US20030102221A1

US20030102221A1 - Multi-capillary electrophoresis apparatus

Info

Publication number: US20030102221A1
Application number: US10/245,492
Authority: US
Inventors: Miho Ozawa; Masaya Kojima; Ryoji Inaba; Yoshitaka Kodama; Motohiro Yamazaki; Eric Nordman
Original assignee: Hitachi High Technologies Corp; Applera Corp
Current assignee: Hitachi High Tech Corp; Applied Biosystems LLC
Priority date: 2001-12-04
Filing date: 2002-09-18
Publication date: 2003-06-05
Also published as: CA2469197A1; EP2587256A2; EP1451567B1; AU2002351220A1; JP2003166976A; JP3979830B2; EP2587256A3; US20030127328A1; EP1451567A1; EP1451567A4; WO2003048755A1; US7459068B2

Abstract

Errors upon analysis caused by, fluctuation in electrophoresis time among plural capillaries in a multi-capillary electrophoresis apparatus is reduced. The multi-capillary electrophoresis apparatus contains a multi-capillary array that has an isolation medium filled therein for isolating a sample, has a sample injecting end on one end thereof, and has, at a position remote from the sample injecting end, a detector part for acquiring information depending on the sample, a voltage applying part for applying a voltage to an electrification path containing the sample injecting end and the detector part, a thermostat oven containing all or a part of the multi-capillary array except for the sample injecting end, a buffer container containing a buffer solution, in which the sample injecting end is immersed, and a temperature controlling part for controlling a temperature of the buffer solution.

Description

BACKGROUND OF THE INVENTION

The present invention relates to a multi-capillary electrophoresis apparatus having a multi-capillary array formed with plural capillaries each having a sample and an isolation medium filled therein, and more particularly, to an apparatus that can suppress measurement fluctuation upon analysis with a multi-capillary electrophoresis apparatus.

In recent years, a capillary electrophoresis apparatus having a capillary having an electrophoretic medium (isolation medium) filled therein, such as a high-polymer gel and a polymer solution, has been developed as shown in Japanese Laid-open Patent Publication JP-A 6-138037. A capillary electrophoresis apparatus has high heat dissipation capacity and can be applied with a high voltage, in comparison to the conventional slab gel electrophoresis apparatus, and therefore, it has such an advantage that electrophoresis can be carried out at a high rate.

FIG. 11 shows a schematic structure of an ordinary capillary electrophoresis apparatus. As shown in FIG. 11, the capillary electrophoresis apparatus B has a

capillary part

103, a thermostat oven 105, a detector part 107 and a buffer container 111.

The

capillary part

103 is formed with plural capillaries 103 a. The buffer container 111 is filled with a buffer solution 111 a. A sample and an isolation medium for isolating the sample are filled in the capillary 103 a. One end 103 b of the capillary 103 a is immersed in the buffer solution 111 a. The other end 103 c of the capillary 103 a is also immersed in, for example, a buffer solution.

The

detector part

107 is retained with a retaining part 107 b for retaining the capillaries 103 a. The detector part 107 is housed in the retaining part 107 b and a cover member 108. A high voltage is applied between the end 103 b and the other end 103 c of the capillary 103 a, whereby the sample is electrophoresed in the isolation medium. The sample thus isolated by electrophoresis is detected in the detector part 107 with an optical means. The retaining part 107 b has a window part 107 c for taking out fluorescence excited by the optical means.

In the capillary electrophoresis apparatus, Joule heat is generated upon applying a high voltage between the both

ends

103 b and 103 c of the capillary 103 a. In particular, air dissolved in the liquid of the isolation medium forms bubbles upon local increase of the temperature to rise the resistance of the isolation medium. When the isolation medium has high resistance, the electrophoresis migration velocity is lowered to cause adverse affects, such as deterioration in resolution power of the sample. In general, in order to prevent local generation of heat in the capillaries 103 a, it has been widely operated that the capillaries 103 a are placed in the thermostat oven 105, whereby the electrophoresis is carried out under the conditions where the temperature is maintained constant.

However, it is actually difficult to place the entire length of the capillaries in the thermostat oven. For example, the

end

103 b forming a sample injecting end for injecting the sample and the detector part 107 for detecting the sample with an optical means are not placed in the thermostat oven 105.

The

sample injecting end

103 b is difficult to be placed in the thermostat oven because maintenance is necessary for injecting the sample from the sample injecting end 103 b. Furthermore, the

ends

103 b and 103 c of the capillary 103 a are immersed in the buffer solution 111 a for electrophoresis. Therefore, it has been difficult to place the

ends

103 b and 103 c and the vicinity of the detector part 107 in the thermostat oven 105 that contains the central part of the capillaries (electrophoresis part).

In a multi-capillary apparatus having plural capillaries, the temperature of the isolation medium in the radial direction of the capillaries are liable to be fluctuated among the plural capillaries. In particular, it is often the case that the temperature is different between the capillaries arranged in the periphery and the capillaries arranged in the central part. It is considered that this is because the capillaries arranged in the periphery are liable to be affected by the outside air.

The temperature of the isolation medium in the capillaries at the

sample injecting end

103 b and a detecting part 103 d is liable to be differentiated from the temperature thereof inside the thermostat oven 105, and therefore, there is a possibility that the electrophoresis time is fluctuated.

A light emission part emitting, for example, laser light to the

detector part

107 is provided, and a CCD image sensor (or a CCD camera having the same) is also provided for receiving the laser light incident on the detecting part 103 d. Thermal noise of the CCD image sensor is increased by the influence of heat.

In order to reduce the thermal noise, it is necessary that the CCD image sensor arranged in the vicinity of the

detector part

107 is maintained at a low temperature as much as possible. Accordingly, it is not preferred that both the detector part 107 and the CCD image sensor are placed in the thermostat oven 105.

Therefore, in the

detector part

107, the temperature of the isolation medium in the radial direction of the capillaries is liable to be fluctuated among the plural capillaries.

The temperature of the isolation medium in the electrophoresis part that is maintained constant with the

thermostat oven

105 is sharply decreased in the vicinity of the detector part 107. When there is temperature fluctuation in the longitudinal direction of the capillaries, there is a possibility that the electrophoresis time is fluctuated.

SUMMARY OF THE INVENTION

An object of the invention is to provide a multi-capillary electrophoresis apparatus having a multi-capillary array containing plural capillaries in that fluctuation of the temperature, particularly fluctuation of the temperature in the radial direction, is reduced, whereby errors upon analysis caused by, for example, fluctuation of the electrophoresis time are reduced.

The invention relates to, as one aspect, a multi-capillary electrophoresis apparatus containing a multi-capillary array that has an isolation medium filled therein for isolating a sample, a sample injecting end on one end thereof, and a detector part for acquiring information depending on the sample at a position remote from the sample injecting end, a voltage applying part for applying a voltage to an electrification path containing the sample injecting end and the detector part, a thermostat oven containing all or a part of the multi-capillary array except for the sample injecting end, a buffer container containing a buffer solution, in which the sample injecting end is immersed, and a temperature controlling part for controlling a temperature of the buffer solution.

The invention also relates to, as another aspect, a multi-capillary electrophoresis apparatus containing a multi-capillary array that has an isolation medium filled therein for isolating a sample, a sample injecting end on one end thereof, and a detector part for acquiring information depending on the sample at a position remote from the sample injecting end, a voltage applying part for applying a voltage to an electrification path containing the sample injecting end and the detector part, a thermostat oven containing all or a part of the multi-capillary array except for the detector part, and a temperature controlling part for controlling a temperature of the detector part.

The invention also relates to, still another aspect, a multi-capillary electrophoresis apparatus containing a multi-capillary array that has an isolation medium filled therein for isolating a sample, a sample injecting end on one end thereof, and a detector part for acquiring information depending on the sample at a position remote from the sample injecting end, a voltage applying part for applying a voltage to an electrification path containing the sample injecting end and the detector part, a thermostat oven containing all or a part of the multi-capillary array except for the sample injecting end and the detector part, a buffer container containing a buffer solution, in which the sample injecting end is immersed, a temperature controlling part for controlling a temperature of the buffer solution, and a temperature controlling part for controlling a temperature of the detector part.

The temperature of the capillaries is controlled by the temperature controlling part, whereby fluctuation of the electrophoresis time in the radial direction among the plural capillaries is particularly reduced, and thus accurate analysis can be carried out for plural samples.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing the overall structure of the multi-capillary electrophoresis apparatus according to one embodiment of the invention, and FIG. 1 also shows the structure of the temperature controlling part (E). [0020]
FIGS. 2A and 2B are diagrams showing the structure of the electrode in the multi-capillary electrophoresis apparatus according to one embodiment of the invention, and FIG. 2C is a diagram showing the structure of the capillary in the vicinity of the sample injecting end inserted in the electrode. [0021]
FIGS. 3A and 3B are diagrams showing the structures of the buffer container (A) and the temperature controlling part (A) installed therein for controlling the temperature of the buffer solution (A) in the multi-capillary electrophoresis apparatus according to one embodiment of the invention. [0022]
FIGS. 4A to [0023] 4C are diagrams showing the structure of the detector part in the multi-capillary electrophoresis apparatus according to one embodiment of the invention.
FIGS. 5A to [0024] 5C are diagrams showing the structures of the container part containing the detector part and the temperature controlling part (B) controlling the temperature of the detector part in the multi-capillary electrophoresis apparatus according to one embodiment of the invention, and also showing the arrangement of the optical means.
FIGS. 6A and 6B are diagrams showing the structure of the temperature controlling part (C) controlling the temperatures among the capillaries in the vicinity of the outlet of the thermostat oven in the multi-capillary electrophoresis apparatus according to one embodiment of the invention. [0025]
FIG. 7A is a diagram showing the structures of the gel block, the buffer container (B) having the buffer solution (B) filled therein, and the conduit connecting them in the multi-capillary electrophoresis apparatus according to one embodiment of the invention, and particularly showing the structure of the temperature controlling part (D) controlling the temperature of the conduit and the buffer container (B). FIG. 7B is a side view of the cover member of the buffer container (B). [0026]
FIG. 8 is a diagram showing the relationship between the controlling part and the respective temperature controlling parts in the multi-capillary electrophoresis apparatus according to one embodiment of the invention. [0027]
FIG. 9 is a graph showing a standard deviation of the electrophoresis time among the capillaries upon using the multi-capillary electrophoresis apparatus according to one embodiment of the invention. [0028]
FIG. 10 is a diagram showing the structure of the detector part in the multi-capillary electrophoresis apparatus according to a modified embodiment of the invention. [0029]
FIG. 11 is a diagram showing the schematic structure of an ordinary multi-capillary electrophoresis apparatus.[0030]

DESCRIPTION OF THE PREFERRED EMBODIMENTS

As a result of earnest experimentation and investigations made by the inventors with respect to a multi-capillary electrophoresis apparatus, it has been found that fluctuation in analytical result among the capillaries of the multi-capillary array having plural capillaries can be suppressed by controlling the temperature of the buffer solution, in which the sample injecting end is immersed, with, for example, a heater. [0031]
Furthermore, it has been also found that fluctuation in analytical result among the plural capillaries can be suppressed by controlling the temperature of the detector part, particularly the temperature in the vicinity of the detecting part of the capillaries to be inspected. [0032]
A multi-capillary electrophoresis apparatus according to one embodiment of the invention will be described with reference to FIG. 1 to FIG. 9. [0033]
FIG. 1 is a diagram showing the overall structure of the multi-capillary electrophoresis apparatus according to one embodiment of the invention. [0034]
As shown in FIG. 1, the multi-capillary electrophoresis apparatus A according to one embodiment of the invention has a [0035] multi-capillary array 3 containing plural capillaries 3 a installed in a container part CS in a thermostat oven 5. The multi-capillary array 3 has plural, for example, 16 capillaries 3 a.
A [0036] sample 4 a containing, for example, specimens of DNA molecules, and an isolation medium 4 b functioning as a medium for isolating the DNA molecules in the sample 4 a has been filled in the capillaries 3 a. The isolation medium 4 b is constituted with, for example, a polymer in a gel form (FIG. 2C).
The DNA fragment sample contained in the [0037] sample 4 a can be distinguished by labeling the primer or the terminator with a fluorescent substance using the Sangar dideoxy method. The DNA fragment sample thus labeled with a fluorescent substance can be distinguished by the optical means described later.
One end of the capillary [0038] 3 a constitutes an injecting end 3 b for injecting the sample 4 a by protruding from the bottom of the thermostat oven 5. The injecting end 3 b is immersed in a buffer solution (A) 11 a. The buffer solution (A) 11 a is contained in a buffer container (A) 11. A electrode (A) 6 a is mounted on the introducing part 3 b.
The other end of the capillary [0039] 3 a protrudes from the side of the thermostat oven 5, and through a detector part 1 for acquiring information depending on the sample 4 a, forms an end part 3 d of the capillaries by packing the plural capillaries 3 a at a capillary fixing part 35. The end part 3 d is connected to an upper gel block 34. The upper gel block 34 is connected to a buffer container (B) 15 having a buffer solution (B) 15 a filled therein, a gel storage container 25 having a gel (isolation medium) 34 c filled therein, and a syringe 31.
As shown with the broken line, a thermostat oven RH may be provided to contain at least one of the [0040] upper gel block 34, the buffer container 15 and the syringe 31.
The multi-capillary electrophoresis apparatus A according to the embodiment has at least one temperature controlling part among first to temperature controlling part (E)s TCM[0041] 1 to TCM5, in addition to a temperature controlling part TCM0, which has been provided in an ordinary multi-capillary electrophoresis apparatus, for controlling the temperature of the capillaries 3 a with the thermostat oven 5.
The multi-capillary electrophoresis apparatus according to the embodiment will be described in detail below with a focus on the temperature controlling parts. [0042]
The temperature controlling part (A) will be described with reference to FIGS. [0043] 1 to 3B. The end of the sample injecting end 3 b of the capillary 3 a is immersed in the buffer solution (A) 11 a. The buffer solution (A) 11 a is filled in the buffer container (A) 11. As shown in FIG. 2A, a electrode (A) 6 a as an electrode on the side of the sample injecting end 3 b is formed by pressing stainless-steel tubes 6 a-1 made of stainless steel into a metallic plate 6 a-2.
As shown in FIG. 2B, the [0044] sample injecting end 3 b is inserted in the stainless-steel tubes 6 a-1 to integrate the sample injecting end 3 b and the electrode (A) 6 a. A positive electrode of a direct current power supply 21 (FIG. 1) is connected to the electrode (A) 6 a through an electrode (not shown in the figure) of the apparatus. Under the conditions where the sample injecting end 3 b is inserted in the stainless-steel tubes 6 a-1, the electrode (A) 6 a is installed in a cover PC made with a resin. As shown in FIG. 2C, the isolation medium 4 b is filled in the capillaries 3 a, and the sample 4 a is filled in the vicinity of the sample injecting end 3 b.
As shown in FIG. 1 and FIG. 3A, the [0045] sample injecting end 3 b and the electrode (A) 6 a are immersed in the buffer solution (A) 11 a filled in the buffer container (A) 11. The buffer solution (A) 11 a is prepared with, for example, TBE (a mixed solution of tris (hydroxymethyl) aminomethane, boric acid and EDTA (ethylenediaminetetraacetic acid)) or TAPS (N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid).
The buffer container (A) [0046] 11 is installed in an adapter AD having an opening on an upper part thereof. The adapter AD has a rubber heater 12 laid on the inner bottom surface thereof.
The [0047] rubber heater 12 and the adapter AD are waterproofed by sealing with silicone rubber SG. An opening 12 c is formed on an outer bottom surface 12 b′ of the adapter AD. A thermistor (temperature monitor) TM is attached to the back surface of the rubber heater 12 b exposed from the opening 12 c, and a first cable CB1 connected to the thermistor TM. Furthermore, a second cable CB2 connected to a power supply PS for the heater and a fuse FS is attached to the back surface of the rubber heater 12 b.
The temperature within the surface of the buffer container (A) [0048] 11 in contact with the rubber heater 12 b can be made constant by using the rubber heater 12 b. Maintenance operations, such as replacement of the heater, can be conveniently carried out by using such a structure that the buffer container (A) 11 is placed on the adapter AD lined with the rubber heater 12 b.
While the [0049] rubber heater 12 b is arranged in contact with the buffer container (A) 11, the term “contact” herein is not limited to such a constitution that both the members are physically and directly in contact with each other, but both the members may be, for example, in indirectly contact with each other. In other words, a sheet having a high heat conductance may be inserted between both the members. In essence, the term means that both the members are thermodynamically connected.
