JP3456070B2 - Capillary array electrophoresis device - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to DNA, RNA,
Alternatively, the present invention relates to a capillary array electrophoresis apparatus that belongs to the technical field relating to an apparatus for separating and analyzing proteins and the like, and particularly for determining the base sequences of DNA and RNA, or measuring the polymorphism of restriction enzyme fragments based on the diversity of individual base sequences. Belong to the technical field concerned.

[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. In particular, recently, development of a high-speed, high-throughput DNA analysis device has been progressing, particularly in connection with a genome analysis project. In DNA analysis, a fluorescently labeled sample is subjected to gel electrophoresis to separate its molecular weight, the sample is irradiated with a laser to detect fluorescence emitted from the fluorescent label, and a series of detected signals is analyzed. 0.3m by gel electrophoresis
A flat gel in which acrylamide is polymerized between two glass plates held at an interval of about m is widely used (slab gel electrophoresis). The sample injected at the migration start point of the upper end (one end) of the slab gel is migrated toward the lower end (the other end) while the molecular weight is separated by the electric field applied to both ends of the slab gel. At a certain distance from the migration start point, laser is irradiated from the side of the flat gel to irradiate all migration paths at once, and excites the separated components of the fluorescently labeled sample passing through the laser irradiation part. To do. The fluorescence emitted by the laser irradiation is continuously and periodically detected at fixed time intervals. D by analyzing this detection result
The NA base sequence has been determined.

Recently, instead of a flat gel, a capillary gel in which a gel is polymerized in a capillary tube made of fused silica has been used. Capillary gel electrophoresis has attracted attention as a method capable of applying a large electric field and capable of high speed and high separation as compared with the above slab gel electrophoresis (Analytical Chemistry).
62, 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 expose the glass (US Pat. No. 5,312,535 (May 17, 1994)). By the laser applied to the position where the glass is exposed, the components separated by electrophoresis of the fluorescently labeled sample that migrates inside the capillary by electrophoresis are excited to emit fluorescence.
The DNA base sequence is determined by continuously detecting this fluorescence at regular time intervals and analyzing it. However, in the above-described on-column measuring device, since the refraction of the laser beam on the capillary surface is large, only one capillary can be used at a time, and the throughput cannot be increased. Therefore, recently, several high-throughput capillary array gel electrophoresis apparatuses have been reported which form a plurality of capillaries in an array and simultaneously analyze a large number of samples at high speed.

The first report example is a capillary array scan system (Nature, 359, 167 (199).
2)), in which a plurality of capillaries are sequentially irradiated with laser one by one, and on-column fluorescence measurement is performed. This device employs a confocal structure in which the position where the laser beam is most narrowed in the capillaries and the position of the light source that enters the fluorescence receiving optical system coincide, and one capillary can be measured independently. 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 report example is a capillary array sheath flow method (Nature, 361, 565-).
566 (1993), Japanese Patent Laid-Open No. 06-138037). In this device, the lower end of the capillary array is immersed in a buffer solution, and the components of the sample whose molecular weight has been separated by gel electrophoresis are eluted as they are in the buffer solution, and the fluorescence emitted from the components of the sample is emitted in the space where no capillaries are present. Off-column measurement for detection (In contrast to on-column measurement in which a capillary is irradiated with a laser to detect a sample component, in the present specification, a method of irradiating a space in which no capillary is present with a laser to detect a sample component is simple. Therefore, we will call it off-column measurement.).

Further, by slowly flowing the buffer solution in the migration direction, the separated components eluted from different capillary gels are mixed in the buffer solution, or two different components separated in one capillary gel are separated. Prevents mixing in buffer. In the vicinity of the exit of the capillary array, the space portion where there is no capillary and buffer solution is irradiated with laser to avoid the problem of laser beam scattering on the surface of the capillary, and the components eluted from multiple capillaries are It is possible to excite substantially all at once and detect fluorescence at substantially the same time.

In the third report example, laser light from a single laser light source is split by a beam splitter or the like in order to perform on-column measurement of a plurality of capillaries simultaneously without mechanically scanning a plurality of capillaries. ,
Irradiate multiple capillaries simultaneously (A
analytical Chemistry 65, 95
6 (1993)).

In the fourth report example, the laser light is expanded by a cylindrical lens in the direction of the capillary arrangement, and a plurality of capillaries are collectively irradiated (Analytic).
alChemistry 66, 1424 (199
4)).

[0008]

In the capillary array scanning method of the first report example, since the fluorescence measurement is performed in order for each capillary, the time required for detecting fluorescence per one capillary is reduced. It will be reduced compared to the usual on-column measurement used. When using n capillary arrays, the time required to detect fluorescence per line is 1 / n at the maximum, compared with the usual on-column measurement, and the glass part of the capillaries where the separated components of the sample do not actually pass through Since it is scanned, it becomes less than 1 / n. As a result, there arises a problem that the fluorescence detection sensitivity decreases. That is, when using n capillary arrays, in order to obtain fluorescence detection sensitivity equivalent to that of normal on-column measurement using one capillary, fluorescence in normal on-column measurement should be n times or more the detection time. It will take time.

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. Furthermore, the present apparatus has a stage movable portion for mechanically performing scanning, and has a structure in which many failures occur.

In the capillary array sheath flow system of the second reported example, the fluorescence intensity obtained from the components separated in molecular weight is compared with the case of on-column fluorescence measurement,
There is a problem that the molecular weight decreases as the molecular weight increases. This problem occurs for the following reasons. In order to prevent the separated components eluted from the lower end of the capillary gel into the buffer solution from mixing in the buffer solution due to diffusion or the like, it is necessary to constantly flow the buffer solution at a certain speed or more in the migration direction. . On the other hand, the migration rate of the sample component that migrates in the capillary gel while being separated by molecular weight decreases as the molecular weight increases. For the flow rate of the buffer,
The smaller the migration speed of the sample component in the capillary gel, 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 measurement sensitivity.

In the third and fourth reported 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 various problems, an object of the present invention is to perform a plurality of capillaries without mechanically scanning a plurality of capillaries or scanning a laser while performing on-column fluorescence measurement. The present invention aims to provide a capillary array electrophoresis apparatus capable of simultaneously and simultaneously irradiating laser light to simultaneously measure the fluorescence emitted from sample components in a plurality of capillaries substantially simultaneously.

