JP3539014B2 - Capillary array electrophoresis device - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to DNA, RNA,
Or separation and analysis equipment for proteins and the like, particularly DNA, RN
The present invention relates to a capillary array electrophoresis apparatus that is effective for determining the nucleotide sequence of A or measuring the polymorphism of restriction enzyme fragments based on the diversity of individual nucleotide sequences.

[0002]

2. Description of the Related Art Analysis techniques for DNA, RNA and the like are becoming increasingly important in the fields of medicine and biology, including gene analysis and genetic diagnosis. In particular, recently, in connection with the genome analysis plan, the development of a high-speed, high-throughput DNA analyzer has been advanced. In DNA analysis, a fluorescently labeled sample is subjected to molecular weight separation by gel electrophoresis, the sample is irradiated with a laser to detect fluorescence emitted from the fluorescent label, and a series of obtained signals is analyzed. In gel electrophoresis, a flat gel obtained by polymerizing acrylamide between two glass plates with a spacing of about 0.3 mm is widely used (slab gel electrophoresis). The sample injected into the upper end of the slab gel is electrophoresed toward the lower end while being separated in molecular weight by an electric field applied to both ends of the slab gel. The position of the flat gel at a certain distance from the starting point of the electrophoresis is irradiated at once with the laser on all the electrophoresis paths on the side surfaces of the flat gel to excite the separated components of the fluorescently labeled sample passing through the laser irradiation part. The detection of fluorescence by laser irradiation is performed continuously and periodically at fixed time intervals. The DNA base sequence is determined by analyzing the detection results.

Recently, capillary gels obtained by polymerizing gels in fused quartz capillaries have been used instead of flat gels. Capillary gel electrophoresis has attracted attention as a method capable of applying a large electric field as compared with the above-described slab gel electrophoresis and capable of high speed and high separation (Analytical Chemistry 6).
2,900 (1990)). Normally, a capillary gel electrophoresis apparatus uses a single capillary tube, irradiates the inside of the capillary near the lower end with a laser, and performs on-column measurement for detecting fluorescence emitted from a fluorescently labeled sample. Since the entire outer surface of the capillary is coated with polyimide, the coating at the position where fluorescence is detected is removed to leave a window with exposed glass (US Pat. No. 5,31,535 (May 17,199).
4)). The separated component of the fluorescently labeled sample that migrates inside the capillary by electrophoresis is excited by the laser irradiated to the position where the glass is exposed, and emits fluorescence. The DNA base sequence is determined by measuring and analyzing the fluorescence continuously and periodically at fixed time intervals.

However, in the above-mentioned on-column measuring device,
Due to the large refraction of the laser beam on the surface of the capillary, only one capillary can be used at a time, and there is a problem that the throughput is not increased. Recently, several examples of a high-throughput capillary array gel electrophoresis apparatus in which a plurality of capillaries are arrayed to simultaneously analyze many samples at high speed have been reported.

In the first example, a capillary array scanning method (Nature, 359, 167 (1992))
In this method, a plurality of capillaries are sequentially irradiated with a laser one by one to measure on-column fluorescence. This device employs a confocal structure in which the position where the laser beam is most converged in the capillary and the position of the light source that enters the fluorescence measuring instrument (fluorescence receiving optical system) match, and each capillary is measured independently. it can. The laser irradiation and fluorescence receiving optical systems are fixed, and the stage holding the capillary array is moved in a one-dimensional direction to irradiate each capillary sequentially with the laser.

A second example is a capillary array sheath flow method (Nature, 361, 565-566 (1)).
993) and JP-A-6-138037). In this device, the lower end of the capillary array is immersed in a buffer solution, and the sample components whose molecular weights have been separated by gel electrophoresis are eluted as they are into the buffer solution, and fluorescence emitted from the sample components is detected in a space where no capillary exists. In contrast to the on-column measurement in which the capillary is irradiated with a laser and the fluorescence emitted from the fluorescence-labeled sample component is detected, in the present specification, the fluorescence is labeled in the space where no capillary is present. The method of detecting the fluorescence emitted from the sample component is called off-column measurement for simplicity).

[0007] Also, by slowly flowing the buffer in the direction of migration of the sample components, the separated components eluted from different capillary gels are mixed with each other in the buffer, or
It prevents mixing of the two components separated in the capillary gel of the book in the buffer solution. In the vicinity of the capillary array exit, the space where the buffer does not exist without the capillary is irradiated with laser to avoid the problem of laser beam scattering on the capillary surface, and the components eluted from the multiple capillaries are substantially eliminated. And the fluorescence can be detected substantially simultaneously.

In a third example, a laser beam from a single laser light source is divided by a beam splitter or the like in order to simultaneously measure a plurality of capillaries on-column without mechanically scanning the plurality of capillaries. Simultaneously irradiating each of a plurality of capillaries (Ana
lytical Chemistry 65,956
(1993)).

In the fourth example, a laser beam is spread in a capillary array direction by a cylindrical lens and is simultaneously irradiated on a plurality of capillaries (Analytical).
Chemistry 66, 1424 (199
4)).

[0010]

In the capillary array scanning method of the first example, the fluorescence measurement is performed one capillary at a time, so that the time allocated to the detection of fluorescence per one capillary is determined by one capillary. It is reduced compared to the usual on-column measurement used. When n capillary arrays are used, the time allocated to fluorescence detection per line is at most 1 / n compared to ordinary on-column measurement, and the glass part of the capillary through which the separated components of the sample do not actually pass is also used. Because it is scanned, 1
/ N or less. As a result, there is a problem that the fluorescence detection sensitivity is reduced. That is, in the first example, in order to obtain the fluorescence detection sensitivity equivalent to the normal on-column measurement using one capillary, it is necessary to take n times or more the time of detecting the fluorescence in the normal on-column measurement. Will do.

The time interval between adjacent peaks in the electrophoresis pattern of the components separated in one capillary decreases as the analysis speed increases, but the time interval between adjacent peaks becomes n times smaller than the time interval between adjacent peaks. If the time required for one scan of the capillary array becomes too long to be ignored, there arises a problem that the resolution of the electrophoresis pattern of the separated components is reduced. Further, the device of the first example has a stage movable portion for mechanically performing scanning, and has a structure in which failures occur frequently.

The capillary array sheath flow method of the second example has a problem that the fluorescence intensity obtained from the components separated from the molecular weight decreases as the molecular weight increases as compared with the case of on-column fluorescence measurement. is there. This problem arises for the following reasons. In order to prevent separation components eluted into the buffer from the lower end of the capillary gel from mixing in the buffer by diffusion, etc., it is necessary to constantly flow the buffer at a constant speed or higher in the direction of sample component migration. is there. On the other hand, the migration speed of a sample component that migrates while being separated by molecular weight in a capillary gel decreases as the molecular weight increases. The lower the migration speed of the sample component in the capillary gel relative to the flow rate of the buffer, the longer the sample component is elongated in the migration direction when the sample component is eluted from the capillary gel into the buffer solution. . As a result, the fluorescence intensity decreases, which causes a decrease in the measurement sensitivity of the fluorescence emitted from the sample component that has been subjected to the fluorescence table.

In the above third and fourth examples, the intensity of the laser beam irradiated per one capillary is reduced, so that there is a problem that the sample components separated by electrophoresis cannot be detected with high sensitivity.

An object of the present invention is to solve the above-mentioned problems of the prior art by performing on-column fluorescence measurement without mechanically scanning a plurality of capillaries or optically scanning a laser beam. To provide a capillary array electrophoresis apparatus that can simultaneously and simultaneously collectively measure the fluorescence emitted from a fluorescently labeled sample component that irradiates a capillary with laser at the same time and migrates through a plurality of capillaries. .

[0015]

In the capillary array electrophoresis apparatus of the present invention, a plurality of capillaries are arranged on the same plane, and a plurality of capillaries are simultaneously excited by irradiating a laser beam in a direction parallel to this plane. It has a feature in a multi-focus capillary array electrophoresis apparatus for performing on-column measurement by performing on-column measurement. More specifically, the capillary array electrophoresis apparatus of the present invention has the following features.

(1) In an electrophoresis apparatus for performing fluorescence measurement by irradiating a plurality of capillaries with a laser, on-column fluorescence measurement is performed by irradiating a laser at a position where the inside of the capillary is filled with an electrophoresis separation medium, and at least the laser irradiation position of the capillary is measured. (The cross section of the capillary of the transparent part to be irradiated with the laser is circular and the outside of the transparent part of the capillary is a transparent gas) is made of a transparent material, at least the laser irradiation position of the capillary is arranged in a plane, and the laser is Irradiation is performed from the side of the array to be arranged in parallel to the array plane, and a plurality of capillaries are simultaneously irradiated. The ratio of the outer diameter to the inner diameter of the capillary at the laser irradiation position is 1 to 7, or 3 to 5. The spacing between the capillaries in the transparent portion is set to four times or less the outer diameter of the capillaries. The outside of the transparent part of the capillary can replace the transparent gas with the transparent liquid. In this case, the ratio of the outer diameter to the inner diameter of the capillary of the transparent part is set to 2 or less, and the interval between the capillaries of the transparent part is outside the capillary. 4 times or less of the diameter.

(2) In an electrophoresis apparatus for performing fluorescence measurement by irradiating a plurality of capillaries with a laser, on-column fluorescence measurement is performed by irradiating a laser at a position where the inside of the capillary is filled with an electrophoresis separation medium, and at least the laser irradiation position of the capillary is measured. (The cross section of the capillary of the transparent part to be irradiated with the laser is elliptical) is made of a transparent material, and at least the laser irradiation position of the capillary is arranged in a plane, and the laser is arranged on the arrangement plane from the side of the array in which the capillary is arranged. Irradiate in parallel to irradiate multiple capillaries simultaneously. Arrange the multiple capillaries in a plane with the minor axis of the ellipse parallel to the capillary array plane and the major axis perpendicular to the capillary array plane, or arrange the minor axis of the ellipse parallel to the capillary array plane and the minor axis to the capillary array A plurality of capillaries are arranged in a plane perpendicular to the plane. The outside of the capillary of the transparent portion is made a transparent gas or a transparent liquid.

(3) In an electrophoresis apparatus for performing fluorescence measurement by irradiating a plurality of capillaries with a laser, on-column fluorescence measurement is performed by irradiating a laser at a position where the inside of the capillary is filled with an electrophoretic separation medium, and at least the laser irradiation position of the capillary is measured. (The cross section of the capillary of the transparent portion to be irradiated with laser is a square or a rectangle or a shape formed by at least two sets of substantially parallel lines.) A transparent material is used, and at least the laser irradiation position of the capillary is arranged in a plane. The laser is irradiated parallel to the array plane from the side of the array in which the capillaries are arrayed (arranged so that one side of the cross-sectional shape is parallel to the capillary array plane), and the plurality of capillaries are simultaneously irradiated. The outside of the transparent portion of the capillary is any of a transparent gas, a transparent liquid, and a transparent solid. When a transparent solid is used, the outside is preferably made of glass, and particularly preferably made of fused quartz.

Further, in the electrophoresis apparatus of (1) to (3), an acrylamide gel or a derivative thereof, or a polymer is filled in the capillary as an electrophoresis medium. In the above electrophoresis apparatus, at least two types of capillaries having different cross-sectional shapes may be connected between the sample injection end and the position where fluorescence is detected. Also,
In the above-mentioned electrophoresis apparatus, the surface of at least the measurement portion of the capillary for detecting fluorescence is coated with antireflection single-layer deposition coating.

In the apparatus described above, the laser irradiation
The laser transmitted by irradiating all the capillaries may be turned back by the total reflection mirror, and all the capillaries may be irradiated again in the reverse path. Alternatively, the laser beam may be split into two using a beam splitter, and the capillary arrangement may be performed. Irradiation may be carried out so as to be opposed from two sides of the body.
The two laser beams from the two laser light sources may be used to irradiate them in opposite directions from the two sides of the array in the capillary array, and the transmittance of the excitation laser wavelength of the fluorescence measurement filter is 10 ⁻⁵ or less. And

[0021]

FIG. 1 shows a plurality of capillaries 1;
(FIG. 1 shows six examples) on the same plane,
By irradiating two laser beams from a direction parallel to this plane and substantially perpendicular to the migration direction of the sample components, the fluorescence-labeled sample components migrating in the plurality of capillaries are excited substantially simultaneously, and the sample components are excited. 2 shows a main part of a multi-focus capillary array electrophoresis apparatus for detecting by measuring on-column. FIG. 1A is a view of a plurality of capillaries as viewed from an array plane direction, and FIG. 1B is a sectional view thereof. The plurality of capillaries irradiated with the laser 2 may be either a separation capillary for separating sample components or a measurement capillary connected to each of the plurality of separation capillaries and detecting the electrophoretically separated sample components. It is. The laser 2 irradiates a portion at a predetermined distance from the starting point of the sample in the capillary filled with the gel (separation capillary) or a predetermined portion of the measurement capillary filled with the gel.

In the multi-focus capillary array electrophoresis apparatus of the present invention, the sample subjected to electrophoretic separation analysis or analysis contains a plurality of fluorescently labeled components. or,
When the component contained in the sample itself emits fluorescence by laser irradiation, the fluorescent label is unnecessary. In each of the drawings referred to below including FIG. 1, a power source for applying an electric field for causing the sample components to migrate, each means such as an electrode, an electrode tank for storing an electrolyte, and the inside of the capillary 1 are shown. An arithmetic processing unit that processes these detection signals after detecting the fluorescence from the sample components to be electrophoresed, a display that displays the results of the arithmetic processing, a control device that controls each part of the multi-focus capillary array electrophoresis device, a laser light source, and the like Is omitted. In the following description, a laser 2 narrowly narrowed in a direction connecting the central axes of the capillaries of a plurality of measurement capillaries or a plurality of separation capillaries arranged on a plane is incident, and this laser 2 is refracted. The axis on which the laser beam 2 travels when it is assumed that the laser beam has traveled ideally without scattering is called the capillary array axis.

(Embodiment 1) In this embodiment, a cylindrical capillary having a circular cross section is used. The features of the multi-focus capillary array electrophoresis apparatus of the present invention include:
(1) making the laser power reach a large number of capillaries; and (2) reducing background light due to scattering of the laser beam.

First, conditions for realizing (1) are as follows:
This was clarified by a simulation experiment described below. FIG. 2 is a cross-sectional view of one cylindrical capillary having an outer radius R and an inner radius r. Laser beam 2 of infinitesimal width incident on a position separated by a distance x from capillary array axis 4
2 shows how the optical path changes due to refraction by the capillary 1. The laser beam has one boundary point where the refractive index changes.
Make 4 passes (or 2 passes) per book capillary.
That is, the incident position P ₁ from the outside of the capillary 1 to the capillary 1, the incident position P ₂ from the capillary 1 to the inside of the capillary 1, the emission position P ₃ from the inside of the capillary 1 to the capillary 1, the position of the capillary 1 from the capillary 1 at four points of the exit position P ₄ to the outside, the laser beam 2 is subjected to refraction. However, since the laser beam 2 does not pass through the inside of the capillary depending on the optical path, P ₂ and P ₃ may not exist. The incident angles and refraction angles of the laser beam at P ₁ , P ₂ , P ₃ , and P ₄ are θ ₁ , θ ₂ , θ ₃ ,
_{θ 4, θ 5, θ 6} , θ 7, a theta _8. The refractive index of the medium outside the capillary is n ₁ , the refractive index of the material of the capillary is n ₂ , and the refractive index of the medium inside the capillary is n ₃ .

Using the geometric relationship shown in FIG. 2 and Snell's law at each interface, (Equation 1) to (Equation 8) are obtained.

[0026]

[Equation 1] θ ₁ = sin ⁻¹ {x / R} (Equation 1)

[0027]

[Equation 2] θ ₂ = sin ⁻¹ {(n ₁ / n ₂ ) sin θ ₁ } (Equation 2)

[0028]

[Equation 3] θ ₃ = sin ⁻¹ {(R / r) sin θ ₂ } (Equation 3)

[0029]

[Equation 4] θ ₄ = sin ^-1 {(n ₂ / n ₃ ) sin θ ₃ } (Equation 4)

[0030]

Equation ₅ θ ₅ = θ ₄ (Equation 5)

[0031]

Equation ₆ θ ₆ = θ ₃ (Equation 6)

[0032]

[Equation 7] θ ₇ = θ ₂ (Equation 7)

[0033]

Θ ₈ = θ ₁ (Equation 8) Assuming that the angle between the laser beam before entering the capillary and the laser beam after transmission is the refraction angle Δθ, Δθ is given by (Equation 9) from FIG.

[0034]

Δθ = (θ ₁ −θ ₂ ) − (θ ₃ −θ ₄ ) + (θ ₅ −θ ₆ ) + (θ ₇ −θ ₈ ) = 2 (−θ ₁ + θ ₂ −θ ₃ + θ ₄₎ ^{) = 2 {-sin -1 (x} / R) + sin -1 (xn 1 / (Rn 2)) -sin -1 (xn 1 / (rn 2)) + sin -1 (xn 1 / (rn 3)) Θ (Equation 9) Δθ varies depending on the incident position x of the laser beam, and the outer radius R and inner radius r of the capillary, the refractive index n ₁ outside the capillary, the refractive index n ₂ of the capillary material, and the refractive index n ₃ inside the capillary. Can be controlled with. Δθ = 0 for a laser beam of x = 0, and | Δθ | monotonically increases as x increases (here, x
≤ r). In other words, the capillary itself has the function of a lens whose focus cannot be determined. If Δθ> 0, it becomes a concave lens, and if Δθ <0, it becomes a convex lens. n ₁ = n ₂
When n = ₃ , of course, Δθ = 0 regardless of x.

When an array in which a plurality of capillaries are arranged in one plane is irradiated with a laser beam from the side of the array so as to pass through the plurality of capillaries, the laser power reaches a number of capillaries. Is more advantageous as the angle of refraction Δθ due to the capillary is smaller, and in particular, as each capillary has a convex lens action of Δθ <0. This is because, when focusing on one capillary, if the transmitted laser beam is narrower than the incident laser beam, the laser power can efficiently reach the adjacent capillary. Under normal electrophoresis conditions, n ₂
> Since n is _3, [Delta] [theta] smaller the n ₁ from (9) becomes small. That is, the outside of the capillary is n ₁ = 1.0
A preferable condition is that the gas is filled with a transparent gas such as air which is zero.

On the other hand, the laser beam is also reflected at each boundary point. That is, part of the laser power of the incident light becomes reflected light, and the rest becomes refracted light (transmitted light). The ratio of the laser power of the reflected light to the laser power of the incident light is represented by a reflectance R.
_ef , where the ratio of the laser power of the refracted light to the laser power of the incident light is the transmittance T _ra , these are functions of the incident angle θ _i and the refraction angle θ _t (that is, the refractive index of both media), and
0) and (Equation 11).

[0037]

2R _ef = tan ² (θ _i −θ _t ) / tan ² (θ _i + θ _t ) + sin ² (θ _i −θ _t ) / sin ² (θ _i + θ _t ) (Equation 10)

[0038]

2T _ra = sin (2θ _i ) sin (2θ _t ) {1 / cos ² (θ _i −θ _t ) +1} / sin ² (θ _i + θ _t ) ( _Equation 11) where R _ef + T _ra = 1. However, the case where the incident light has no polarization is shown here. Each time the laser power passes through the boundary point, it gradually decreases according to (Equation 10) and (Equation 11). Therefore, the optical path length of the laser beam inside the capillary through which the sample component passes during electrophoresis (point P _{2 in} FIG. ₂₎
By multiplying the laser power at the position in length) which connects the door point P _3, the excitation efficiency in this capillary laser beam (excitation efficiency of labeling by the laser beam of the labeled sample components migrate to the capillary) Was estimated. Further, the position where the laser beam having an infinitesimal width is incident is defined as a distance x from the capillary array axis (x = 0), and by changing this distance x, the excitation efficiency of the laser beam having a finite width in this capillary was estimated. . Furthermore, when a plurality of capillaries were arranged in a plane, the above calculation was continuously performed to examine how the excitation efficiency of each of the plurality of capillaries changes.

As a specific example, the capillary has an outer diameter of 0.375 mm (R = 0.1875 mm) and an inner diameter of 0.
A fused quartz cylindrical tube (n ₂ = 1.46) having a length of 1 mm (r = 0.05 mm) and a length of 50 cm was used. 7M as denaturing agent
4% T (Total monomer) containing urea
concentration), 5% C (Cross
linking material concentr
), and gelled (n ₃ = 1.36). At a position 30 cm from the end of the capillary on the sample injection side, the polyimide coating of each capillary was removed over the entire circumference at a length of 10 mm, and a window for measuring fluorescence was prepared in advance to serve as a laser irradiation site.

As shown in FIG. 3, the measurement portions of these ten capillary gels were arranged at a pitch of 0.375 mm, and the capillaries 1 were closely attached to each other and fixed on a glass plate 5 to be arranged in a plane. The outside of the capillary at the fluorescence measurement position was air (n ₁ = 1.00). The fluorescence measurement is performed from the direction perpendicular to the capillary array plane through the first lens 6, the band-pass filter 7, and the second lens 8 to the two-dimensional detector 9.
Performed by Under the present experimental conditions, since the reflection of the laser beam 2 by the capillary was large, it was necessary to make the transmittance of the laser wavelength lower than that of a band-pass filter used for fluorescence measurement such as slab gel electrophoresis. Therefore, a band-pass filter having a transmittance of 10 ⁻⁵ or less for the laser wavelength was used.

FIG. 4 is a cross-sectional view of the fluorescence measurement portion with respect to the capillary axis. A laser beam was focused to a beam diameter of 0.1 mm, and was made incident along the capillary array axis from the side of the capillary array. At this time, from equation (9), Δθ <0 was always satisfied for the laser beam of x ≦ r, and each capillary had a convex lens function. The beam width of 0.1 mm was considered by dividing it into 11 infinitesimally small beam components at 0.01 mm intervals. That is, the beam component is the capillary array axis (the center axis of the laser beam, x =
0) is 0.00, ± 0.01, ± 0.0
2. The light enters the first capillary at positions of ± 0.03, ± 0.04, and ± 0.05 mm. FIG. 5 shows a result of a simulation of what optical path the capillary transmitted light of each beam component travels in the capillary array. FIG. 5 is a cross-sectional view of the fluorescence measurement portion with respect to the capillary axis. The upper part shows five capillaries on the laser light source side, and the lower part shows five capillaries on the laser emission side. That is, the laser beam enters the upper left capillary and exits from the lower right capillary.

The calculation of all the optical paths was performed continuously assuming that ten capillaries were arranged as described above.
Since the beam component (x = 0.00) located at the center axis of the laser beam has no incident angle at all the boundary points, it is not refracted at all, Δθ = 0, and Progress linearly. Beam components other than x = 0.00 are basically refracted in the direction toward the capillary array axis due to the convex lens action of the capillary, and the optical path travels through the capillary array while vibrating up and down about the capillary array axis. did. The amplitude and period of the vibration differed depending on the beam component, but the optical path of each beam component was vertically symmetric with respect to the capillary array axis. As a result, it was found that all the beam components transmitted through the inside of the capillary and contributed to the excitation of the sample components migrating inside the capillary without departing from the outside of the capillary for all of the ten capillaries. That is, in the capillary electrophoresis apparatus of the present invention, since the laser beam irradiated on each capillary gel is almost converged, and each of the plurality of capillary gels forms an optical path of the laser beam which is approximately converged, so that the It can be said that it is appropriate to be called a focus capillary electrophoresis apparatus.

When the number of capillaries used was changed, the length of the optical path (optical path length) passing through the inside of each capillary was determined for each capillary in order to evaluate the degree of reduction in the excitation efficiency in each capillary. This optical path length was multiplied by the laser power at that position, integrated for all beam components, and the integrated value was used as the excitation efficiency in each capillary. FIG. 6 shows the excitation efficiency of each capillary determined in this manner (the excitation efficiency of each capillary is standardized by the excitation efficiency of the first capillary on the laser light source side). In FIG. 6, the capillaries are numbered from 1 to 10 in order from the laser light source side.

Since the laser power of all beam components is attenuated by reflection each time the laser beam passes through the boundary point where the refractive index changes, the excitation efficiency of each capillary increases as the distance from the laser light source increases as shown in FIG. Decreased. The actually measured fluorescence intensity from each capillary is proportional to this excitation efficiency. As shown in FIG. 6, the excitation efficiency of the tenth capillary reached 54.1% of the first capillary. From this value, the average transmittance of the laser beam per capillary was found to be as high as 93.4% (0.934 ⁹ = 0.541). From the relationship between the sensitivity of the detector and the dynamic range, assuming that the decrease in excitation efficiency that can be measured simultaneously is up to 20%, in this calculation experiment condition,
Simultaneous measurement of capillaries up to this was possible (0.
934 ²³ = 0.21).

As another specific example, the inner diameter of the capillary is 0.1 mm (r = 0.05 mm), and the outer diameter of the capillary is 0.11 mm (R = 0.055 mm) and 0.2 mm.
(R = 0.1 mm), 0.375 mm (R = 0.75 m
m), 0.75 mm (R = 0.375 mm), 1.0 m
For the case of m (R = 0.5 mm), the same calculation as above was performed (all other conditions were the same as above). However, the arrangement pitch of the ten capillaries was matched with the outer diameter of each capillary, and the capillaries were closely arranged. FIG. 7 shows a change in the pumping efficiency of the tenth capillary arranged farthest from the laser light source with respect to the ratio of the outer diameter / inner diameter of the capillary (R / r). It is standardized by the excitation efficiency of the first capillary arranged closest to the light source). In this calculation experiment, the capillary inner diameter was unified to 0.1 mm. However, even when capillaries of other inner diameters were used, the capillary diameter was essentially the same if the outer diameter / inner diameter ratio (R / r) was the same. (The incident laser beam width is assumed to be substantially the same as the inner diameter of the capillary.)

