an “intersection 14”), and through holes 16 are formed in the upper transparent plate 15 at positions corresponding to the respective ends of the grooves 12 and 13 respectively. I have. The grooves 12 and 13 are formed on the surface of the transparent flat plate 11 by etching, and the width of the groove portion is 10 to 100.
It is formed so as to have a thickness of about μm and a depth of about 5 to 50 μm. In addition, for convenience of handling, the chip of this embodiment has a rectangular shape with a size of about 1 to 3 cm on a side, but the size and shape are not limited thereto.

The electrophoresis chip 10 includes first and second reservoirs R1 and R2 formed by through holes 16 formed by bonding the pair of transparent flat plates 11 and 15 with the grooves 12 and 13 inside. Thus, a sample introduction channel 20 communicating with the outside and a separation channel 21 communicating with the outside by the third and fourth reservoirs R3 and R4 are formed inside. As shown in FIG. 1, each of the reservoirs R1 to R4 is adapted to receive a capillary for introducing a liquid such as an electrophoresis liquid from the outside, and to apply a voltage to the electrophoresis liquid stored therein. (Not shown) are provided. This electrode may be formed of, for example, a conductive thin film formed on the surface of one transparent flat plate by photolithography, and formed continuously to the end face of the transparent flat plate.

In the electrophoresis chip 10, the fourth reservoir R4 is a detection point by a chemiluminescence method. In the microchip electrophoresis apparatus of this embodiment, as shown in FIG. Photomultiplier tube 2 just below
2 are provided.

Next, the configuration of a voltage control circuit for applying a voltage to the electrodes will be described with reference to FIG. The electrodes provided in the first to fourth reservoirs R1 to R4 are connected to one ends of six first to sixth switches S1 to S6 which are turned on and off independently. The other end of the second switch S2 is the first switch
The other ends of the first, third and sixth switches S1, S3, S6 are connected to a voltage source (HV1) 23 and a second voltage source (HV2).
24, and the other ends of the fourth and fifth switches S4 and S5 are grounded. The on / off operations of the switches S1 to S6 and the voltages generated by the voltage sources 23 and 24 are controlled by the control unit 25.

An example of the analysis using the microchip electrophoresis apparatus of the present embodiment is as follows. (1) Preparation for Analysis First, an electrophoresis running solution (0.1 M Tris-borate buffer) is pressure-injected from one or more reservoirs (a plurality of reservoirs may be used), and injected into the sample introduction channel 20 and the entire separation channel 21. Fill the running solution. Then, a predetermined amount of a small amount of a sample (for example, 1 × 10 ⁻⁴ M Dns-leucine, or a mixture of Dns-lysine and Dns-glycine) is supplied to the first reservoir (sample injection point) R1.
And an electrophoresis running solution (0.1 M Tris-borate buffer) is injected into the second and third reservoirs R2 and R3.
Further, 4 mM TD was used as a luminescent reagent in the fourth reservoir R4.
A mixture of PO and 100 mM hydrogen peroxide solution is added.

(2) Introduction of Sample into Sample Introducing Channel 20 The controller 25 closes the switches S1, S2, S3, and S4, and sets the first voltage source 23 to 0.3 kV and the second voltage source 24 to 0. A voltage of 6 kV is generated for 15 seconds. This allows
As shown in FIG. 4A, the first and fourth reservoirs R1,
0.6 kV for R4, 0.3 kV for the third reservoir R3
Is applied, and the second reservoir R2 is grounded. Then, due to the potential difference, the sample injected into the first reservoir R1 enters the sample introduction flow path 20, passes through the intersection 14, and moves toward the second reservoir R2. At this time, since the electrophoresis running also moves in the direction of the second reservoir R2 from the third and fourth reservoirs R3 and R4, it is difficult for the sample to diffuse to the separation channel 21 during such a sample introduction period. Absent.

(3) Separation of Sample Next, the control unit 25 controls the switches S1, S2, S3, S
4, the switches S1, S2, S5 and S6 are immediately closed, and the first voltage source 23 generates a voltage of 0.5 kV and the second voltage source 24 generates a voltage of 0.2 kV. As a result, FIG.
As shown in (b), the first and second reservoirs R1, R2
, 0.2 kV is applied to the third reservoir R3, and 0.5 kV is applied to the third reservoir R3, and the fourth reservoir R4 is grounded. Then, the sample present at the intersection 14 is introduced into the separation channel 21 due to the potential difference, and migrates in the direction of the fourth reservoir R4. In the sample introduction channel 20, the first reservoir R
The sample existing on one side moves back to the first reservoir R1, and the sample existing on the second reservoir R2 side from the intersection 14 moves toward the second reservoir R2. That is, only a predetermined amount of the sample depending on the volume of the intersection 14 is introduced into the separation channel 21.

