CAPILLARY ELECTROPHORESIS DEVICE AND THERMOSTAT

Abstract

Capillary electrophoresis device comprising: a thermostat (1) with a heat source, a first heat conduction layer and a detection window (41) for detecting a sample, wherein the thermostat (1) is designed to keep a capillary (6) at a predetermined temperature; a capillary holder (5) with a second heat conduction layer to sandwich the capillary (6) between the second heat conduction layer and the first heat conduction layer, wherein the capillary holder (5) is designed to hold the capillary (6); and a detection unit designed to detect the sample to be electrophoresed in the capillary (6), wherein in the heat source, a heat generation amount of a periphery of the detection window (41) and / or an end of the capillary (6) is higher than a heat generation amount of another portion, and wherein the capillary electrophoresis device is characterized in that the capillary holder (5) has recesses (55) arranged along a section in which the capillary (6) is held.

Description

Technical area

[0001] The present invention relates to a thermostat in a capillary electrophoresis device for separating and analyzing nucleic acids, proteins and the like. State of the art

[0002] In a capillary electrophoresis device in which a capillary is filled with an electrophoresis medium such as a polymer gel or a polymer solution, and electrophoresis is performed by applying a high voltage to both ends of the capillary, Patent Literature 1 describes that "...a thermostat device includes a body frame and a door frame, and the temperature control element is mounted in the body frame." and "...a capillary is held by being sandwiched between a temperature control element of the body frame and a capillary assembly press sponge of the door frame, so that the temperature is always kept constant." Furthermore, US Pat. No. 7,883,613 B2 (see Patent Literature 2) describes an invention in the field of capillary electrophoresis used for the separation and analysis of nucleic acids and proteins.The invention comprises a capillary electrophoresis device that houses a capillary array in a thermostatic device. It includes mechanisms for easily filling the capillaries with a separation medium and preventing contamination. A special feature is the efficient temperature control of the capillaries and the optical detection unit, which is ensured by a single temperature control system. This improves the accuracy of the analysis and prevents temperature fluctuations that could affect the results. JP 2002 - 71 642 A (cf. Patent Literature 3) describes an invention in the field of electrophoresis, in particular a method and apparatus for capillary electrophoresis and a capillary array. The invention aims to effectively dissipate the heat generated by Joule heat in the capillaries.This is achieved through a combination of Peltier elements for temperature control and a gas circulation system that cools the capillaries. A solid body with high thermal conductivity, such as metal or polymeric material, is used to dissipate heat from the capillaries. These measures improve temperature stability and thus the resolution and accuracy of the analysis. Finally, JP 2011 - 112 375 A (see Patent Literature 4) also describes an invention in the field of capillary electrophoresis, focusing on a thermostat for temperature control of the capillary of a capillary electrophoresis device. The invention comprises a flexible thermostat that adapts to the shape of the capillary and is in direct contact with it to ensure efficient temperature control. This reduces the size of the thermostat and the entire device.The invention aims to improve temperature stability and facilitate handling of the capillary. Literature listPatent literature Patent literature 1: JP 2007 - 322 367 A Patent literature 2: US 7,883,613 B2 Patent literature 3: JP 2002 - 071 642 A Patent literature 4: JP 2011 - 112 375 A SUMMARY OF THE INVENTIONTechnical Problem

[0003] Patent Literature 1 describes a thermostat device that efficiently dissipates the heat generated by a capillary itself by bringing the capillary into contact with a temperature control element. However, in the thermostat device described in Patent Literature 1, the temperature of the thermostat device drops locally in the area around a detection unit attached to the thermostat device, and thus the temperature distribution of a temperature control element becomes uneven.

[0004] The present invention has been made to solve such problems, and its object is to provide a capillary electrophoresis apparatus with high analytical performance by keeping the temperature constant in the longitudinal direction of the capillary. Solution to the problem

[0005] The problem is solved by the features of claims 1 and 13. Particular embodiments are described in the dependent claims. Advantageous effects of the invention

