Chemistry: electrical and wave energy – Apparatus – Electrophoretic or electro-osmotic apparatus
Reexamination Certificate
2001-03-05
2004-10-19
Noguerola, Alex (Department: 1743)
Chemistry: electrical and wave energy
Apparatus
Electrophoretic or electro-osmotic apparatus
C422S070000, C204S601000, C204S605000
Reexamination Certificate
active
06805784
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to capillary arrays used in capillary array electrophoresis equipment that separates and analyzes samples such as DNA, proteins, etc.
2. Description of Prior Art
The technology is well known of configuring an array by combining a plurality of capillaries, supplying to each capillary and moving electrophoresis medium and the sample to be separated or analyzed, and using the target sample for carrying out separation or analysis. The samples such as DNA, protein, etc., that are labeled by fluorescent materials are supplied to the capillaries. Such technology has been disclosed in U.S. Pat. Nos. 5,366,608, 5,529,679, 5,516,409, 5,730,850, 5,790,727, 5,582,705, 5,439,578, and 5,274,240, etc. From the point of view of the throughput of separation and analysis, the method using multiple capillaries has many advantages compared with the electrophoresis method using a flat plate gel.
A capillary array electrophoresis equipment has been disclosed in JP A 9-96623. The capillaries have an external diameter of 0.1 to 0.7 mm, an internal diameter of 0.02 to 0.5 mm, with the outer covering being a Polyimide resin coating. Each capillary itself is quartz pipe, with a number of capillaries in the array (generally, a few to several tens of capillaries are common.).
This equipment is provided with a loading head that takes the DNA into the capillary by electrophoresis from the sample tray containing DNA sample labeled by a fluorescent material, an excitation optical system that irradiates excitation light beam from electromagnetic waves, for example, the laser light source via the mirror, beam splitter, and focusing lens to the detection sections that are arranged and fixed in the order of the sampling number of the loading header, and a detection lens system and CCD camera that detect the fluorescence light which is the signal light. In this example, laser light is irradiated from both side surfaces of the capillary array containing the electrophoretic DNA, focusing the laser light by the lens effect of the capillary, thereby irradiating all the capillaries with laser light, and detecting the fluorescent light from each capillary using the detection optical system.
SUMMARY OF THE INVENTION
An object of the present invention is related to the structure of the capillary head section, and to eliminate air bubble generated when a gel is introduced, and also to ensure that there is no adherence of air bubbles to the liquid surface.
The present invention relates to capillary arrays used in capillary array electrophoresis equipment that separates and analyzes samples such as DNA, proteins, etc., and in particular to the prevention of air bubble generation at the capillary head section of the capillary array and to the improvement of the sealing construction.
In more detailed terms, the present invention divides the capillary head into three parts of the pipe-shaped capillary holder
32
, the sealing peg
33
, and the tightening knob
34
. Because of this, even when the tightening knob is rotated, the sealing surface that maintains the hermeticity does not rotate but only the sealing pressure is applied. The capillaries are bundled together, passed through the capillary holder, and adhered together, the capillaries and the capillary holder are cut simultaneously so that they are in the same plane. Further, the roughness of the surfaces of the capillaries and the capillary holder in the same plane is made so that the surfaces each have an approximately mirror-finished surface.
According to other preferred embodiments, in the shared plane of the capillaries and the capillary holder, the internal diameter area of the capillary holder is made so that the capillary filling rate becomes 60 to 73% indicating the ratio of the area of the capillaries and the adhesive surface to the internal diameter area of the capillary holder. Because of the above, there is no generation of craters appearing in the cut surface because voids are introduced in the adhesive material during the adhesion of the capillary head. In addition, since there are no undulations in the liquid contacting surface, it has been possible to eliminate the adhesion of air bubbles to that surface.
In the present invention, the hardness of the capillary holder, of the tightening knob, and of the buffer liquid container is larger than the hardness of the sealing peg. The area of the surface of contact between the capillary holder and the sealing peg is made larger than the area of the surface of contact between the sealing peg and the tightening knob. Further, electrophoresis is carried out by applying a voltage of about 10 to 20 kV from the high voltage power supply between the electrode attached to the capillary front tip of the loading header and the electrode provided in the buffer liquid container that injects the buffer liquid, which is the electrophoretic medium in the capillary, from the capillary head into the detection section. The capillaries, the loading header, the electrodes, the detection section, and the capillary head part prepared in an integrated manner are called the capillary array on the whole. The attaching and detaching of the capillary array are done as a single unit. Further, gel has been introduced inside the capillary that becomes the resistance to electrophoresis, the speed of electrophoresis changes depending on the size of the electrophoretic DNA or protein molecule.
The detection time t (called the electrophoresis time) of this detection system is given by the following equation:
t
=
KL
ET
Where,
k: Proportionality constant
E: Electrophoresis voltage
L: Length of the capillary
T: Ambient temperature.
The detection time of the equipment at present is generally very long being in the range of 0.5 to 3 hours.
From the temporal variation pattern of the detection light intensity obtained from the above equation, the analysis of the DNA or the protein is done by the signal processing and computation equipment. However, the resolution of the signal pattern varies due to different factors, and in particular, the effect of the temperature changes of the equipment is large. One such example is explained below in detail. When injecting a gel in the capillary array, the high viscosity gel put in the buffer liquid container is applied a high pressure of 5 to 10 MPa using a pump, etc., thereby injecting the gel into the capillary. The completion of gel injection can be confirmed when the gel comes out from the electrode section. The injecting pressure is large because the viscosity of the gel is high and the internal diameter of the capillary is small. After injecting the gel, the buffer liquid for causing electrophoresis inside the gel is made to permeate from the buffer liquid container and from the electrode side of the loading header. In this type of analyzing equipment, the following problems will be generated if air bubbles are included in the part filled with the gel.
(1) If an air bubble with a diameter larger than the internal diameter of the capillary is injected into the capillary, the conduction of electricity inside the capillary will be cut off making electrophoresis impossible and hence the analysis also becomes impossible.
(2) While the capillary head is mounted in the buffer liquid container or affixed to the buffer liquid container at the high-pressure resisting and hermetic sealing part, if an air bubble is present in the vicinity of the mounting part 30, the volume of the air bubble changes due to the system temperature changes during analysis, and the buffer liquid comes out and goes back in via the capillary at the open end which is the electrode end. The amount of buffer liquid coming out and going back in will be different for each capillary because there will be differences in the pressure loss. Since this increases or decreases the time taken for electrophoresis, it will have direct effect on the analysis. The number 26 refers to the adhesive material that bonds the bundle of capillary tubes to the capillary head.
Because
Kasai Syozo
Kita Toshiaki
Morioka Tomonari
Tsukada Kiyoshi
Hitachi , Ltd.
Noguerola Alex
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