The temperature difference of the [0050] isolation medium 4 b in the plural sample injecting ends 3 b can be suppressed by using the temperature controlling part (A) TCM1.
The temperature controlling part (B) TCM[0051] 2 controlling the temperature of the detector part 1 will be described with reference to FIGS. 4A to 5C.
As shown in FIGS. 4A to [0052] 4C, the multi-capillary 3 formed with plural capillaries 3 a is supported by clipping between a capillary supporting part 77 made with, for example, a glass plate, and a pressing member 78. An outer periphery of the capillaries 3 a is covered with a light shielding resin 51 a, such as polyimide. A region that is not coated with the light shielding resin 51 a is provided on the outer periphery of the capillaries 3 a between the capillary supporting part 77 and the pressing member 78.
The region is irradiated with laser light L. The region is referred to as a detecting [0053] part 3 c. An opening 78 a is formed in the region containing the detecting part 3 c in the pressing member 78. Excitation light K generated upon irradiating the sample with the laser light is radiated to the exterior through the opening 78 a. The structures described herein are totally referred to as a detector part.
Fluctuation in intensity depending on the position of the laser light L incident on the [0054] capillaries 3 a can be suppressed by irradiating the capillaries 3 a with the laser light L from both above and beneath.
As shown in FIGS. 5A to [0055] 5C, the capillary supporting part 77 and the pressing member 78, as well as the multi-capillary 3 supported therebetween, are contained in a container part 7. The container part 7 is constituted with a main body 7 a and a cover member 7 b. The main body 7 a and the cover member 7 b are rotatablly connected with a hinge HG as a central axis.
The [0056] main body 7 a has a container part (A) 7 a-3, which can contain the capillary supporting part 77 and the pressing member 78, a groove (A) 7 a-1 and a groove (B) 7 a-2, which are connected to the container part (A) 7 a-3 and extend toward both sides, and a protrusion part 7 a-4, which is connected to the groove (B) 7 a-2 and protrudes from the side surface of the main body. The groove (A) 7 a-1, the groove (B) 7 a-2 and the protrusion (A) part 7 a-4 have a flat plane, on which the multi-capillary 3 can be arranged. The container part (A) 7 a-3 forms a concave part that is deeper than the flat plane. An opening 7 a-5 penetrating the main body 7 a is formed in the concave part.
In the [0057] cover member 7 b, on the other hand, a spring member SP is provided at a position corresponding to the container part (A) 7 a-3. Upon closing the cover member 7 b, the spring member SP presses the capillary supporting part 77, whereby the multi-capillary array 3 is strongly supported. A convex part (A) 7 b-1 and a convex part (B) 7 b-2 engaging with the groove (A) 7 a-1 and the groove (B) 7 a-2, respectively, are provided at positions corresponding to the grooves on both sides of the spring member SP. A protrusion (B) part 7 b-3 extending from the convex part (B) 7 b-2 is also provided at a position corresponding to the protrusion (A) part 7 a-4.
In the case where the [0058] cover member 7 b is closed, the protrusion part formed with the protrusion (A) part 7 a-4 and the protrusion (B) part 7 b-3 is contained in a concave part 35′ formed in the upper gel block 34 (FIG. 1) to connect the container part 7 and the upper gel block 34.
FIG. 5B shows such a state that the [0059] capillary supporting part 77 and the pressing member 78 are contained in the container part (A) 7 a-3, and the multi-capillary array 3 is contained in the container part (A) 7 a-3, the groove (A) 7 a-1 and the groove (B) 7 a-2. Laser devices 61 emitting laser light are provided above and beneath the main body 7 a. A through hole (A) 7 c-1 and a through hole (B) 7 c-2 are formed, whereby laser light L reaches a position corresponding to the detecting part 3 c (FIG. 4B).
Upon emitting the laser light L from the [0060] laser devices 61, the sample 4 a in the detecting part 3 c is irradiated with the laser light L to generate excitation light K. The excitation light K is emitted from the opening 7 a-5 and detected with a photo-accepting unit, such as a CCD camera 71 having a CCD image sensor 73.
The temperature controlling part (B) TCM[0061] 2 provided in the vicinity of the detector part 1 contains, for example, at least one of a rubber heater (A) 8 b-1 and a rubber heater (B) 8 b-2, which are attached to the surfaces of the groove (A) 7 a-1 and the groove (B) 7 a-2 facing the cover part 7 b, respectively, and a rubber heater (C) 8 a-1 and a rubber heater (D) 8 a-2, which are attached to the surfaces of the protrusion (A) part 7 b-1 and the protrusion (B) part 7 b-2 facing the main body 7 a, respectively. Furthermore, a temperature monitor 8 c is provided in the vicinity of the container part (A) 7 a-3. A thermal conductor sheet may be attached instead of the rubber heaters, or in alternative, a rubber heater and a thermal conductor sheet may be accumulated and attached.
Upon closing the [0062] cover part 7 b, the cover part 7 b and the main body 7 a may be fixed with a fixing screw FS. The term “vicinity” of the detector part 1 herein means a region where the temperature of the detector part 1 can be directly or indirectly controlled. For example, the heater may be provided on an outer peripheral surface of the container part 7.
The temperature difference of the isolation medium among the capillaries of the multi-capillary array at the [0063] detector part 1 can be suppressed by the temperature controlling part (B) TCM2.
The temperature controlling part (C) TCM[0064] 3 will be described with reference to FIGS. 6A and 6B. The multi-capillary 3 in the capillary containing part CS in the thermostat oven 5 is connected to the detector part 1 through the thermostat oven 5. An opening is formed on a side (outlet) of the detector part 1 in the thermostat oven 5. A concave part 5 a-1 is formed on a main body 5 a of the thermostat oven 5, and a convex part 5 b-1 engaging with the concave part 5 a-1 is formed on a cover part 5 b of the thermostat oven 5. The multi-capillary array 3 is inserted in a gap formed between the concave part 5 a-1 and the convex part 5 b-1 formed upon closing the cover part 5 b.
The temperature controlling part (C) TCM[0065] 3 contains rubber heaters HS1 and HS2 attached to the surfaces of the concave part 5 a-1 and the convex part 5 b-1 facing each other, and a temperature monitor HS′ provided in the vicinity thereof. The rubber heater may be attached to one of the facing surfaces of the concave part 5 a-1 and the convex part 5 b-1. A thermal conductor sheet may be attached instead of the rubber heater, or in alternative, both of them may be accumulated and attached.
The temperature difference of the isolation medium among the capillaries of the multi-capillary array directed from the [0066] thermostat oven 5 to the detector part 1 can be suppressed by the temperature controlling part (C) TCM3.
The fourth and temperature controlling part (E) TCM[0067] 4 and TCM5 will be described with reference to FIGS. 7A and 7B.
An [0068] upper gel block 34 is, for example, a block formed with an acrylic resin. A syringe 31, a gel storage container 25 and a buffer container (B) 15 are connected to the upper gel block 34. First to flow path (E)s 31 a to 31 e are formed in the upper gel block 34.
A [0069] fresh gel 34 c is filled in the gel storage container 25. The gel storage container 25 is connected to an end of the flow path (B) 31 b through a tube path (A) 34 b. A first valve (check valve) V1 is provided between an end of the tube path (A) 34 b and the flow path (B) 31 b to allow only the flow of the gel from the gel storage container 25 toward the upper gel block 34.
The [0070] syringe 31 and the upper gel block 34 are connected at a connecting part 31′. When a pin valve PV is closed, and a plunger of the syringe 31 is withdrawn for reducing pressure, the fresh gel 34 c in the gel storage container 25 is filled in the syringe 31 through the tube path (A) 34 b, the flow path (B) 31 b and the flow path (A) 31 a. When the pin valve PV is closed, and the plunger of the syringe 31 is pressed, the gel filled in the syringe 31 can be injected into the capillaries 3 a through the flow path (A) 31 a, the flow path (C) 31 c and the flow path (D) 31 d. The gel functions as the isolation medium 4 b inside the capillaries 3 a. The isolation medium 4 b after analysis can be discharged outside the capillaries 3 a through the sample injecting end 3 b of the capillaries by again charging the fresh gel by the foregoing operation.
A tube path (B) [0071] 15 b is provided between the flow path (E) 31 e in the upper gel block 34 and a flow path (F) 15 e of a lower gel block 15 c to connect them. The lower gel block 15 c has a protrusion part 15 c′ protruding downward. The pin valve PV for opening and shutting an end opening 15 d of the flow path (F) 15 e is attached to the lower gel block 15 c. A tip end of the pin valve PV reaches the interior of the protrusion part 15 c′. The isolation medium 4 b is filled in the flow path (E) 31 e in the upper gel block 34, the tube path (B) 15 b, and the flow path (F) 15 e in the lower gel block 15 c. A buffer solution (B) 15 a is filled in a buffer container (B) 15. The isolation medium 4 b may be filled in the buffer container (B) 15 instead of the buffer solution. The isolation medium 4 b and the buffer solution (B) 15 a are in contact with each other at the end opening 15 d of the flow path (F) 15 e.
Upon carrying out electrophoresis, the pin valve PV is moved in the withdrawing direction (upward in the figure). A [0072] tip end 6 b′ of a electrode (B) 6 b is grounded. Upon opening the pin valve PV, an electrification path between the electrode (A) 6 a and the electrode (B) 6 b through the buffer solution 11 a between the electrode (A) 6 a and the sample injecting end 3 b of the capillaries, the isolation medium 4 b filled in the sample injecting end 3 b of the capillaries, the capillaries 3 a, the end part 3 d of the capillaries, the flow path (E) 31 e in the upper gel block 34, the tube path (B) 15 b and the flow path (F) 15 e in the lower gel block 15 c, and the buffer solution (B) 15 a between the end opening 15 d of the flow path (F) 15 e and the electrode (B) 6 b.
Therefore, when the pin valve PV is opened, and a voltage is applied between the electrode (A) [0073] 6 a and the electrode (B) 6 b with the direct current power supply 21 (FIG. 1), such a voltage can be applied between both the ends of the electrification path (to be precise, the voltage is applied to the buffer solutions positioned on both ends of the isolation medium, which are filled in the electrification path). Consequently, an electric current can be ran in the isolation medium 4 b filled in the capillaries 3 a.
Upon opening the valve, the [0074] isolation medium 4 b is filled to the electrode of the apparatus through a hole 15 b′ and a hole 15 b″. Therefore, an electric current can be ran in the isolation medium 4 b filled in the capillaries 3 a.
In the case where the gel is filled in the [0075] capillaries 3 a, the pin valve PV is pressed. The electrification path formed with the isolation medium between the capillaries 3 a and the electrode on the apparatus is cut off with the pin valve PV. At this time, the isolation medium can be injected from the gel storage container 25 to the capillaries 3 a with the syringe 31.
As the temperature controlling part (D) TCM[0076] 4, a rubber heater 36 arranged on an outer surface of the tube path (B) 15 b and a temperature monitor 36 b attached to the outer surface of the tube path (B) 15 b exposed from an opening formed on the rubber heater 36. In alternative, it is possible to provide a rubber heater HT attached to an outer surface of the buffer container (B) 15 and a temperature monitor HT′ attached to an exposed surface of the buffer container (B) 15 exposed from an opening formed on the rubber heater HT. Both of them may be provided. A heater may also be provided on an outer surface of the upper gel block 34 or in the interior thereof.
The buffer solution (A) [0077] 11 a and the buffer solution (B) 15 a are prepared with, for example, TBE (a mixed solution of tris(hydroxymethyl)aminomethane, boric acid and EDTA (ethylenediaminetetraacetic acid)) or TAPS (N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid). The tube path (B) is similarly immersed in the buffer solution 15 a.
The [0078] buffer solutions 11 a and 15 a are filled in the buffer containers 11 and 15, respectively. The electrode (A) 6 a and the electrode (B) 6 b are immersed in the buffer solution 11 a and the buffer solution 15 a, respectively. The buffer solutions 11 a and 15 a connect electrically between electrodes and the separation medium in the capillary.
In FIGS. 7A and 7B, an upper surface of the buffer solution (B) [0079] 15 a is positioned above the end opening 15 d of the flow path (F) 15 e. Therefore, at least a part of the protrusion part 15 c′ of the lower gel block 15 c is immersed in the buffer solution (B) 15 a.
The temperature difference of the isolation medium in the capillaries of the multi-capillary array in the vicinity of the buffer container (B) can be suppressed by the temperature controlling part (D) TCM[0080] 4.
The temperature controlling part (E) TCM[0081] 5 will be described with reference to FIG. 1. The temperature controlling part (E) TCM5 controls the temperature of at least one of the upper gel block 34, the buffer solution (B) 15 a and the tube path (B) 15 b. It is preferred that the temperature controlling part (E) TCM5 is constituted with a thermostat oven RH and a temperature monitor RH′ equipped therein.
The temperature difference of the isolation medium in the capillaries of the multi-capillary array in at least one region of the [0082] upper gel block 34, the buffer solution (B) 15 a and the tube path (B) 15 b can be suppressed by the temperature controlling part (E) TCM5.
A temperature controlling function provided in the electrophoresis apparatus A will be described with reference to FIG. 8. [0083]
A [0084] temperature controlling part 26 is provided for carrying out the entire temperature control, for example, PID control. The temperature controlling part 26 carries out the entire temperature control of the capillary electrophoresis apparatus A.
As described in the foregoing, a [0085] temperature monitor 31 is provided in the thermostat oven 5 for monitoring the temperature inside the thermostat oven 5. The temperature monitor 31 sends a signal S1 to the controlling part 26, and the controlling part 26 sends a control signal S2 controlling the temperature in the thermostat oven 5 based on the signal S1, whereby the basic temperature controlling part TCM0 is constituted.
The temperature controlling part (A) TCM[0086] 1 contains the rubber heater 12 b and the temperature monitor 12 d. The temperature monitor 12 d sends a signal S3 to the controlling part 26, and the controlling part 26 sends a control signal S4 controlling the temperature of the buffer solution (A) 11 a based on the signal S3 through the rubber heater 12 b.
The temperature controlling part (B) TCM[0087] 2 contains the rubber heaters 8 a-1 and 8 a-2, the rubber heaters 8 b-1 and 8 b-2, and the temperature monitor 8 c. The temperature monitor 8 c sends a signal S5 to the controlling part 26, and the controlling part 26 sends a control signal S6 controlling the temperature of the isolation medium 4 b in the vicinity of the detector part 1 (in the detecting part 3 c of the capillaries) based on the signal S5.
The temperature controlling part (C) TCM[0088] 3 contains the rubber heaters HS1 and HS2 and the temperature monitor HS′. The temperature monitor HS′ sends a signal S7 to the controlling part 26, and the controlling part 26 sends a control signal S8 controlling the temperature in the vicinity of the outlet of the 1 oven 5 based on the signal S7 through the rubber heaters HS1 and HS2.
The temperature controlling part (D) TCM[0089] 4 contains the heater 36 and the temperature monitor 36 b, and further contains the heater HT and the temperature monitor HT′, which are in contact with the outer peripheral surface of the buffer container (B) 15. The temperature monitor 36 b and the temperature monitor HT′ send a signal S9 to the controlling part 26, and the controlling part 26 sends a control signal S10 controlling the temperature of the isolation medium 4 b in the vicinity of the upper gel block 34 based on the signal S9 through the heaters 36 and HT.
The temperature controlling part (E) TCM[0090] 5 contains the thermostat oven RH and the temperature monitor RH′ provided inside the thermostat oven RH. The temperature monitor RH′ sends a signal S11 to the controlling part 26, and the controlling part 26 sends a control signal S12 controlling the temperature of the thermostat oven RH based on the signal S11.
While the [0091] rubber heater 12 b is provided in contact with the bottom surface of the buffer container (A) 11, it may be provided on the outer side surface of the buffer container (A) 11. The rubber heater 12 b may be provided on one of the bottom surface and the side surface, and also may be provided both of them. Furthermore, the buffer container (A) 11 may be placed on a heater to carry out the temperature control.
The [0092] temperature controlling part 26 sends the temperature control signals S2, S4, S6, S8, S10 and S12 to the thermostat oven or the heaters based on the signals S1, S3, S5, S7, S9 and S11 sent from the temperature monitors to the controlling part 26, whereby the temperature control is accomplished. As the temperature monitor, for example, a platinum resistance thermometer and a thermocouple can be used.
As a method for temperature control, for example, the PID (proportional integral derivation) control may be used as described in the foregoing. That is, such a method can be used that the detection output from the temperature monitor is subjected to feedback to the heater and a Peltier element. [0093]
The temperature control is carried out to reduce the difference of the temperatures in the radial direction of the [0094] plural capillaries 3 a (i.e., the temperatures of the isolation medium in the capillaries at the positions that are distant from the ends thereof by the same distance). For example, when the temperature measured by the temperature monitor 31 attached to the thermostat oven 5 and the temperatures of the other parts are controlled to reduce the difference therefrom, there is such a tendency that the temperature difference in the radial direction is reduced.
A method for using the multi-capillary electrophoresis apparatus A (i.e., a method for analyzing a sample) will be briefly described below. [0095]
The [0096] isolation medium 4 b is filled in the capillaries 3 a by using the syringe 31. For example, 16 capillaries 3 a are used. Subsequently, a sample 4 a containing plural kinds of DNA molecules having different base lengths (DNA fragment sample) is introduced to the isolation medium 4 b filled in the capillaries 3 a through the side of the sample injecting end 3 b. The sample injecting end 3 b is immersed in the buffer solution (A) 1 a filled in the buffer container (A) 11. The PID control is carried out with the temperature controlling part 26 to reduce the temperature difference among the plural capillaries 3 a.
The temperature control is carried out to reduce the temperature difference in the radial direction of the [0097] plural capillaries 3 a by using at least one of the temperature controlling parts (A) to (E). Under the continued temperature control, a high voltage, for example, about from 10 to 20 kV, is applied between the electrode (A) 6 a (cathode) and the electrode (B) 6 b (anode) with the direct current power supply 21.
