WO2003058228A1

WO2003058228A1 - Electrophoresis-use microchip

Info

Publication number: WO2003058228A1
Application number: PCT/JP2002/013410
Authority: WO
Inventors: Minoru Adachi; Isamu Takeuchi; Yoshinobu Baba
Original assignee: Cluster Technology Co., Ltd.; Millennium Gate Technology Co., Ltd.
Priority date: 2001-12-28
Filing date: 2002-12-20
Publication date: 2003-07-17
Also published as: JP2005164242A; AU2002367338A1

Abstract

An electrophoresis-use microchip, which analyzes or determines chemical substances or biological components, is provided with micro-channels each up to 200 μm wide and 100 μm deep, has no sample load-use micro-channel that makes use of the intersection of micro-channels, and is provided at the opposite ends of the micro-channel with electrodes respectively connectable with a power supply. Accordingly, the electrophoresis-use microchip is comparatively compact, has a quantitative feature, and can be analyzed using a small amount of a sample.

Description

Microchip for electrophoresis

Technical field

The present invention relates to a microchip for electrophoresis. For more information, see Microelectrophoresis for electrophoresis for measuring or quantifying trace chemicals or biological components.

The present invention relates to a tip, an electrophoresis apparatus including the microchip, and a method for detecting or quantifying a trace amount of a chemical substance or a biological component using the electrophoresis apparatus.

Background art

In recent years, with the remarkable progress of biotechnology, methods for analyzing and measuring target substances in minute samples have been developed. In particular, in the genetic field, human genome analysis has been almost completed, and diseases, particularly genetic diseases, have been diagnosed by detecting DNA, and the use of DNA chips has been increasing.

At present, microchip-based genomic analysis technologies such as DNA chips, microarrays, and microcapillary electrophoresis chips are rapidly advancing, and devices have already been sold. In addition to chips with only one channel for electrophoresis, microchip-type capillary array electrophoresis in which 96 channels are arrayed has been developed (Shi, Y. et al., Anal. Chera. , 7 1: 5354-5361, 1999; Baba et al., Experimental Medicine, 18: 12 (extra number) 1595-1601, 2000). In this method, the microchannel array is arranged radially, and the center can be scanned while rotating with a laser.

In these conventional microcapillary electrophoresis methods, a method for more accurately loading a sample into a microcapillary has been developed (Japanese Patent Application Laid-Open No. 10-507506). In this method, multiple channels A chip having a structure for adjusting the sample amount is used, which is designed so that only the sample present in the intersection of the channels is fed into the microchannel by forming the intersection and controlling the potential of each channel extending from the intersection. Most microcapillary electrophoresis chips currently on the market or under development have the sample volume control structure described above. For example, this structure for adjusting the sample amount is also employed in microchip-type capillary array electrophoresis in which 96 channels are arrayed. This structure requires wiring and a voltage controller to control the potential for each sample, and a small-sized microchip eventually becomes a large-scale device. Also, a large amount of sample to be supplied to the sample amount adjusting structure is required. Therefore, there is a need for a microchip for electrophoresis that is relatively compact and quantitative, and can be analyzed with a small amount of sample. Disclosure of the invention

SUMMARY OF THE INVENTION The present invention has been made to solve the above problems, and the present inventors have developed a microchip for electrophoresis having no structure for adjusting a sample amount by using a droplet discharge device having a fine structure. Have been found to be available, and have completed the present invention.

The present invention provides a microchip for electrophoresis for analyzing or quantifying a chemical substance or a biological component. The microchip has a width of 200 μππ or less and a depth of 100 μm / zm or less. It has a channel, does not have a microchannel for sample loading using an intersection of microchannels, and has electrodes connectable to a power source at both ends of the microphone opening channel.

In a preferred embodiment, the microchip further includes a sample loading hole having an opening having a size of 200 111 or less.

In a preferred embodiment, the microchannel contains a support for electrophoresis. A gel capable of separating the sample.

In a preferred embodiment, a microstructure capable of separating samples in electrophoresis is provided in the microchannel.

In a preferred embodiment, the surfaces of the microchannel and the sample loading hole are water-repellent.

The present invention also provides an electrophoresis apparatus for analyzing or quantifying a chemical substance or a biological component, comprising any one of the microchips described above, a voltage controller for electrophoresis, and a droplet discharge device having a fine structure. I do.

In a preferred embodiment, the droplet discharging device is a device having an ink jet head capable of discharging a sample of 10 picoliters (pL) or less. The present invention further provides a method for analyzing or quantifying a chemical substance or a biological component by electrophoresis, the method comprising the steps of: providing a microchannel or a sample loading hole in a microchip of any of the above microchips; Discharging a sample using a droplet discharging device; electrophoresis of the discharged sample in the microchannel; and detecting or quantifying a sample separated by the electrophoresis. . BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a conceptual diagram of a top view of a microchip having a sample loading hole according to the present invention, in which (a) is an example in which microchannels are arranged in parallel, (b) is arranged in columns, and (c) is arranged radially. Is shown.

