Electrophoresis chip

Details Electrophoresis chip Electrophoresis chip

2002-02-28

2004-05-11

Olsen, Kaj K. (Department: 1753)

Chemistry: electrical and wave energy

Apparatus

Electrophoretic or electro-osmotic apparatus

C204S616000, C204S620000

Reexamination Certificate

active

06733648

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to an apparatus for separating and analyzing a biological sample. More particularly, the invention relates to an electrophoresis chip suitably used for separation and analysis of a DNA fragment originated from a genome, polynucleotide fragment originated from RNA, protein, peptide, and the like, and to an electrophoresis apparatus.
2. Description of the Related Art
For analysis and division of a biological material, a separation technology using electrophoresis has been used most widely. For example, in a field of DNA analysis, DNA sequencing has frequently been carried out by using polyacrylamide gel electrophoresis. All genomic sequences of microbes such as
Escherichia coli
or yeast already have been unraveled. In the case of multicellular organisms, all genomes of
Caenorhabditis elegance
and
Drosophila melanogaster
have nearly been unraveled. Analysis of all human genomes will be completed in early 2000s. For an electrophoresis medium of electrophoresis having such high resolution, other than polyacrylamide gel, or high polymer consisting of derivatives of methyl cellulose or acrylamide polymer can be used.
In electrophoresis, generally, polymer is used as an electrophoresis medium, often by being filled a capillary made of silica-base material or plastic (Anal. Chem. (1992) 64,967-972). For DNA sample preparation and PCR product checking, an electrophoresis apparatus using agarose gel as an electrophoresis medium has frequently been used. Recently, a technology has been developed for forming a capillary structure by using glass or plastic for substrate, forming a very small groove in this substrate, and adhering another substrate as a surface cover to the substrate, and an electrophoresis chip using this technology has reached a stage of being put to practical use (Anal. Chem, (1992) 64,1926-1932, Anal. Chem. (1995) 67,3676-3680). In all of those methods, a structure is employed, in which an electrophoresis medium is formed in a plotted region substantially having a capillary form that is formed inside a capillary or a substrate (glass).
SUMMARY OF THE INVENTION
Conventionally, in electrophoresis for performing electrophoretic separation of a DNA fragment by high resolution as in the case of base sequence determination, an electrophoresis medium has formed in an area plotted in a groove form in the capillary or the substrate (glass). Generally, it is because when a gel is formed, a method of pouring gel precursor or polymer into a mold of a capillary or the like is an easy preparation method. In the conventional gel preparation method, a gel precursor or polymer must be poured for each capillary having an inner diameter of about 50 &mgr;m, and generally the following processes are necessary: (1) pouring of a gel precursor or polymer by connecting a pump to each capillary, and (2) disconnecting of a joined portion between the pump and the capillary through a valve. Particularly, there remains a problem in filling of a self-organization gel such as agarose. That is, since agarose has a property of being dissolved at a high temperature of 70 to 90° C., and gelled at a low temperature of 40 to 60° C., a problem of the agarose being formed into a gel in the pump occurs, and its processing is difficult. Also in the case of the polyacrylamide gel, since gelling occurs in the pump, there is a problem that maintaining a gel precursor flow passage including the pump, the valve and the like becomes extremely difficult. Consequently, the polyacrylamide gel has not been put to practical use yet. As another problem, there has been a serious one caused by inevitable presence of a pipe wall, while a gel shape can be easily reproduced according to accuracy of an inner diameter of the capillary as the mold. Specifically, since a sectional area is narrow when the gel precursor is gelled, and a surface area of a wall surface is wide with respect to a gel volume, gel shrinkage occurs following gelling to cause application of hysteresis. Thus, depending on an electrophoretic state, problems including a reduction in separation and, as a worst case, cutting-off of the gel have occurred.
In the case of the capillary system, although a sample capacity used for real electrophoretic separation is in a range of several tens to several hundreds of nL (nano-liter), because a technology of pouring a sample into the capillary is still poor, a sample capacity of several tens &mgr;L (micro-liter) is necessary at present. In other words, samples of several tens to several hundreds times as large as a sample capacity necessary for electrophoresis are now wasted. Thus establishment of a technology of handling a very small quantity of samples is an important technical task.
On the other hand, in the case of conventional agarose electrophoresis of a horizontal flat plate type, a gel may only be formed by pouring dissolved agarose into a mold having an open structure on an upper surface. Accordingly, gel preparation is easy. In the conventional agarose electrophoresis, gel is used by being dipped in buffer solution for electrophoresis. Since at least an upper surface of the gel is not in contact with a solid interface such as glass or plastic, an effect of the interface can be reduced. For sample injection, a method of adding a sample of increased specific gravity to a position having a concave surface formed during gel preparation is employed. Normally, a sample of several &mgr;L is used.
In the conventional agarose electrophoresis, a sample volume is in order of micro-liter, which is not in line with a tendency of technology that intends to perform analysis with high sensitivity by using a very small quantity of samples in the future. Actually, electrophoresis having separation performance similar to that of the agarose electrophoresis and using electrophoresis chips considered to be capable of being supplied in great quantity at low costs may become a mainstream. However, as in the case of the capillary system, a method of filling a groove (capillary) formed in a substrate with an electrophoresis medium (gel) and a technology for pouring a very small quantity of samples into the electrophoresis medium have not been established yet. Sample injection thus has taken a lot of effort, and there has been a problem of inevitably supplying a great quantity of samples more than necessary.
Objects of the present invention are to provide an electrophoresis chip capable of easily performing injection of a very small quantity of samples, an electrophoresis apparatus using the same, and a method of manufacturing the electrophoresis chip.
According to the invention, an electrophoresis chip is provided, which is constructed in a manner that a thin and long hydrophilic region, and a hydrophobic region are formed on a surface of an electrical insulating substrate, the hydrophobic region surrounding the hydrophilic region, a gel (electrophoresis medium) is formed in the hydrophilic region, and thus a shape of the gel can be formed with good reproducibility. As it is hydrophilic, gel precursor solution is adhered only to the hydrophilic region on the surface of the substrate. In the hydrophobic region, as it is repelled, the gel precursor solution can be easily removed. A quantity of the gel precursor solution left in the hydrophilic region is decided by hydrophobicity of the hydrophobic region, hydrophilicity of the hydrophilic region, and hydrophilicity of the gel precursor solution on the surface of the substrate. Thus, a fixed quantity of electrophoresis gel (electrophoresis medium) is formed in the hydrophilic region.
The produced electrophoresis chip can be used in a submarine form as in the case of agarose electrophoresis. The electrophoresis chip is placed in a humidifying box, and electrophoresis can be carried out in a manner that one surface of the gel is in contact with air or inert gas. Thus, compared with the conventional method of using a capillary, it is possible to suppress a reduction in resolution, which affects presence

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