FIG. 8 shows the average transmittance of one capillary of the laser beam from the results of FIG. 7, and the maximum number of capillaries which can be measured simultaneously to reduce the excitation efficiency up to 20%. The results are shown below. 7 and 8, the ratio R / r of the outer diameter / inner diameter of the capillary is 4
It has been found that when a value near the value (accurately, 3.75) is taken, a large number of capillaries can be measured most efficiently, and the number of capillaries that can be measured simultaneously decreases as the value departs from this value. Capillary outer / inner diameter ratio R /
When r is small, the number of capillaries that can be measured simultaneously decreases because the laser beam width and the outer diameter of the capillary are close to each other, and there are beam components that deviate from the outer diameter of the capillary. The reason why the number of capillaries that can be measured simultaneously when the ratio R / r of the outer diameter / inner diameter of the capillary is large is that a beam component that transmits only the glass part of the capillary and does not transmit inside the capillary exists.

When a large number or a plurality of types of samples to be analyzed or analyzed are prepared and processed, a microtiter plate is generally widely used. This microtiter plate is a plate with 96 holes arranged in a 12 × 8 lattice and has become a global standard.
Therefore, it is very important to make the number of samples simultaneously processed in electrophoresis measurement a multiple of 12 or 8, when linking sample preparation and mass processing of electrophoresis measurement.
That is, the number of samples processed and measured simultaneously is 8, 12, 1
6, 24,... From the results of FIG. 8, the outer diameter of the capillary / the diameter of the capillary to enable simultaneous measurement with a decrease in excitation efficiency of each capillary of up to 20% is possible.
When the number of samples to be processed and measured at the same time is 8, 12, 16, and 24, the conditions regarding the inner diameter ratio R / r are 1 <R / r ≦ 10, 1 <R / r ≦ 10, 1 ≤R /
r ≦ 7 and 3 ≦ R / r ≦ 5.

Next, another key point of the present invention, a condition for reducing background light due to scattering of a laser beam, will be described. The scattering of the laser beam is mainly caused by the reflection of the laser beam at the boundary point where the refractive index of each capillary changes. The reflected light travels in a direction where the incident angle and the reflection angle are equal according to the law of reflection. As a result, the reflected light travels in various directions, and some reflected light is directly incident on a photodetection system arranged in a direction perpendicular to the plane in which the capillaries are arranged. This significantly increases the background light at the time of fluorescence measurement, has a large effect, and lowers the measurement sensitivity. Therefore, the influence was estimated by integrating the laser power of the reflected light directly incident on the photodetection system for all capillaries.

FIG. 9 shows the results obtained by integrating the laser power of the reflected light directly incident on the photodetection system for the ten capillaries under the same calculation and experimental conditions as those for obtaining the results of FIGS. 7 and 8 above. The change in outer diameter / inner diameter ratio (R / r) of the resulting capillary is shown. As described above, the laser power of the reflected light that is directly incident on the light detection system significantly increases the background light during fluorescence measurement and leads to a decrease in measurement sensitivity. Therefore, the smaller the value, the more sensitive the fluorescence measurement. Can be. From the results in FIG. 9, it can be seen that when the ratio R / r of the outer diameter / inner diameter of the capillary is around 4, the background light becomes the smallest and the fluorescence can be measured with high sensitivity. As the distance from this value increases, the background light increases and the measurement sensitivity decreases. did. The reason why the background light increased greatly when the ratio R / r of the outer diameter / inner diameter of the capillary was small is that the value of the laser beam width and the outer diameter of the capillary were close to each other. This is because the ratio has increased. From the above results, it was found that the condition of 2 ≦ R / r was effective for high sensitivity measurement.

In order to reduce the laser power of the reflected light that is directly incident on the light detection system, the reflectance at each boundary point where the refractive index changes may be reduced. As one means for that,
The outside of the capillary at the position where the laser is irradiated may be filled with a transparent liquid such as water or an aqueous solution to increase the refraction angle of the capillary. That is, it acts in the direction of the concave lens, which is disadvantageous in that the laser power reaches many capillaries. Therefore, conditions should be evaluated in consideration of these. As a result of the simulation, the effect of reducing the background light by filling the outside of the capillary with the liquid was larger for the capillary satisfying R / r ≦ 2. As a result, a large number of capillaries can be arranged and analyzed simultaneously, so that high-speed, high-throughput, high-sensitivity analysis can be realized.

Even if a cylindrical capillary other than the size used in this embodiment is used, the same effect can be obtained.

Second Embodiment In the first embodiment, the capillaries arranged in a plane are brought into close contact with each other, but here, the spacing between the capillaries is set to be equal to or larger than the outer diameter of the capillaries. As a specific example, an outer diameter of 0.2 mm (R
= 0.1mm), inner diameter 0.1mm (r = 0.05m)
m), a fused quartz cylindrical tube having a length of 50 cm (n ₂ = 1.4)
6) were used. The arrangement interval is 1 time of the capillary outer diameter (the same as in Embodiment 1), 2 times, 3 times, 4 times, 5 times,
That is, each case of 0.2, 0.4, 0.6, 0.8, and 1.0 mm was examined. Other experimental conditions were the same as in (Embodiment 1).

FIG. 10 shows the pumping efficiency of the tenth capillary array furthest from the laser light source as a variable of the arrangement interval of the capillaries. Is standardized by the excitation efficiency of the first capillary arranged in the first capillary). From the results in FIG. 10, the excitation efficiency in the tenth capillary was reduced as the array spacing of the capillaries was increased, which became a disadvantageous condition for simultaneous measurement of many capillaries. In other words, it has been found that the simultaneous measurement of a large number of capillaries can be performed most efficiently when the arrangement interval of the capillaries is matched with the outer diameter of the capillaries, that is, when the capillaries are closely arranged. The S / N of the tenth capillary under the optimum conditions (capillaries are closely arranged) is the S / N of the first capillary.
Assuming that the condition of halving is within the practical range, the excitation efficiency is 1 /
It is within the allowable range up to the condition of 4. This excitation efficiency is 1 /
From FIG. 10, the condition that the array spacing is 4 is 0.8 mm or less, that is, the array spacing is 4 mm of the capillary outer diameter.
If it is less than twice, it is satisfied.

(Embodiment 3) Based on the simulation result of (Embodiment 1), in this embodiment, the electrophoresis of the DNA sample was actually performed. First, 5 arranged in a plane
Electrophoresis was performed using the capillary gel of the book, the capillary gel of the portion for fluorescence detection was placed in water, and the DNA base sequence was determined by measuring fluorescence.
FIG. 11 shows the basic configuration of the used apparatus. The capillary gel has a length of 50 cm and an outer diameter of 0.2 mm (R = 0.1
mm), a fused quartz tube (n ₂ = 1.46) having an inner diameter of 0.1 mm (r = 0.05 mm) is filled with 4% T (Totalmonomer conc) containing 7M urea as a modifier.
entry), 5% C (Crosslinki)
ng material concentratio
gelation after injecting acrylamide at a concentration of n) (n ₃
= 1.36). At a position 30 cm from the end of the capillary on the sample injection side, the polyimide coating was removed over the entire circumference, 10 mm in length, and a window for measuring fluorescence was prepared in advance to serve as a laser irradiation site. As shown in FIG. 11, five capillary gels were arranged in a plane at a pitch of 0.2 mm, and fixed in a fluorescence measurement cell 12 filled with water (n ₁ = 1.33). However, FIG. 11 shows only the capillary gel 1 near the measurement window of the capillary gel.

FIG. 12 is a sectional view of the fluorescence measurement cell 12 with respect to the capillary axis, and shows a YAG laser (532 nm,
20 mW) and a He-Ne laser (594 nm, 10 m
After W) was made coaxial, it was condensed to a beam diameter of 0.1 mm to form a beam 2, which was incident from the side of the capillary array along the capillary array axis. The capillary 1 filled with the acrylamide gel 10 is arranged on a plane inside the fluorescence measurement cell 12, and the outside of the capillary 1 is filled with water 14.