FIG. 2 is a cross-sectional view (a) along the line AA and a cross-sectional view (b) along the line BB of the microchip having the sample loading hole of the present invention shown in FIG. 1 (a). BEST MODE FOR CARRYING OUT THE INVENTION The microchip for electrophoresis of the present invention is a microchip for electrophoresis for analyzing or quantifying a chemical substance or a biological component, and the intersection of the microchannels that a conventional microchip for electrophoresis has. A small amount of sample can be electrophoresed in an accurate amount without the use of a sample volume adjustment structure that utilizes the method. Hereinafter, the electrophoresis microchip of the present invention will be described.

(Substrate of the microchip for electrophoresis of the present invention)

The substrate of the microchip for electrophoresis of the present invention is not particularly limited as long as it is a material usually used for electrophoresis, and examples thereof include glass, fused quartz, crystal quartz, silica, and plastic. It is preferable to use a non-conductive material such as glass and fused silica because a relatively high voltage can be applied. On the microchip substrate, microchannels described in detail below are formed by a fine processing technique. For example, it can be formed by irradiating synchrotron radiation, ion beam, microwave laser, femtosecond laser, and the like. Alternatively, it can be formed by optical lithography and chemical etching.

The substrate may be used as it is, or may be used after subjecting the surface to various treatments according to the purpose. Although there is no particular limitation on the surface treatment method, for example, sputtering, CVD (microwave plasma vapor phase synthesis), ion plating, electroless plating, and the like can be mentioned. In addition, when a sample is ejected by a droplet ejection device having a fine structure, a minute droplet may not be accurately placed at a target position due to an influence of static electricity. Therefore, it is preferable that the surface of the microchip base material has been subjected to an antistatic treatment. Alternatively, a plastic or the like into which an antistatic agent is previously kneaded may be used as the base material. Further, the substrate may be used by being covered with a cover plate. The material of the cover plate can be selected as well as the substrate. (Micro channel)

The microchip for electrophoresis of the present invention is provided with a microchannel for separating and detecting a sample by electrophoresis. The microchannel may be groove-shaped, slit-shaped or tunnel-shaped. The shape of the cross section of the microchannel is not particularly limited, and is appropriately determined depending on the purpose, and may be a concave shape, a U shape, a V shape, or the like. For example, in the cross section taken along the line AA of the microchip shown in FIG. 1 (a), the microchannel can be shaped as shown in FIG. 2 (a). The width of the microchannel is less than 200 / xm and the maximum width is 0.3 μπ! 200200 // m, more preferably 12 μm to 150 μm. The depth is 以下 Ι Ιμπι or less, preferably 50 ππ or less, more preferably 0.3 / zm to 50 μπι, and 12 μπ! More preferably, it is 50. The length can be determined as appropriate depending on the sample to be analyzed, the medium used, and the like. The number and arrangement of the microchannels are not particularly limited, but a plurality of microchannels are preferably provided. For example, they can be arranged arbitrarily, such as parallel, tandem, radial, etc. as shown in (a) to (c) of FIG.

Droplet force discharged from a droplet discharge device having a fine structure into a microchannel The droplet force adheres to the inner wall of the microchannel due to surface tension, and may not reach the electrophoresis channel in the microchannel. In order to prevent this, the surface of the microchannel or the surface of the microchip may be subjected to a water-repellent treatment as necessary. The water-repellent treatment may be performed by any method. For example, a method in which a resin film is formed on a substrate and a water-repellent resin layer is formed on the resin film by a physical vapor deposition method is used. No. The microchip of the present invention treated in this way can precisely arrange particularly a very small amount of sample.

The microchip for electrophoresis of the present invention includes a microphone such as that found in conventional products. There is no microchannel for sample loading using the intersection of the low channels. Therefore, the microchip of the present invention can be provided as a microarray in which a large number of microchannels are formed on a microchip having a fixed area. Further, microchannels for other uses may be provided according to the purpose. Further, the microchannel may be branched in the middle as necessary.

An electrophoresis medium is provided inside the electrophoresis microchannel. Examples of the medium include gels, porous polymers, aqueous solutions, and microstructures. Microstructure refers to a three-dimensional structure (Baba et al., Supra) processed by ultra-fine processing technology, which may be engraved in the microchannel in advance. The medium can be appropriately selected depending on the sample to be separated and analyzed.

Samples that can be separated and analyzed include, but are not limited to, chemical substances and biological components, and specifically include amino acids, peptides, proteins, nucleic acids (DNA, RNA), polysaccharides, and Alkyroy. And charged substances such as dyes and dyes. Even a neutral substance can be used as a sample by selecting a medium that can be converted to an ionic state. In the microchip of the present invention, in particular, DNA and protein can be suitably analyzed. In the case of DNA and protein analysis, the medium is preferably a polyacrylamide gel, agarose gel, or the like.