The DNA molecules migrate toward the electrode (B) [0098] 6 b (electrophoresed) because they are negatively filled. Differences in electrophoresis migration velocity of the DNA molecules occur corresponding to the base lengths thereof. The molecules having smaller base lengths exhibit larger electrophoresis migration velocities to require shorter periods of time to reach the detecting part 3 c. Upon irradiating the sample (DNA molecules) reaching the detecting part 3 c with laser light L, identification markers attached to the DNA molecules are excited to cause fluorescence. The fluorescence is subjected to photoelectric transfer with a photo acceptance unit (CCD image sensor) provided in a CCD camera 71. The DNA molecules can be distinguished by electric signals obtained from the CCD camera 71, and thus the species of DNA can be distinguished. Consequently, a sample containing DNA fragments is subjected to electrophoresis, and fluorescence from the sample is detected in the course of electrophoresis, whereby the DNA base sequencing can be carried out for determining the base sequence.
The [0099] isolation medium 4 b and the sample 4 a can be discharged to the outside through the path 31 e as described in the foregoing. It is preferred that the isolation medium 4 b is replaced per analysis of one sample, and a fresh isolation medium 4 b is used for analysis of a new sample.
FIG. 9 shows standard deviations of electrophoresis time in the case where [0100] 16 capillaries 3 a are used, in which a sample is injected, and the capillaries are simultaneously subjected to electrophoresis under the same conditions. The data shown in FIG. 9 are experimental results in the case where the temperature controlling part (A) TCM1 and the temperature controlling part (B) TCM2 are attached to the buffer container (A) 11 and the detector part 1, respectively.
In the case where no temperature controlling part is provided on the buffer container (A) [0101] 11 and the detector part 1 (i.e., an ordinary electrophoresis apparatus), the standard deviation of electrophoresis time among the 16 capillaries 3 a is about 0.62. On the other hand, in the case where only the temperature controlling part (A) TCM1 is provided, the standard deviation of electrophoresis time of the 16 capillaries 3 a is about 0.16. In the case where only the temperature controlling part (B) TCM2 is provided, the standard deviation of electrophoresis time of the 16 capillaries 3 a is about 0.13. In the case where both the temperature controlling part (A) TCM1 and the temperature controlling part (B) TCM2 are provided, the standard deviation of electrophoresis time of the 16 capillaries 3 a is about 0.13.
It can be understood from the results that the difference of the electrophoresis time among the 16 capillaries can be reduced by providing the temperature controlling part on one of the buffer container (A) [0102] 11 and the detector part 1 for carrying out temperature control.
A multi-capillary electrophoresis apparatus according to a modified embodiment of the invention will be described with reference to FIG. 10. FIG. 10 is a cross sectional view showing the structure of the [0103] detector part 1 of the multi-capillary electrophoresis apparatus.
As shown in FIG. 10, in the [0104] detector part 1 of the multi-capillary electrophoresis apparatus according to the modified embodiment, a pressing plate 78 and a good thermal conductor 7 d, such as Al, covering at least a part of the outer side surface of the pressing plate 78 are provided on the side opposite to a capillary supporting part 77 with the capillaries 3 a being inserted therebetween. A Peltier element 81 is attached in contact with the outer peripheral surface of the good thermal conductor 7 d. The other constitutions than those noted herein are the same as in the multi-capillary electrophoresis apparatus described in the foregoing.
The [0105] Peltier element 81 is connected to a direct current power supply 91. More specifically, the Peltier element 81 has an n-type semiconductor layer 81 a, a p-type semiconductor layer 81 b, an electrode 81 d that is formed on one surface of the n-type semiconductor layer 81 a and is connected to a negative electrode of a variable direct current power supply 91 capable of changing the output voltage, an electrode 81 c that is formed on one surface of the p-type semiconductor layer 81 b and is connected to a positive electrode of the variable direct current power supply 91, and a common electrode 81 e that is formed on the surfaces of the n-type semiconductor layer 81 a and the p-type semiconductor layer 81 b opposite to the one surfaces thereof and is commonly connected to both the semiconductor layers 81 a and 81 b.
A good [0106] thermal conductor 75 is formed in contact with a CCD image sensor 73. The CCD image sensor 73 and the common electrode 81 e carry out mutual heat exchange through the good thermal conductor 75. A temperature monitor is provided on the good thermal conductor 75. An electric signal based on the temperature measured by the temperature monitor is sent to the controlling part 26 (FIG. 1). The controlling part 26 sends a control signal for determining the voltage to be applied based on the electric signal to the variable direct current power supply 91.
In the case where the temperature measured by the temperature monitor [0107] 8 a′ is too low, for example, the controlling part 26 sends such a signal to the variable direct current power supply 91 that the voltage applied to the Peltier element 81 is increased. Upon increasing the voltage applied to the Peltier element 81, the temperature on the side of the electrode 81 c and the electrode 81 d is increased, and therefore, the temperature of the capillaries 3 a is increased. On the other hand, the temperature of the electrode 81 e is decreased, and thus the CCD solid image pickup element 73 can be cooled through the good thermal conductor 75. Accordingly, the noise of the CCD solid image pickup element 73 can be reduced.
The invention has been described with reference to the specific embodiments, but the invention is not construed as being limited thereto. It is apparent to a skilled person in the art that other various changes, improvements and combinations can be applied to the invention without deviating from the spirit thereof. [0108]
According to the multi-capillary electrophoresis apparatus of the invention, fluctuation of the electrophoresis migration velocity in the radial direction of plural capillaries can be suppressed. [0109]
Therefore, analysis of a sample can be carried out in a more accurate manner by using the multi-capillary electrophoresis apparatus. [0110]