[0013]

In a capillary array electrophoresis apparatus of the present invention, at least a part of a plurality of capillaries for detecting a component of a sample to be electrophoresed is arranged in a plane, and a laser beam is applied to the plurality of capillaries. In a capillary array electrophoresis apparatus for irradiating, (1) irradiating a plurality of capillaries with a laser beam from a direction parallel to a plane on which at least a part of the array is arranged; One or two of the arranging planes
Irradiating a plurality of capillaries from one side in a direction parallel to the plane, (3) outer radius of the capillaries,
By satisfying a predetermined relationship among the inner radius, the refractive index of the medium outside the capillary, the refractive index of the material of the capillary, and the refractive index of the medium inside the capillary, the refractive index of the laser light by the capillary is made sufficiently small, and Irradiating the capillaries with laser light having sufficient intensity, (4) Irradiating laser light onto a plurality of capillaries substantially simultaneously without scanning the capillary array or laser light, and In order to detect fluorescence emitted from the components of the sample to be migrated at substantially the same time, the laser light is applied to one of the planes on which the at least a part is arranged.
Irradiating a plurality of capillaries from a direction parallel to the front upper plane from one or two sides, and (5) substantially scanning the plurality of capillaries with laser light without scanning the capillary array or the laser light. Simultaneous irradiation, in order to detect the fluorescence emitted from the components of the sample that migrate in each of the above-mentioned capillaries substantially at the same time, the outer radius of the capillary, the inner radius, the refractive index of the medium outside the capillary, the material of the capillary The refractive index and the refractive index of the medium inside the capillaries satisfy a predetermined relationship to sufficiently reduce the refraction of the laser light by the capillaries and irradiate all the capillaries with laser light having sufficient intensity. And a capillary array electrophoresis apparatus having

Further, the capillary array electrophoresis apparatus of the present invention has the features (1) to (5) described above.
(A) The portion of each capillary irradiated with laser light is transparent, the laser light passes through at least three media having different refractive indices, and (b) each capillary through which the laser light passes. The outer radius is R, the inner radius is r, the refractive index is n ₂ , the refractive index of the medium outside each capillary through which the laser light passes is n _1, and the refractive index of the medium inside each capillary through which the laser light passes when the rate as _{n 3, (360 / π)} │sin -1 {r / (2R)} - sin -1
{Rn ₁ / (2Rn ₂ )) + sin ⁻¹ {n ₁ / (2
n ₂ ))}-sin ⁻¹ {n ₁ / (2n ₃ )} │ ≦ 6.2 °, or (c) (360 /
^{π) │sin -1 {r / (} 2R)} - sin -1 {rn 1 /
(2Rn ₂ ))} + sin ⁻¹ {n ₁ / (2n ₂ ))} − s
in ⁻¹ {n ₁ / (2n ₃ )} │ ≦ 1.2 °, and (d) the medium outside the capillaries through which the laser beam passes has a refractive index n ₁ = 1.33 to n. It is water or a buffer solution in the range of ₁ = 1.37, the ratio R / r of the outer radius to the inner radius is R / r = 2 ± 0.2, and each capillary through which the laser beam passes is Refractive index n ₂ = 1.46 ±
It is made of quartz glass of 0.02, and the refractive index of the medium inside each capillary through which the laser light passes is n ₃ = 1.3.
7 ± 0.04, and this medium is made of acrylamide gel, and (e) the medium outside each capillary through which the laser light passes has a refractive index of n ₁ = 1.33 to n ₁ = 1.3.
Water or buffer solution in the range of 7, the ratio R / r of the outer radius to the inner radius is R / r = 2 ± 0.2, and each capillary through which the laser light passes has a refractive index n ₂ = 1.46 ±
It is made of quartz glass of 0.02, and the refractive index of the medium inside each capillary through which the laser light passes is n ₃ = 1.4.
0 ± 0.04, and the medium is made of acrylamide gel mixed with formamide, and (f) the medium outside each capillary through which the laser light passes has a refractive index n ₁
= 1.00, the ratio of the outer radius to the inner radius is R /
r is R / r = 5 ± 0.5, and each capillary through which the laser light passes is made of silica glass having a refractive index n ₂ = 1.46 ± 0.02. The refractive index of the internal medium is n ₃ = 1.37 ± 0.04
And that this medium consists of acrylamide gel,
(G) The medium outside each capillary through which the laser light passes is air having a refractive index n ₁ = 1.00, and the ratio R / r of the outer radius to the inner radius is R / r = 3 ± 0.3. And each capillary through which the laser light passes has a refractive index n ₂ = 1.60.
It is made of glass of ± 0.02, and the refractive index of the medium inside each capillary through which the laser light passes is n ₃ = 1.37.
It is ± 0.04, and is characterized in that this medium is made of acrylamide gel.

[0015]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings. In FIG. 1, a plurality of capillaries 1 (in FIG. 1, six examples are shown) are arranged on the same plane, and a laser 2 is irradiated from a direction parallel to this plane and substantially orthogonal to the migration direction of sample components. Then, the main part of the capillary array electrophoresis apparatus for exciting a plurality of capillaries substantially at the same time and measuring and detecting the sample components separated in each capillary is shown. Figure 1
(A) is a plan view of a plurality of capillary arrays, FIG.
(B) is the sectional view. The plurality of capillaries irradiated with the laser 2 are either separation capillaries for separating sample components or measurement capillaries connected to the separation capillaries for detecting sample components electrophoretically separated. When the plurality of capillaries irradiated with the laser 2 are separation capillaries, the laser 2 is irradiated at a predetermined distance from the migration start point of the sample.
Each of these capillaries is filled with gel.
Electrophoresis analysis by the capillary array electrophoresis apparatus of the present invention,
Alternatively, the sample to be analyzed contains a plurality of fluorescently labeled components. Further, when the component itself contained in the sample has a property of emitting fluorescence upon irradiation with laser, the fluorescent label is unnecessary. In addition, in each drawing referred to below including FIG. 1, in order to apply an electric field for migration,
Each unit such as a power source, an electrode, an electrode layer containing an electrode and an electrolytic solution, and further, after detecting fluorescence from a sample component migrating in the capillary gel 1, an arithmetic processing unit and an arithmetic processing for processing these detection signals. A display for displaying the results, a controller for controlling each part of the capillary array electrophoresis apparatus, a laser light source, etc. are omitted.

(First Embodiment) This embodiment is an apparatus for determining a DNA base sequence by electrophoresis of five capillary gels, and the basic configuration of the apparatus is shown in FIG. However, FIG. 2 shows only the vicinity of the fluorescence measurement portion for detecting the sample components that migrate in the five capillary gels 1. Capillary gel 1 has a length of 50 cm and an outer diameter of 0.2.
mm (outer radius, R = 0.1 mm), inner diameter 0.1 mm (inner radius, r = 0.05 mm) fused silica tube (refractive index = 1.
46), 4% T containing 7M urea as a modifier
(Totalmonomer concentrati
on), 5% C (Crosslinking material)
It was prepared by injecting acrylamide at a concentration of rial concentration and then gelling (refractive index = 1.37). 30 from the capillary end on the sample injection side
The polyimide coating of each capillary was removed at a position of cm over a length of 10 mm to form a laser irradiation part. Figure 2
As described above, the five capillary gels were kept parallel to each other, aligned at a pitch of 0.2 mm, fixed on the glass flat plate 3, and arranged in a plane. As shown in FIG. 3, the medium surrounding the capillary gel is air 9 (refractive index = 1.00).