As shown in FIG. 11, the fluorescence measurement was performed in a direction perpendicular to the plane on which the capillary gels were arranged. An image splitting prism for horizontally dividing a group of five fluorescent light emitting points in a line with a width of 0.8 mm in the horizontal direction by the first camera lens 6 and dividing the image of the fluorescent light emitting point group into four in the vertical direction; The light was transmitted through four types of combination filters 14 corresponding to the light fluxes connecting the four images, and was imaged by the second camera lens 8. This fluorescence sorting method is described in
No. 269936 describes this in detail. 5 × 4 = 2
The fluorescent light emitting point group developed into 0 two-dimensionally was subjected to exposure measurement at a time by a cooling type two-dimensional CCD camera 9. Continuous measurement was performed at an exposure time of 1.0 second and a data acquisition interval of 1.5 seconds.

The nucleotide sequences of the five DNA samples were determined according to the Sanger method. The prepared DNA sample components include four kinds of phosphors Cy3 (maximum emission wavelength 565 nm), TRITC (maximum emission wavelength 580 nm), and Texas Red (maximum emission wavelength) corresponding to the terminal base species C, G, A, and T. 615 nm), Cy5 (maximum emission wavelength 665 nm)
Was labeled. Here, Cy3 and Cy5 are sold by Biological Detection Systems (USA), and TRITC and Texas Red are sold by Molecular Probes. Four kinds of combination filters were used so that these four kinds of fluorescence were wavelength-selected and imaged on a two-dimensional CCD camera. After mixing the four kinds of reactants corresponding to the terminal base type for each sample type, the mixture was concentrated 10-fold by ethanol precipitation, and the sample solvent was replaced with formamide. The sample injection ends of the five capillary gels were respectively immersed in five types of DNA sample solutions, and a sample was injected by applying a constant electric field intensity of 100 V / cm (5 kV) to both ends of the capillary gel for 2 seconds. Electrophoresis was performed at a constant electric field intensity of 100 V / cm (5 kV) for about 3 hours. The time change of four kinds of signals corresponding to the four kinds of fluorescence obtained by the two-dimensional CCD camera was analyzed by a computer, and the DNA base sequences of five kinds of samples were determined.

FIG. 13 shows, among the DNA sequence results obtained in this embodiment, A labeled with TexasRed.
It is an electrophoresis pattern of the components, and migration time is 30 minutes to 60 minutes.
In the electrophoresis pattern, the pattern of the component A from the primer to about 130 bases is shown. However, all of the analyzed samples are M13mp18, which is a standard sample.
The capillary -1 at the top of FIG. 13 is the capillary closest to the laser light source, and indicates the electrophoresis pattern of the capillary at a position distant from the laser light source in the numerical order below. The peak intensity decreased with increasing distance from the laser light source. The peak intensity of Capillary-5 is 1/3 or less of Capillary-1, but it is still sufficiently high S / N (S / N = 7 at the position corresponding to base length 91).
34), and peak detection was possible. The S / N at the peak of capillary-1 was 91 base length.
, S / N = 3300.

In this experimental condition, since the outside of the capillary at the position where the laser is irradiated is made of water, the laser reflection at the capillary interface is reduced and the background light signal is reduced by almost one digit as compared with the case where the outside is made of air. Diminished. This means that what has been described in the first embodiment has been experimentally confirmed. Electrophoresis patterns of components labeled with other fluorescent materials were obtained in the same manner, and DNA sequencing by five capillary gel electrophoresis was realized with high sensitivity.

Next, electrophoresis is performed using ten capillary gels arranged in a plane, and the fluorescence emitted from the sample components to be electrophoresed is detected and measured in the air, thereby determining the DNA base sequence. Carried out. The capillary used has an outer diameter of 0.375 mm (R = 0.1875 m
m), a fused silica tube (n ₂ = 1.46) having an inner diameter of 0.1 mm (r = 0.05 mm), and ten capillary arrays were arranged on a glass plane at 0.375 mm as shown in FIG.
It is fixed at a pitch, and the fluorescence measurement cells shown in FIGS. 11 and 12 are not used. That is, the medium outside the capillary is air (n
₁ = 1.00). Other experimental conditions were the same as in the previous experiment.

FIGS. 14 and 15 show the D obtained in this embodiment.
It is an electrophoresis pattern of the A component labeled with Texas Red in the NA sequence result, and the migration time is 30 minutes.
1 to 60 minutes electrophoresis pattern
The pattern of the A component up to the length of 00 bases is shown. However, the analyzed samples were M13mp of the standard sample for all 10 types.
Eighteen. 14 and 15, the uppermost capillary -1 shown in FIG. 14 is the capillary closest to the laser light source, and the electrophoresis pattern of the capillary moving away from the laser light source is shown in the numerical order below. All ten capillaries in the lower capillary-10 could be measured with sufficient S / N. In the peak giving a base length of 9, the S / N was 237 for Capillary-1, 237 for Capillary-4, 110 for Capillary-7, and 140 for Capillary-10 (the S / N at Capillary-10 was lower). It is considered that the S / N ratio of Capillary-7 is larger than the variation of measurement). Electrophoresis patterns of components labeled with other fluorophores were obtained in the same manner as in FIGS. 14 and 15, and DNA base sequence determination by 10 capillary gel electrophoresis was realized with high sensitivity.

(Embodiment 4) In the present embodiment, an elliptical cylindrical capillary having an elliptical cross section is used. By using this capillary, 2
There are two different effects. A first arrangement method is to make the minor axis of the ellipse of the capillary cross section parallel to the arrangement plane of the capillary. In this case, the angle of incidence of the laser beam on the capillary is smaller than when a cylindrical capillary is used, so that the laser power of the reflected light directly incident on the photodetection system for detecting fluorescence is reduced, and the background light signal is reduced. Decrease. This has the effect of increasing the fluorescence detection sensitivity. A second arrangement method is to make the major axis of the ellipse of the capillary cross section parallel to the arrangement plane of the capillary. In this case, since the spacing between the migration paths (inside the capillaries) of the adjacent capillaries can be increased while the capillaries are arranged in close contact, crosstalk between the capillaries is eliminated, which is advantageous for independent measurement of a plurality of capillaries.
As shown in (Embodiment 3), the close-contact arrangement of the capillaries is effective for efficient simultaneous measurement of many capillaries. Next, a specific example using the first arrangement method will be described.

As a specific example, the cross section has an outer major axis of 0.75 mm.
(Outer major radius R ₁ = 0.375 mm), outer minor axis (outer minor radius R ₂ = 0.1875 mm), inner major axis 0.2 mm (inner major radius r ₁ = 0.1 mm), inner minor axis 0.05 mm ( An elliptical capillary with an inner minor radius r ₂ = 0.025 mm) was used. The inner area of this capillary cross section is 0.375 mm in outer diameter (R = 0.1875) used in (Embodiment 1).
mm), equal to the internal area of the cross section of a cylindrical capillary having an inner diameter of 0.1 mm (r = 0.05 mm) (7.85).
× 10 ^-3 mm ² ). Capillaries of this shape can be manufactured by Polymicro (USA). Other experimental conditions were the same as in (Embodiment 1), the material of the capillary having a total length of 50 cm was fused quartz (n ₂ = 1.46), and the inside of the capillary was 4% T containing 7M urea and 5% C
Of acrylamide gel (n ₃ = 1.36). At a position 30 cm from the end of the capillary on the sample injection side, a length of 10 mm, a window for measuring the fluorescence by removing the polyimide coating over the entire circumference was prepared in advance and used as a laser irradiation site.

The sites for measuring the fluorescence of the ten capillary gels were arranged in a plane as in FIGS. 3 and 4. In this case, the minor diameters of the ellipses of all the capillaries were parallel to the arrangement plane of the capillaries. And the major axis was arranged perpendicular to the arrangement plane of the capillaries. This arrangement method is effective in reducing reflected light directly incident on the photodetection system as described above. The arrangement pitch of the capillaries was made to correspond to the minor axis of the ellipse, and a close contact arrangement was adopted. The outside of the capillary at the fluorescence measurement site was air (n ₁ = 1.00). In the same manner as in FIG. 3, the fluorescence measurement is performed in the direction perpendicular to the capillary arrangement plane from the first lens, the bandpass filter, and the second lens.
The measurement was performed by a two-dimensional detector through a lens. The laser was condensed to a beam diameter of 0.1 mm, and was incident from the side of the capillary array along the capillary array axis.