(Sample loading hole)

The microchip substrate for electrophoresis of the present invention may be provided with a sample loading hole for placing a sample. Sample loading holes are provided on the microchannel to facilitate sample placement in the microchannel.

The shape, size and depth of the sample loading hole to be provided The size (ie, the volume of the concave portion) is not particularly limited, and may be determined according to the sample amount. In general, the sample loading hole can easily dispense 10 pL into the hole, and is shaped and sized so that the discharged sample reaches the flow path of the microchannel electrophoresis. It is. For example, in the microchip shown in Fig. 1, the cross section BB passing through the sample loading hole is as shown in Fig. 2 (b). In other words, it has a slightly larger opening than the microchannel shown in Fig. 2 (a), and has a shape that allows easy placement of the sample. In addition, the sample loading hole is preferably subjected to a water-repellent treatment in the same manner as the above-described microchannel, in that the sample can surely reach the flow channel of the microchannel. The shape of the opening may be, for example, a circle, a square, a diamond, or an ellipse. When a cover plate is used, it is preferable that a through hole having the same shape and size as the opening of the sample loading hole is formed in the cover plate. (Electrode connectable to power supply)

The microchannel is typically provided at both ends with electrodes connectable to a power supply. By using the side on which the sample is loaded as the cathode and the other end as the anode, the sample can be moved as in the usual electrophoresis. The material of the electrode is not particularly limited as long as it is usually used as an electrode. The electrodes may be individually connected to the power supply, but the wires on the cathode side and the anode side may be combined into one, and each microchannel may be connected in parallel to the power supply. In this way, the connection to the power supply is easy, and the wiring of the microchip itself can be simplified and the microchip can be made compact. (Droplet ejection device with fine structure)

Placing a small amount of sample in an accurate amount on the microchip for electrophoresis of the present invention For this purpose, a droplet discharge device having a fine structure is used. In the present invention, the droplet discharge device having a fine structure may be any device that can discharge a sample of 10 pL or less, preferably 4 pL or less with high accuracy. For example, there is a device having an ink jet head as described in Semiconductor FPD World 2001. 6, 154-156. Further, it may be a bubble jet (registered trademark) device, a micropump, a micropitter, or the like. By changing the number of droplets ejected from these devices into the microchannel, the amount of sample can be adjusted. (Sample detection or quantification)

The sample separated by electrophoresis can be detected by a commonly used detection method. Typically, UV absorption, fluorescence, chemiluminescence, change in refractive index, etc. are measured. In the present invention, fluorescence detection is preferred because a small amount of sample is measured. In addition, since the sample can be arranged in an accurate amount, quantification by measuring the intensity of the detected signal is possible. Industrial availability '14

ADVANTAGE OF THE INVENTION The microchip for electrophoresis of this invention can arrange | position a small amount of target sample accurately on a base material using the droplet discharge apparatus which has a microstructure, and enables accurate measurement of a target substance. For example, analysis of a single molecule protein is also possible. In particular, because there is no structure for adjusting the sample amount, there is no wasteful sample placement on the chip compared to the conventional one, and measurement can be performed with a small sample amount, and cost performance is good. Also, there is no need for an electrode for adjusting the sample amount, and measurement can be performed with a simple device. In addition, the simple structure of the microchip allows many microchannels to be placed on the microchip. Thus, a compact and highly accurate microchip for electrophoresis can be easily provided.

Claims

The scope of the claims

1. An electrophoresis microphone orifice chip for analyzing or quantifying chemical substances or biological components, which has a microchannel with a width of 200 m or less and a depth of 100 μm or less. A microchip which does not have a microchannel for sample loading using an intersection and has electrodes connectable to a power source at both ends of the microchannel.

2. The microchip according to claim 1, further comprising a sample loading hole having an opening having a size of 200 / im or less.

3. The microchip according to claim 1, wherein a gel capable of separating a sample in electrophoresis is provided in the microchannel.

4. The microchip according to claim 1, further comprising a microstructure capable of separating a sample in electrophoresis in the microchannel.

5. The microchip according to claim 1, wherein the surfaces of the microchannel and the sample loading hole are subjected to a water-repellent treatment.

6. For analyzing or quantifying a chemical substance or a biological component, comprising the microchip according to any one of claims 1 to 5, a voltage controller for electrophoresis, and a droplet discharge device having a fine structure. Electrophoresis device.

7. The droplet ejection apparatus is capable of ejecting a sample of 10 pL or less. The electrophoresis device according to claim 6, wherein the electrophoresis device has a head.

8. A method for analyzing or quantifying chemical substances or biological components by electrophoresis,

A step of discharging a sample into a microchannel or a sample loading hole of a microchip according to any one of claims 1 to 5, using a droplet discharge device having a fine structure;

Electrophoresing the discharged sample in the microchannel;

Detecting or quantifying the sample separated by the electrophoresis.