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).
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 xcexcm, 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 90xc2x0 C., and gelled at a low temperature of 40 to 60xc2x0 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 xcexcL (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 xcexcL 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 of a charged interface such as an electroosmotic flow.
A problem of injecting a very small quantity of sample solution can be solved by contriving pattern shapes of the hydrophobic region and the hydrophilic region formed on the surface of the substrate. The hydrophobic region and the hydrophilic region are provided on the surface of the substrate, the hydrophilic region with a thin and long shape being divided in a longitudinal direction. That is, first and second hydrophilic regions, which are thin and long in the longitudinal direction, are formed, and one tail end of the first hydrophilic regions is placed adjacently to that of the second hydrophilic region with a gap. The hydrophobic region is formed to surround the first and second hydrophilic regions. Gels (electrophoresis media) are formed in the first and second hydrophilic regions to construct first and second electrophoresis lanes.
Sample solution containing a sample is supplied to a region of the gap, a solution lane is formed by the sample solution between the gels (electrophoresis media) of the first and second hydrophilic regions, and thus the first and second electrophoresis lanes are connected with each other. The supplying of the sample solution to the region of the gap can be carried out by directly dropping the solution to a gap portion with a micro-dispenser. In another method of supplying sample solution, a thin pin (needle) hydrophilic only in a small part of its tip, and hydrophobic in other parts is dipped in the sample solution, lifted to form a droplet in the tip of the pin, and this droplet is brought into contact with the hydrophobic region on the substrate. Then, the droplet is held in a gap between the pin and the hydrophobic region formed on the substrate. By moving the pin, the droplet can be moved in an arbitrary direction. When the pin is passed through the gap portion provided in the gel (electrophoresis medium), the droplet is moved to the gels (electrophoresis media) of the first and second hydrophilic regions, the droplet is substantially held in the gap portion, and thus the supplying of the sample solution is completed.
Immediately after the formation of the solution lane by the droplet, electrophoresis can be carried out. By increasing/reducing a size of the gap, i.e., a width of the gap (widths of the first and second hydrophilic regions) and a length of a gap (gap length), and by holding the sample solution in the form of the droplet in the tip of the pin to supply the droplet to the gap, a sample of sub 1L can be injected.
Another electrophoresis chip of the invention is constructed in a manner that a plurality of thin and long hydrophilic regions are formed at predetermined intervals on surfaces of two electrical insulating substrates, and a hydrophobic region is formed on a surface other than the plurality of hydrophilic regions. The two substrates are fixed in parallel with a gap of several tens of xcexcm to about 1 mm so as to place the hydrophilic regions oppositely to each other. Then, gel precursor solution may be poured into the gap to form a gel (electrophoresis medium) between the opposing hydrophilic regions of the two substrates. In this case, an effect of an interface is increased, but drying and shrinkage of the gel (electrophoresis medium) during electrophoresis can be effectively prevented.
In the foregoing, the hydrophobic region and the hydrophilic region were formed on the electrical insulating substrate. However, by reforming the surface of the electrical insulating hydrophobic substrate, it is possible to form a hydrophilic region of a desired shape on the hydrophobic substrate. Conversely, by reforming the surface of the electrical insulating hydrophilic substrate, it is possible to form a hydrophobic region so as to leave a hydrophilic region of a desired shape.
Embodiments of the invention are summarized as follows. An electrophoresis chip is provided, which includes an electrical insulating substrate and a electrophoresis medium formed to be linear on a surface of the substrate. In this case, a region adjacent to the electrophoresis medium on the surface of the substrate is hydrophobic.
(2) An electrophoresis chip is provided, which includes: an electrical insulating substrate having a linear hydrophilic region and a hydrophobic region adjacent to the hydrophilic region on a surface of the substrate; an electrophoresis medium formed on the hydrophilic region of the substrate by providing a gap of a predetermined length in one place in a longitudinal direction; and a pair of electrodes connected to both ends of the electrophoresis medium in the longitudinal direction.
(3) In the electrophoresis chip of (1) or (2), the substrate is glass.
(4) In the electrophoresis chip of (1) or (2), the electrophoresis medium is a gel.
(5) In the electrophoresis chip of (2), a sample is held in the gap.
(6) In the electrophoresis chip of (2), the gap is provided in a position close to one end from a center of the electrophoresis medium in the longitudinal direction.
(7) In the electrophoresis chip of (6), a length of a longer element medium of two element media of the electrophoresis medium divided into two parts is set in a range of 10 mm to 100 mm.
In the electrophoresis chip of the invention, to perform real electrophoresis separation, a necessary length of the electrophoresis medium is 10 mm at a minimum. In this case, it is possible to detect a difference between base lengths, one being twice as long as the other. In addition, to perform separation with accuracy of 10% of a length of a DNA, a length of 100 mm is enough.
(8) In the electrophoresis chip of (1) or (2), a width of the electrophoresis medium is set in a range of 0.1 mm to 5 mm.
To form a gel to be linear by a method of the invention, a width of 0.1 mm or more, preferably 0.2 mm or more, is necessary. If a width of an electrophoresis medium exceeds 5 mm, there is not much difference from a case where a planar gel is formed, losing an advantage of the method of the invention.
(9) In the electrophoresis chip of (1), a length of the gap in the longitudinal direction is set in a range of 0.2 mm to 1 mm.
To form a gap, a ratio between a width of the gel and the length of the gap is important. With a minimum width of 0.1 mm of the gel, a gap may be set in a range of about 0.1 mm to 0.2 mm. However, as the width is larger, an upper limit of the gap length becomes about 1 mm. If the gap length exceeds 1 mm, it is difficult to hold aqueous solution.
(10) An electrophoresis chip is provided, which includes an electrical insulating substrate having a plurality of linear hydrophilic regions formed almost in parallel on a surface and a hydrophobic region adjacent to the hydrophilic regions; a plurality of electrophoresis media, each formed on one of the plurality of hydrophilic regions of the substrate by providing a gap of a predetermined length in one place in a longitudinal direction; and a pair of electrodes, one being connected to one ends of the plurality of electrophoresis media and the other being connected to the other ends thereof.
(11) An electrophoresis chip is provided, which includes: an electrical insulating substrate having a plurality of linear hydrophilic regions formed almost in parallel on a surface of the substrate and a hydrophobic region adjacent to the hydrophilic regions; a plurality of electrophoresis media, each formed on one of the hydrophilic regions of the substrate by providing a gap of a predetermined length in one place in a longitudinal direction; and plural pairs of electrodes individually connected to both ends of the plurality of electrophoresis media.
(12) An electrophoresis chip is provided, which includes: an electrical insulating substrate having a thin and long hydrophilic region formed on a surface of the substrate and a hydrophobic region formed surrounding the hydrophilic region; and an electrophoresis medium formed on the hydrophilic region of the substrate by providing a gap of a predetermined length in one place in a longitudinal direction. In this case, an electrophoresis lane is formed by the electrophoresis medium and sample solution supplied to the gap.