Claims

What is claimed is:

1. A multi-capillary electrophoresis apparatus comprising:

a multi-capillary array having an isolation medium filled therein for isolating a sample, a sample injecting end on one end thereof, and a detector part for acquiring information depending on the sample at a position remote from the sample injecting end;

a voltage applying part for applying a voltage to an electrification path comprising the sample injecting end and the detector part;

a thermostat oven containing all or a part of the multi-capillary array except for the sample injecting end;

a buffer container containing a buffer solution, in which the sample injecting end is immersed; and

a temperature controlling part for controlling a temperature of the buffer solution.

2. A multi-capillary electrophoresis apparatus as claimed in claim 1, wherein the temperature controlling part comprises a heater in contact with the buffer container.

3. A multi-capillary electrophoresis apparatus comprising:

a thermostat oven containing all or a part of the multi-capillary array except for the detector part; and

a temperature controlling part for controlling a temperature of the detector part.

4. A multi-capillary electrophoresis apparatus as claimed in claim 3, wherein the temperature controlling part comprises a heater arranged in a vicinity of the detector part.

5. A multi-capillary electrophoresis apparatus comprising:

a thermostat oven containing all or a part of the multi-capillary array except for the sample injecting end and the detector part;

a buffer container containing a buffer solution, in which the sample injecting end is immersed;

a first temperature controlling part for controlling a temperature of the buffer solution; and

a second temperature controlling part for controlling a temperature of the detector part.

6. A multi-capillary electrophoresis apparatus as claimed in claim 5,

wherein the first temperature controlling part comprises a first heater for heating the buffer solution and a first sensor for measuring a temperature of the buffer solution; and

the second temperature controlling part comprises a heater for heating the detector part and a second sensor for measuring a temperature of the detector part.

7. A multi-capillary electrophoresis apparatus comprising:

a temperature controlling part for controlling a temperature in a vicinity of an outlet of the thermostat oven on a side of the detector part.

8. A multi-capillary electrophoresis apparatus comprising:

a thermostat oven containing all or a part of the multi-capillary array;

a gel block arranged outside the thermostat oven and charging the isolation medium in the capillary array; and

a temperature controlling part for controlling a temperature of the gel block.

9. A multi-capillary electrophoresis apparatus comprising:

a thermostat oven containing all or a part of the multi-capillary array; a flow path connected to the multi-capillary array and having the isolation medium filled therein; and

a temperature controlling part for controlling a temperature of the flow path.

10. A multi-capillary electrophoresis apparatus comprising:

a thermostat oven containing all or a part of the multi-capillary array;

a flow path connected to the multi-capillary array and having the isolation medium filled therein;

a buffer container containing a buffer solution, in which the flow path is immersed; and

11. A multi-capillary electrophoresis apparatus as claimed in claim 10, wherein the temperature controlling part comprises a heater in contact with the buffer container.

12. A multi-capillary electrophoresis apparatus as claimed in claim 1, wherein the detector part comprises a photo accepting unit receiving excitation light generated upon irradiating the sample with laser light, and a Peltier element cooling the photo acceptance unit or heating the detector part.