FIG. 3 is a cross-sectional view perpendicular to the capillary axis of the fluorescence measurement portion, showing a YAG laser (532 nm, 20).
mW), and He-Ne laser (594 nm, 10 m
W) was made coaxial, the diameter of the beam 2 was reduced to 0.1 mm, and the beam was made incident from a direction parallel to the plane in which the capillaries are arranged and substantially perpendicular to the capillary axis (that is, from the side of the capillary array). 2 is incident). The fluorescence was measured through the glass flat plate 3 from the direction perpendicular to the plane on which the capillary gel is arranged. As shown in FIG. 3, fluorescence can be measured from the opposite side of the glass flat plate 3, and a group of 5 fluorescent emission points arranged in a row at a width of 0.8 mm in the horizontal direction at intervals of 0.2 mm. With the configuration shown in FIG.
The camera lens 3 converts the light into almost parallel light beams, and the image is vertically changed to 4
The image dividing prism that divides the image into four and four types of combination filters 5 corresponding to the light flux that connects the four images are transmitted,
An image was formed on the two-dimensional CCD camera 7 by the second camera lens 6. Incidentally, this fluorescence selection method is disclosed in JP-A-02-26993.
This is described in detail in Japanese Patent No. Two-dimensionally developed fluorescent light emission point groups consisting of 5 × 4 = 20 pieces were exposed to a cooling type two-dimensional CCD camera 7 at one time and measured. This measurement was continuously 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. All five types of samples are standard samples, M
13 mp18. The prepared DNA sample components include
Four types of phosphors Cy corresponding to the terminal base species C, G, A and T
3 (maximum emission wavelength 565 nm), TRITC (maximum emission wavelength 580 nm), Texas Red (maximum emission wavelength 615 nm), and Cy5 (maximum emission wavelength 665 nm) were labeled. Here, the phosphors Cy3 and Cy5 are phosphors sold by Biological Detection System (USA), and TRITC and Texas Red are phosphors sold by Molecular Probes. Four kinds of combination filters 5 were used so that the fluorescence emitted from these four kinds of phosphors was wavelength-selected and imaged on the two-dimensional CCD camera 7. 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. Each sample injection end of each of the 5 capillary gels was dipped into 5 kinds of DNA sample solutions correspondingly, and a constant electric field strength of 100 V / cm (3 kV) was applied to both ends of the capillary gel for 2 seconds to inject the sample. I did. As a result, different DNA samples are added to the migration starting points of the five capillary gels.

Electrophoresis has a constant electric field strength of 100 V / cm.
It was carried out at (3 kV) for about 3 hours. Two-dimensional CCD camera 7
It was possible to determine the base sequence by analyzing the time variation of the four types of signals corresponding to the four types of fluorescence obtained in step 4 above, but the fluorescence intensity from the two capillaries on the laser 2 emission side was determined. Was almost an order of magnitude lower than the capillary on the laser incident side.

(Second Embodiment) In order to realize more efficient simultaneous laser irradiation for a plurality of capillaries, an example in which the focusing condition of laser light is examined and the measurement is performed under the optimum condition, This will be described below. Since the capillaries have a cylindrical shape, each capillary itself has a lens function, and the irradiated laser beam is largely refracted when passing through the first capillary. Since refraction occurs in all capillaries, the intensity of the laser light that reaches and enters each capillary decreases almost exponentially, and therefore the intensity of the fluorescence emitted from the sample component in each capillary is also used. It will decrease almost exponentially according to the number of.

FIG. 4 shows an outer radius R (23) and an inner radius r (2
FIG. 4 is a cross-sectional view of a capillary having 4), showing a state of refraction by one capillary of an infinitely small laser beam 2 incident at a position separated by a distance x (10) from the axis of the capillary. In FIG. 4, a cross section of one capillary having the axis O of the capillary on the capillary array axis 19 on which the axes of the plurality of capillaries are arranged is shown, and the other plurality of capillaries are omitted.

The refractive index of the medium 20 outside the capillary is n
₁ , the material 21 of the capillary has a refractive index of n ₂ , and the medium 22 inside the capillary has a refractive index of n ₃ . If the angle formed by the laser beam before entering the capillary and the laser beam after passing through the capillary is the refraction angle Δθ, then Δθ
Is given by (Equation 1) from FIG. Note that θ ₁ is the incident angle 11 from the medium outside the capillary to the capillary,
θ ₂ is the refraction angle 12 from the medium outside the capillary to the capillary, θ ₃ is the incident angle 13 from the capillary material to the medium inside the capillary, θ ₄ is the refraction angle 14 from the capillary material to the medium inside the capillary, and θ ₅ is Incidence angle 1 from the medium inside the capillary to the capillary material
5, θ ₆ is the angle of refraction 16 from the medium inside the capillary to the capillary material, θ ₇ is the angle of incidence 17 from the capillary material to the medium outside the capillary, and θ ₈ is the angle of refraction 18 from the capillary material to the medium outside the capillary. is there.

[0023]

[Formula 1] 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 (Equation 1) Here, the relationship of θ ₁ = θ ₈ , θ ₂ = θ ₇ , θ ₃ = θ ₆ , and θ ₄ = θ ₅ was used. The geometrical relationship of the optical path followed by the laser beam 2 shown in FIG. 4 and Snell at the interface of different media
By using the law of (Equation 1) to (Equation 5),
(Equation 6) is obtained.

[0024]

[Equation 2] sin θ ₁ = x / R (Equation 2)

[0025]

Equation 3 sin θ ₁ / sin θ ₂ = n ₂ / n ₁ (Equation 3)

[0026]

Equation 4 sin θ ₂ / sin θ ₃ = r / R (Equation 4)

[0027]

Equation 5 sin θ ₃ / sin θ ₄ = n ₃ / n ₂ (Equation 5)

[0028]

Δθ = 2 {sin ⁻¹ (x / R) −sin ⁻¹ (xn ₁ / (Rn ₂ )) + sin ⁻¹ (xn ₁ / (rn ₂ )) − sin ⁻¹ (xn ₁ / ( rn ₃ ))} (Equation 6) If the magnitude of Δθ given by (Equation 6) is sufficiently small, the attenuation of the laser light intensity is further reduced, and by the configuration shown in FIG. 1 to FIG. It is possible to realize more efficient simultaneous laser irradiation to the capillaries and to excite the sample components migrating in each capillary substantially at the same time.

Usually, the laser irradiation to the capillaries is performed by narrowing the laser to the inner diameter of the capillaries. In the usual case, the laser intensity has a Gaussian distribution with the central axis of the laser beam as the axis of symmetry. That is, when the central axis of the laser beam is incident on the central axis of the capillaries, in the direction perpendicular to the array axis where the central axes of the plurality of capillaries are arrayed, at a position at a distance of approximately half the inner radius of the capillaries,
The laser intensity is almost the intensity that gives the full width at half maximum of the Gaussian distribution. Therefore, for the sake of simplicity, the alignment axis of the capillary 1
Focusing only on the laser beam (that is, x = r / 2) that is incident at a position ½ of the inner radius r from 9 (Equation 6),

[0030]

[Equation 7] | Δθ | = (360 / π) | sin ⁻¹ {x / (2R)} −sin ⁻¹ {rn ₁ / (2Rn ₂ )} + sin ⁻¹ {n ₁ / (2n ₂ )} − sin ⁻¹ {n ₁ / (2n ₃ )} │ (Expression 7)

Consider how spatially the laser beam spreads in order to simultaneously excite the sample that migrates in a plurality of capillaries. First, the displacement of the laser beam in the direction perpendicular to the direction of the array axis where the central axes of the capillaries are arrayed at the position of the Nth capillary from the first capillary on which the laser beam is incident is approximately |
It is given by NRΔθ│. The laser intensity in the Nth capillary is (1/10) or more of the laser intensity in the first capillary, so that simultaneous excitation conditions for simultaneously exciting the sample migrating in the Nth capillary , The above displacement is r
In other words, it can be restated that it is not more than {√ (10) -1} times / 2. That is, (Equation 8) holds,

[0032]

[Equation 8] | NRΔθ | ≦ {√ (10) -1} r / 2 (Equation 8) (Equation 8) is the above-mentioned simultaneous excitation condition of N capillaries.

Here, the number of capillaries to be excited at the same time is set to 5 or more (N ≧ 5). Considering that the ratio R / r of the outer shape to the inner diameter of the capillary is usually R / r ≧ 2, (Equation 8) becomes | Δθ | ≦ 6.2 °, and (Equation 7)
Therefore, the simultaneous excitation condition can be expressed as (Equation 9).

[0034]

Equation 9] ^{(360 / π) │sin -1 {} r / (2R)} - sin -1 {rn 1 / (2Rn 2))} + sin -1 {n 1 / (2n 2))} - sin - ¹ {n ₁ / (2n ₃ )} │ ≦ 6.2 ° (Equation 9) The leather strength of the N-th capillary is (1/2) or more of the leather strength of the first capillary. , And the simultaneous excitation condition for the N-th capillary, this condition is the same as (Equation 8) and (Equation 1
0).

[0035]

[Equation 10] | NRΔθ | ≦ {√ (2) -1} r / 2 (Equation 10) | Δθ | ≦ 1.2 in consideration of N ≧ 5 and R / r ≧ 2.
From (Equation 7), the above simultaneous excitation condition is (Equation 11)
Can be expressed as

[0036]

Equation 11] ^{(360 / π) │sin -1 {} r / (2R)} - sin -1 {rn 1 / (2Rn 2))} + sin -1 {n 1 / (2n 2))} - sin - ¹ {n ₁ / (2n ₃ )} │ ≦ 1.2 ° (Equation 11) FIG. 5 is the result of calculating the change in | Δθ | using (Equation 7), and the outer radius R of the capillary is R = 0.1 mm, inner radius r = 0.05 mm, the material of the capillary is quartz (n ₂
= 1.46) and the medium outside the capillary is air (n ₁ = 1.00), water (n ₁ = 1.33), and quartz (n ₁ = 1.46). , Changes in | Δθ | with respect to changes in the refractive index (n ₃ ) of the medium outside the capillary are shown for each medium outside the capillary. The refractive index of acrylamide gel is estimated to be about 1.37.

In the case of (first embodiment), n ₁ = 1.0
Since 0 (air) and n ₃ = 1.37 (acrylamide gel), | Δθ | = 6.48 ° is calculated from (Equation 7). From the results of FIG. 5, if the medium outside the capillary is filled with water (n ₁ = 1.33), then | Δθ | = 1.26 °, and the laser beam before entering the capillary and the laser beam after passing through the capillary are obtained. The refraction angle Δθ formed by the beam becomes small, and the fluorescence from the sample component that migrates in more capillaries can be simultaneously measured. The medium outside the capillary is made of the same material as the capillary, namely quartz glass (n
Assuming that ₁ = 1.46) is satisfied, | Δθ |
= 4.40 °, it is more advantageous to use water as the medium outside the capillary.

From the analysis by (Equation 7), if the outside of the capillary can be filled with a medium having a refractive index between air and water, specifically, a medium with n ₁ = 1.28, | Δθ | Can be small, but in practice such a medium cannot normally be present, so under the above conditions it is best to fill the outside of the capillary with water. Here further | [Delta] [theta] | for the reduced from 5, it is understood that it do it by increasing the refractive index of the acrylamide gel to n ₃ = 1.40 from n ₃ = 1.37. N ₃ = in (Equation 7)
If it is 1.40, | Δθ | = 0.10 °, and the refraction becomes considerably small. As a method of increasing the refractive index of acrylamide gel, for example, formamide (refractive index 1.
45) may be mixed with an appropriate concentration. Formamide, like urea, is a denaturing agent, and a mixture of about 10% has an effect of increasing the resolution of electrophoresis, and a mixture of 20% or more has a drawback of decreasing the migration rate (Select).
Rhophoresis, 13, 484-486 (199
2)). However, the refractive index of acrylamide gel is 1.3
A 10% formamide incorporation is sufficient to increase from 7 to 1.40.

FIG. 6 shows the refraction of the medium outside the capillary when the outer radius of the capillary is changed from R = 0.1 mm to R = 0.25 mm among the conditions assumed when obtaining the result of FIG. The change in | Δθ | with respect to the change in the ratio (n ₃ ) was calculated using (Equation 7), and is shown for each medium outside the capillary. Comparing FIG. 6 and FIG. 5,
By increasing the outer radius R by 2.5 times while keeping the inner radius r of the capillary, n ₁ = 1.00, n ₁ = 1.
It can be seen that the curve for 33 is shifted to the right. As a result, the refractive index of the acrylamide gel n ₃ = 1.
37, the outside of the capillary is air (n ₁ = 1.0
When 0) is satisfied, the refraction becomes as small as | Δθ | = 0.87 °, and it can be determined that the condition is appropriate for simultaneous measurement of fluorescence from sample components migrating in a larger number of capillaries.

FIG. 7 shows that the material of the capillary has a refractive index n ₂
= 1.46, the medium inside the capillary has a refractive index n ₃ =
When the acrylamide gel of 1.37 is used, the medium outside the capillary is set to air (refractive index n ₁ = 1.0).
0), a virtual medium (refractive index n ₁ = 1.28), and water (refractive index n ₁ = 1.33), the outer diameter of the capillary is 2
The change of | Δθ | with respect to the ratio R / r of R and inner diameter 2r is
The result calculated using (Equation 7) is shown. When the medium outside the capillary is air (refractive index n ₁ = 1.00), when R / r is about 6.6, | Δθ | becomes minimum,
| Δθ | increases as R / r decreases. 5 and 6
Similar to the result shown in, when R / r = 5, | Δθ | = 0.8
At 7 ° and R / r = 2, | Δθ | = 6.48 °, and R
The case of / r = 5 is a condition suitable for simultaneously irradiating more capillaries.

The medium outside the capillary is water (refractive index n ₁
= 1.33), when R / r is about 1.4, |
Δθ | becomes the minimum, and | Δθ | increases as R / r increases. Normally available capillaries are 2 ≦ R /
Capillaries with r ≦ 8 and R / r = 1.4 are practically unusable. Similar to the results shown in FIGS. 5 and 6, when R / r = 2, | Δθ | = 1.26 °, R / r = 5
Then, | Δθ | = 2.86 °, and the case of R / r = 2 is a condition suitable for simultaneously irradiating more capillaries. When the medium outside the capillary is a virtual medium (refractive index n ₁ = 1.28), when R / r = 2,
Although │Δθ│ is the minimum, a medium having a refractive index n ₁ = 1.28 cannot normally exist, and thus such a virtual medium cannot be used in practice.

FIG. 8 shows the conditions assumed for obtaining the results shown in FIG. 5, in which the outer radius of the capillary is changed from R = 0.1 mm to R = 0.15 mm, and the refractive index of the material of the capillary is n.
The change in | Δθ | with respect to the change in the refractive index (n ₃ ) of the medium outside the capillary when changing from ₂ = 1.46 to n ₂ = 1.60 is calculated using (Equation 7), and the capillary is calculated. Is shown for each medium outside. Refractive index = 1.6
For materials with 0, P sold by OHARA CORPORATION
There is HM53 (glass product number). Similar to the case of FIG. 6, the result of FIG. 8 shows the refractive index n ₃ = 1.37 of the acrylamide gel.
In contrast, the outside of the capillary is air (n ₁ = 1.00)
When the above condition is satisfied, the refractive index becomes as small as | Δθ | = 0.84 °, and it can be determined that the condition is suitable for simultaneously measuring the fluorescence from the sample components that migrate in a larger number of capillaries.

In this embodiment, DNA base sequence determination was carried out by electrophoresis of five capillary gels. The apparatus used uses a fluorescence measuring cell 25 in place of the glass flat plate 3 in the basic configuration of the apparatus shown in FIG. 2 (FIG. 9). Capillary gel has a length of 50 cm and an outer diameter of 0.2
mm (outer radius, R = 0.1 mm), inner diameter 0.1 mm (inner radius, r = 0.05 mm) fused silica tube (n ₂ = 1.4)
In 6), 4% T containing 7 M urea as a modifier (T
total monomer concentratio
n), 5% C (Crosslinking material)
It was prepared by injecting acrylamide at a concentration of ial concentration and then gelling (n ₃ =
1.37). Substituting the values of R, r, n ₁ , n ₂ and n ₃ into (Equation 7) gives | Δθ | = 1.26 °. In this embodiment, the refraction by the capillary gel is sufficiently small,
The conditions are suitable for simultaneous measurement of fluorescence emitted from sample components that migrate in five or more capillaries. At a position 30 cm from the end of the capillary on the sample injection side, length 10
The polyimide coating of each capillary was removed over a range of mm to make a laser irradiation part. As shown in FIG. 9, the five capillary gels were arranged at a pitch of 0.2 mm and fixed in a fluorescence measurement cell 25 filled with water (n ₁ = 1.33). However, FIG. 9 shows only the vicinity where the fluorescence measuring cell 25 of the capillary gel is arranged.

FIG. 10 is a sectional view of the fluorescence measuring cell 25, which is perpendicular to the capillary axis. In this cross-sectional view, at least the surface on the side where the laser beam enters and exits (the entire surface, or only the part where the laser beam enters and exits) and the surface on the side that detects the fluorescence emitted from the sample component that migrates in the capillary (the entire surface). , Or only a plurality of portions for detecting fluorescence from each capillary) may be made of a transparent material. In the fluorescence measuring cell 25, the axes of the capillaries are arranged in parallel, and the axes of the capillaries are fixed so that they are substantially flush with each other.
₁ = 1.33). (First embodiment)
After making the same two kinds of lasers coaxial with those used in 1., the beam diameter was narrowed down to 0.1 mm and made to enter from the side of the plane where the axes of each capillary gel were arranged, through the transparent wall of the fluorescence measurement cell 25. It was In the first embodiment, the fluorescence measurement is performed through the glass flat plate 3 from the direction perpendicular to the plane in which the capillary gel is arranged, whereas in the present embodiment, the transparent material of the fluorescence measurement cell 25 is used. The fluorescence is measured from the part composed of. Except for this point, the configuration and conditions for measuring fluorescence and the method for measuring fluorescence are exactly the same as in the first embodiment. In addition, 5 used
The electrophoretic conditions including the addition of the seed DNA sample, the labeled fluorescent substance, the sample to the migration start point, and the like are exactly the same as those in the first embodiment. In this manner, electrophoresis was performed for about 3 hours, and the time changes 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, and the DNA base sequences of the five types of samples were analyzed. Were determined.

FIG. 11 shows that among the DNA sequence results obtained in this embodiment, A labeled with Texas Red was used.
The electrophoretic pattern of the components is shown. Figure 11 shows migration time 3
The electrophoretic pattern in the range of 0 to 60 minutes is shown, and the pattern of the A component from the primer to about 130 bases long is shown (5 samples analyzed were M13 of the standard sample).
mp18). The numbers shown in the right column of FIG. 11 indicate capillary numbers, and the laser beam is incident in the order of capillaries number 1, 2, 3, 4, 5. That is, the laser beam first enters the uppermost capillary 1, then the laser beam that has passed through the capillary 1 enters the next capillary 2, and finally the capillary 5 passes through the capillaries 1 to 4. The laser beam is incident. Figure 11
Shows the electrophoretic pattern in the order of the capillaries that are separated from the capillary 1 on which the laser beam first enters. The peak intensity decreases with distance from the capillary 1, which is caused by the fact that the refraction of the laser by the capillary is not completely zero. The peak intensity of the capillary 5 is 1/3 or less that of the capillary 1, but the S / N (base length 9
At the position corresponding to 1, S / N = 734), the migration peak can be detected. The S / N at the peak of the capillary 1 is S / N at the position corresponding to the base length 91.
N = 3300.

Electrophoresis patterns of sample components labeled with other fluorophores were obtained in the same manner, and the fragments were detected with high sensitivity by electrophoresis using 5 capillary gels to determine the DNA base sequence. It was realized. In FIG. 11, the vertical axis represents the relative fluorescence intensity standardized with the peak of the predetermined base length of the capillary 1 as 1, and the migration patterns obtained from the respective capillaries do not show strictly similar shapes. The reason is that the capillaries do not have exactly the same length, and the gels filled in the capillaries do not have exactly the same concentration (the same applies to FIGS. 13 and 14 described later).

(Third Embodiment) This embodiment is based on (second embodiment).
Embodiment), the number of capillaries was increased to 10 and 4% T (Total monomer con) containing 7M urea was used as a denaturant for the capillary gel.
5% C (Crosslink)
ing material concentratio
It is an embodiment prepared by adding 10% formamide to acrylamide having a concentration of n) and polymerizing. As a result of making the composition of the capillary gel different from that of the second embodiment, the refractive index of the acrylamide gel increased to n ₃ = 1.40. Experimental conditions other than these are (second embodiment)
Exactly the same as. From (Equation 7), | Δθ | = 0.10.
Is calculated, and as in the case of the fourth embodiment described below, the refraction by the capillaries becomes smaller, and the fluorescence emitted from the migrating sample component can be measured with high sensitivity.
DNA sequencing could be achieved by electrophoresing the seed samples using 10 capillary gels.

(Fourth Embodiment) This embodiment is an embodiment in which capillaries are arranged in the air. Figure 12
A cross-sectional view perpendicular to the capillary axis of a portion for irradiating a laser beam 2 to 10 capillaries 1 filled with gel 8 and arranged in air 9 to measure fluorescence is shown. The capillary used has an outer diameter of 0.5 mm (outer radius, R =
0.25 mm) and an inner diameter of 0.1 mm (inner radius, r = 0.
05 mm) fused quartz tube (n ₂ = 1.46),
The arrangement of the ten capillaries 1 is 0.5 mm pitch on the glass flat plate 3 as shown in FIG. 12, and similarly to FIG. 3, the respective capillary axes are arranged in parallel so that the respective capillary axes form substantially the same plane. Fixed as. In this embodiment,
The fluorescence measurement cell was not used, and the medium outside the capillary was air (n ₁ = 1.00). The experimental conditions other than these were exactly the same as those of the second embodiment. From (Equation 7),
Since | Δθ | = 0.87 ° is calculated and the refraction by the capillaries becomes sufficiently small, this experimental condition is that 10 or more capillaries can be used without significantly attenuating the laser light intensity applied to each capillary. This is a condition under which fluorescence emitted from the sample components that migrate can be simultaneously excited.
As shown in FIG. 12, fluorescence is detected from the glass flat plate 3 side or the opposite side of the glass flat plate 3.

13 and 14 show D obtained in this embodiment.
It is the electrophoresis pattern of the A component labeled with Texas Red in the NA sequence results. This is an electrophoretic pattern of a migration time of 30 minutes to 60 minutes, and shows the pattern of the A component from the primer to about 120 bases in length (10 samples analyzed were M13 of the standard sample).
mp18). The numbers in the right columns of FIGS. 13 and 14 indicate the capillary numbers as in FIG. 11, and the laser beam irradiates the respective capillaries in the order of capillary 1, capillary 2, ..., Capillary 10. The meaning of the relative fluorescence intensity on the vertical axis is the same as in FIG. The laser beam is first irradiated onto the capillary 1, then the laser beam that has passed through the capillary 1 is irradiated onto the capillary 2, ..., And the laser beam that has passed through the capillary 9 from the capillary 1 is irradiated onto the capillary 10. Figure 1
3 and FIG. 14 show the electrophoretic patterns obtained from the capillaries 2, 3, ..., 10 which are moved away from the capillary 1 which is first irradiated with the laser beam, and 10 capillaries have sufficient S / N (base length). The S / N in the peak giving 91 is 237 in capillary 1, 237 in capillary 4, 110 in capillary 7, and 142 in capillary 10 (S / N in capillary 10
The reason why N is larger than S / N in the capillary 7 is considered to be variation in measurement)), and fluorescence could be measured. The electrophoretic patterns of sample components labeled with other fluorophores are also shown in FIG.
3, obtained in the same manner as in FIG. 14, the fluorescence emitted from the sample components to be electrophoresed can be measured with high sensitivity by electrophoresis using 10 capillary gels, and DNA base sequence determination can be realized.

(Fifth Embodiment) In the present embodiment, (fourth embodiment)
Embodiment), the outer diameter of the capillary used is 0.3 mm (outer radius, R = 0.15 mm), and the inner diameter is 0.
1 mm (inner radius, r = 0.05 mm), the refractive index of the material of the capillary is n ₂ = 1.60. Specifically, PHM53 (n ₂ = 1.60) sold by OHARA INC. Was used as the material of the capillary. The experimental conditions other than these were exactly the same as those in (Fourth Embodiment). From (Equation 7), | Δθ | = 0.84 ° is calculated,
The refraction by the capillary is smaller than that of | Δθ | = 0.87 ° in the fourth embodiment. Therefore,
A laser beam can excite sample components that migrate in 10 capillaries substantially at the same time, and 10 samples can
By using electrophoresis using a book capillary gel, fluorescence emitted from the sample components to be migrated can be measured with high sensitivity.
A sequencing could be realized.

(Sixth Embodiment) In this embodiment, FIG.
As shown in FIG. 5, the capillaries used are composed of two types of capillaries, and each capillary has a different diameter dimension. Each of the 10 capillaries used is
In the electrophoretic separation part, the outer diameter is 0.2 mm (outer radius, R =
0.1 mm), inner diameter 0.1 mm (inner radius, r = 0.05)
The outer diameter of the capillary (separation capillary 27) having a size of 0.5 mm and the portion for measuring the fluorescence by irradiating the laser beam 2 is 0.5 mm (outer radius, R = 0.25 m).
m) and an inner diameter of 0.1 mm (inner radius, r = 0.05 mm). As shown in FIG. 15, the separation capillaries 27 and the measurement capillaries 28 are connected to each other, and the axes of the measurement capillaries 28 are parallel to each other at a pitch of 0.5 mm on the glass plane in the same manner as in FIGS. 3 and 12. The measurement capillaries 28 were arranged and fixed on a flat glass plate so that the axes of the respective measurement capillaries 28 formed a flat surface. At this time, the central axes of the 10 sets of separation capillaries and the measurement capillaries facing each other were made to coincide with each other. The material for both capillaries was quartz (n ₂ = 1.46), and the connecting surface was previously polished to be flat.

Both capillaries contained 4% T (Totalmonomer c) containing 7M urea as a denaturant.
oncentration), 5% C (Crossli
nking material concentrat
(ion) was injected and then gelled. The connecting portion of the capillary was immersed in a buffer solution so as not to be electrically disconnected, and the separation capillary was electrophoresed so that the separated sample components were electrophoresed on the opposing measurement capillary.

The separation capillary had a length of 25 cm, and the measurement capillary had a length of 15 cm. However, FIG. 15 shows only the vicinity of the connecting portion of both capillaries. 5c from the connecting position of the separation capillary and the measurement capillary
At position m, the polyimide coating of the measuring capillary was previously removed over a length of 10 mm, and the position was irradiated with the laser beam 2 as shown in FIG. At this time, the migration distance is (25 + 5) cm = 30 cm. The experimental conditions other than these were exactly the same as those of the fourth embodiment.
That is, the apparatus configuration conditions in the present embodiment are such that the refraction by the capillaries of the laser is sufficiently small, and the sample component that migrates 10 capillaries without significantly attenuating the laser light intensity applied to each capillary. Is a condition capable of being excited at substantially the same time, and the fluorescence emitted from the migrating sample components can be measured with high sensitivity by electrophoresis using 10 capillary gels of 10 types of samples.
The sequencing could be realized. Although not shown in FIG. 15, fluorescence is detected from the glass flat plate side or the opposite side of the glass flat plate.

Generally, the price of a capillary becomes higher as the outer diameter is larger and the inner diameter is smaller, that is, as the wall thickness is larger, in proportion to the volume of glass. That is, the price per unit length of the separation capillaries and the measurement capillaries used in the present embodiment is eight times higher. In this embodiment, since the separation capillary and the measurement capillary can be attached and detached, only the separation capillary can be exchanged after the measurement of one sample is completed, and the measurement of the next sample can be performed, and the cost can be reduced. This is another effect of this embodiment.

The capillary used in the present embodiment, which is composed of two types of capillaries shown in FIG. 15 each having a different diameter, has been described with reference to FIG.
It is needless to say that the same result as that of the present embodiment can be obtained by applying the above-described embodiment.

(Seventh Embodiment) In the above (first embodiment) to (sixth embodiment), each capillary axis is parallel to the plane formed by the planarly arranged capillary gels from one direction. The configuration of irradiating the laser beam so as to pass through has been described. However, in (First Embodiment) to (Sixth Embodiment), as shown in FIG. 16, the laser beam is passed through each capillary axis from two directions. It may be configured to irradiate. In the configuration of FIG. 16A, the laser beam 2 (a laser beam having a single wavelength or a laser beam obtained by mixing laser beams having a plurality of wavelengths from a plurality of laser light sources to be coaxial) may be used as a semitransparent mirror 29. , And 30-1, 30-2,
The configuration is such that the laser beam is radiated so as to pass through each capillary axis from two directions by 30-3. The configuration of FIG. 16B has two laser beams 2-1 and 2-
The laser beams 2-1 and 2-2 are laser beams having a single wavelength or laser beams having a plurality of wavelengths from a plurality of laser light sources, respectively. The laser light mixed and coaxial may be used. In the configurations of FIGS. 16A and 16B, in (First Embodiment) to (Sixth Embodiment), it is possible to further improve the nonuniformity of the intensity of the laser light with which each capillary is irradiated. It is possible to irradiate each capillary with laser light of such intensity. As a result, it becomes possible to perform capillary array electrophoresis using a larger number. Note that FIG.
In the configuration of (a), since the laser beam is branched so that the laser beam is irradiated from two directions so as to pass through each capillary axis, the irradiation intensity of the laser beam is reduced, so that it is desirable to use a high-power laser light source. Further, when the configuration of FIG. 16B is applied to (the first embodiment) to (the sixth embodiment), the wavelength of a single laser beam or a plurality of laser beams included in the laser beam 2-1 and The laser beam 2-2 may be configured to have the same wavelength or a plurality of wavelengths of laser light, or may have different wavelengths.

(Eighth Embodiment) In the above (first embodiment) to (seventh embodiment), acrylamide gel is used for all, but other separation medium may be used. The capillary itself may be reused by using a replaceable separation medium. However, optimization of the refractive index of each medium and the diameter dimension of the capillary based on, for example, (Equation 7) according to the refractive index of the separation medium is generated from the sample component that migrates in the plurality of capillaries. This is important for simultaneous high-sensitivity fluorescence measurements. Also, from (Equation 7), | Δθ | is not determined by the outer diameter 2R and the inner diameter 2r of the capillary independently, but | Δθ | is determined by the ratio R / r of the outer diameter to the inner diameter.
Therefore, in each of the above-described embodiments, even when the capillaries having different outer diameters 2R and inner diameters 2r are used, substantially the same effect can be obtained as long as the ratio of the outer diameter to the inner diameter (R / r) is equal. .

(Ninth Embodiment) In the above (first embodiment) to (eighth embodiment), a plurality of capillary gels are used, but a single capillary gel may be used. Needless to say. Even when a single capillary gel is used, the refraction | Δθ | of the laser beam due to the capillary gel given by (Equation 7) is minimized and the gel beam on which the sample component migrates is irradiated with the laser beam to improve efficiency. Fluorescence from the sample components can be detected well.

Finally, the present invention will be briefly summarized. In a capillary array electrophoresis apparatus, a plurality of capillaries are irradiated with laser light at substantially the same time without scanning the capillary array or laser light. On-column fluorescence measurement is performed by detecting fluorescence emitted from the sample component that migrates at substantially the same time. That is, a plurality of capillaries are arrayed on one plane, and a laser is irradiated from one or two sides of the array plane parallel to the array plane. The refractive index of the laser capillary is sufficiently small by satisfying a predetermined relationship among the outer radius of the capillary, the inner radius, the refractive index of the medium outside the capillary, the refractive index of the material of the capillary, and the refractive index of the medium inside the capillary. By irradiating all the capillaries with laser light of sufficient intensity, high sensitivity, high speed, and high throughput can be easily realized.

[0060]

According to the present invention, in a capillary array electrophoresis apparatus, a plurality of capillaries are irradiated with laser light substantially at the same time without scanning the capillary array or laser light, and each capillary is electrophoresed. The fluorescence emitted from the sample components can be detected at substantially the same time, and on-column fluorescence measurement that can easily achieve highly sensitive, high-speed, and high-throughput analysis can be realized.

