Chemistry: molecular biology and microbiology – Apparatus – Including measuring or testing
Reexamination Certificate
2000-02-04
2001-11-20
Nguyen, Bao-Thuy L. (Department: 1641)
Chemistry: molecular biology and microbiology
Apparatus
Including measuring or testing
C204S450000, C204S451000, C204S556000, C204S600000, C204S601000, C204S603000, C204S194000, C204S400000, C422S068100, C422S070000, C422S082050, C422S051000, C422S051000, C422S067000, C422S062000, C422S063000, C422S082080, C422S082090, C422S098000, C422S105000, C422S110000, C435S287300, C435S288200, C435S288500, C435S288700, C436S514000, C436S805000, C436S806000, C436S807000, C436S809000, C436S824000
Reexamination Certificate
active
06319705
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates to a device for electrophoresis capable of analyzing extremely small quantities of samples at a very high speed and with a high resolution. More particularly, the invention relates to a device for electrophoresis referred to as a “microchip” having a separation flow route formed inside transparent planar members and throughholes provided to one of the surfaces at positions corresponding to both ends of the separation flow route and reaching to this flow route.
Devices for electrophoresis have been in use for analyzing a very small quantity of protein or nucleic acid, and those using a capillary have been representative examples. Devices of this type have a glass capillary with internal diameter less than 100 &mgr;m and, after it is filled with a buffer and a sample is introduced at one of its ends, a high potential difference is applied between its ends and the target substance of analysis is dispersed inside the capillary. The application of a high potential is possible because the interior of the capillary has a relatively large surface area compared to its small volume and hence has a high cooling efficiency, and hence even a very small quantity of a sample such as DNA can be analyzed speedily and at a high resolution.
Since capillaries are easily breakable, having outer diameters as smaller as several 10-100 &mgr;m, it is not an easy job for the user to exchange them. In view of this problem capillary devices for electrophoresis comprising two base plates joined together (referred to as the microchip) have been proposed as a form of device for electrophoresis which can take the place of the capillaries of the conventional kind, being capable of carrying out an analysis speedily and allowing the device to be easily miniaturized, as shown, for example, by D. J. Harrison et al (Anal, Chem. Acta 283 (1993) 361-366).
FIGS. 1A
,
1
B and
1
C show an example of such a microchip
1
, characterized as comprising a pair of transparent base plates
1
a
and
1
b
(for example, of glass, quartz or a resin material), mutually intersecting capillary grooves
3
and
5
being formed on a surface of one of these base plates (
1
b
) and the other base plate (
1
a
) being provided with reservoirs
7
in the form of a throughhole at positions corresponding to the end points of these grooves
3
and
5
.
When the microchip
1
thus structured is used for carrying out an analysis, the two base plates
1
a
and
1
b
are stacked one on top of the other as shown in
FIG. 1C
, and a migration liquid is injected into the grooves
3
and
5
from one of the reservoirs
7
. After a sample is injected into one of the reservoirs
7
at one of the ends of the shorter groove
3
serving as the “sample introducing flow route”, a high potential difference is applied between the reservoirs at both ends of this groove
3
such that the sample is dispersed throughout the groove
3
.
Thereafter, a migration potential difference is applied between the reservoirs
7
at both ends of the longer groove
5
serving as the “separation flow route” such that the portion of the sample at the intersecting area
9
of the two grooves
3
and
5
begins to migrate inside the longer groove
5
. If an optical detector is disposed at a suitable position on the longer groove
5
, the separated portions of the sample transported through electrophoresis can be sequentially detected thereby.
A problem with such a microchip is that the detection sensitivity is relatively low because the optical path length inside the separation flow route is short. In view of this problem, there has been proposed a new kind of microchip device for electrophoresis, as shown in
FIG. 2
, adapted to stop the application of the migration potential when target components in the sample to be detected have been separated or while they are being separated, to expose the entire separation flow route to a light beam, to use a linear image sensor or the like to repeatedly measure the light absorption of fluorescence and to carry out a multi-point averaging process on the measured values at different positions along the separation flow route such that the detection sensitivity can be improved.
Explained more in detail with reference to
FIG. 2
, a first mirror
13
, a slit
15
and a second mirror
17
are disposed along the optical path of the light from a light source
11
for obtaining a parallel beam of light from the source
11
. The parallel beam of light thus prepared is passed through a dispersion grating
19
. There is provided a third mirror
21
serving to lead only a selected portion of the light dispersed by the grating
19
having a specified wavelength to the microchip
1
. Disposed on the opposite side of the microchip
1
away from the light-incident side is a photodiode array (PDA)
23
with a plurality of linearly aligned photodiodes for measuring the light from the separation flow route. A high-voltage electric power source
25
is connected to the microchip
1
. The PDA
23
is provided with a data processor
29
adapted to receive signals from the individual photodiodes of the PDA
23
through an analog-to-digital (A/D) converter
27
and to carry out multi-point averaging of signals for each photodiode.
When a sample is to be analyzed with such a device, the sample is injected at one end of the sample introducing flow route
3
of the microchip
1
and potential differences are applied from the power source
25
between the ends of the flow routes
3
and
5
as explained above to firstly bring the sample to the intersection area
9
of the flow routes
3
and
5
and then to complete the separation of target components to be analyzed inside the separation flow route
5
. Thereafter, the application of the potential difference is stopped and a beam of monochromatic light is made incident within a specified region along the separation flow route
5
. At each of specified positions along the separation flow route
5
, the distribution of mutual interaction between the separated components and the monochromatic light such as absorption or fluorescence is repeatedly measured by the PDA
23
, and the signals from the PDA
23
are analyzed by a multi-point averaging routine by the date processor
29
for each of the photodiode to achieve a detection with high sensitivity.
The microchip device for electrophoresis shown in
FIG. 2
may be characterized as irradiating a specified area along the separation flow route
5
with a monochromatic beam of light with a wavelength selected by the grating
19
. If a spectrum over a certain range of wavelengths is desired, however, it is necessary to vary a parameter (such as the angle of orientation) of the grating
19
and to thereby sequentially measure the reaction between the monochromatic light of different wavelengths and separated components. If it is desired to maintain the same level of detection sensitivity over the given range of wavelengths, measurement must be repeated at different wavelengths but the time required for the measurements becomes inconveniently long. Not only is it impossible to conclude the analysis quickly, but if the time for the measurements is prolonged under a separated condition, the level of separation is also adversely affected due to the natural diffusion of the separated components.
SUMMARY OF THE INVENTION
It is therefore an object of this invention to provide a microchip device for electrophoresis with which a light spectrum from different positions along the separation flow route can be obtained quickly and with a high level of sensitivity.
A microchip device for electrophoresis embodying this invention, with which the above and other objects can be accomplished, may be characterized as comprising not only a microchip having a separation flow route formed between transparent planar members and throughholes which are formed through one of these transparent planar members at positions corresponding to end points of the separation flow route so as to reach the separation flow route and an electrical power source f
Coudert Brothers
Nguyen Bao-Thuy L.
Shimadzu Corporation
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