[0006] The capillary electrophoresis apparatus according to the present invention can maintain a temperature uniformly in the longitudinal direction of the capillaries, so that the separation performance of the capillary electrophoresis apparatus can be stabilized and the analysis performance can be improved. Short description of the drawings Fig. Figure 1 is an exploded view of a thermostat in a capillary electrophoresis apparatus. Fig. Figure 2A is a perspective view of the thermostat. Fig. Figure 2B is a cross-sectional view of the thermostat. Fig. Figure 3A is a top view of a capillary holder. Fig. Figure 3B is a cross-sectional view of the capillary holder. Fig. 4 is a plan view of a heater. Fig. 5 is a diagram for explaining a temperature difference in the radial direction of the capillary in a thermostat to which the present invention is not applied. Fig. 6 is a diagram for explaining a temperature difference in the radial direction of the capillary. Fig. Figure 7A is a schematic perspective view of a thermostat. Fig. Figure 7B is a cross-sectional view of a thermostat. Fig. Figure 8A is a cross-sectional view of a thermostat. Fig. Figure 8B is a cross-sectional view of a thermostat. Fig. 9 is a perspective view of a capillary electrophoresis apparatus. Description of embodiments

[0007] Fig. 9 illustrates a schematic view of a capillary electrophoresis apparatus according to the present embodiment. The apparatus can be roughly divided into two units, namely an automatic sampling unit 150 arranged at a lower part of the apparatus and an irradiation detection / thermostat unit 160 arranged at an upper part of the apparatus.

[0008] In the automatic sampling unit 150, a Y-axis drive 85 is mounted on a sampler base 80. Additionally, a Z-axis drive 90 is mounted on the Y-axis drive 85. A sample tray 100 is mounted on the Z-axis drive 90, and a container 20 containing an electrophoretic medium, an anode buffer container 30, a cathode buffer container 40, and a sample container 50 can be placed on the sample tray 100. The sample container 50 is placed on the X-axis drive 95 mounted on the sample tray 100 and can be driven along the X-axis. A liquid feed mechanism 62 is also mounted on the Z-axis drive 90. The liquid feed mechanism 62 is arranged below the container 20 containing the electrophoretic medium 20.

[0009] The irradiation detection / thermostat unit 160 is provided with the thermostat 1, and the interior can be maintained at a constant temperature. An irradiation detection unit 3 is mounted behind the thermostat 1 to enable detection during electrophoresis. The capillary 6 is inserted into the thermostat 1, electrophoresis is performed in the thermostat 1 while the capillary 6 is maintained at a constant temperature, and detection is performed by the irradiation detection unit 3. In addition, an electrode 115 is also mounted on the thermostat 1 to provide a ground connection when a high voltage is applied for electrophoresis.

[0010] As described above, the capillary 6 is attached to the thermostat 1. The electrophoretic medium container 20, the anode buffer container 30, the cathode buffer container 40, and the sample container 50 can be driven along the Y-axis and Z-axis by the automatic sampling unit 150, and the sample container 50 can be additionally driven along the X-axis. The electrophoretic medium container 20, the anode buffer container 30, the cathode buffer container 40, and the sample container 50 can be connected to the attached capillary 6 while switching between them by the movement of the sampling unit 150.

[0011] Fig. Figure 1 is a schematic view of the thermostat 1 in the capillary electrophoresis device. The thermostat 1 comprises a housing body 2a, a capillary holder 5 that holds the capillary 6, and a housing cover 2b. Here, the capillary 6 and the capillary detection unit 56 are arranged on a surface that is not actually visible, but they are in Fig. 1 is shown by dashed lines for explanation. The irradiation detection unit 3 and the temperature control unit 4 are arranged in the housing body 2a. The irradiation detection unit 3 irradiates the capillary 6 with light from a light source (not shown), such as a laser or an LED, and detects the fluorescence or the like emitted by the capillary 6. Therefore, the temperature control unit 4 is provided with a detection window 41.

[0012] Furthermore, a temperature sensor 42 for measuring a temperature of the temperature control unit 4 is arranged in the temperature control unit 4, a surface temperature of the temperature control unit 4 is measured, and the output of the temperature control unit 4 is adjusted to obtain a desired temperature. The capillary 6 is fixed to the capillary holder 5. At a lower end of the capillary holder 5, both ends of the capillary 6 are fixed via an electrode holder 57 and a capillary head 58, and a cathode end 51 and an anode end 52 of the capillary 6 protrude.