As described above, when the sample migrates in the separation channel 21, the components contained in the sample are separated and reach the fourth reservoir R4 with a time lag. As shown in FIG. 4C, the sample component that has reached the fourth reservoir R4 comes into contact with the luminescent reagent stored therein, causing a chemical reaction to emit light. This emitted light is received by the photomultiplier tube 22 located immediately below the fourth reservoir R4, and an electric signal corresponding to the emission intensity is extracted from the photomultiplier tube 22.

FIG. 5 is a graph showing an example of a detection result when Dns-leucine is used as a sample, and FIG. 6 is an example of a detection result when a mixed solution of Dns-lysine and Dns-glycine is used as a sample. It is a graph. As can be seen from FIGS. 5 and 6, the intensity signal for the sample component shows a sharp peak, and these components are detected with high sensitivity. As can be seen from FIG. 6, Dns-lysine and D
It is almost completely separated from ns-glycine while having an adjacent peak, and can quantitatively analyze each component accurately.

In such a quantitative analysis, a calibration curve created by using a result of previously measuring a plurality of standard samples having known concentrations is used. FIG. 7 shows that Dns-lysine and D
It is a graph which shows the calibration curve obtained by analysis of the liquid mixture with ns-glycine. When performing quantitative analysis of a component whose content is unknown, the concentration of the component can be determined by illuminating the peak height obtained by the above-described measurement with a calibration curve.

The luminescent reagent is not limited to the above description, and an appropriate reagent is used depending on the sample. For example, when a transition metal ion or heme protein is used as a sample, a mixed solution of luminol and aqueous hydrogen peroxide can be used.

In the above embodiment, the chemiluminescence detector according to the present invention is applied to a microchip electrophoresis apparatus, but the scope of the present invention is not limited to electrophoresis.
That is, the present invention is not limited to the one in which the sample components are separated by a potential difference. It may perform separation analysis.

The above embodiment is merely an example, and it is apparent that changes and modifications can be made within the spirit of the present invention.

[Brief description of the drawings]

FIG. 1 is an external perspective view of a microchip electrophoresis apparatus according to one embodiment of the present invention.

FIG. 2 is a configuration diagram of a voltage control circuit of the microchip electrophoresis apparatus according to the embodiment.

FIG. 3 is a plan external view of the electrophoresis chip in the present embodiment.

FIG. 4 is a schematic state diagram illustrating an analysis operation of the microchip electrophoresis apparatus according to the embodiment.

FIG. 5 is a graph showing an example of a detection result when Dns-leucine is used as a sample in the microchip electrophoresis apparatus according to the present embodiment.

FIG. 6 is a graph showing an example of a detection result when a mixed solution of Dns-lysine and Dns-glycine is used as a sample in the microchip electrophoresis apparatus according to the present embodiment.

FIG. 7 is a graph showing a calibration curve obtained by analyzing a mixture of Dns-lysine and Dns-glycine in the microchip electrophoresis apparatus according to the present embodiment.

FIG. 8 is a configuration diagram of a conventional capillary electrophoresis apparatus using a chemiluminescence detector.

FIG. 9 is an enlarged view of a portion A in FIG. 8;

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 10 ... Electrophoresis chip 11, 15 ... Transparent flat plate 12 ... Sample extension groove 13 ... Migration groove 14 ... Intersection 16 ... Through-hole 20 ... Sample introduction channel 21 ... Separation channel 22 ... Photomultiplier tube 23, 24 ... Voltage Source 25 ... Control unit R1, R2, R3, R4 ... Reservoir S1, S2, S3, S4, S5, S6 ... Switch

 ────────────────────────────────────────────────── ─── Continued on the front page (72) Inventor Riichiro Nakajima 27-20 Terayacho, Takatsuki-shi, Osaka F-term (reference) 2G054 AA06 AB07 BB03 CA21 CE08 EA01 FA06 FA07 FA21 JA06 2G057 AA14 AC01 BA03 BB02

Claims (2)

[Claims] 1. A pair of substantially the same flat plates, a groove through which liquid flows is formed on the surface of at least one of the flat plates, and a through hole is formed at a position substantially corresponding to an end of the groove on the other flat plate, A liquid sample component is separated using a separation / analysis microchip in which the two flat plates are bonded together with the groove inside and a separation channel corresponding to the groove is formed, and the chemistry for detecting each separated component is used. In a luminescence detection method, a luminescent reagent is introduced into a through-hole at the exit end of a separation channel, and the luminescent reagent and a component eluted into the through-hole are sequentially reacted with each other,
A method for detecting chemiluminescence, comprising detecting light emitted thereby. 2. A microchip electrophoresis apparatus using the chemiluminescence detection method according to claim 1, wherein a voltage applying means for electrophoresis for applying a predetermined potential difference to both ends of the separation channel, and A light-receiving means disposed under the through-hole to detect light generated by a chemical reaction between the component eluted in the through-hole at the exit end by electrophoresis in the separation channel and the luminescent reagent; A microchip electrophoresis apparatus, comprising: