JP2003270149A - Capillary array electrophoretic device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a capillary array electrophoretic device irradiating a plurality of capillaries by a laser at the same time and carrying out on-column fluorometry. <P>SOLUTION: The plurality of capillaries 1 are arranged on a plane of glass 5, and the laser 2 is irradiated from a side of the array along an array axis. By relationships among an outside radius and inside radius of the capillary, a refractive index of a medium 11 of a capillary outer part, a refractive index of a material 1 of the capillary and a refractive index of a medium 10 of a capillary interior, the capillary has a convex lens effect. A laser beam is repeatedly focused and it travels through the capillary array without deviating from the array axis of the capillaries. Sufficient laser power reaches each capillary interior, concurrent fluorometry is made possible, and high sensitiveness, high speed and high throughput can be realized. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to DNA, RNA,
Or a separation / analysis apparatus for proteins, etc.
The present invention relates to a capillary array electrophoresis apparatus effective for determining the base sequence of A or measuring the polymorphism of restriction enzyme fragments based on the diversity of individual base sequences.

[0002]

2. Description of the Related Art Analysis techniques for DNA, RNA, etc. are becoming more and more important in the fields of medicine and biology including gene analysis and gene diagnosis. Particularly in recent years, the development of a high-speed, high-throughput DNA analysis device has been advanced in relation to the genome analysis project. In DNA analysis, a fluorescently labeled sample is subjected to gel electrophoresis to separate its molecular weight, and the sample is irradiated with a laser to detect the fluorescence emitted from the fluorescent label, and the resulting series of signals is analyzed. In gel electrophoresis, a slab gel in which acrylamide is polymerized between two glass plates with a spacing of about 0.3 mm is widely used (slab gel electrophoresis). The sample injected at the upper end of the slab gel is electrophoresed toward the lower end while being separated in molecular weight by the electric field applied to both ends of the slab gel. The position of the slab gel at a certain distance from the migration start point is irradiated with a laser on all the migration paths of the side face of the slab gel at one time to excite the separation component of the fluorescently labeled sample passing through the laser irradiation part. Fluorescence detection by laser irradiation is continuously and periodically performed at fixed time intervals. The DNA base sequence is determined by analyzing the detection result.

Recently, instead of a flat gel, a capillary gel in which a gel is polymerized in a fused silica capillary tube has been used. Capillary gel electrophoresis has attracted attention as a method capable of applying a large electric field as compared with the above-mentioned slab gel electrophoresis and capable of high speed and high separation (Analytical Chemistry 6).
2,900 (1990)). Usually, in a capillary gel electrophoresis apparatus, one capillary tube is used, the inside of the capillary near the lower end of the capillary tube is irradiated with a laser, and on-column measurement is performed to detect fluorescence emitted from a fluorescence-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 glass-exposed window (US Pat. No. 5,321,535 (May 17,199).
4)). The separated component of the fluorescence-labeled sample, which migrates inside the capillary by electrophoresis, is excited by the laser applied to the position where the glass is exposed to emit fluorescence. The DNA base sequence is determined by continuously measuring and measuring this fluorescence at regular time intervals.

However, in the above 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 the throughput cannot be increased. Recently, some examples of a high-throughput capillary array gel electrophoresis apparatus in which a plurality of capillaries are arrayed and a large number of samples are simultaneously analyzed at high speed have been reported.

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

The second example is a capillary array sheath flow system (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, the sample components whose molecular weight has been separated by gel electrophoresis are eluted as they are in the buffer solution, and the fluorescence emitted from the sample components is detected in the space where no capillaries are present. In contrast to on-column measurement in which fluorescence emitted from a fluorescence-labeled sample component is detected by irradiating a capillary with a laser, in the present specification, fluorescent labeling is performed in a space portion where no capillary is present. The method of detecting fluorescence emitted from sample components is called off-column measurement for simplicity).

Further, by slowly flowing the buffer solution in the migration direction of the sample components, the separated components eluted from different capillary gels are mixed with each other in the buffer solution, or 1
The mixing of the two components separated in the book's capillary gel in buffer is prevented. In the vicinity of the exit of the capillary array, laser irradiation is applied to the space where there are no capillaries and buffer is present to avoid the problem of laser beam scattering on the capillary surface, and the components eluted from multiple capillaries are substantially eliminated. It is possible to excite all together and detect fluorescence at substantially the same time.

In the third example, in order to perform on-column measurement of a plurality of capillaries simultaneously without mechanically scanning a plurality of capillaries, laser light from a single laser light source is split by a beam splitter or the like, Simultaneous irradiation of each of a plurality of capillaries (Ana
Lytical Chemistry 65,956
(1993)).

In the fourth example, the laser light is expanded by a cylindrical lens in the direction in which the capillaries are arranged, and the plurality of capillaries are collectively irradiated (Analytical).
Chemistry 66, 1424 (199
4)).

[0010]

In the capillary array scanning method of the first example, the fluorescence measurement is performed one by one in order, so that the time allotted for fluorescence detection per one capillary It is reduced compared to the usual on-column measurement used. When using n capillary arrays, the maximum time allotted for fluorescence detection per line is 1 / n as compared to normal on-column measurement, and the glass part of the capillaries where the separated components of the sample do not actually pass through 1 because it is scanned
/ N or less. As a result, there arises a problem that the fluorescence detection sensitivity decreases. That is, in the first example described above, in order to obtain fluorescence detection sensitivity equivalent to that of ordinary on-column measurement using one capillary, a time that is n times or more the fluorescence detection time in ordinary on-column measurement is required. Will be done.

Further, the time interval between adjacent peaks of the electrophoretic pattern of the components separated in one capillary becomes smaller as the analysis becomes faster, but the time interval between adjacent peaks is n. 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 electrophoretic pattern of the separated components decreases. Further, the apparatus of this first example has a stage movable portion for mechanically performing scanning, and has a structure in which failures frequently occur.

In the capillary array sheath flow system of the above second example, there is a problem that the fluorescence intensity obtained from the components whose molecular weights have been separated becomes smaller as the molecular weight becomes larger than that in the case of on-column fluorescence measurement. is there. This problem occurs for the following reasons. In order to prevent the separated components eluted in the buffer solution from the lower end of the capillary gel from mixing in the buffer solution by diffusion etc., it is necessary to constantly flow the buffer solution at a certain speed or more in the migration direction of the sample components. is there. On the other hand, the migration speed of the sample component that migrates in the capillary gel while being separated in molecular weight decreases as the molecular weight increases. The smaller the migration rate of the sample component in the capillary gel with respect to the flow rate of the buffer solution, the more the sample component is stretched in the migration direction when it is eluted from the capillary gel into the buffer solution. . As a result, the fluorescence intensity becomes small, which causes a decrease in the measurement sensitivity of the fluorescence emitted from the sample components conducted by the fluorescence table.

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

In order to solve the above-mentioned problems of the prior art, an object of the present invention is to perform on-column fluorescence measurement without mechanically scanning a plurality of capillaries or optically scanning a laser beam. To provide a capillary array electrophoresis apparatus capable of irradiating a plurality of capillaries with a laser at substantially the same time to measure the fluorescence emitted from a fluorescently labeled sample component that migrates in a plurality of capillaries substantially at the same time. .

[0015]

In a capillary array electrophoresis apparatus of the present invention, a plurality of capillaries are arranged on the same plane, and a laser beam is irradiated from a direction parallel to the plane to simultaneously excite the plurality of capillaries. It is characterized by a multi-focus capillary array electrophoresis apparatus that performs on-column measurement after that. More specifically, the capillary array electrophoresis apparatus of the present invention has the following features.

(1) In an electrophoretic device for irradiating a plurality of capillaries with a laser to measure fluorescence, on-column fluorescence measurement is performed by irradiating a position where the inside of the capillaries is filled with an electrophoretic separation medium with a laser, and at least a laser irradiation position of the capillaries. (The cross section of the capillary of the transparent part irradiated with laser is circular, and the outside of the transparent part of the capillary is transparent gas) 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 Irradiation is performed from the side of the array to be arrayed 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 arrangement interval of the capillaries in the transparent portion is not more than 4 times the outer diameter of the capillaries. It is also possible to replace the transparent gas with a transparent liquid outside the transparent portion of the capillary. In this case, the ratio of the outer diameter to the inner diameter of the capillary of the transparent portion is set to 2 or less, and the arrangement interval of the capillaries in the transparent portion is outside the capillary. The diameter is 4 times or less.

(2) In an electrophoretic device for irradiating a plurality of capillaries with a laser to measure fluorescence, on-column fluorescence measurement is performed by irradiating a position where the inside of the capillaries is filled with an electrophoretic separation medium with a laser, and at least a laser irradiation position of the capillaries. (The cross-section of the laser-irradiated transparent capillaries is elliptical) is made of a transparent material, at least the laser irradiation positions of the capillaries are arranged in a plane, and the lasers are arranged on the array plane from the side of the array where the capillaries are arranged. Irradiate in parallel and simultaneously irradiate multiple capillaries. Plural capillaries are arranged in a plane with the minor axis of the ellipse parallel to the capillary array plane and the major radius perpendicular to the capillary array plane, or the major axis of the ellipse is parallel to the capillary array plane and the minor radius is the capillary array. A plurality of capillaries are arranged in a plane so as to be perpendicular to the plane. The outside of the capillary of the transparent portion is made into a transparent gas or a transparent liquid.

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

Further, in the electrophoretic device of (1) to (3), the inside of the capillary is filled with acrylamide gel or its derivative or polymer as an electrophoretic medium. Further, in the 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 electrophoretic device, at least the surface of the measurement portion for detecting fluorescence of the capillary is coated with antireflection single layer vapor deposition.

In the apparatus described above, the laser irradiation is
A laser that irradiates all of the capillaries and passes through may be folded back by a total reflection mirror, and then all of the capillaries may be irradiated in the reverse path again. Alternatively, the laser beam may be split into two using a beam splitter, and the capillary array may be arranged. Irradiation may be made to face each other from two sides of the body, or 2
Irradiation may be performed by using two laser beams from a single laser light source so as to oppose each other in two lateral directions of the array body in which the capillary array is provided, and the transmittance of the excitation laser wavelength of the fluorescence measurement filter is 10 ^-5 or less. And

[0021]

DESCRIPTION OF THE PREFERRED EMBODIMENTS FIG. 1 shows a plurality of capillaries 1.
(In FIG. 1, six examples are shown) are arranged on the same plane,
By irradiating two laser beams from a direction which is parallel to this plane and is substantially orthogonal to the migration direction of the sample components, the fluorescently labeled sample components that migrate in the plurality of capillaries are excited substantially at the same time, and The main part of the multi-focus capillary array electrophoresis apparatus which detects and measures on-column is shown. FIG. 1A is a view seen from the array plane direction of a plurality of capillaries, and FIG. 1B is a sectional view thereof. The plurality of capillaries irradiated with the laser 2 are either separation capillaries for separating sample components, or measurement capillaries connected to each of the plurality of separation capillaries for detecting sample components electrophoretically separated. Is. The laser 2 irradiates a part of the capillary filled with gel (separation capillary) at a predetermined distance from the migration start point of the sample or a predetermined part of the measurement capillary filled with gel.

In the multi-focus capillary array electrophoresis apparatus of the present invention, the sample to be subjected to electrophoretic separation analysis or analysis contains a plurality of fluorescently labeled components. or,
When the component itself contained in the sample 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 migrating a sample component, each means such as an electrode and an electrode tank accommodating an electrolytic solution, and the inside of the capillary 1 are shown. An arithmetic processing device that processes the detection signals after detecting the fluorescence from the sample components to be electrophoresed, a display that displays the arithmetic processing result, a control device that controls each part of the multi-focus capillary array electrophoresis device, a laser light source, etc. Is omitted. In the following description, a laser 2 narrowed in a direction connecting the central axes of the capillaries of the plurality of measurement capillaries or the plurality of separation capillaries arranged on a plane is incident, and the laser 2 is refracted. The axis along which the laser 2 travels when it is assumed that the laser beam ideally goes straight without being scattered is called a 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 are:
There are two points: (1) to make the laser power reach many capillaries, and (2) to reduce the background light due to the scattering of the laser beam.

First, the conditions for realizing (1) are
It 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. An infinitely small laser beam 2 incident at a position separated by a distance x from the capillary array axis 4.
2 shows how the optical path changes due to refraction by the capillary 1. The laser beam has a boundary point 1 where the refractive index changes.
Pass four (or twice) 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 exit position P ₃ from the inside of the capillary 1 to the capillary 1, and the capillary 1 to the capillary 1 The laser beam 2 is refracted at four points P _{4 that} are emitted to the outside. However, depending on the optical path of the laser beam 2, P ₂ and P ₃ may not exist because they do not pass through the inside of the capillary. The incident angle and the refraction angle of the laser beam at P ₁ , P ₂ , P ₃ , P ₄ are θ ₁ , θ ₂ , θ ₃ , respectively.
θ ₄ , θ ₅ , θ ₆ , θ ₇ , and θ ₈ . Let n _{1 be} the refractive index of the medium outside the capillary, n _{2 be} the refractive index of the material of the capillary, and n _{3 be} the refractive index of the medium inside the capillary.

Using the geometrical 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]

Equation 8] θ ₈ = θ ₁ ... (8) and the laser beam before the capillary incident the laser beam after transmission to the angle of refraction [Delta] [theta] an angle, [Delta] [theta] by 2, from equation (9).

[0034]

[Formula 9] 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 )) } (Equation 9) Δθ varies depending on the incident position x of the laser beam, and is the outer radius R of the capillary, the inner radius r, 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 always for a laser beam with x = 0, and | Δθ | monotonically increases as x increases (however, here, x
≦ r is assumed). That is, the capillary itself has a function of a lens whose focus is not fixed, and becomes a concave lens if Δθ> 0, and becomes a convex lens if Δθ <0. n ₁ = n ₂
When = n ₃ , of course, Δθ = 0 regardless of x.

When a laser beam is radiated from the side of the array so that the array is composed of a plurality of capillaries arranged in a plane, the laser power reaches a large number of capillaries. In particular, it is advantageous that the smaller the refraction angle Δθ by the capillaries, and in particular, that each capillary has a convex lens action of Δθ <0. This is because, when focusing on one capillary, if the transmitted laser beam is narrowed down than the incident laser beam, the laser power can be made to reach the adjacent capillary efficiently. Under normal electrophoresis conditions, always n ₂
Since> n ₃ , according to (Equation 9), the smaller n ₁ is, the smaller Δθ is. That is, n ₁ = 1.0 outside the capillary
A suitable condition is to be 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 reflectance R is the ratio of the laser power of the reflected light to the laser power of the incident light.
_{Let ef be} the transmittance T _ra , which is the ratio of the laser power of refracted light to the laser power of incident light, and these are functions of the incident angle θ _i and the refraction angle θ _t (that is, the refractive indices of both media), and
0) and (Equation 11) are given.

[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) Here, R _ef + T _ra = 1. However, here, the case where the incident light is not polarized is shown. Each time the laser power passes through the boundary point, the laser power is sequentially reduced 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).
And the point P ₃ ) is multiplied by the laser power at that position to obtain the excitation efficiency of the laser beam in this capillary (the excitation efficiency of the labeling of the labeled sample component migrating in this capillary with the laser beam). Estimated. Further, the position at which a laser beam with an infinitely small width is incident is set as a distance x from the capillary array axis (x = 0), and by changing this distance x, the excitation efficiency of the laser beam with a finite width in this capillary is estimated. . Further, when a plurality of capillaries are arranged in a plane, it was investigated how the above-described calculation is continuously performed to change the excitation efficiency in each capillary of the plurality of capillaries.

As a concrete example, the capillaries have an outer diameter of 0.375 mm (R = 0.1875 mm) and an inner diameter of 0.
A fused silica 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 a modifier
4% T (Total monomer containing urea)
5% C (Cross)
linking material concentr
cation) and then gelled (n ₃ = 1.36). A window for measuring fluorescence by removing the polyimide coating of each capillary with a length of 10 mm at a position 30 cm from the end of the capillary on the sample injection side was prepared in advance as a laser irradiation site.

As shown in FIG. 3, the measurement portions of these 10 capillary gels were arranged at a pitch of 0.375 mm, and the capillaries 1 were brought into close contact with each other and fixed on the glass flat plate 5 and 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 bandpass filter 7, and the second lens 8 to the two-dimensional detector 9
It was done by. Under the conditions of this experiment, since the reflection of the laser beam 2 by the capillary is large, it is necessary to lower the transmittance of the laser wavelength than that of the bandpass filter used for fluorescence measurement such as flat gel electrophoresis. Therefore, a bandpass filter having a laser wavelength transmittance of 10 ⁻⁵ or less was used.

FIG. 4 is a cross-sectional view of the fluorescence measuring portion with respect to the capillary axis, in which a laser was focused to a beam diameter of 0.1 mm and was made incident from the side of the capillary array along the capillary array axis. At this time, from (Equation 9), Δθ <0 always holds for the laser beam of x ≦ r, and each capillary has a convex lens function. A beam width of 0.1 mm was divided into a total of eleven infinitely small beam components with 0.01 mm intervals. That is, the beam component is a capillary array axis (laser beam central axis, x =
0) distance x is 0.00, ± 0.01, ± 0.0
The light enters the first capillary at the positions of 2, ± 0.03, ± 0.04, and ± 0.05 mm. FIG. 5 shows a result of simulating what optical path the capillary transmitted light of each beam component travels in the capillary array. FIG. 5 shows a cross-sectional view of the fluorescence measurement portion with respect to the capillary axis. The upper stage shows five capillaries on the laser light source side, and the lower stage 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.

Calculation of all optical paths was carried out continuously assuming that 10 capillaries were arranged as described above.
The beam component (x = 0.00) located on the central axis of the laser beam is not refracted at all because the incident angle at all boundary points is 0 degree, and Δθ = 0, which is on the axis of the capillary array. Go straight. The beam components other than x = 0.00 are basically refracted in the direction toward the capillary array axis due to the action of the convex lens of the capillary, and the optical path travels in the capillary array while vibrating up and down around the capillary array axis. did. Although the amplitude and period of vibration differed depending on the beam component, the optical path of each beam component was vertically symmetrical with the capillary array axis as the axis of symmetry. As a result of the above, it was found that all of the 10 beam capillaries contributed to the excitation of the sample components that passed through the capillaries and migrated inside the capillaries without deviating to the outside of the capillaries. That is, in the capillary electrophoresis apparatus of the present invention, since the laser beam irradiated on each capillary gel is substantially converged, an optical path of the convergent laser beam is formed in each of the plurality of capillary gels. It can be said that it is suitable to be called a focus capillary electrophoresis device.

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

Since the laser powers of all the beam components are attenuated by reflection every time they pass through the boundary point where the refractive index changes, the pumping efficiency of each capillary increases as the distance from the laser 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 10th capillary reached 54.1% of the 1st capillary. The average transmittance of the laser beam per capillary was calculated from this value, and the efficiency was 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 the excitation efficiency that can be measured simultaneously is up to 20%, under the conditions of this calculation experiment, 24
Capillary up to books could be measured simultaneously (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), 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
The same calculation as above was performed for m (R = 0.5 mm) (all other conditions were the same as above). However, the arrangement pitch of 10 capillaries was made to coincide with the outer diameter of each capillary, and the capillaries were closely arranged. FIG. 7 shows a change in the excitation efficiency of the tenth capillary arranged farthest from the laser light source with respect to the outer diameter / inner diameter ratio (R / r) of the capillary. It is standardized by the excitation efficiency of the first capillary arranged at the position closest to the light source). In this calculation experiment, the inner diameter of the capillaries was unified to 0.1 mm, but even if the capillaries with other inner diameters are used, it is essential that the outer diameter / inner diameter ratio (R / r) of the capillaries is the same. The same result can be obtained (however, the incident laser beam width is almost the same as the inner diameter of the capillary).

In FIG. 8, the average transmittance of each laser beam capillary is calculated from the results of FIG. 7, and the maximum number of capillaries that can be simultaneously measured is determined in the same manner as the above, which reduces the excitation efficiency to 20%. The results are shown below. From the results of FIGS. 7 and 8, the outer diameter / inner diameter ratio R / r of the capillary is 4
It has been found that when a value in the vicinity (correctly, 3.75) is taken, a large number of capillaries can be measured at the same time most efficiently, and the number of capillaries that can be measured at the same time decreases as the value deviates from this value. Capillary outer diameter / inner diameter ratio R /
The reason why the number of capillaries that can be simultaneously measured decreases when r is small is that there are beam components that deviate from the outer diameter of the capillary because the laser beam width and the outer diameter of the capillary are close. When the ratio R / r of the outer diameter / the inner diameter of the capillaries is large, the reason why the number of capillaries that can be simultaneously measured decreases is that there is a beam component that transmits only the glass portion of the capillaries and does not pass through the inside of the capillaries.

When a large amount of a sample to be analyzed or a plurality of types of samples to be 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 grid pattern, and has become a worldwide standard.
Therefore, setting the number of samples to be simultaneously processed in the electrophoretic measurement to be a multiple of 12 or 8 is very important when linking the sample preparation and the large-scale processing of the electrophoretic measurement.
That is, the number of samples to be processed and measured simultaneously is 8, 12, 1
It is preferably 6, 24, .... From the results of FIG. 8, the outer diameter of the capillary / the outer diameter of the capillary for enabling simultaneous measurement with a decrease in the excitation efficiency of each capillary up to 20%.
The conditions relating to the ratio R / r of inner diameters are 1 <R / r ≦ 10, 1 <R / r ≦ 10, 1 when the numbers of samples to be processed and measured simultaneously are 8, 12, 16, and 24, respectively. ≤R /
r ≦ 7 and 3 ≦ R / r ≦ 5.

Next, the condition for reducing the background light due to the scattering of the laser beam, which is another key point of the present invention, will be described. The scattering of the laser beam is mainly due to the reflection of the laser beam at the boundary point where the refractive index of each capillary changes. According to the law of reflection, the reflected light travels in a direction in which the incident angle is equal to the reflected angle. As a result, although the reflected light travels in various directions, there is reflected light that is directly incident on the photodetection system arranged in the 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 influence, and leads to a decrease in 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 laser power of the reflected light directly incident on the photodetection system for ten capillaries, which is calculated and calculated under the same experimental conditions as those for obtaining the results shown in FIGS. 7 and 8. The change in the outer diameter / inner diameter ratio (R / r) of the resulting capillaries 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 higher the sensitivity of fluorescence measurement. You can From the result of FIG. 9, when the outer diameter / inner diameter ratio R / r of the capillary is around 4, the background light becomes the smallest, and fluorescence measurement can be performed with high sensitivity. The background light increases and the measurement sensitivity decreases as the value deviates from this value. 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 was that 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 is effective for high sensitivity measurement.

In order to reduce the laser power of the reflected light which is directly incident on the photodetection system, the reflectance at each boundary point where the refractive index changes may be lowered. 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 is applied in the direction of the concave lens, which is disadvantageous in that the laser power reaches many capillaries. Therefore, the conditions should be evaluated based on these tradeoffs. As a result of the simulation, regarding the capillaries with R / r ≦ 2, the effect of reducing the background light was larger by filling the outside of the capillaries with the liquid. As a result, a large number of capillaries can be arranged side by side for simultaneous analysis, so that high-speed, high-throughput, high-sensitivity analysis can be realized.

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

(Embodiment 2) In (Embodiment 1), the capillaries arranged in a plane are in close contact with each other, but here, the intervals between the capillaries are made equal to or larger than the outer diameter of the capillaries. As a specific example, the capillary has an outer diameter of 0.2 mm (R
= 0.1 mm), inner diameter 0.1 mm (r = 0.05 m
m), a fused silica cylindrical tube with a length of 50 cm (n ₂ = 1.4
10) of 6) were used. The array spacing is 1 times the outer diameter of the capillary (the same as in the first embodiment), 2 times, 3 times, 4 times, 5 times,
That is, the cases of 0.2, 0.4, 0.6, 0.8 and 1.0 mm were examined. The other experimental conditions were the same as in (Embodiment 1).

FIG. 10 shows the excitation efficiency of the tenth capillary arrayed at the position farthest from the laser light source, with the array interval of the capillaries as a variable (the excitation efficiency is the position closest to the laser light source as in FIG. 6). Is standardized by the excitation efficiency in the first capillary arranged in (1). From the result of FIG. 10, as the arrangement interval of the capillaries increased, the excitation efficiency of the tenth capillary decreased, which was a disadvantageous condition for simultaneous measurement of a large number of capillaries. That is, it has been found that when the arrangement interval of the capillaries is made to coincide with the outer diameter of the capillaries, that is, when the capillaries are closely arranged, the simultaneous measurement of a large number of capillaries can be performed most efficiently. The S / N of the 10th capillary under this optimum condition (where the capillaries are closely arranged) is the S / N of the 1st capillary.
If the practical range is set to half the
The allowable range is up to 4. This excitation efficiency is 1 /
The condition of 4 is that the arrangement interval of the capillaries is 0.8 mm or less from FIG. 10, that is, the arrangement interval is 4 which is the outer diameter of the capillary.
If less than twice, it is satisfied.

(Embodiment 3) Based on the simulation result of (Embodiment 1), electrophoresis of a DNA sample was actually performed in this embodiment. 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 fluorescence was measured to determine the DNA base sequence.
FIG. 11 shows the basic configuration of the device used. The capillary gel has a length of 50 cm and an outer diameter of 0.2 mm (R = 0.1
mm) and an inner diameter of 0.1 mm (r = 0.05 mm) in a fused silica tube (n ₂ = 1.46) containing 4% T (Totalmonomer conc) containing 7M urea as a modifier.
5% C (Crosslinki)
ng material concentratio
gelation (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 a length of 10 mm and the entire circumference, and a window for measuring fluorescence was prepared in advance and used as a laser irradiation site. As shown in FIG. 11, five capillary gels were aligned in a 0.2 mm pitch and arranged in a plane, and fixed in a fluorescence measurement cell 12 filled with water (n ₁ = 1.33). However, in FIG. 11, the capillary gel 1 is shown only in the vicinity of the measurement window of the capillary gel.

FIG. 12 is a cross-sectional view of the fluorescence measuring cell 12 with respect to the capillary axis, showing a YAG laser (532 nm,
20 mW) and 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 made 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 the direction perpendicular to the plane on which the capillary gel was arranged. An image splitting prism that splits the image of the fluorescent light emitting point group into four in the vertical direction by making five fluorescent light emitting point groups, each of which has a width of 0.8 mm in the horizontal direction and arranged in one row, into substantially parallel light beams by the first camera lens 6. The four types of combination filters 14 corresponding to the light fluxes forming the four images were transmitted, and an image was formed by the second camera lens 8. Incidentally, this fluorescence selection method is described in JP-A-2-
This is described in detail in Japanese Patent No. 269936. 5 × 4 = 2
The fluorescent light emission point group developed into two zero-dimensional was subjected to exposure measurement at once with a cooling type two-dimensional CCD camera 9. Continuous measurement was performed with an exposure time of 1.0 second and a data acquisition interval of 1.5 seconds.

The nucleotide sequences of the 5 DNA samples were determined according to the Sanger method. The prepared DNA sample components include four types 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. Four kinds of reaction products corresponding to the terminal base species were mixed for each sample species and then concentrated 10 times by ethanol precipitation to replace the sample solvent with formamide. The sample injection ends of the five capillary gels were respectively immersed in five kinds of DNA sample solutions, and a constant electric field strength of 100 V / cm (5 kV) was applied to both ends of the capillary gel for 2 seconds to perform the sample injection. Electrophoresis was performed at a constant electric field strength of 100 V / cm (5 kV) for about 3 hours. The time bases of the four types of signals corresponding to the four types of fluorescence obtained by the two-dimensional CCD camera were analyzed by a computer to determine the DNA base sequences of the five types of samples.

FIG. 13 shows the results of DNA sequencing obtained in this embodiment, in which A labeled with Texas Red was used.
It is an electrophoretic pattern of the components, and the migration time is from 30 minutes to 60 minutes.
The minute electrophoretic pattern shows the pattern of the component A from the primer to about 130 bases in length. However, the analyzed samples are M13mp18 which is a standard sample for all five types.
Capillary-1 at the top of FIG. 13 is the capillary closest to the laser light source, and the capillary patterns at positions farther from the laser light source are shown in numerical order below. The peak intensity decreased as the distance from the laser light source increased. The peak intensity of Capillary-5 is 1/3 or less of that of Capillary-1, but it is still sufficiently high S / N (S / N = 7 at the position corresponding to base length 91).
34) was possible. The S / N at the peak of capillary-1 is 91
S / N = 3300 at the position corresponding to.

Under the conditions of this experiment, since the outside of the capillary at the position where the laser is irradiated is water, the laser reflection at the capillary interface is reduced and the background light signal is close to one digit compared to the case where the outside is air. Diminished. This means that what has been described in (Embodiment 1) has been experimentally confirmed. The electrophoretic patterns of the components labeled with other fluorophores were obtained in the same manner, and DNA base sequence determination by 5 capillary gel electrophoresis could be realized with high sensitivity.

Next, 10 capillary gels arranged in a plane are used for electrophoresis, and the fluorescence emitted from the sample components to be migrated is detected and measured in the air to determine the DNA base sequence. Carried out. The capillary used has an outer diameter of 0.375 mm (R = 0.1875 m
m), an inner diameter of 0.1 mm (r = 0.05 mm), a fused silica tube (n ₂ = 1.46), and 10 capillary arrays have a diameter of 0.375 mm on the glass plane as shown in FIG.
The pitch is fixed and the fluorescence measurement cell shown in FIGS. 11 and 12 is not used. That is, the medium outside the capillary is air (n
₁ = 1.00). The other experimental conditions were the same as those of the previous experiment.

14 and 15 show D obtained in this embodiment.
Of the results of NA sequencing, it is the electrophoresis pattern of A component labeled with Texas Red, and the migration time was 30
Minute to 60 minutes electrophoresis pattern, about 1 from the primer
The pattern of A component up to 00 base length is shown. However, the analyzed samples are M13mp which is a 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 in the following numerical order, the electrophoresis patterns of the capillaries moving away from the laser light source are shown. It was possible to measure with sufficient S / N for any of the 10 capillaries of the lower capillary-10. In the peak giving the base length 9, the S / N was 237 in capillary-1, 237 in capillary-4, 110 in capillary-7, and 140 in capillary-10 (S / N in capillary-10 was Larger than S / N of Capillary-7 is considered to be measurement variation). The electrophoretic patterns of the components labeled with other fluorophores were also obtained in the same manner as in FIGS. 14 and 15, and DNA base sequence determination by 10 capillary gel electrophoresis could be realized with high sensitivity.

(Embodiment 4) In this embodiment, an elliptic cylindrical capillary having an elliptical cross section is used. By using this capillary, 2
Three different effects are created. The first arrangement method is to make the minor axis of the ellipse of the capillary cross section parallel to the arrangement plane of the capillaries. In this case, the incident angle of the laser beam on the capillary is smaller than that when a cylindrical capillary is used, so the laser power of the reflected light directly incident on the photodetection system that detects fluorescence is reduced, and the background light signal is reduced. Decrease. This has the effect of increasing the fluorescence detection sensitivity. The second arrangement method is to make the major axis of the ellipse of the capillary cross section parallel to the arrangement plane of the capillaries. In this case, since the migration paths (inside the capillaries) of adjacent capillaries can be increased while closely aligning the capillaries, mutual crosstalk 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 a large number of capillaries. Next, a specific example using the first arrangement method will be shown.

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 radius (outer minor radius R ₂ = 0.1875 mm), inner major radius 0.2 mm (inner major radius r ₁ = 0.1 mm), inner minor radius 0.05 mm ( An elliptical capillary with an inner short radius r ₂ = 0.025 mm) was used. The internal area of the cross section of this capillary is 0.375 mm (R = 0.1875) used in (Embodiment 1).
mm) and an inner diameter of 0.1 mm (r = 0.05 mm), which is equal to the internal area of the cross section of the cylindrical capillary (7.85 mm).
× 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 silica (n ₂ = 1.46), and the inside of the capillary contained 4% T and 5% C containing 7 M urea.
The acrylamide gel (n ₃ = 1.36) having the concentration of was loaded. A window for measuring fluorescence by removing the polyimide coating was prepared in advance at a position 30 cm from the end of the capillary on the sample injection side and having a length of 10 mm over the entire circumference to be a laser irradiation site.

The sites for measuring fluorescence of the 10 capillary gels were arranged in a plane as in FIGS. 3 and 4. At this time, the minor axes of the ellipses of all the capillaries were parallel to the plane of the arrangement of the capillaries. And the major axis was arrayed so as to be perpendicular to the capillary array plane. This arrangement method is effective in reducing the reflected light that directly enters the photodetection system as described above. The arrangement pitch of the capillaries was made to match the minor axis of the ellipse to form a close contact arrangement. 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 from the direction perpendicular to the capillary array plane from the first lens, the bandpass filter, and the second lens.
It was performed by a two-dimensional detector through a lens. The laser was focused to a beam diameter of 0.1 mm and was made incident from the side of the capillary array body along the axis of the capillary array.

Under these experimental conditions, the same simulation experiment as in (Embodiment 1) was conducted. 16 is 1
It is the result of calculating the optical path of one beam component. FIG. 16 is a cross-sectional view of the fluorescence measurement portion with respect to the capillary axis. The leftmost capillary is the capillary closest to the laser light source, and the capillary moving to the right below is the capillary farther from the laser light source. The calculation of all optical paths was carried out continuously assuming that 10 capillaries had the above arrangement. In this calculation experimental condition,
Since the refraction angle of the laser beam by the capillaries is Δθ˜0, that is, the lens action of the capillaries is small, each beam component travels through the capillary array without being significantly refracted. All beam components are for all 10 capillaries,
It was found that it contributes to the excitation of the sample components that pass through the inside of the capillary and migrate inside the capillary without deviating to the outside of the capillary.

As in FIG. 6, in order to evaluate the change in the degree of decrease in excitation efficiency depending on the number of capillaries, the length of the optical path (optical path length) passing through the inside of the capillary is calculated for each capillary, and this optical path is calculated. 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 efficiency of each capillary thus obtained (normalized by the value of the first capillary on the laser light source side). Figure 1
7, the capillaries are numbered 1 to 10 in order from the laser light source side. Similar to FIG. 6, the excitation efficiency for each capillary decreased exponentially as the distance from the laser light source increased. The excitation efficiency of the 10th capillary reached 53.4% of the 1st capillary, and the degree of this attenuation was almost the same as in the case of FIG. In other words, the average transmission rate per one capillary is 93.3% (0.933 ⁹ =
0.536), it was possible to simultaneously measure up to 24 capillaries under the conditions of this calculation experiment (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 10 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 is sufficiently small. Therefore, it was found that the background light can be significantly reduced and the fluorescence measurement can be performed with higher sensitivity by changing the cylindrical capillary to the elliptic cylindrical capillary.

The same effect can be obtained by using an elliptic cylindrical capillary having a shape other than that used in this embodiment. 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 the elliptic cylindrical capillary. That is, by reducing the reflectance of the laser beam incident on the capillaries, it becomes possible to efficiently measure a large number of capillaries simultaneously.

(Embodiment 5) In the present embodiment, a calculation experiment was performed under the calculation experiment conditions of (Embodiment 4) by applying antireflection coating to the laser irradiation portion of each capillary. A window for measuring the fluorescence of an elliptic cylindrical capillary (the part where the coating is removed) before filling the acrylamide gel.
Magnesium fluoride (MgF ₂ ) (refractive index n ₀ = 1.3
8) was deposited as a single layer in a thickness of 184 nm. By this treatment, the average transmittance per capillary after gel filling was increased from 93.3% to 97.0%. At this time,
The excitation efficiency in the 10th capillary relative to the excitation efficiency in the 1st capillary from the laser light source is
Increased from 53.4% to 76.0% (0.970 ⁹ =
0.76). Along with this, the maximum number of capillaries that can be simultaneously measured with the decrease in excitation efficiency up to 20% has increased from 24 to 53 (0.970 ⁵² = 0.21).

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

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

Under the conditions of this calculation experiment, since the incident angle of the laser beam on each capillary is always zero, the influence of 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 10th capillary from the laser light source is
The excitation efficiency in the first capillary was 51.5% (0.929 ⁹ = 0.515). Assuming that the decrease in the excitation efficiency that can be measured simultaneously is 20% due to the relationship between the detector sensitivity and the dynamic range, the calculation experimental condition is 22
It was found that it is possible to simultaneously measure capillaries up to a book (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 10 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 tube-shaped capillary, background light can be significantly reduced, and fluorescence measurement can be performed with higher sensitivity.

(Embodiment 7) In this embodiment, under the calculation experiment conditions of (Embodiment 6), water (n ₁ = 1.33) was used outside the capillary at the position where fluorescence was measured. The other calculation experiment conditions were the same as those in (Embodiment 6). Under the conditions of this calculation experiment, since the difference between the refractive index outside the capillary and the refractive index of the capillary was smaller than that in the case of (Embodiment 6), the reflectance of the laser beam at each boundary point where the refractive index changes. Was significantly reduced. As a result, the average transmittance per capillary was 92.9% to 9%.
It increased to 9.3%. At this time, the excitation efficiency in the tenth capillary with respect to the excitation efficiency in the first capillary from the laser light source is 51.5% to 93.
It was increased to 9% (0.993 ⁹ = 0.939). Along with this, the maximum number of capillaries that can be simultaneously measured with the decrease in excitation efficiency up to 20% has increased from 22 to 230 (0.993 ²²⁹ = 0.20).

Next, under the calculation experiment conditions of (Embodiment 6), quartz glass (n ₁ = 1.46) was used outside the capillary at the position where fluorescence was measured. This calculation experiment condition may be realized by welding the measurement portions of a plurality of rectangular tube-shaped capillaries, or may be realized by forming a plurality of holes corresponding to the inside of the capillaries in a quartz glass block. The other calculation experiment conditions were the same as those in (Embodiment 6). Under the conditions of this calculation experiment, since the difference between the refractive index outside the capillary and the refractive index of the capillary was smaller than that in the case of (Embodiment 6), the reflectance of the laser beam at each boundary point where the refractive index changes. Was significantly reduced. As a result, the average transmittance per capillary was 92.9% to 99.8%.
Increased to. At this time, the excitation efficiency in the 10th capillary with respect to the excitation efficiency in the 1st 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 simultaneously measured with the decrease in excitation efficiency up 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.
Even if it is a liquid or a solid other than this, if it is a transparent material, the same effect is exhibited.

(Embodiment 8) In this embodiment, as shown in FIG. 19, under the calculation experiment conditions of (Embodiment 6),
The total reflection mirror 15 was arranged at a position where the laser beam 2 was transmitted through the ten capillaries, the laser beam 2 was turned back, and the ten capillaries were transmitted again through the reverse path.
The other calculation experiment conditions were the same as those in (Embodiment 6).