[0013] In the capillary electrophoresis device, electrophoresis is performed by contacting the cathode end 51 with a sample solution (not shown), contacting the anode end 52 with a buffer solution (not shown), and applying a high voltage to both ends. The capillary holder 5 is sandwiched between the housing cover 2b and the housing body 2a and pressed against the temperature control unit 4. An opening portion 21 is provided in the lower part of the housing body 2a so that the cathode end 51 and the anode end 52, which are exposed to the outside of the housing body 2a, can be in liquid contact with the sample solution and the buffer solution, respectively.

[0014] Fig. Fig. 2A is a perspective view when the capillary holder 5 is attached to the housing body 2a via the housing cover 2b, and Fig. Figure 2B is a cross-sectional view along the line AA' of the thermostat 1 with the capillary 6 in Fig. 2A.

[0015] In the Fig. In the thermostat 1 shown in Figure 2A, the capillary holder 5 is pressed against the temperature control unit 4 by the housing cover 2b and is fixed to the housing body 2a. In this configuration, although the structures of the temperature control unit 4 and the capillary holder 5 are not visible, the arrangement of the capillary 6 and the capillary detection unit 56 is schematically shown by dashed lines. Furthermore, although an example in which four capillaries 6 are provided is given in the present embodiment, the number of capillaries is not limited to four.

[0016] As in Fig. 2B, the temperature control unit 4 provided in the housing body 2a comprises a heat insulation layer 43, a heating layer 44, a heat diffusion plate 45 and a first heat conduction layer 46.

[0017] The capillary holder 5 comprises a carrier substrate 53, a flexible second heat conduction layer 54 attached to the carrier substrate 53, and the capillary 6 with a diameter (d) attached to a surface of the second heat conduction layer 54. The carrier substrate 53 is provided with recesses 55 with a height (h1). The height (h1) of the recess 55 formed in the carrier substrate 53 is smaller than the diameter (d) of the capillary 6 (h1 < d). The second heat conduction layer 54 is flexible and thus deforms along the recesses 55 of the carrier substrate 53. In addition, the capillary 6 is arranged along the recesses 55 formed on the carrier substrate 53.

[0018] Here, by attaching the capillary holder 5 to the housing body 2a with the housing cover 2b, the second heat conduction layer 54, which is attached to the support substrate 53, is brought into contact with the first heat conduction layer 46, which constitutes a surface of the temperature control unit 4. The capillary 6 arranged in the recess 55 of the support substrate 53 contacts the first heat conduction layer 46 and the second heat conduction layer 54, which is deformed along the recesses 55 of the support substrate 53.

[0019] The first heat conduction layer 46 and the second heat conduction layer 54 play a role in transferring heat from the heating layer 44 to the capillary 6, while absorbing heat generated by the capillary 6 and preventing an excessive temperature rise in the capillary 6. Therefore, the heat conduction layers must have thermal conductivity and insulation and are formed, for example, using a material such as thermally conductive rubber.

[0020] The structure of the capillary holder 5 is described with reference to the Fig. 3A and Fig. 3B described in detail. Fig. Fig. 3A is a plan view indicating the position of the capillary 6 arranged in the capillary holder 5, and Fig. Figure 3B is a cross-sectional view of the capillary holder 5 (the cross section BB' in Fig. 3A). The capillary holder 5 is formed by attaching the flexible second heat conduction layer 54 to the support substrate 53 and disposing the capillary 6 on the second heat conduction layer 54. The second heat conduction layer 54 is flexible and can be deformed according to the surface shape of the support substrate 53. Furthermore, the end of the capillary 6 is attached to the electrode holder 57, and the capillary 6 is arranged along the recesses 55 formed by the second heat conduction layer 54. The capillary 6 is attached to the capillary detection unit 56 provided in the capillary holder 5, and the other end is connected to the capillary head 58. The capillary detection unit 56 is attached to the detection window 41 of the temperature control unit 4.

[0021] Although the support substrate 53 presses the capillary 6 against the first heat conduction layer 46, the housing cover 2b has no temperature control function and serves as a heat radiation path of the temperature control unit 4. Therefore, the thermal conductivity is desirably low, and the housing cover 2b is preferably formed of, for example, a plastic material. Furthermore, a thermal insulation layer may be provided between the support substrate 53 and the housing cover 2b. Furthermore, the recess 55 provided in the support substrate 53 need not be a single recess 55 for a plurality of the capillaries 6, and the recess 55 may be formed for each individual capillary.

[0022] Fig. 4 illustrates a pattern of a resistance heating wire when the heating layer 44 of the temperature control unit 4 is a film heater 70. The film heater 70 is formed of a base part 49, a first resistance heating wire 48a, and a second resistance heating wire 48b. For the base part 49, for example, a polyimide film, silicone rubber, ceramic, or the like is used. In the present embodiment, a heater terminal 47 is provided on the base part 49, and the first resistance heating wire 48a and the second resistance heating wire 48b are connected in series to the heater terminal 47. The first resistance heating wire 48a is a resistance heating wire that is wider than the second resistance heating wire 48b.

[0023] Here, a first high-heat generation region 60 in which the second resistance heating wire 48b is provided with narrow pitches in the fixed portion of the electrode holder 57, and a second high-heat generation region 61 in which the periphery of the detection window 41 is provided as the second resistance heating wire 48b, thus increasing the heat generation amount by arranging the second resistance heating wire 48b with narrow pitches are provided. The other region is provided as the first resistance heating wire 48a, and reducing the heat generation amount by arranging the first resistance heating wire 48a with wide pitches. That is, the heating layer 44 is formed by dividing the in-plane heat generation amount into at least two regions, a large region and a small region.

[0024] However, it is not necessary to adjust the heat generation amount solely by the thickness or density of the resistance heating wire. For example, the heating can be divided into individual zones, and the temperature can be controlled individually. Furthermore, the heat generation amount of the heater is not limited to two levels and can be divided into three or more levels according to the heat radiation conditions of a temperature control structure.

[0025] Furthermore, since the heat diffusion plate 45 constituting the temperature control unit 4 needs to distribute the heat generation of the heater evenly to the first heat conduction layer 46, it is preferable that the heat diffusion plate 45 is a metallic material with high thermal conductivity and is formed of, for example, aluminum or copper.

[0026] According to the embodiment described above, the following practical effects can be obtained. (1) Reduction of temperature fluctuation in the longitudinal direction of the capillary 6 In the capillary 6, since the opening portion 21 in the housing body 2a is provided near the electrode holder 57 and the capillary head 58, which are located outside the thermostat 1, the amount of heat radiation is large, and the temperature of the temperature control unit 4 drops near the opening portion 21. Similarly, at the detection window 41, the irradiation detection unit 3 is arranged at the back of the heater, the heater can be omitted, and the irradiation detection unit 3 serves as a heat radiation path, and therefore the temperature of the heater surface drops locally.Since the amount of heat generation is large in the vicinity of the detection window 41 and the opening portion 21 provided in the case body 2, in the heating layer 44 of the present embodiment, by providing the first high-heat generation region 60 and the second high-heat generation region 61 near the opening portion 21 and the detection window 41 in the temperature control unit 4, a temperature drop can be suppressed, the temperature fluctuation on the surface of the first heat conduction layer 46 can be reduced, and the temperature fluctuation in the longitudinal direction of the capillary 6 can be reduced. (2) Reducing the temperature fluctuation in the radial direction of the capillary 6. Since the capillary 6 is arranged along the recesses 55 provided in the support substrate 53 of the capillary holder 5, the second heat conduction layer 54 is in close contact with the first heat conduction layer 46 provided in the temperature control unit 4, and the temperature difference between the second heat conduction layer 54 and the first heat conduction layer 46 is reduced. Since the capillary 6 is sandwiched between the first heat conduction layer 46 and the second heat conduction layer 54, the temperature difference in the radial direction of the capillary 6 becomes small because the temperature difference becomes small.

[0027] Fig. 5 shows the temperature distribution from the housing body 2a to the housing cover 2b in a thermostat in which no recess 55 is provided in the carrier substrate 53. Fig. 6 illustrates the temperature distribution in the radial direction of the capillary 6 in the thermostat 1 according to the present embodiment.

[0028] As in Fig. 5, when the temperature of the first heat conduction layer 46 is controlled to become a temperature Th without providing the recess 55 in the support substrate 53, the temperature Tc of the second heat conduction layer 54 becomes lower than the temperature Th of the first heat conduction layer 46 (Th > Tc) because the second heat conduction layer 54 is not in contact with the first heat conduction layer 46, and a temperature difference Th-Tc is generated in the radial direction of the capillary 6. In addition, the temperature of the case lid 2b is indicated by Tcase1, and the temperature of the case body 2a is indicated by Tcase2.

