JP3536851B2 - Capillary array electrophoresis device - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to DNA, RNA,
Or, it belongs to the technical field related to separation and analysis equipment for proteins and the like, and particularly to a capillary array electrophoresis apparatus for determining the base sequence of DNA and RNA, or measuring the polymorphism of restriction enzyme fragments based on the diversity of individual base sequences. It belongs to such a technical field.

[0002]

2. Description of the Related Art Analysis techniques for DNA, RNA and the like are becoming increasingly important in the fields of medicine and biology, including gene analysis and genetic diagnosis. Particularly recently, particularly in connection with a genome analysis plan, development of a high-speed, high-throughput DNA analyzer has been progressing. In DNA analysis, a fluorescently labeled sample is subjected to molecular weight separation by gel electrophoresis, the sample is irradiated with a laser to detect fluorescence emitted from the fluorescent label, and a series of detected signals is analyzed. 0.3m for gel electrophoresis
A flat gel obtained by polymerizing acrylamide between two glass plates held at an interval of about m is widely used (slab gel electrophoresis). The sample injected into the migration start point at the upper end (one end) of the slab gel is migrated toward the lower end (the other end) while being separated in molecular weight by an electric field applied to both ends of the slab gel. At a certain distance from the starting point of the electrophoresis, a laser is irradiated from the side of the slab gel to irradiate all the electrophoresis paths at once, and excites the electrophoretically separated components of the fluorescently labeled sample passing through the laser irradiation part. I do. Fluorescence emitted by laser irradiation is detected continuously and periodically at fixed time intervals. By analyzing this detection result, D
The NA base sequence has been determined.

Recently, capillary gels obtained by polymerizing gels in capillaries made of fused quartz have been used instead of flat gels. 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-described slab gel electrophoresis (Analytical Chemistry).
62, 900 (1990)). Normally, a capillary gel electrophoresis apparatus uses a single capillary tube, irradiates the inside of the capillary near the lower end with a laser, and performs on-column measurement for detecting fluorescence emitted from a fluorescently labeled sample. Since the entire outer surface of the capillary is coated with polyimide, the coating at the position where fluorescence is detected is removed to expose the glass (US Pat. No. 5,315,535 (May 17, 1994)). The laser that irradiates the position where the glass is exposed excites the electrophoretically separated components of the fluorescently labeled sample that migrates inside the capillary by electrophoresis, and emits fluorescence.
The DNA base sequence is determined by detecting and analyzing the fluorescence continuously and periodically at fixed time intervals. However, in the above-described on-column measurement device, since the laser beam has a large refraction on the surface of the capillary, only one capillary can be used at a time, and there is a problem that the throughput does not increase. Thus, recently, several high-throughput capillary array gel electrophoresis apparatuses for arraying a plurality of capillaries and simultaneously analyzing many samples at high speed have been reported.

The first report is based on a capillary array scanning method (Nature, 359, 167 (199).
2)), in which a plurality of capillaries are sequentially irradiated with a laser one by one, and on-column fluorescence measurement is performed. This apparatus employs a confocal structure in which the position where the laser beam is most narrowed in the capillary and the position of the light source incident on the fluorescence receiving optical system are adopted, and each capillary can be measured independently. The laser irradiation and fluorescence receiving optical systems are fixed, and the stage holding the capillary array is moved in a one-dimensional direction to irradiate each capillary sequentially with the laser. The second report example is a capillary array sheath flow method (Nature, 361, 565-65).
566 (1993), JP-A-06-138037). In this device, the lower end of the capillary array is immersed in a buffer solution, the components of the sample whose molecular weight has been separated by gel electrophoresis are eluted directly into the buffer solution, and the fluorescence emitted from the components of the sample is released in a space where no capillary exists. In contrast to on-column measurement, in which detection is performed and off-column measurement (which irradiates the capillary with laser to detect sample components), the present specification uses a simpler method to detect sample components by irradiating laser in a space where no capillary exists. Therefore, it will be referred to as off-column measurement.).

[0005] In addition, by slowly flowing the buffer in the direction of electrophoresis, the separated components eluted from different capillary gels are mixed in the buffer, or two different components separated in one capillary gel are separated. Prevents mixing in buffer. In the vicinity of the outlet of the capillary array, the space where the buffer does not exist without the capillary is irradiated with laser to avoid the problem of scattering of the laser beam on the surface of the capillary, and the component eluted from the multiple capillaries, It is possible to excite substantially simultaneously and to detect fluorescence substantially simultaneously.

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

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

[0008]

In the capillary array scanning method of the first report, the fluorescence measurement is performed one capillary at a time, so that the time required for fluorescence detection for one capillary is determined by one capillary. It is reduced compared to the usual on-column measurement used. When n capillary arrays are used, the time required for fluorescence detection per line is at most 1 / n compared to the usual on-column measurement, and the glass part of the capillary through which the separated components of the sample do not actually pass is also used. Since it is scanned, it becomes 1 / n or less. As a result, there is a problem that the fluorescence detection sensitivity is reduced. That is, when n capillary arrays are used, in order to obtain the same fluorescence detection sensitivity as the normal on-column measurement using one capillary, the fluorescence in the normal on-column measurement is n times or more the detection time. It takes time.

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

[0010] In the capillary array sheath flow method of the second reported example, the fluorescence intensity obtained from the components separated in molecular weight is smaller than that in the case of the on-column fluorescence measurement.
There is a problem that as the molecular weight increases, the molecular weight decreases. This problem arises for the following reasons. In order to prevent the separated components eluted into the buffer from the lower end of the capillary gel from mixing in the buffer by diffusion or the like, it is necessary to constantly flow the buffer at a certain speed or higher in the direction of electrophoresis. . On the other hand, the migration speed of a sample component which migrates while being separated in molecular weight in a capillary gel decreases as the molecular weight increases. For the buffer flow rate,
The lower the migration speed of the sample component in the capillary gel, the more the sample component is elongated in the migration direction when eluted from the capillary gel into the buffer solution. As a result, the fluorescence intensity decreases, which causes the measurement sensitivity to decrease.

The third and fourth reports have a problem that the intensity of the laser beam irradiated per capillary decreases, so that the sample components separated by electrophoresis cannot be detected with high sensitivity.

[0012] An object of the present invention is to solve the above-mentioned various problems by substantially scanning a plurality of capillaries without mechanically scanning a plurality of capillaries or scanning a laser while performing on-column fluorescence measurement. It is an object of the present invention to provide a capillary array electrophoresis apparatus capable of simultaneously and simultaneously irradiating a laser beam and simultaneously and collectively measuring fluorescence emitted from a sample component in a plurality of capillaries.

[0013]

According to a capillary array electrophoresis apparatus of the present invention, at least a part of a plurality of capillaries for detecting components of a sample to be electrophoresed is arranged in a plane, and a laser beam is applied to the plurality of capillaries. In the capillary array electrophoresis apparatus for irradiation, (1) irradiating a plurality of capillaries with a laser beam from a direction parallel to a plane on which at least a part is arranged; One or two of the planes to be arranged
Irradiating a plurality of capillaries from one side in a direction parallel to the plane, (3) an outer radius of the capillaries,
The inner radius, the refractive index of the medium outside the capillary, the refractive index of the material of the capillary, the predetermined relationship between the refractive index of the medium inside the capillary, to sufficiently reduce the refraction of the laser light by the capillary, Irradiating the capillary with laser light having a sufficient intensity; (4) irradiating a plurality of capillaries with laser light substantially simultaneously without scanning the capillary array or the laser light, and In order to substantially simultaneously detect the fluorescence emitted from the components of the sample to be electrophoresed, the laser light is applied to one of the planes on which at least a part thereof is arranged.
Irradiating a plurality of capillaries from one or two sides in a direction parallel to the front upper plane; (5) applying a laser beam to a plurality of capillaries substantially without scanning a capillary array or the laser beam; Irradiation 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 to detect the fluorescence emitted from the components of the sample migrating in each of the capillaries substantially simultaneously Satisfying a predetermined relationship between the refractive index and the refractive index of the medium inside the capillary, making the refraction of the laser light by the capillary sufficiently small, and irradiating all the capillaries with laser light having sufficient intensity. Is a capillary array electrophoresis apparatus having the following.

Further, in addition to the above features (1) to (5), the capillary array electrophoresis apparatus of the present invention
(A) that the portion of each of the capillaries irradiated with the laser beam is transparent; that the laser beam passes through at least three media having different refractive indices; (b) each of the capillaries through which the laser beam passes 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 , the refraction 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) ｝ -S
in ^-1 {rn ₁ / (2Rn ₂ ))} + sin ^-1 {n ₁ /
(2n ₂ )｝｝ − sin ⁻¹ {n ₁ / (2n ₃ )} | ≦ 1.
(D) the medium outside each capillary through which the laser beam passes has a refractive index n ₁ = 1.33
From n ₁ = 1.37 to water or a buffer,
The ratio R / r between the outer radius and the inner radius is R / r = 2 ± 0.
2, each of the capillaries through which the laser beam passes is made of quartz glass having a refractive index n ₂ = 1.46 ± 0.02, and the refractive index of the medium inside each of the capillaries through which the laser beam passes is n ₃ = 1.37 ± 0.04, this medium is made of acrylamide gel, and (e) the medium outside each capillary through which laser light passes has a refractive index n ₁ =
Water or buffer in the range of 1.33 to n ₁ = 1.37, wherein 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 is
It is made of quartz glass having a refractive index n ₂ = 1.46 ± 0.02, and the refractive index of the medium inside each capillary through which the laser light passes is n ₃ = 1.40 ± 0.04, and this medium is (F) The medium outside the capillaries through which the laser light passes is air having a refractive index of n ₁ = 1.00, and the ratio of the outer radius to the inner radius R / r is R / r = 5 ± 0.5, and each capillary through which the laser beam passes has a refractive index n ₂
= 1.46 ± 0.02, the refractive index of the medium inside each capillary through which laser light passes is n ₃ = 1.37 ± 0.04, and this medium is made 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 between the outer radius and the inner radius is R / r =
3 ± 0.3, each capillary through which the laser beam passes is made of glass having a refractive index n ₂ = 1.60 ± 0.02, and the refractive index of the medium inside each capillary through which the laser beam passes is n ₃ = 1.37 ± 0.04, and this medium is also made of acrylamide gel.

[0015]

Embodiments of the present invention will be described below in detail with reference to the drawings. In FIG. 1, a plurality of capillaries 1 (six examples are shown in FIG. 1) are arranged on the same plane, and a laser 2 is irradiated from a direction parallel to this plane and substantially perpendicular to the direction of electrophoresis of sample components. 1 shows a main part of a capillary array electrophoresis apparatus that excites a plurality of capillaries substantially at the same time and detects sample components separated in each capillary by on-column measurement. FIG.
(A) is a plan view of a plurality of capillary arrays, FIG.
(B) 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 the separation capillaries and for detecting electrophoretically separated sample components. When the plurality of capillaries to be irradiated with the laser 2 are separation capillaries, the laser 2 is irradiated at a predetermined distance from the starting point of sample migration.
Each of these capillaries is filled with a gel.
Electrophoresis analysis with the capillary array electrophoresis apparatus of the present invention,
Alternatively, the sample to be analyzed contains a plurality of components that are fluorescently labeled. In addition, when the component contained in the sample itself has a property of emitting fluorescence by laser irradiation, the fluorescent label is unnecessary. In each of the drawings referred to below including FIG. 1, an electric field for applying an electric field for electrophoresis is shown.
Each unit such as a power supply, an electrode, an electrode and an electrode layer for accommodating an electrolytic solution, an arithmetic processing unit for detecting fluorescence from a sample component migrating in the capillary gel 1 and then processing these detection signals, an arithmetic processing A display for displaying the result, a controller for controlling each part of the capillary array electrophoresis apparatus, a laser light source, and the like are omitted.

(First Embodiment) This embodiment is an apparatus for DNA base sequence determination by five capillary gel electrophoresis, and the basic configuration of the apparatus is shown in FIG. However, FIG. 2 shows only the vicinity of a fluorescence measurement portion for detecting a sample component migrating 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)
Acrylamide was injected at a concentration of (real concentration) and then gelled (refractive index = 1.37). 30 from the capillary end on the sample injection side
At a position of 10 cm, the polyimide coating of each capillary was removed over a length of 10 mm to obtain a laser irradiated portion. FIG.
As described above, the five capillary gels were fixed on the glass flat plate 3 at a pitch of 0.2 mm while keeping the capillary axes parallel, 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 sectional view perpendicular to the capillary axis of the fluorescence measurement portion, and shows a YAG laser (532 nm, 20 nm).
mW), and a He-Ne laser (594 nm, 10 m
After making the beam W) coaxial, the diameter of the beam 2 was reduced to 0.1 mm, and the beam 2 was incident from a direction parallel to the plane in which the capillaries are arranged and almost perpendicular to the capillary axis (that is, the beam from the side of the capillary array). 2). The fluorescence was measured through the glass flat plate 3 in a direction perpendicular to the plane on which the capillary gels were arranged. As shown in FIG. 3, the measurement of the fluorescence can be performed from the opposite side of the flat glass plate 3 to a width of 0.8 mm in the horizontal direction.
An image splitting prism for converting five fluorescent light emitting point groups arranged in a line at an interval of 0.2 mm into a substantially parallel light flux by the first camera lens 3 by the configuration shown in FIG. The light was transmitted through four types of combination filters 5 corresponding to the light beams forming the four images, and was imaged on the two-dimensional CCD camera 7 by the second camera lens 6. This fluorescence sorting method is described in detail in JP-A-02-269936. The two-dimensionally developed 5 × 4 = 20 fluorescent light emitting points were exposed to the cooled two-dimensional CCD camera 7 at one time and measured. This measurement was continuously performed at an exposure time of 1.0 second and a data acquisition interval of 1.5 seconds.

The nucleotide sequences of the five DNA samples were determined according to the Sanger method. The sample is M
13mp18. 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). Here, the phosphors Cy3 and Cy5 are phosphors sold by Biological Detection Systems Inc. (USA), and TRITC and Texas Red are phosphors sold by Molecular Probes. Four kinds of combination filters 5 were used so that the fluorescences emitted from these four kinds of phosphors were wavelength-selected and imaged on the two-dimensional CCD camera 7. After mixing the four kinds of reactants corresponding to the terminal base type for each sample type, the mixture was concentrated 10-fold by ethanol precipitation, and the sample solvent was replaced with formamide. The sample injection ends of the five capillary gels were immersed corresponding to the five DNA sample solutions, and a constant electric field intensity of 100 V / cm (3 kV) was applied to both ends of the capillary gel for 2 seconds to perform sample injection. Done. As a result, different DNA samples are added to the respective electrophoresis starting points of the five capillary gels.

Electrophoresis is performed at a constant electric field strength of 100 V / cm.
(3 kV) for about 3 hours. 2D CCD camera 7
The time change of the four signals corresponding to the four types of fluorescence obtained in the above was analyzed by a computer, and the base sequence could be determined. However, the fluorescence intensities from the two capillaries on the side where the laser 2 emits were obtained. Was nearly an order of magnitude lower than the capillary on the laser incident side.

(Second Embodiment) In order to realize more efficient simultaneous laser irradiation on a plurality of capillaries, an example in which laser beam convergence conditions are examined and measurement is performed under optimal conditions will be described. 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 greatly refracted when passing through the first capillary. Since refraction occurs in all capillaries, the intensity of the laser light reaching and entering each capillary decreases almost exponentially, and therefore the intensity of the fluorescence emitted from the sample component in each capillary also decreases. 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 how an infinitesimally narrow laser beam 2 incident on a position separated by a distance x (10) from the axis of the capillary is refracted by one capillary. FIG. 4 shows a cross section of one capillary having the axis O of the capillary on the capillary arrangement axis 19 where the axes of the plurality of capillaries are arranged, and the other plurality of capillaries are omitted.

The refractive index of the medium 20 outside the capillary is n
₁ , the refractive index of the material 21 of the capillary is n ₂ , and the refractive index of the medium 22 inside the capillary is n ₃ . If the angle between 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 angle of incidence 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, θ ₅ is Angle of incidence 1 from the medium inside the capillary to the capillary material
5, θ ₆ is the refraction angle 16 from the medium inside the capillary to the capillary material, θ ₇ is the incident angle 17 from the capillary material to the medium outside the capillary, and θ ₈ is the refraction angle 18 from the capillary material to the medium outside the capillary. is there.

[0023]

Equation 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 relationships of θ ₁ = θ ₈ , θ ₂ = θ ₇ , θ ₃ = θ ₆ , and θ ₄ = θ ₅ were used. The geometrical relationship of the optical path of the laser beam 2 shown in FIG. 4 and the Snell at the interface of different media
By using the law of (1), (Equation 1) becomes (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]

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 beam intensity is further reduced, and a plurality of laser beams are reduced by the configuration shown in FIGS. 1 to 3. It is possible to realize more efficient simultaneous laser irradiation on the capillaries, and to excite the sample components that migrate in each capillary substantially simultaneously.

Normally, laser irradiation to the capillary is performed by narrowing the laser to about the inner diameter of the capillary. Normally, the laser intensity has a Gaussian distribution with the center 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 capillary, in a direction perpendicular to the array axis where the central axes of the capillaries are arranged, at a position at a distance of approximately half the inner radius of the capillary,
The laser intensity is approximately an intensity that gives the half width of the Gaussian distribution. Therefore, for simplicity, the capillary arrangement axis 1
Focusing on only the laser beam (that is, x = r / 2) that is incident on a position separated by a distance of 1/2 of the inner radius r from 9 (Equation 6),

[0030]

| 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)

To simultaneously excite a sample migrating in a plurality of capillaries, consider how spatially the laser beam spreads. First, from the first capillary into which the laser beam is incident, the displacement of the laser beam in the direction perpendicular to the direction of the arrangement axis where the central axes of the capillaries are arranged at the position of the N-th capillary is approximately │
NRΔθ |. Simultaneous excitation conditions for simultaneously exciting the sample migrating in the N-th capillary that the laser intensity in the N-th capillary is not less than (1/10) of the laser intensity in the first capillary. If the above condition is satisfied, the above displacement becomes r
{(10) -1} times or less of / 2. That is, (Equation 8) holds,

[0032]

| NRΔθ | ≦ {(10) -1} r / 2 (Equation 8) The (Equation 8) is the condition for simultaneous excitation of the N capillaries.

Here, it is assumed that the number of capillaries to be excited simultaneously is 5 or more (N ≧ 5). Considering that the ratio R / r between the outer shape and the inner diameter of the capillary is usually R / r ≧ 2, (Equation 8) becomes | Δθ | ≦ 6.2 °, and (Equation 7)
Therefore, the above 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 in the N-th capillary is (1/2) or more of the leather strength in the first capillary. , And the simultaneous excitation conditions in the N-th capillary, this condition is obtained by the same manner as in (Equation 8).
0).

[0035]

| NRΔθ | ≦ {(2) -1} r / 2 (Equation 10) Considering N ≧ 5 and R / r ≧ 2, | Δθ | ≦ 1.2
From (Equation 7), the 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 shows the result of calculating the change of | Δθ | using (Equation 7), and the outer radius R of the capillary is 0.1 mm, inner radius r = 0.05 mm, and the material of the capillary is quartz (n ₂
= 1.46) and the medium outside the capillary is assumed to be air (n ₁ = 1.00), water (n ₁ = 1.33), and quartz (n ₁ = 1.46). The change of | Δθ | with respect to the change of the refractive index (n ₃ ) of the medium outside the capillary is shown for each medium outside the capillary. The refractive index of acrylamide gel is estimated to be about 1.37.

In the case of the first embodiment, n ₁ = 1.0
Since 0 (air) and n ₃ = 1.37 (acrylamide gel), it is calculated from Expression 7 as | Δθ | = 6.48 °. From the results in FIG. 5, when the medium outside the capillary is filled with water (n ₁ = 1.33), | 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 is reduced, and the fluorescence from the sample components migrating in more capillaries can be measured simultaneously. The medium outside the capillary is made of the same material as the capillary, that is, quartz glass (n
₁ = 1.46), | Δθ |
= 4.40 °, it is more advantageous to use water as the medium outside the capillary.

According to the analysis by (Equation 7), if it is possible to fill the outside of the capillary with a medium having a refractive index between air and water, specifically, a medium having n ₁ = 1.28, | Δθ | Can be reduced, but in practice such a medium cannot usually 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. In Equation 7, n ₃ =
If it is 1.40, | Δθ | = 0.10 °, and the refraction is considerably reduced. As a method for increasing the refractive index of acrylamide gel, for example, formamide (refractive index 1.
45) may be mixed at an appropriate concentration. Formamide is a denaturing agent like urea, and the contamination of about 10% has the effect of increasing the resolution of electrophoresis, and the contamination of 20% or more decreases the electrophoresis speed (Elect).
rophoresis, 13, 484-486 (199)
2)). However, the refractive index of acrylamide gel is 1.3.
In order to increase from 7 to 1.40, the incorporation of about 10% formamide is sufficient.

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 results of FIG. The change in | Δθ | with respect to the change in the rate (n ₃ ) is calculated using (Equation 7) and is shown for each medium outside the capillary. 6 and FIG. 5,
By increasing the outer radius R by 2.5 times while keeping the inner radius r of the capillary unchanged, n ₁ = 1.00 and n ₁ = 1.
It can be seen that the curve for 33 is shifted to the right. As a result, the refractive index of acrylamide gel n ₃ = 1.
37, air outside the capillary (n ₁ = 1.0
0), the refraction becomes as small as | Δθ | = 0.87 °, and it can be determined that the condition is suitable for simultaneously measuring the fluorescence from the 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 and the medium inside the capillary has a refractive index n ₃ =
When the acrylamide gel is made of 1.37, the medium outside the capillary is 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 2
The change of | Δθ | with respect to the ratio R / r of R and the 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 |
| Δθ | increases as R / r decreases. 5 and 6
As in the results shown in FIG. 5, when R / r = 5, | Δθ |
At 7 ° and R / r = 2, | Δθ | = 6.48 °, and R
The condition of / r = 5 is a more suitable condition for simultaneously irradiating more capillaries.

The medium outside the capillary is water (refractive index n _1).
= 1.33), and when R / r is about 1.4, |
Δθ | becomes minimum, and | Δθ | increases as R / r increases. In a normally available capillary, 2 ≦ R /
Capillaries with r ≦ 8 and R / r = 1.4 cannot be used in practice. As in the results shown in FIGS. 5 and 6, when R / r = 2, | Δθ | = 1.26 ° and R / r = 5
| Δθ | = 2.86 °, and the case of R / r = 2 is a more suitable condition 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 minimal, a medium with a refractive index n ₁ = 1.28 cannot normally be present, so that such a virtual medium cannot be used in practice.

FIG. 8 shows that, among the conditions assumed when obtaining the results of FIG. 5, 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 set to n.
The change of | Δθ | with respect to the change of 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. Are shown for each of the media outside. Refractive index = 1.6
Materials with 0 include P, which is sold by Ohara Co., Ltd.
There is HM53 (glass part number). The result of FIG. 8 is the same as that of FIG. 6, and the refractive index n ₃ of the acrylamide gel = 1.37.
Air outside the capillary (n ₁ = 1.00)
Is satisfied, the refraction 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 migrating in a larger number of capillaries.

In this embodiment, the DNA base sequence was determined by electrophoresis of five capillary gels. The used apparatus uses a fluorescence measurement cell 25 instead 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)
6), 4% T containing 7M urea as a denaturant (T
total monomer concentratio
n), 5% C (Crosslinking material)
acrylamide at a concentration of ial concentration was injected and then gelled (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 capillary end on the sample injection side, a length of 10
In mm, the polyimide coating of each capillary was removed to obtain a laser irradiated portion. 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 measurement cell 25 of the capillary gel is arranged.

FIG. 10 is a sectional view of a portion of the fluorescence measurement cell 25 perpendicular to the capillary axis. In this cross-sectional view, at least the surface on the side where the laser beam enters and exits (or the entire surface or only the portion where the laser beam enters and exits) and the surface on the side where the fluorescence emitted from the sample component migrating in the capillary is detected (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 measurement cell 25, the axes of the capillaries are arranged in parallel, fixed so that the axes of the capillaries are substantially in the same plane, and the outside of each capillary is water (n).
₁ = 1.33). (1st Embodiment)
After the same two kinds of lasers used in the above were made coaxial, the beam diameter was reduced to 0.1 mm, and the light was made to enter through the transparent wall of the fluorescence measurement cell 25 from the side of the plane where the axes of the capillary gels were arranged. Was. In the first embodiment, the measurement of the fluorescence is performed through the glass plate 3 from a direction perpendicular to the plane on which the capillary gel is arranged. In the present embodiment, the transparent material of the fluorescence measurement cell 25 is used. Fluorescence is measured from the portion consisting of Except for this point, the configuration, conditions, and method of measuring fluorescence are exactly the same as those of the first embodiment. In addition, used 5
The species DNA sample, the labeled fluorophore, the electrophoresis conditions including the addition of the sample to the electrophoresis start point, and the like are exactly the same as those in the first embodiment. In this way, the electrophoresis is performed for about 3 hours, the time change of four kinds of signals corresponding to the four kinds of fluorescence obtained by the two-dimensional CCD camera is analyzed by a computer, and the DNA base sequences of the five kinds of samples are analyzed. Were determined.

FIG. 11 shows, among the DNA sequence results obtained in the present embodiment, A labeled with TexasRed.
3 shows the electrophoresis pattern of the components. FIG. 11 shows migration time 3
It shows the electrophoresis pattern in the range of 0 to 60 minutes, and shows the pattern of the A component from the primer to about 130 bases in length (the analyzed samples were M13 of the standard sample for all five species).
mp18). The numbers shown in the right column of FIG. 11 indicate the capillary numbers, and the laser beams are incident in the order of the capillary numbers 1, 2, 3, 4, and 5. That is, the laser beam first enters the uppermost capillary 1, then the laser beam passing through the capillary 1 enters the next capillary 2, and finally passes through the capillaries 5 through the capillaries 1 to 4. Laser beam is incident. FIG.
Shows the electrophoresis pattern in the order of the capillaries moving away from the capillary 1 on which the laser beam first enters. The peak intensity decreases with increasing 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 of that of the capillary 1, but the S / N (base length 9
At the position corresponding to 1, S / N = 734), it is possible to detect the migration peak. The S / N at the peak of the capillary 1 was S / N at a position corresponding to a base length of 91.
N = 3300.

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

(Third Embodiment) In this embodiment, the second embodiment
In the embodiment), the number of capillaries is increased to ten, and 4% T (Total monomer copolymer) containing 7 M urea using a capillary gel as a denaturant is used.
centration), 5% C (Crosslink)
ing material concentratio
This is an embodiment prepared by adding 10% formamide to acrylamide having a concentration of n) and polymerizing it. As a result of changing the composition of the capillary gel 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)
And exactly the same. From equation (7), | Δθ | = 0.10
°, and as described below (the fourth embodiment), the refraction by the capillary becomes smaller, and the fluorescence emitted from the sample component to be migrated can be measured with high sensitivity.
DNA sequencing of the seed samples was achieved by electrophoresis using 10 capillary gels.

(Fourth Embodiment) This embodiment is an embodiment in which a capillary is arranged in the air. FIG.
A cross-sectional view perpendicular to the capillary axis of a portion for measuring fluorescence by irradiating a laser beam 2 to ten capillaries 1 filled with a gel 8 arranged in air 9 is shown. The capillary used has an outer diameter of 0.5 mm (outer radius, R =
0.25 mm), inner diameter 0.1 mm (inner radius, r = 0.
05 mm) and a fused quartz tube (n ₂ = 1.46)
As shown in FIG. 12, the arrangement of the ten capillaries 1 is arranged at 0.5 mm pitch on the glass flat plate 3 in the same manner as in FIG. 3, and the respective capillary axes are arranged in parallel, and the respective capillary axes form substantially the same plane. So fixed. In the present embodiment,
The fluorescence measurement cell was not used, and the medium outside the capillary was air (n ₁ = 1.00). The other experimental conditions were exactly the same as those of the second embodiment. From (Equation 7),
Is calculated as | Δθ | = 0.87 °, and the refraction by the capillary becomes sufficiently small. Therefore, this experimental condition is that the laser light intensity applied to each capillary is not greatly attenuated, and that the light intensity in 10 or more capillaries can be measured. This is a condition under which the fluorescence emitted from the sample components to be migrated can be simultaneously excited.
As shown in FIG. 12, the detection of the fluorescence is performed from the side of the glass flat plate 3 or the opposite side of the glass flat plate 3.

FIGS. 13 and 14 show the D obtained in this embodiment.
It is an electrophoresis pattern of the A component labeled with Texas Red in the NA sequence results. This is an electrophoresis pattern having a migration time of 30 minutes to 60 minutes, and shows a pattern of the A component from the primer to about 120 bases in length.
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 each capillary 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 applied to the capillary 1, then the laser beam passing through the capillary 1 is applied to the capillary 2,..., The laser beam passing from the capillary 1 to the capillary 9 is applied to the capillary 10. FIG.
3 and FIG. 14 show the electrophoresis patterns obtained from the capillaries 2, 3,..., 10 moving away from the capillary 1 which is first irradiated with the laser beam, and all of the ten capillaries have a sufficient S / N (base length). In the peak giving 91, the S / N is 237 for capillary 1, 237 for capillary 4, 110 for capillary 7, and 142 for capillary 10 (S / N for capillary 10).
The reason why N is larger than the S / N in the capillary 7 is considered to be measurement variation). The electrophoresis pattern of sample components labeled with other fluorescent substances is also shown in FIG.
3. Obtained in the same manner as in FIG. 14, the fluorescence generated from the sample components to be electrophoresed can be measured with high sensitivity by electrophoresis using ten capillary gels, and the DNA base sequence can be determined.

(Fifth Embodiment) In this embodiment, the (fourth embodiment)
In the embodiment), the capillary used has an outer diameter of 0.3 mm (outer radius, R = 0.15 mm) and an inner diameter of 0.3 mm.
1 mm (inner radius, r = 0.05 mm), and the refractive index of the material of the capillary is n ₂ = 1.60. Specifically the material of the capillary, with a PHM53 being released from Ltd. Ohara (n ₂ = 1.60). The other experimental conditions were exactly the same as in (Fourth Embodiment). From (Equation 7), it is calculated that | Δθ | = 0.84 °,
The refraction by the capillary is smaller than that of | Δθ | = 0.87 ° in the fourth embodiment. Therefore,
The sample components migrating in the ten capillaries can be excited substantially simultaneously by the laser beam, and ten kinds of samples can be excited by one.
Electrophoresis using a capillary gel can measure fluorescence emitted from sample components to be migrated with high sensitivity,
A base sequencing could be realized.

(Sixth Embodiment) In the present embodiment, FIG.
As shown in FIG. 5, the capillary used is composed of two types of capillaries, each having a different diameter. Each of the ten capillaries used is
In the electrophoresis 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)
mm), and a portion for measuring fluorescence by irradiating the laser beam 2 with a capillary having a dimension of 0.5 mm (outer radius, R = 0.25 m)
m) and a capillary (measurement capillary 28) having a diameter of 0.1 mm (inner radius, r = 0.05 mm). As shown in FIG. 15, the separation capillary 27 and the measurement capillary 28 are connected, and the axes of the measurement capillary 28 are parallel to each other at a 0.5 mm pitch on the glass plane in the same manner as in FIGS. The array was fixed on a glass flat plate such that the axis of each measurement capillary 28 was flat. At this time, the central axes of the ten sets of the separation capillaries and the measurement capillaries were made to coincide with each other. The material of both capillaries was quartz (n ₂ = 1.46), and the connection surfaces were polished and flattened in advance.

Both capillaries were provided with 4% T (Totalmonomer c) containing 7M urea as a denaturing agent.
concentration, 5% C (Crossli
nking material concentrat
acrylamide at a concentration of (ion) was injected and gelled. The connecting portion of the capillary was immersed in a buffer solution so as not to be electrically disconnected, and the sample component separated by migrating the capillary for separation was allowed to migrate to the opposing measurement capillary as it was.

The separation capillary was 25 cm long, and the measurement capillary was 15 cm long. However, FIG. 15 shows only the vicinity of the connection portion between both capillaries. 5c from the connection position between the separation capillary and the measurement capillary
At position m, the polyimide coating of the measurement capillary was removed in advance over a length of 10 mm, and this position was irradiated with a laser beam 2 as shown in FIG. At this time, the migration distance is (25 + 5) cm = 30 cm. The other experimental conditions were exactly the same as in the fourth embodiment.
That is, the apparatus configuration conditions in the present embodiment are such that the refraction of the laser by the capillary is sufficiently small, and the sample components for migrating the ten capillaries without greatly attenuating the intensity of the laser light applied to each capillary. Can be excited at substantially the same time, and the fluorescence emitted from the sample components to be electrophoresed can be measured with high sensitivity by electrophoresis of 10 kinds of samples using 10 capillary gels.
The sequencing was realized. Although not shown in FIG. 15, the detection of fluorescence is performed from the side of the flat glass plate or from the opposite side of the flat glass plate.

In general, the price of the capillary increases as the outer diameter increases and the inner diameter decreases, that is, as the wall thickness increases, the cost increases almost in proportion to the volume of the glass. That is, the price per unit length of the separation capillary and the measurement capillary used in the present embodiment is eight times higher than that of the latter. In the present embodiment, the separation capillary and the measurement capillary can be attached and detached. Therefore, only the separation capillary is replaced after the measurement on one sample is completed, and the measurement on the next sample can be performed, and the cost can be reduced. This is another effect of the present embodiment.

The capillary used in this embodiment, which is shown in FIG. 15 and is composed of two types of capillaries having different diameters, 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 present embodiment.

(Seventh Embodiment) In the above (First Embodiment) to (Sixth Embodiment), each capillary axis is set in one direction parallel to the plane formed by the capillary gels arranged in a plane. The configuration for irradiating the laser beam so as to pass the laser beam has been described. However, in the first embodiment to the sixth embodiment, as shown in FIG. 16, the laser beam passes through each capillary axis from two directions as shown in FIG. May be applied. In the configuration of FIG. 16A, the laser beam 2 (which may be a single-wavelength laser beam or a laser beam obtained by mixing laser beams of a plurality of wavelengths from a plurality of laser light sources and made coaxial) may be converted into a semi-transparent mirror 29. , And 30-1, 30-2,
According to 30-3, a laser beam is irradiated so as to branch and pass through each capillary axis from two directions. FIG. 16B shows a configuration in which two laser beams 2-1 and 2-
2 is applied to a plurality of capillaries from two opposite directions, and each of the laser beams 2-1 and 2-2 emits a single-wavelength laser beam or a plurality of laser beams from a plurality of laser light sources. Laser light which is mixed and coaxial may be used. In the configurations of FIGS. 16A and 16B, in (First Embodiment) to (Sixth Embodiment), the non-uniformity of the intensity of the laser light applied to each capillary is improved to improve the efficiency. Each capillary can be irradiated with laser light having a similar intensity. As a result, capillary array electrophoresis using a larger number of tubes becomes possible. Note that FIG.
In the configuration (a), a laser beam is irradiated from two directions so as to pass through each capillary axis, so that the laser light irradiation intensity is reduced. Therefore, it is desirable to use a high-power laser light source. Further, when the configuration of FIG. 16B is applied from (first embodiment) to (sixth embodiment), the wavelengths of one or more laser beams included in the laser beam 2-1 are determined. The wavelength of one or more laser beams included in the laser beam 2-2 may be the same or different.

(Eighth Embodiment) In the above (First Embodiment) to (Seventh Embodiment), acrylamide gel is used in all cases, but other separation media may be used. Using a replaceable separation medium, the capillary itself may be reused. However, optimization of the refractive index of each medium and the diameter of the capillary based on, for example, (Equation 7) according to the refractive index of the separation medium originates from a sample component that migrates in a plurality of capillaries. This is important for efficient simultaneous measurement of fluorescence with high sensitivity. From equation (7), | Δθ | is not determined by the outer diameter 2R and the inner diameter 2r of the capillary alone, but | Δθ | is determined by the ratio R / r between the outer diameter and the inner diameter.
Therefore, in each of the embodiments described above, even when 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. However, 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 on which the sample component migrates is irradiated with the laser beam to improve the efficiency. Fluorescence from sample components can be detected well.

Lastly, the present invention will be briefly summarized. In a capillary array electrophoresis apparatus, a plurality of capillaries are irradiated with laser light substantially simultaneously without scanning the capillary array or laser light, and each capillary is electrophoresed. Fluorescence emitted from the sample components that migrate is detected substantially simultaneously, and on-column fluorescence measurement is performed. That is, a plurality of capillaries are arranged on one plane, and a laser is irradiated from one or two sides of the arrangement plane in parallel with the arrangement plane. By satisfying a predetermined relationship between the outer radius and inner radius of the capillary, 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 refraction of the laser by the capillary is sufficiently reduced. 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 to migrate through each capillary. Fluorescence emitted from sample components to be detected can be detected substantially simultaneously, and on-column fluorescence measurement that can easily achieve high-sensitivity, high-speed, high-throughput analysis can be realized.