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

T _eff = T _ram ↑ (n−1) + T _ram ↑ (2N−n) (Equation 12) The first term of (Equation 12) is excitation by a laser beam traveling in a direction away from the laser light source. The second term represents excitation by a laser beam that is folded back by the total reflection mirror and advances in a direction approaching the laser light source. (Equation 12) is standardized by the excitation efficiency in 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 in the first capillary in the case of (Embodiment 6).
According to ( _Equation 12), the excitation efficiency T _eff is (Embodiment 4)
Similarly, it is monotonically decreasing with respect to n, and is minimum at n = N, that is, 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 in the case of (Embodiment 6), that is, when the total reflection mirror is not used. It means double. Under the conditions of this calculation experiment, N = 10 and T _ra = 0.929, so the excitation efficiency in the 10th 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 simultaneously measured with the decrease in excitation efficiency up to 20% has increased from 22 to 32 (0.929 ³¹
+0.929 ³² = 0.20). Even if the method shown in the present embodiment is applied to a cylindrical capillary or an elliptic cylindrical capillary, the same effect can be obtained.

(Embodiment 9) In this embodiment, as shown in FIG. 20, under the calculation experiment conditions of (Embodiment 6),
After splitting the laser beam 2 into two by a beam splitter such as a half mirror 16, reflection mirrors 17-1, 17-
Irradiation was performed by using Nos. 2 and 17-3 so as to oppose each other from both sides of two side surfaces in the plane direction of the capillary array body in which 10 capillaries were arrayed. The laser beams emitted from both sides were both adjusted to have a beam diameter of 0.1 mm by a lens (not shown) so as to be coaxial with each other. The other calculation experiment conditions were the same as those in (Embodiment 6). When N capillaries are arrayed on a plane, the excitation efficiency T _eff in the nth (n ≦ N) capillary from one side of the array of capillaries is T _ram , where the average transmittance of each laser beam 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 divided into two, respectively.
7 shows excitation with a laser beam of a book. (Equation 14) is standardized by the excitation efficiency in the first capillary in the case of (Embodiment 6). According to (Equation 14), the excitation efficiency T _eff is minimum for n = (N + 1) / 2, that is, for the capillaries in the center of the array where a plurality of capillaries are arrayed, and the excitation efficiency in other capillaries is the above array center position. Symmetrical about. Minimum value of excitation efficiency T
_effmin is ( _Equation 15).

T _effmin = T _ram ↑ ((N + 1) / 2) ( _Equation 15) ( _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) (T _ram ≦ 1) is obviously larger. Under the calculation experimental conditions, N = 10 and T _ram = 0.9.
Since it is 29, the excitation efficiency is the minimum in the fifth capillary, which is 71.8%. On the other hand, in (Embodiment 6), the excitation efficiency was the smallest in the 10th capillary, which was 51.5%. Therefore, it was found that this method can perform simultaneous measurement of a large number of capillaries more efficiently. Along with this, the maximum number of capillaries that can be simultaneously measured with the decrease in excitation efficiency up to 20% has increased from 22 to 43 (0.929 ²¹ =
0.21). Even if the method shown in the present embodiment is applied to a cylindrical capillary or an elliptic 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 while facing each other from both sides of two side faces of a capillary array body in which 10 capillaries were arrayed. Both of the laser beams 2-1 and 2-2 were adjusted so that their beam widths would be 0.1 mm by a lens (not shown) and they would be coaxial with each other. The other calculation experiment conditions were the same as those in (Embodiment 6). When N capillaries are arrayed, the nth (n
The excitation efficiency T _eff in a capillary of ≦ N) is the average transmittance of the laser beam per capillary T
Let _{ram be} expressed by ( _Equation 16), and T _eff = T _ram ↑ (n−1) + T _ram ↑ (N−n) ( _Equation 16) ( _Equation 14) It is possible to excite a sample component that migrates in a plurality of capillaries with an excitation efficiency that is twice as high as the excitation efficiency shown in FIG. First of (Equation 16)
The term and the second term respectively indicate excitation by laser beams from two laser light sources. (Equation 16) is (Embodiment 6)
Is standardized by the excitation efficiency in the first capillary in the case of, and T _eff > 1 means that (Equation 1
It means that the excitation efficiency becomes higher than the excitation efficiency shown in 4). According to (Equation 16), the excitation efficiency T _eff is n
= (N + 1) / 2, that is, the capillary located at the center position of the arrangement of the plurality of capillaries has the minimum value, and the excitation efficiency in the other capillaries is symmetrical with respect to the center position. 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 ↑ of the excitation efficiency when one laser light source is used. (N-1)
(When n = N) It is obviously larger than (T _ram ≦ 1).
Under the conditions of this calculation experiment, N = 10 and T _ram = 0.929, so that the excitation efficiency is the minimum in the fifth capillary, which is 143.7%. On the other hand, (Embodiment 4)
Then, the excitation efficiency was the smallest in the 10th capillary and was 51.5%. Therefore, it was found that this method can perform simultaneous measurement of a large number of capillaries more efficiently. Along with this, decrease in excitation efficiency
The maximum number of capillaries that can be simultaneously measured up to 0% has increased from 22 to 63 (2 x 0.929 ³¹
= 0.20). Even if the method shown in the present embodiment is applied to a cylindrical capillary or an elliptic cylindrical capillary, the same effect can be obtained.

(Embodiment 11) In this embodiment, FIG.
As shown in, a specific example of a method for connecting two capillaries having different shapes to form one electrophoresis path and arranging the electrophoresis paths on a plurality of planes and measuring fluorescence from each capillary simultaneously is shown. . The electrophoretic separation portion of each of the 10 capillaries has a cross-section with an outer diameter of 0.2 mm (R =
(0.1 mm) and an inner diameter of 0.1 mm (r = 0.05 mm), a circular capillary 19 for separation is used, and the fluorescence measuring portion has a square cross section with an outer circumference of 0.2 mm on each side. The inner circumference is 0.1
A square tube-shaped capillary 20 of a measuring capillary having a square shape of mm was used. The separation capillaries and the 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 10 sets of separated capillaries and the measurement capillaries were made to coincide with each other. Both capillaries were made of quartz (n ₂ = 1.46), the connecting surface was previously polished to be flat, and 4% T (Total monomer co-polymer) containing 7M urea as a modifier was used.
5% C (Crosslin)
king material concentrati
on) acrylamide was injected and then gelled. The connecting portion of the capillaries was immersed in a buffer solution (not shown) so as not to be electrically disconnected, and the sample separation components that had migrated through the separation capillaries migrated as they were to the opposing measurement capillaries. 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 in advance over a length of 10 mm over the entire circumference to form a window for measuring fluorescence, and a laser was irradiated at this position. In other words, the migration distance is 25 + 5
= 30 cm. The other calculation experiment conditions were the same as those in (Embodiment 6). That is, the average transmittance per capillary was as high as 92.9%, and the excitation efficiency of the 10th capillary from the laser light source was 51.5% of that of the 1st capillary (0.929 ^9).
= 0.515). From the relationship between the sensitivity of the detector and the dynamic range, if the decrease in the excitation efficiency that can be measured simultaneously is up to 20%, it was found that up to 22 capillaries can be measured simultaneously under the conditions of this calculation experiment (0.929 ²¹ =
0.21).