Under these experimental conditions, a simulation experiment similar to that of the first embodiment was performed. FIG.
It is the result of having calculated the optical path of one beam component. FIG. 16 is a cross-sectional view of the fluorescence measurement site with respect to the capillary axis. The leftmost capillary is the capillary closest to the laser light source, and the capillary going further to the right is farther from the laser light source. All calculations of the optical path were performed continuously assuming that the ten capillaries had the above arrangement. In this calculation experiment condition,
Since the angle of refraction of the laser beam due to the capillary was Δθ０0, that is, the lens action of the capillary was small, each beam component traveled in the capillary array without receiving much refraction. All beam components for all 10 capillaries
It has been found that, without deviating from the outside of the capillary, the light passes through the inside of the capillary and contributes to the excitation of the sample components migrating inside the capillary.

As in FIG. 6, in order to evaluate the change in the degree of reduction in the excitation efficiency due to the number of capillaries, the length of the optical path (optical path length) passing through the inside of the capillary is determined for each capillary. The length was multiplied by the laser power at that position and integrated for all beam components to obtain the excitation efficiency.

FIG. 17 shows the excitation efficiencies of the respective capillaries obtained in this way (normalized by the values of the first capillary on the laser light source side). FIG.
At 7, each capillary was numbered from 1 to 10 in order from the laser light source side. As in FIG. 6, the excitation efficiency for each capillary decreased exponentially with distance from the laser light source. The excitation efficiency of the tenth capillary was as high as 53.4% of the first capillary, and the degree of this attenuation was almost equal to that of FIG. That is, the average transmittance per capillary is 93.3% (0.933 ⁹ =
0.536), under the present calculation experiment conditions, simultaneous measurement of up to 24 capillaries was possible (0.933 ²³ =
0.20).

On the other hand, the result of integrating the laser power of the reflected light directly incident on the photodetection system for ten capillaries was zero, unlike the case of (Embodiment 1). This is because the incident angle of the laser beam at each boundary point where the refractive index changes becomes sufficiently small. Therefore, it has been found that by changing from a cylindrical capillary to an elliptical cylindrical capillary, background light can be greatly reduced and fluorescence measurement can be performed with higher sensitivity.

Even if an elliptical cylindrical capillary other than the shape and size used in the present embodiment is used, the same effect can be obtained. Further, as described for the cylindrical capillaries in (Embodiments 1 and 3), the effect of filling the outside of the capillary at the laser irradiation position with a transparent liquid such as water can be similarly obtained for an elliptical cylindrical capillary. That is, since the reflectivity of the laser beam incident on the capillary is reduced, a large number of capillaries can be efficiently and simultaneously measured.

(Embodiment 5) In the present embodiment, a calculation experiment was carried out under the conditions of the calculation experiment of (Embodiment 4) by applying a reflection-reducing coating to the laser-irradiated portion of each capillary. Window for measuring fluorescence of elliptical cylindrical capillary before acrylamide gel filling (part where coating is removed)
To magnesium fluoride (MgF ₂ ) (refractive index n ₀ = 1.3
8) was deposited in a single layer with a thickness of 184 nm. By this treatment, the average transmittance per capillary after gel filling increased from 93.3% to 97.0%. At this time,
The excitation efficiency in the tenth capillary relative to the excitation efficiency in the first capillary from the laser light source is:
Was increased from 53.4% to 76.0% (0.970 ⁹ =
0.76). Along with this, the maximum number of capillaries that can be measured simultaneously to reduce the excitation efficiency to 20% increased from 24 to 53 (0.970 ⁵² = 0.21).

The anti-reflection coating applied to the laser-irradiated portion of the capillary used in this embodiment can be applied to the cylindrical capillary described in (Embodiment 1) or the square-tube capillary described in (Embodiment 6). Of course, the same effect can be obtained. Further, the coating method and type are not limited to those used in the present embodiment.

(Embodiment 6) In this embodiment, a rectangular cylindrical capillary 1 as shown in FIG. 18 is used. The cross section of the rectangular tube-shaped capillary is a square having an outer periphery of 0.2 mm on a side and a square having an inner periphery of 0.1 mm on a side. Capillaries of this shape can be manufactured by Polymicro (USA). Other experimental conditions were the same as in (Embodiment 1), and the material of the capillary having a total length of 50 cm was fused quartz (n
₂ = 1.46), and the inside of the capillary was filled with acrylamide gel 10 (n ₃ = 1.36) containing 7M urea at a concentration of 4% T and 5% C. At a position 30 cm from the end of the capillary on the sample injection side, a length of 10 mm, a window for measuring the fluorescence by removing the polyimide coating over the entire circumference was prepared in advance and used as a laser irradiation site. The area of the window for measuring the fluorescence of the ten capillary gels is 0.4 mm.
They were fixed on a glass flat plate 5 at the same pitch and arranged in a plane. The outside of the capillary at the position where the fluorescence was measured was air (n ₁ = 1.00) 11. As in FIG. 3, the fluorescence measurement was performed by a two-dimensional detector through a first lens, a band-pass filter, and a second lens from a direction perpendicular to the arrangement plane of the capillaries. FIG. 18 is a cross-sectional view of a portion for measuring the fluorescence with respect to the capillary axis. The laser was condensed to a beam diameter of 0.1 mm, and was made to enter the beam 2 along the capillary array axis from the side of the capillary array.

Under the conditions of this calculation experiment, the angle of incidence of the laser beam on each capillary is always zero, so that the influence of the refraction of the laser beam can be ignored. Considering the influence of reflection at each boundary point where the refractive index changes, the average transmittance per capillary is as high as 92.9%, and the excitation efficiency in the tenth capillary from the laser light source is:
It was pumping efficiency 51.5% in the first run of the capillary (0.929 ⁹ = 0.515). Assuming that the excitation efficiency that can be measured simultaneously can be reduced up to 20% from the relationship between the sensitivity of the detector and the dynamic range, under the conditions of this calculation experiment, 22%
It has been found that simultaneous measurement of capillaries up to this is possible (0.929 ²¹ = 0.21).

On the other hand, the result of integrating the laser power of the reflected light directly incident on the photodetection system for the ten capillaries was zero, as in the case of (Embodiment 4).
This is because the incident angle of the laser beam at each boundary point where the refractive index changes is always zero. Therefore, by changing from a cylindrical capillary to a rectangular cylindrical capillary, background light can be significantly reduced, and fluorescence measurement can be performed with higher sensitivity.

(Embodiment 7) In the present embodiment, under the calculation experimental conditions of (Embodiment 6), the outside of the capillary at the position where fluorescence is measured is water (n ₁ = 1.33). Other calculation experiment conditions were the same as in (Embodiment 6). Under the conditions of this calculation experiment, the difference between the refractive index outside the capillary and the refractive index of the capillary was smaller than in the case of (Embodiment 6), so that the reflectance of the laser beam at each boundary point where the refractive index changed was changed. Has been greatly reduced. As a result, the average transmittance per capillary was 92.9% to 9%.
Increased to 9.3%. At this time, the excitation efficiency of the tenth capillary relative to the excitation efficiency of the first capillary from the laser light source is 51.5% to 93.
Increased to 9% (0.993 ⁹ = 0.939). Along with this, the maximum number of capillaries that can be measured simultaneously to reduce the excitation efficiency to 20% increased from 22 to 230 (0.993 ²²⁹ = 0.20).

Next, under the calculation experiment conditions of (Embodiment 6), the outside of the capillary at the position where fluorescence was measured was made of quartz glass (n ₁ = 1.46). The present calculation experiment conditions may be realized by welding measurement portions of a plurality of rectangular cylindrical capillaries, or may be realized by making a plurality of holes corresponding to the inside of the capillaries in a block of quartz glass. Other calculation experiment conditions were the same as in (Embodiment 6). Under the conditions of this calculation experiment, the difference between the refractive index outside the capillary and the refractive index of the capillary was smaller than in the case of (Embodiment 6), so that the reflectance of the laser beam at each boundary point where the refractive index changed was changed. Has been greatly reduced. As a result, the average transmittance per capillary was 92.9% to 99.8%.
Increased. At this time, the excitation efficiency of the tenth capillary with respect to the excitation efficiency of the first capillary from the laser light source increased from 51.5% to 98.2% (0.998 ⁹ = 0.982). Along with this, the maximum number of capillaries that can be measured simultaneously to reduce the excitation efficiency to 20% increased from 26 to 804 (0.998 ⁸⁰³ = 0.20).

In this embodiment, the outside of the capillary at the position where fluorescence is measured is filled with water or quartz glass.
Other liquids or solids can exhibit the same effects as long as they are made of a transparent material.

(Embodiment 8) In this embodiment, under the calculation experiment conditions of (Embodiment 6), as shown in FIG.
The total reflection mirror 15 was arranged at a position where the laser beam 2 passed through the ten capillaries, and the laser beam 2 was turned back and transmitted through the ten capillaries again in the reverse path.
Other calculation experiment conditions were the same as in (Embodiment 6).

