For quantitative determination of a micro-amount of biological material such as DNA, chemical amplification such as a PCR (Polymerase Chain Reaction) method is generally utilized. In the utilization of the chemical amplification method, the amount of the biological material is estimated by quantitative determination of products after the amplification. However, since amplification efficiency always fluctuates, estimation in the quantitative determination for the biological material is inacurate. In order to solve the problem, it has been desired for direct quantitative determination of the micro-amount of the biological material without using the chemical amplification. One of the methods of attaining the same is a method referred to as single molecule counting.
The single molecule counting means a method of bonding a fluorescence-label with a biological material of an object for the quantitative determination and counting fluorescence-labeled molecules by laser excitation (for example, refer to “Analytical Chemistry”, vol. 74, No. 19 (2002), p. 5033). “Analytical Chemistry”, vol. 74, No. 19 (2002), p. 5033 describes that DNA molecules by the number of about 1,000 in 0.3 μL of a sample solution can be detected. FIG. 1 shows a constitutional view of a detection system for practicing the existent method. A laser light for exciting fluorescence-labeled molecules is emitted from a laser light source 1, passed through a shutter 2 for adjusting an exposure time, focused through a lens 3 and then inputted to a capillary 12. A sample solution containing a biological material intended for quantitative determination (hereinafter referred to as a target) is filled in the capillary. The target molecule is fluorescence-labeled before introduction into the capillary 12. Accordingly, in a case where the target molecule is contained in the sample solution, fluorescence is emitted by a light incident to the capillary. In this case, the target molecule is dispersed in the sample solution, and the target molecule in an irradiated region with the laser (volume: 5×10−11L) in the capillary appears as a minute luminous body. For detecting such a minute luminous body, images of the laser irradiation region is focused by an objective lens 6 on a CCD (Charge Coupled Device) 8 in a camera 7 to obtain fluorescent images. In the acquired image data, the luminous body is distinguished from the background in the laser irradiation region and the number thereof is counted to conduct quantitative determination of concentration of the target molecules.
Then, FIG. 2 shows an enlarged view for a region in the capillary to be irradiated with a laser. A laser light 13 the spot of which is shaped into an elliptic shape by two cylindrical lenses is irradiated to a capillary 12 filled with the sample solution and having a square cross section. The fluorescence-labeled target molecule 14 is electrophoretically moved by 90 μm/sec in the direction 15.
As can be seen from FIG. 1 and FIG. 2, an electrophoretic direction 15 and an incident direction 5 of a laser light are perpendicular to each other. This is a constitution necessary for measuring the concentration of the electrophoretically moved target molecule on every moving distance. Such a constitution is also described in JP-A Nos. 21556/1988, 134101/1995, 105834/1996, 43197/1997, and 210910/1997. Further, in the existent examples described above, gel is inserted between the two sheets of glass substrates and molecules in the gel are separated on every molecular size due to the electrophoresis of the molecules in the gel. Target molecules on every molecular size are excited by a laser beam passing between the two sheets of glass substrates to quantitatively determine the concentration on every molecular size by measurement of fluorescent intensity.
Further, in the method disclosed in the “Analytical Chemistry”, vol. 74, No. 19 (2002), p. 5033 and in JP-A Nos. 21556/1988, 134101/1995, 105834/1996, 43197/1997, and 210910/1997, the measuring direction by fluorescence and the incident direction of the laser light are set perpendicular to each other. Further, JP-A No. 229859/1995, JP-W Nos. 513555/1998, and 513556/1998 disclose a constitution in which the direction of the laser excitation and the measuring direction of fluorescence are perpendicular to each other although not using the electrophoresis. Particularly, in JP-A No. 229859/1997, a fluorescence material or a sample containing a fluorescence material intended for fluorescence measurement is filled in the sample container and the laser light is incident from one end of the sample container, passed in the inside and then emitted at the other end. The sample container has a shape elongated along the propagating direction of the laser light and formed of a transparent material such as glass. It is described that the accumulated value of the fluorescence intensity is detected in the direction substantially perpendicular to the pulse excited light based on the signal in synchronization with the output timing of the pulse excited light by using this constitution, to calculate the fluorescence life. Further, JP-A Nos. 513555/1988 and 513556/1998 also describe that a gel is filled between two sheets of glass plates and a laser light is incident from the end of the glass plate to the inside of the gel. The concentration of fluorescence present in the gel is measured with this constitution.
Further, JP-A No. 177097/2003 (Patent Document 10) discloses a constitution in which a laser light is entered in a chip for optical measurement and the direction of the fluorescence measurement and the direction of the incident laser light are made different.
On the other hand, JP-A No. 528714/2002 discloses that a sample solution containing a target is held on a plane and excited by a laser light from the rear face or the upper surface of the sample solution to detect fluorescence from the fluorescence-labeled target molecules. Particularly, it is described that the sample can be scanned (moved) to a detector for quantitative determination of target concentration in many sample solutions by fluorescence measurement in this system.