Generally, the larger the outer diameter and the smaller the inner diameter of a capillary, that is, the thicker it is, the more expensive the capillary becomes in proportion to the volume of glass. In addition, the elliptic cylinder shape and the rectangular cylinder shape capillary are more expensive than the cylindrical shape capillary. That is, as shown in the present embodiment, it is advantageous for simultaneous measurement of a large number of capillaries, but if an expensive capillary is used for the measurement capillary and an inexpensive capillary is used for the separation capillary, the running cost can be reduced. Further, since the separation capillary and the measurement capillary can be attached and detached, the cost can be further reduced by exchanging only the separation capillary after the measurement and performing the next measurement. This is another effect of this embodiment. Of course, the same effect can be obtained by combining capillaries having shapes other than the above.

In the above embodiments, acrylamide gel was used for all the electrophoretic separation media, but other separation media such as solutions or polymers may be used. Polymers include polymers of acrylamide and its derivatives and polymers of polysaccharides such as cellulose and its derivatives. If the polymer in the capillaries is made replaceable, the capillaries can be used repeatedly, so that the cost and labor can be reduced.

Although fused silica is used as the material of the capillaries in the above embodiments, other materials may of course be used. In particular, glass can be made of materials having various refractive indexes, and the lens action of the capillary can be controlled as well as the shape of the capillary. That is, more efficient simultaneous measurement of a large number of capillaries is possible. Further, the shape of the capillary is limited to the cylinder, the elliptic cylinder, and the rectangular cylinder, but of course, other shapes may be used. Further, it is needless to say that the present invention is not limited to the above embodiments, and the characteristic items described in the above embodiments may be combined.

Capillary gel electrophoresis enables high-speed analysis because it can apply a high voltage as compared with flat plate gel electrophoresis. Multi-focus capillary array electrophoresis, in which a plurality of capillaries are arranged and analyzed at one time, is a high-throughput analysis method capable of simultaneously analyzing a large number of samples at high speed. Since the introduction of the sample into the capillary can be performed by the electric field injection in the capillary array electrophoresis, it is very simple as compared with the slab gel electrophoresis. Further, on-column measurement in which fluorescence is measured by directly irradiating the capillary with a laser has few factors that reduce the electrophoretic resolution,
In addition, it is a highly sensitive fluorescence measurement method because it has good excitation efficiency.

[0091]

According to the present invention, while performing on-column fluorescence measurement, a plurality of capillaries can be irradiated with laser at substantially the same time without performing laser beam scanning, and fluorescence measurement can be performed at once. 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 repeatedly used without moving, so that the effect in terms of practical use that the cost and labor can be significantly reduced is great.

[Brief description of drawings]

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

FIG. 4 is a sectional view of a portion for measuring fluorescence according to the first embodiment of the present invention.

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

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

FIG. 7 is a diagram showing the relationship between the outer diameter / inner diameter ratio 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 outer diameter / inner diameter of the capillaries and the number of capillaries that can be simultaneously measured in Embodiment 1 of the present invention.

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

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

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

FIG. 12 is a sectional view of a portion for measuring fluorescence according to the third embodiment of the present invention.

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

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

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

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

FIG. 17 is a diagram showing the relationship between the capillary position and the excitation efficiency in the fourth embodiment of the present invention.

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

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

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

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

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

[Explanation of symbols]

1 ... Capillary, 2 ... Laser, 3 ... Distance of deviation of incident laser from the capillary array axis, 4 ... Capillary array axis, 5 ... Glass plane, 6 ... First lens, 7 ... Filter, 8 ... Second lens, 9 … Two-dimensional CCD camera, 10…
Acrylamide gel, 11 ... Air, 12 ... Fluorescence measuring cell, 13 ... Image division 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 tube-shaped capillary of measurement capillary.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) G01N 27/26 325A (72) Inventor Hideki Kamihara 1-280 Higashi Renegakubo, Kokubunji, Tokyo Hitachi Research Center Co., Ltd. In-house F-term (reference) 2G043 AA03 BA16 CA03 DA02 DA05 EA01 EA19 GA07 GB19 HA01 HA02 HA09 HA15 KA02 KA05 KA09 LA03 2G057 AA04 AB01 AB04 AB06 AC01 BA01 BA05 BB04 DC07

Claims (5)

[Claims] 1. A plurality of at least partially arranged in a plane.
Of capillaries, One parallel to the plane formed by each axis of the plurality of capillaries
Irradiate a laser to the measurement position of the capillary from the direction
Means and Means for detecting fluorescence emitted inside each of the capillaries
Has and Each of the capillaries with respect to the axis of the measurement position
The capillaries are circular and have a position where the laser passes.
The outer radius of the Lee is R, the inner radius is r, and the refractive index is n._TwoAnd then before
The medium outside the capillary at the position where the laser passes
Quality index n ₁And in front of the position where the laser passes
The refractive index of the medium inside the capillary is n_ThreeAnd the above
Each of the capillaries at the position of incidence of the laser on the capillaries.
Let x be the distance from the plane formed by each axis of the pillary, and x
When ≦ r, {-sin^-1(X / R) + sin
^-1(Xn₁/ (Rn_Two))-Sin^-1(Xn₁/
(Rn _Two)) + Sin^-1(Xn₁/ (Rn_Three)) <0
Capillary array characterized by satisfying the relationship
Electrophoresis device. 2. The capillary array electrophoresis apparatus according to claim 1, wherein the n ₂ is larger than the n ₃ . 3. The capillary array electrophoresis apparatus according to claim 2, wherein the n ₁ is 1.00. 4. The capillary array electrophoresis apparatus according to claim 1, wherein the external medium is any one of a transparent gas, a transparent liquid and a transparent solid. 5. The capillary array electrophoresis apparatus according to claim 1, wherein the capillary contains an electrophoretic separation medium, and the electrophoretic separation medium is replaceable.