When N capillaries are arranged, the excitation efficiency T _eff in the n-th (n ≦ N) capillary from the laser light source is given by T _ram , where T _ram is the average transmittance of the laser beam per capillary. 12) (Note that in the following description, XＹ (Y) represents X to the Yth power).

T _eff = T _ram ↑ (n−1) + T _ram ↑ (2N−n) (Equation 12) The first term of (Equation 12) is the excitation by the laser beam traveling in the direction away from the laser light source. The second term expresses the excitation by the laser beam that is turned by the total reflection mirror and travels in the direction approaching the laser light source. (Equation 12) is standardized by the excitation efficiency of the first capillary in the case of (Embodiment 6), and T _eff > 1 means that
This means that the excitation efficiency is equal to or higher than the excitation efficiency of the first capillary in the case of (Embodiment 6).
According to ( _Equation 12), the excitation efficiency T _eff is (Embodiment 4)
Is monotonically decreasing with respect to n, and becomes minimum at n = N, that is, at the capillary farthest from the laser light source. The excitation efficiency T _{effmin at} this time is ( _Equation 13).

T _effmin = T _ram ↑ (N−1) + T _ram ↑ (N) ( _Equation 13) ( _Equation 13) is about the case of (Embodiment 6), that is, the case of not using a total reflection mirror. It means twice. Under the conditions of this calculation experiment, N = 10 and T _ra = 0.929, so that the excitation efficiency of the tenth capillary is 5
Increased from 1.5% to 99.4% (0.929 ⁹ +
0.929 ⁹ = 0.994). Along with this, the maximum number of capillaries that can be measured simultaneously to reduce the excitation efficiency to 20% has increased from 22 to 32 (0.929 ³¹
Tasu0.929 ³² = 0.20). Even if the method described in the present embodiment is applied to a cylindrical capillary or an elliptical cylindrical capillary, the same effect can be obtained.

(Embodiment 9) In this embodiment, under the calculation experiment conditions of (Embodiment 6), as shown in FIG.
After splitting the laser beam 2 into two beams by a beam splitter such as a half mirror 16, the reflection mirrors 17-1 and 17-
By using 2, 17-3, irradiation was carried out so as to be opposed from both sides of two side surfaces in a planar direction of a capillary array having ten capillaries. The laser beams irradiated from both sides were adjusted to have a beam diameter of 0.1 mm by a lens (not shown) and to be coaxial with each other. Other calculation experiment conditions were the same as in (Embodiment 6). When N capillaries are arranged on a plane, the excitation efficiency T _eff in the n-th (n ≦ N) capillaries from one side of the capillary arrangement is given by T _ram , where the average transmittance of the laser beam per capillary is T _ram. , (Equation 14).

2T _eff = T _ram ↑ (n−1) + T _ram ↑ (N−n) ( _Equation 14) The first and second terms of (Equation 14) are respectively divided into 2
3 illustrates excitation by a laser beam of a book. (Equation 14) is standardized by the excitation efficiency of the first capillary in the case of (Embodiment 6). According to (Equation 14), the excitation efficiency T _eff is n = (N + 1) / 2, that is, the minimum at the capillary at the center of the array where a plurality of capillaries are arranged, and the excitation efficiency at the other capillaries is the above-described center of the array. Is symmetric with respect to Minimum value of excitation efficiency T
_effmin is ( _Equation 15).

T _effmin = T _ram ↑ ((N + 1) / 2) ( _Equation 15) The ( _Equation 15) is the minimum value T of the excitation efficiency in the case of (Embodiment 6), that is, when the laser beam is not divided. _ram ↑ (n
-1) (when n = N) is clearly larger than (T _ram ≦ 1). Under the conditions of this calculation experiment, N = 10 and T _ram = 0.9
Since it is 29, the excitation efficiency becomes the minimum at the fifth capillary and is 71.8%. On the other hand, in (Embodiment 6), the excitation efficiency was minimum at the tenth capillary, and was 51.5%. Therefore, it was found that simultaneous measurement of many capillaries can be performed more efficiently by this method. Along with this, the maximum number of capillaries that can be measured simultaneously to reduce the excitation efficiency to 20% has increased from 22 to 43 (0.929 ²¹ =
0.21). Even if the method described in the present embodiment is applied to a cylindrical capillary or an elliptical cylindrical capillary, the same effect can be obtained.

(Embodiment 10) In this embodiment, under the calculation experiment conditions of (Embodiment 6), as shown in FIG.
Using two laser light sources 18-1 and 18-2, laser beams 2-1 and 2-2 were irradiated from both sides of two sides of a capillary array in which ten capillaries were arrayed. Both laser beams 2-1 and 2-2 were adjusted to have a beam width of 0.1 mm with a lens (not shown) and to be coaxial with each other. Other calculation experiment conditions were the same as in (Embodiment 6). When N capillaries are arranged, the n-th (n
≦ N), the pumping efficiency T _eff is the average transmittance of a laser beam per capillary.
_Assuming that _ram is represented by ( _Equation 16), T _eff = T _ram ↑ (n−1) + T _ram ↑ (N−n) ( _Equation 16) Obtained in the case of (Embodiment 9) (Equation 14) ) Can excite a sample component migrating in a plurality of capillaries with an excitation efficiency twice as high as the excitation efficiency shown in (2). The first of (Equation 16)
The term and the second term indicate excitation by laser beams from two laser light sources, respectively. (Equation 16) is (Embodiment 6)
Is standardized by the excitation efficiency of the first capillary, and T _eff > 1 means that (Equation 1)
This means that the pumping efficiency becomes higher than the pumping efficiency shown in 4). According to (Equation 16), the excitation efficiency T _eff is n
= (N + 1) / 2, that is, the minimum is obtained at the capillary located at the center of the arrangement of a plurality of capillaries, and the excitation efficiencies of the other capillaries are symmetric with respect to the center. The minimum value T _effmin of the excitation efficiency is ( _Equation 17). T _effmin = 2 · T _ram ↑ ((N + 1) / 2) ( _Equation 17) This is the case of (Embodiment 6), that is, the minimum value T _{ram の} of the excitation efficiency when one laser light source is used. (N-1)
(When n = N) is clearly larger than (T _ram ≦ 1).
Under the conditions of this calculation experiment, since N = 10 and T _ram = 0.929, the excitation efficiency becomes the minimum at the fifth capillary, and is 143.7%. On the other hand (Embodiment 4)
In the figure, the excitation efficiency became the minimum in the tenth capillary and was 51.5%. Therefore, it has been found that simultaneous measurement of many capillaries can be performed more efficiently by this method. As a result, the decrease in excitation efficiency is reduced by 2
The maximum number of simultaneously measurable capillaries up to 0% increased from 22 to 63 (2 × 0.9929 ³¹
= 0.20). Even if the method described in the present embodiment is applied to a cylindrical capillary or an elliptical cylindrical capillary, the same effect can be obtained.

(Embodiment 11) In this embodiment, FIG.
As shown in the figure, a specific example of a method of connecting two capillaries having different shapes to form one electrophoresis path, arranging them on a plurality of planes, and simultaneously measuring the fluorescence from each capillary is shown. . Each electrophoretic separation part of the ten capillaries has an outer diameter of 0.2 mm (R =
0.1 mm) and a cylindrical capillary 19 of a circular separation capillary having an inner diameter of 0.1 mm (r = 0.05 mm). , Inner circumference 0.1 per side
A square cylindrical capillary 20 of a measurement capillary having a square shape of mm was used. Separation capillaries and measurement capillaries constituting each electrophoresis path were connected and arranged on a glass plane at a pitch of 0.2 mm. The central axes of the ten sets of connected separation capillaries and the measurement capillaries were made to coincide with each other. The material of both capillaries is quartz (n ₂ = 1.46), the connection surface is polished and flattened in advance, and 4% T (Total monomer co.) Containing 7M urea as a modifier is used.
ncentration), 5% C (Crosslin
king material concentrati
acrylamide at a concentration of (on) was injected and gelled. The connecting portion of the capillary was immersed in a buffer solution (not shown) so as not to be electrically disconnected, so that the sample separation component that had migrated through the separation capillary migrated directly to the measurement capillary facing the same. The separation capillary had a length of 25 cm, and the measurement capillary had a length of 15 cm. At a position 5 cm from the position where the measurement capillary is connected,
The polyimide coating of the measurement capillary was removed beforehand over the entire circumference of a length of 10 mm to form a window for measuring fluorescence, and this position was irradiated with laser. That is, the migration distance is 25 + 5
= 30 cm. Other calculation experiment conditions were the same as in (Embodiment 6). That is, the average transmittance per capillary was as high as 92.9%, and the excitation efficiency of the tenth capillary from the laser light source was 51.5% of that of the first capillary (0.929 ^9).
= 0.515). From the relationship between the sensitivity of the detector and the dynamic range, if the decrease in excitation efficiency that can be measured simultaneously is assumed to be 20%, it was found that simultaneous measurement of up to 22 capillaries is possible under this calculation experiment condition (0.929 ^21). =
0.21).