[Brief description of the 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 a migration direction of sample components. Showing the main parts of
(A) A plan view of a plurality of capillary arrays, and (b) a cross-sectional view thereof.

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

FIG. 3 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, according to the first embodiment of the present invention.

FIG. 4 is a cross-sectional view of a capillary according to a second embodiment of the present invention having an outer radius R and an inner radius r, and showing an infinitesimal laser beam incident at 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 by the capillary gel. Magnitude of refraction | Δθ |
FIG.

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

FIG. 7: | 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 |.

FIG. 8 shows | Δθ | with respect to changes 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 among the conditions assumed when obtaining the results of FIG. FIG. 4 is a diagram showing changes in the medium for each medium outside the capillary.

9 is 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. 2; .

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

FIG. 11 shows A of the DNA sequence results obtained in the second embodiment of the present invention, which is labeled with TexasRed.
The figure which shows the electrophoresis pattern of a component.

FIG. 12 is a fourth embodiment of the present invention, in which 10 capillaries filled with gel, which are placed in air,
FIG. 4 is a cross-sectional view of a portion perpendicular to a capillary axis of a portion where fluorescence is measured by irradiation with a laser beam.

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

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

FIG. 15 is a view showing a laser irradiation part of a capillary electrophoresis apparatus according to a sixth embodiment of the present invention, which uses 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. 16 (a) divides the laser beam with a translucent mirror; FIG. 3B is a diagram illustrating a configuration in which the laser beam is irradiated from two directions to pass through each capillary axis, and FIG. 4B illustrates a configuration in which two laser beams are irradiated to a plurality of capillaries from two opposite directions.

[Explanation of symbols]

1. Capillary, 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 of the incident laser from the capillary axis (x), 11: angle of incidence from the medium outside the capillary to the capillary (θ ₁ ), 12: refraction angle from the medium outside the capillary to the capillary (θ ₂ ), 13: Angle of incidence (θ ₃ ) from the capillary material to the medium inside the capillary, 14: Refraction angle (θ ₄ ) from the capillary material to the medium inside the capillary, 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: a capillary array axis in which the axes of the respective capillaries are arranged; 20: a refractive index (n ₁ ) of a medium outside the capillary;
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: cell for fluorescence measurement, 26: water , 27 ... Capillary for separation,
28: Capillary for measurement, 29-1, 29-2, 29
-3, 29-4: Translucent mirror.

──────────────────────────────────────────────────続 き Continuation of the front page (56) References JP-A-5-60728 (JP, A) JP-A-5-281134 (JP, A) JP-A-5-79469 (JP, U) 506424 (JP, A) U.S. Pat. No. 5,324,401 (US, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) G01N 27/447 G01N 21/64

Claims (5)

(57) [Claims] 1. A plurality of at least some of which are arranged in a plane
And the plurality of capillaries arranged in a plane.
Each of the capillaries is viewed from a direction parallel to the plane defined by the axes of the pillar.
Means for irradiating a transparent part of the pillary with a laser;
Fluorescence emitted from the components of the sample migrating the capillary is detected.
Means for emitting the laser beam, and
The outer radius of the pillar is R, the inner radius is r, and the refractive index is n.₂When
And the outside of each capillary through which the laser passes
Let the refractive index of the medium be n₁And the laser passes through each
Let the refractive index of the medium inside the capillary be n₃When (360 / π) | sin^-1{R / (2R)}-sin
^-1｛Rn₁/ (2Rn ₂)｝ + Sin^-1｛N₁/
(2n₂)｝-Sin^-1｛N₁/ (2n₃)｝ | ≦
6.2 ° Capillary array characterized by satisfying the following relationship:
Electrophoresis device. 2. A plurality of at least some of which are arranged in a plane
And the plurality of capillaries arranged in a plane.
Each of the capillaries is viewed from a direction parallel to the plane defined by the axes of the pillar.
Means for irradiating a transparent part of the pillary with a laser;
Fluorescence emitted from the components of the sample migrating the capillary is detected.
Means for emitting the laser beam, and
The outer radius of the pillar is R, the inner radius is r, and the refractive index is n.₂When
And the outside of each capillary through which the laser passes
Let the refractive index of the medium be n₁And the laser passes through each
Let the refractive index of the medium inside the capillary be n₃When (360 / π) | sin^-1{R / (2R)}-sin
^-1｛Rn₁/ (2Rn ₂)｝ + Sin^-1｛N₁/
(2n₂)｝-Sin^-1｛N₁/ (2n₃)｝ | ≦
1.2 ° Capillary array characterized by satisfying the following relationship:
Electrophoresis device. 3. The capillary array electrophoresis apparatus according to claim 1, wherein a medium outside the capillary through which the laser passes is air. 4. The capillary array electrophoresis apparatus according to claim 1, wherein a medium outside the capillary through which the laser passes has a refractive index in a range of 1.00 to 1.33. 5. The capillary array electrophoresis apparatus according to claim 1, wherein a medium outside the capillary through which the laser passes has a refractive index of 1.28.