Chemistry: electrical and wave energy – Apparatus – Electrophoretic or electro-osmotic apparatus
Reexamination Certificate
2001-03-23
2004-10-26
Nguyen, Nam (Department: 1753)
Chemistry: electrical and wave energy
Apparatus
Electrophoretic or electro-osmotic apparatus
C204S601000, C356S344000
Reexamination Certificate
active
06808610
ABSTRACT:
BACKGROUND OF THE INVENTION
Field of the Invention
The present invention relates to an electrophoresis apparatus in which specimens such as DNA and protein are separated and analyzed through electrophoresis by making use of a capillary array constituted by collecting a plurality of capillaries.
Electrophoresis which makes use of capillaries has been used for the purpose of determining, for example, base sequencing and base length of DNA. When a specimen containing DNA which is the object of measurement is injected into gel such as polyacrylamide within a glass capillary and a voltage is applied between both ends of the capillary, DNA compounds in the specimen move in the capillary and are separated depending on such as their molecular weights to form DNA bands within the capillary. Since a fluorescent coloring matter is added for the respective DNA bands, when laser beam is irradiated thereto, light is emitted therefrom, thus through reading of the emitted light by means of a fluorescent measurement means the sequencing of DNA is determined. The separation and analysis of protein are performed in a like manner to examine the structure of the protein.
One such laser beam irradiation method is as follows. In a capillary array constituted by a plurality of capillaries, a cover coating such as polyamide on the surface of the capillaries is removed to form a detection portion, laser beam is irradiated to a capillary located at one side or capillaries located at both sides in the detection portion and the laser beam irradiated in such a manner passes across the plurality of capillaries.
In the above explained conventional laser beam irradiation method, when the laser beam is irradiated from one side of the capillary array, reflection beam from the surface of the capillary array returns to a laser oscillator which causes a problem of instabilizing the laser oscillation. Further, when irradiating laser beam from both sides of the capillary array, other than the reflected beam from the surface of the capillary array, the beam transmitted through the capillary array returns to the laser oscillator which causes a problem of instabilizing the laser oscillation.
According to the present invention, at least the following three methods which resolve the problems of the returning beam and the reflected beam from the capillary array are used. In strict sense no plane faces exist in the capillary array, however, when a plurality of capillaries are aligned in parallel, the center axes of the respective capillaries are substantially aligned on a certain plane face which is called hereinbelow as “a capillary array plane face” or “an array plane face”
(1) An irradiation optical axis making incident in parallel direction with respect to the capillary array plane face is inclined in non-perpendicular direction with respect to the longitudinal direction of the capillaries. Thus, the reflected beam from the capillaries is not overlapped on the laser beam axis, thereby, no noises are introduced.
(2) An irradiation optical axis making incident in parallel direction with respect to the capillary array plane face is inclined with respect to the array plane face. In this instance, since the laser beam is irradiated from both sides of the array, when looking at one of the beam irradiation spots, the beam transmitted through the array and the incident beam align side by side.
(3) Incident angles of two laser beam optical axes irradiated from both sides of the capillary plane face which cross each other in parallel direction with respect to the capillary array plane face are differentiated from each other.
In the present invention, at least one of the above arrangements (1) through (3) is employed. In particular, through combination of above two or three arrangements the disturbance caused by the returning beam of the irradiated beam is desirably eliminated.
Accordingly, one embodiment of the present invention which resolves the above problems is to provide a capillary array electrophoresis apparatus in which laser beam is irradiated to either one or both end capillaries at both sides of a capillary array and the laser beam passes through the plurality of capillaries is characterized, in that between a laser beam source and a laser beam condensing means which is disposed on an optical axis between the capillaries and the laser beam source at the remotest position from the capillaries, an overlapping of reflected laser beam by a capillary face to which the laser beam makes incident with the incident laser beam is prevented.
The above condition is fulfilled when the optical axis of the incident laser beam is not perpendicular to the center axis of the capillaries. In some of conventional electrophoresis apparatus having a single capillary, the optical axis of the incident beam is not exactly perpendicular to the center axis of the capillary. However, the purpose of such arrangement in the electrophoresis apparatus having the single capillary is to possibly prevent direct incidence of reflected beam to a detection system by inclining the optical axis. The purpose of inclining the laser beam optical axis according to the present invention is fundamentally different from that of the above referred to conventional art. In the above conventional art, it is necessary to incline the laser beam optical axis greatly to the extent that the direct reflection beam sufficiently offsets from a condenser lens for fluorescent light. For example, when F value of a detection system is 1.4, it is necessary to give an incident angle of more than 20°. On the other hand, according to the present invention, it is enough that when the focal distance of a condenser lens is 50 mm, the inclination of the laser beam optical axis of about 1°~2° is sufficient.
Further, another embodiment of the present invention is to provide a capillary array electrophoresis apparatus in which laser beam is irradiated to both end capillaries at both sides of a capillary array and the two laser beams respectively pass through the plurality of capillaries is characterized, in that the plane face formed by the capillary array is not in parallel with the incident laser beams. When branching the laser beam and arranging the same so as to oppose coaxially each other, a problem of returning laser beam fundamentally arises. However, in the present capillary array, the beam passing the capillaries has an optical axis around a straight line defined by a crossing line of two plane faces one formed by the capillary array and the other formed by an optical axis of the beam advancing in the capillaries and the inclined incident laser beam. Accordingly, with this measure, in the capillary array of the present invention such a condition is produced that the opposing laser beams are coaxial within the capillaries, but are not coaxial in the space out of the capillaries.
Further, still another embodiment of the present invention is to provide a capillary array electrophoresis apparatus in which laser beam is irradiated to both end capillaries at both sides of a capillary array and the two laser beams respectively pass through the plurality of capillaries is characterized, in that the orthogonal projections of the two incident laser beams with respect to the plane face formed by the capillary array are not in parallel.
When the orthogonal projections of the two incident laser beams with respect to the plane face formed by the capillary array are not parallel as referred to above, the following problem may arise. Since the laser beam diameter when adding the two laser beams becomes large in comparison with when the two laser beams are coaxial, therefore, it is feared that spatial resolution in a fluorescent detection is reduced. Namely, in an electrophoresis, DNA compounds in the specimen move in the capillary and are separated depending on, for example, their molecular weights to form DNA bands within the capillary. In such an instance, it is possible that the resolution detection capability of these DNA bands is reduced. In order to avoid those possibilities, it is preferab
Inaba Ryoji
Kodama Yoshitaka
Ozawa Miho
Takahashi Satoshi
Dickstein , Shapiro, Morin & Oshinsky, LLP
Hitachi , Ltd.
Mutschler Brian L.
Nguyen Nam
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