Generally, the larger the outer diameter and the smaller the inner diameter, that is, the thicker the capillary, the more expensive it becomes in proportion to the volume of the glass. In addition, an elliptic cylindrical shape or a rectangular cylindrical capillary is more expensive than a cylindrical capillary. That is, as shown in the present embodiment, although it is advantageous for simultaneous measurement of many capillaries, running costs can be reduced by using expensive capillaries for measurement capillaries and using inexpensive capillaries for separation capillaries. Further, since the separation capillary and the measurement capillary can be detached from each other, the cost can be further reduced by exchanging only the separation capillary after the measurement is completed and performing the next measurement. This is another effect of the present embodiment. Of course, a similar effect can be obtained by combining capillaries with shapes other than those described above.

In the above embodiment, acrylamide gel is used as the electrophoresis separation medium, but other separation medium may be used in the form of a solution or a polymer. Examples of the polymer include polymers of acrylamide and its derivatives, and polymers of polysaccharides such as cellulose and its derivatives. If the polymer in the capillary is made exchangeable, the cost and labor can be reduced because the capillary body can be used repeatedly.

In the above embodiment, fused silica is used for the material of the capillary, but other materials may be used. In particular, glass having various refractive indices can be used, and the lens function of the capillary can be controlled similarly to the shape of the capillary. That is, more efficient simultaneous measurement of many capillaries is possible. In addition, the shape of the capillary is limited to a cylinder, an elliptical cylinder, and a square cylinder, but may be other shapes. Furthermore, the present invention is not limited to the above embodiments, and it goes without saying that the features described in the above embodiments may be combined.

Capillary gel electrophoresis can apply a higher voltage than plate gel electrophoresis, so that high-speed analysis is possible. Multi-focus capillary array electrophoresis, in which a plurality of capillaries are arranged and analyzed at once, is a high-throughput analysis technique capable of simultaneously analyzing a large number of samples at high speed. The sample can be introduced into the capillary by capillary array electrophoresis by electric field injection, which is very simple as compared with slab gel electrophoresis. In addition, on-column measurement that directly irradiates the capillary with a laser to measure fluorescence has few factors that lower the electrophoretic resolution,
In addition, it is a highly sensitive fluorescence measurement method because of its high excitation efficiency.

[0091]

According to the present invention, a plurality of capillaries can be irradiated with laser beams substantially simultaneously without performing laser beam scanning while performing on-column fluorescence measurement, and fluorescence measurement can be performed collectively. High throughput analysis can be achieved. Moreover, the device configuration and the capillary arrangement are very simple and practical. If a refillable material such as a polymer is used as the electrophoretic medium, the capillary array itself can be used repeatedly without moving, so that the cost and labor can be greatly reduced, and the practical effect is enormous.

[Brief description of the drawings]

FIG. 1A is a plan view and FIG. 1B is a cross-sectional view showing a configuration of a main part of a capillary array electrophoresis apparatus for performing on-column measurement of a capillary array according to the present invention.

FIG. 2 is a sectional view showing an optical path of a laser beam in a capillary of the present invention.

FIG. 3 is a diagram illustrating a device configuration according to the first embodiment of the present invention.

FIG. 4 is a sectional view of a part for measuring fluorescence in the first embodiment of the present invention.

FIG. 5 is a diagram showing a calculation result of an optical path in a capillary array according to the first embodiment of the present invention.

FIG. 6 is a diagram showing a relationship between a capillary position and an excitation efficiency according to the first embodiment of the present invention.

FIG. 7 is a diagram showing a relationship between the ratio of the outer diameter / inner diameter of the capillary and the excitation efficiency in the first embodiment of the present invention.

FIG. 8 is a diagram showing the relationship between the ratio of the capillary outer diameter / inner diameter and the number of capillaries that can be simultaneously measured in the first embodiment of the present invention.

FIG. 9 is a diagram showing a calculation result of a ratio of the outer diameter / inner diameter of the capillary and the intensity of reflected light directly incident on the photodetection system in the first embodiment of the present invention.

FIG. 10 is a diagram showing the relationship between the capillary arrangement interval and the excitation efficiency in the second embodiment of the present invention.

FIG. 11 is a diagram illustrating a main configuration of an apparatus according to a third embodiment of the present invention.

FIG. 12 is a sectional view of a part for measuring fluorescence in Embodiment 3 of the present invention.

FIG. 13 is a view showing an example of an electrophoresis pattern obtained in Embodiment 3 of the present invention.

FIG. 14 is a diagram showing an example of an electrophoresis pattern obtained in Embodiment 3 of the present invention.

FIG. 15 is a view showing an example of an electrophoresis pattern obtained in Embodiment 3 of the present invention.

FIG. 16 is a diagram showing a calculation result of an optical path in a capillary array according to the fourth embodiment of the present invention.

FIG. 17 is a diagram illustrating a relationship between a capillary position and an excitation efficiency according to a fourth embodiment of the present invention.

FIG. 18 is a sectional view of a part for measuring fluorescence according to the sixth embodiment of the present invention.

FIG. 19 is a sectional view of a part for measuring fluorescence according to the eighth embodiment of the present invention.

FIG. 20 is a sectional view of a part for measuring fluorescence according to the ninth embodiment of the present invention.

FIG. 21 is a sectional view of a part for measuring fluorescence according to the tenth embodiment of the present invention.

FIG. 22 is a sectional view of a part for measuring fluorescence according to the eleventh embodiment of the present invention.

[Explanation of symbols]

DESCRIPTION OF SYMBOLS 1 ... Capillary, 2 ... Laser, 3 ... Distance of deviation of incident laser from capillary arrangement axis, 4 ... Capillary arrangement axis, 5 ... Glass plane, 6 ... First lens, 7 ... Filter, 8 ... Second lens, 9 ... 2D CCD camera, 10 ...
Acrylamide gel, 11 air, 12 fluorescence measurement cell, 13 image splitting prism and combination filter, 14 water, 15 total reflection mirror, 16 half mirror, 17-1, 17-2, 17-3 reflection Mirror, 18
-1, 18-2: laser light source, 2-1, 2-2: laser beam, 19: cylindrical capillary of separation capillary, 20: square cylindrical capillary of measurement capillary.

──────────────────────────────────────────────────続 き Continuation of the front page (56) References JP-A-6-174694 (JP, A) JP-A-61-66947 (JP, A) JP-A 4-110959 (JP, U) JP-A 56-174694 21748 (JP, U) Japanese Utility Model Hei 5-79469 (JP, U) (58) Field surveyed (Int. Cl. ⁷ , DB name) G01N 27/447 G01N 21/64

Claims (7)

(57) [Claims] A plurality of capillaries at least partially arranged in a plane, and a laser positioned at a measurement position of the capillary from a direction parallel to a plane formed by each axis of the plurality of capillaries arranged in a plane. Means for irradiating the capillary, and means for detecting fluorescence emitted inside the capillary, wherein the capillary has a circular cross section with respect to the axis of the measurement position, and the outside is filled with gas at the measurement position. A capillary array electrophoresis apparatus, wherein an arrangement interval is four times or less the outer diameter of the capillary. 2. The capillary array electrophoresis apparatus according to claim 1, wherein the measurement position is provided with an anti-reflection coating. 3. The capillary array electrophoresis apparatus according to claim 2, wherein said coating is a single layer deposition. 4. The apparatus according to claim 1, further comprising a total reflection mirror, wherein the laser which has irradiated and transmitted the plurality of capillaries is turned back by the total reflection mirror, and irradiates the plurality of capillaries again along a path opposite to the transmission. The capillary array electrophoresis apparatus according to claim 1, wherein 5. The apparatus according to claim 1, further comprising a beam splitter, wherein the beam splitter divides the laser into two and irradiates the laser from two directions parallel to and opposed to the surface. Item 2. A capillary array electrophoresis apparatus according to item 1. 6. The capillary has an electrophoresis separation section and a fluorescence measurement section connected to the electrophoresis separation section, wherein the electrophoresis separation and the fluorescence measurement section have different cross sections with respect to the axis. The capillary array electrophoresis apparatus according to claim 1, wherein 7. The capillary array electrophoresis apparatus according to claim 1, wherein the capillary contains an electrophoresis separation medium, and the electrophoresis separation medium is replaceable.