Device for electrophoresis

Details Device for electrophoresis Device for electrophoresis

2001-03-13

2004-06-29

Noguerola, Alex (Department: 1753)

Chemistry: electrical and wave energy

Apparatus

Electrophoretic or electro-osmotic apparatus

C204S601000, C422S070000

Reexamination Certificate

active

06755952

ABSTRACT:

TECHNICAL FIELD
The present invention relates to a method and a device for electrophoresis by and with which a fluorescence- or chemiluminescence-labeled biological sample such as DNA or a protein is separated and detected.
BACKGROUND ART
Electrophoresis is a method for separating a biological sample such as DNA or a protein according to its molecular weight. Among such electrophoresis, capillary electrophoresis is known as a method having an enhanced sample resolving power.
FIGS. 12A and 12B
are illustrations of an exemplary electrophoresis device using a gel-injected capillary. First, as shown in
FIG. 12A
, one end of the capillary
121
injected with a gel
122
is immersed in a sample solution
123
containing DNAs to be separated while the other end is immersed in a buffer bath
124
. A voltage is applied from a power source
125
to the sample solution
123
and the buffer bath
124
such that the potential of the sample solution
123
is negative while the potential of the buffer bath
124
is positive. Since DNAs
127
are negatively-charged, they migrate toward the high potential end and enter into the gel
122
of the capillary
121
. Next, as shown in
FIG. 12B
, the end of the capillary
121
immersed in the sample solution is replaced in a buffer bath
126
with a negative potential such that the buffer bath
124
with the other end of the capillary
121
immersed has a positive potential. A voltage is continuously applied from the power source
125
to the buffer bathes
126
and
124
to direct the DNAs
127
to migrate through the gel
122
toward the high potential end as represented by an arrow. Since DNA
127
a
with a smaller molecular weight migrates faster than DNA
127
b
with a larger molecular weight, DNAs may be separated according to their molecular weights. The separated DNAs
127
a
and
127
b
may be detected by irradiating fluorescent labels pre-bound to the DNAs with excitation light
132
from a light source
131
, and detecting the fluorescence emitted from the excited fluorescent label with a photosensor
133
. In this manner, molecular weights of the DNAs
127
are determined based on the time required from the initiation of the electrophoresis for each of the DNAs to arrive at a detection point (the position of radiating the excitation light).
As described above, capillary electrophoresis determines a molecular weight of a sample component based on the time required from the initiation of the electrophoresis for the component to arrive at a detection point fixed on an electrophoresis pathway. Therefore, even when the components are sufficiently separated before they reach the detection point, electrophoresis has to be continued until all of the components arrive at the detection point. Components with smaller molecular weights have larger mobility and thus arrive at the detection point at an earlier stage. On the other hand, as the molecular weight becomes larger, the mobility becomes smaller in inverse proportion to the logarithm of the molecular weight. As a result, the time required for the component to reach the detection point becomes longer, and thus requiring long time for separation and detection.
The capillary used for capillary electrophoresis is a narrow fused silica tube with inner and outer diameters of as small as 25-100 &mgr;m and 100-200 &mgr;m, respectively, which can very easily be broken. Accordingly, the outer surface of the tube is usually coated with polyimide for reinforcement. Since this polyimide coating, however, prevents sufficient excitation light to enter inside the capillary and prevents sufficient fluorescence to come out, the polyimide coating at the detection point is peeled off. Since it is difficult to read the entire capillary, the sample has to be separated and detected after a predetermined electrophoresis time to determine the molecular weight.
Furthermore, although the capillary is coated with polyimide, it cannot be bent for more than a predetermined curvature. Accordingly, some capillaries used, depending on their lengths, may be difficult to be handled with and may require a large space for setting, rendering miniaturization of the device difficult. In the case of SSCP (Single Strand Conformation Polymorphism) method which requires electrophoresis to be carried out while maintaining an electrophoresis pathway at a predetermined temperature, the temperature of the capillary stretching over a large area needs to be controlled. This requires a very expensive controlling mechanism, rendering this method unpractical.
The present invention has an objective of providing an electrophoresis method and an electrophoresis device which have the same resolving power as when a capillary is used and which allow detection in a shorter time. The present invention also has an objective of providing a compact electrophoresis device whose electrophoresis pathway can be controlled of its temperature with high accuracy.
In order to accomplish the above-described objectives, the electrophoresis device of the invention typically comprises: an electrophoresis board provided with a two-dimensional tubular electrophoresis pathway formed by laminating a plane plate on a plate provided with a narrow groove; an excitation light source capable of radiating excitation light to the entire surface of the electrophoresis board; a two-dimensional sensor for receiving fluorescence or luminescence; and a means for controlling the temperature of the entire surface of the electrophoresis board.
By using the above-described means, the following can be realized. The separated state of a sample can be confirmed during the electrophoresis by reading the entire electrophoresis pathway. Accordingly, there is no need of conducting electrophoresis for a long time than is necessary. The electrophoresis may be ended when the sample has sufficiently been separated to determine the molecular weight by measuring the migration distance. As a result, separation and detection may be conducted within a minimum time. For example, when an electrophoresis pathway with a width of 100 &mgr;m is formed in an electrophoresis board, the length of the pathway may be about 2 m within an area of 2 cm
2
. Accordingly, a compact device can be realized with an inexpensive temperature controlling system. As a result, temperature of the entire electrophoresis pathway can accurately be controlled with a low-cost mechanism.
An electrophoresis method of the invention comprises the steps of: subjecting a fluorescence-labeled sample to electrophoresis through a two-dimensional electrophoresis pathway; and radiating excitation light to the entire electrophoresis pathway to simultaneously detect luminescence emitted from the fluorescent label of the migrating sample with a two-dimensional sensor.
Another electrophoresis method of the invention comprises the steps of: subjecting a mixture, which contains a fluorescence-labeled sample and a substance as an excitation energy donor for the fluorescent substance, to electrophoresis through a two-dimensional electrophoresis pathway; and simultaneously detecting luminescence emitted from the fluorescent label of the migrating sample with a two-dimensional sensor.
According to these electrophoresis methods, there is no need of performing electrophoresis until all of the sample components to be detected reach the detection point as in conventional methods. Molecular weights of the sample components may be determined based on the migration distances when the sample components are sufficiently separated.
An electrophoresis device of the present invention comprises: an electrophoresis board provided with a narrow tubular electrophoresis pathway; an excitation light source for radiating excitation light to the entire electrophoresis board; and a two-dimensional sensor for receiving luminescence emitted from the sample irradiated by the excitation light source and migrating through the electrophoresis pathway. The sample is simultaneously detected from the entire electrophoresis pathway by the two-dimensional sensor. This electrophoresis

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