[Brief description of drawings]

FIG. 1 is an embodiment of the present invention, in which a plurality of capillaries arranged on the same plane are irradiated with a laser beam in a direction parallel to the plane and substantially orthogonal to the migration direction of sample components. Shows the main part of
(A) The top view of a plurality of capillary arrays, (b) The sectional view.

FIG. 2 is a diagram showing a basic configuration of an apparatus for determining a DNA base sequence by electrophoresis using five capillary gels, which is the first embodiment of the present invention.

FIG. 3 is a first embodiment of the present invention, and is a cross-sectional view perpendicular to the capillary axis of a fluorescence measurement portion in which the medium surrounding the capillary gel is air.

FIG. 4 is a second embodiment of the present invention, and is a cross-sectional view of a capillary having an outer radius R and an inner radius r, showing an infinitely small laser beam incident at a position separated by a distance x from the axis of the capillary. The figure which shows the mode of refraction by one capillary.

FIG. 5 is a second embodiment of the present invention, in which the outer radius and inner radius of the capillary, the refractive index of the capillary material, the refractive index of the gel, the refractive index of the medium outside the capillary, and the laser light from the capillary gel are used. Refraction magnitude | Δθ |
And FIG.

FIG. 6 shows the change in | Δθ | with respect to the change in the refractive index of the medium outside the capillary when the outer radius of the capillary is changed, among the conditions assumed to obtain the results in FIG.
The figure shown about each medium outside a capillary.

FIG. 7 shows | Δθ when the ratio of the outer radius to the inner radius of the capillary and the refractive index of the medium outside the capillary are changed.
The figure which shows the change of |.

8 is one of the conditions assumed to obtain the results in FIG. 5, where | Δθ | with respect to the change in the refractive index of the medium outside the capillary when the outer radius of the capillary and the refractive index of the material of the capillary are changed. FIG. 6 is a diagram showing changes in the above for each medium outside the capillary.

9 is a diagram showing a second embodiment of the present invention, showing a configuration of a capillary gel electrophoresis apparatus using a fluorescence measurement cell instead of a glass flat plate in the basic configuration of the apparatus shown in FIG. .

10 is a cross-sectional view of a portion of the fluorescence measurement cell of FIG. 9 which is perpendicular to the capillary axis.

FIG. 11: A of Texas Red-labeled A out of the DNA sequencing results obtained in the second embodiment of the present invention.
The figure which shows the electrophoresis pattern of a component.

FIG. 12 is a fourth embodiment of the present invention, in which 10 gel-filled capillaries placed in air are provided.
Sectional drawing which is perpendicular to the capillary axis of the portion where a laser beam is irradiated to measure fluorescence.

FIG. 13 is a diagram showing an electrophoretic pattern of a component A labeled with Texas Red in the obtained DNA sequencing results in the fourth embodiment of the present invention.

FIG. 14 is a diagram showing an electrophoresis pattern of a component A labeled with Texas Red in the obtained DNA sequencing results in the fourth embodiment of the present invention.

FIG. 15 is a sixth embodiment of the present invention, and is a diagram showing a laser irradiation portion of a capillary electrophoresis apparatus using a capillary composed of two capillaries each having a different diameter dimension.

FIG. 16 is a seventh embodiment of the present invention, showing a configuration in which a laser beam is irradiated from two directions so as to pass through each capillary axis, and FIG. 16A shows a laser beam split by a semitransparent mirror. FIG. 3B is a diagram showing a configuration in which two capillaries are irradiated from two directions so as to pass through each capillary axis, and FIG.

[Explanation of symbols]

1 ... Capillary 2, 2-1, 2-2 ... Laser light, 3
... Glass flat plate, 4 ... First lens, 5 ... Image splitting prism and combination filter, 6 ... Second lens, 7 ... Two-dimensional CCD camera, 8 ... Acrylamide gel, 9 ... Air,
10 ... Distance of deviation from the capillary axis of the incident laser (x), 11 ... Incidence angle (θ ₁ ) from the medium outside the capillary to the capillary, 12 ... Refraction angle (θ ₂ ) from the medium outside the capillary to the capillary, 13 ... Angle of incidence from the capillary material to the medium inside the capillary (θ ₃ ), 14 ... Angle of refraction from the capillary material to the medium inside the capillary (θ ₄ ), 15 ... Angle of incidence from the medium inside the capillary to the capillary material ( θ ₅ ), 16
... refraction angle (θ ₆ ) from the medium inside the capillary to the capillary material, 17 ... incidence angle (θ ₇ ) from the capillary material to the medium outside the capillary, 18 ... refraction angle (θ from the capillary material to the medium outside the capillary) ₈ ),
19 ... Capillary array axis in which the axes of the capillaries are arrayed, 20 ... Refractive index (n ₁ ) of the medium outside the capillaries,
21 ... Refractive index of the capillary material (n ₂ ), 22 ... Refractive index of the medium inside the capillary (n ₃ ), 23 ... Outer radius of the capillary, 24 ... Inner radius of the capillary, 25 ... Fluorescence measuring cell, 26 ... Water , 27 ... Separation capillaries,
28 ... Measuring capillaries, 29-1, 29-2, 29
-3, 29-4 ... Semi-transparent mirror.

Continuation of the front page (56) Reference JP-A-6-138037 (JP, A) JP-A-7-20090 (JP, A) JP-A-5-281134 (JP, A) JP-A-5-60728 (JP , A) JP-A-6-94617 (JP, A) JP-A-6-294739 (JP, A) JP-A-63-231247 (JP, A) JP-A-5-157733 (JP, A) (58) Fields investigated (Int.Cl. ⁷ , DB name) G01N 27/447 G01N 21/64

Claims (6)

(57) [Claims] 1. A plurality of capillaries, at least a part of which are arranged in a plane, and transparent of each of the capillaries, which are parallel to a plane formed by axes of the plurality of capillaries and are opposed to each other. A capillary array electrophoresis apparatus comprising: a unit for irradiating a portion with a laser; and a unit for detecting fluorescence emitted from a component of a sample that migrates through each of the capillaries. 2. The electrophoretic device according to claim 1, wherein
A capillary array electrophoresis apparatus, characterized in that the laser is branched into two and irradiated from the two opposite directions. 3. The electrophoretic device according to claim 1, wherein
A capillary array electrophoretic device, characterized in that laser light having a plurality of wavelengths from a plurality of laser light sources is irradiated from the two facing directions. Wherein the medium outside of the capillary in which the laser to pass through, capillary array electrophoresis device according to any one of claims 1 to 3, characterized in that air. External medium of the capillary wherein said laser passes the capillary array electrophoretic device according to any one of claims 1 to 3, characterized in that its refractive index is in the range of 1.00 1.33. 6. The external medium of the capillary in which the laser passes may capillary array electrophoresis system according to any one of claims 1 to 3, characterized in that the refractive index is 1.28.