[0029] Fig. Fig. 6 shows the temperature distribution in the present embodiment. When the recesses 55 are provided in the support substrate 53, the second heat conduction layer 54 is in contact with the first heat conduction layer 46 in the area except for the recesses 55, whereby the temperature (Tc') of the second heat conduction layer 54 is higher than the temperature (Tc) of the first heat conduction layer 46. Fig. 5 (Tc' > Tc). Therefore, the temperature difference (Th-Tc') that occurred between the first heat conduction layer 46 and the second heat conduction layer 54 is reduced. Consequently, the temperature difference between the two contact points of the capillary 6 that are in contact with both heat conduction layers can be suppressed to a small value ({Th - Tc} > {Th - Tc'}). Thus, the amount of heat generation of the detection window 41 of the heating layer 44 and the opening portion 21 provided in the housing body 2a is increased. By providing the recesses 55 in the support substrate 53, temperature fluctuations in the longitudinal and radial directions of the capillary 6 can be reduced, and a decrease in the separation efficiency of a sample during capillary electrophoresis can be prevented.

[0030] Next, a second embodiment of the present invention will be described. The second embodiment is an example of a structure in which the heights of the recesses provided in the support substrate 53 are in two stages.

[0031] The description of sections with the same functions as the previously described sections with the same reference numbers will not be repeated.

[0032] The Fig. 7 and Fig. 8 shows a thermostat according to the second embodiment and a cross-sectional view thereof. Fig. Figure 7A is a schematic perspective view of the thermostat. Fig. Figure 7B is a cross-sectional view taken along line CC' in the longitudinal direction of the capillary. Fig. Figure 8A is a cross-sectional view along line DD' of Fig. 7B. Fig. 8B is a cross section along the line EE' of Fig. 7B.

[0033] The thermostat 1 according to the second embodiment has a structure in which the height of the recess formed in the support substrate 53 is divided into two stages.

[0034] As in Fig. 7B, the carrier substrate 53 has a first recess 101 and a second recess 102. In the first recess 101, the capillary 6 touches the second heat conduction layer 54 and the first heat conduction layer 46. In contrast, the capillary 6 in the second recess 102 does not touch the second heat conduction layer 54. Here, Fig. 8A a cross-section of the first recess 101, and Fig. 8B shows a cross section of the second recess 102. As in Fig. 8A, the height (h2) of the first recess 101 is smaller than the diameter (d) of the capillary (h2 < d) because the capillary 6 is in contact with the second heat conduction layer 54 and the first heat conduction layer 46 arranged in the first recess 101.

[0035] In addition, as in Fig.8B, the height (h3) of the second recess 102 is greater than the diameter (d) of the capillary (h3 > d) because the capillary 6 is not brought into contact with the second heat conduction layer 54 arranged in the second recess 102.

[0036] The height of the recess provided in the support substrate 53 is not limited to two steps. Considering the deformation when pressing the housing cover 2b against the housing body 2a, the height of the recess can be set to three or more steps, the capillary 6 evenly contacts the first heat conduction layer 46, and the contact area with the second heat conduction layer 54 is reduced.

[0037] According to the above-described embodiment, by reducing the contact area between the second heat conduction layer 54 and the capillary 6 provided on the capillary holder 5, the influence of the temperature drop of the capillary due to the second heat conduction layer 54, whose temperature is lower than that of the first heat conduction layer 46, can be reduced. Therefore, the capillary 6 approaches the temperature of the first heat conduction layer 46, which controls the temperature, and temperature fluctuations in the radial direction of the capillary can be suppressed. List of reference symbols 1 thermostat 2a Housing body 2b Housing cover 3 Irradiation detection unit 4 Temperature control unit 5 Capillary holder 6 capillaries 20 containers with electrophoretic medium 21 Opening section 30 anode buffer tanks 40 cathode buffer tanks 41 recording windows 42 Temperature sensor 43 Thermal insulation layer 44 heating layer 45 Heat diffusion plate 46 first heat conduction layer 47 Heating connection 48a first resistance heating wire 48b second resistance heating wire 49 Base part 50 sample containers 51 Cathode end 52 Anode end 53 Carrier substrate 54 second heat conduction layer 55 Deepening 56 Capillary detection unit 57 Electrode holder 58 Capillary head 60 first area for generating strong warming 61 second area for generating strong warming 62 Liquid supply mechanism 70 foil heating 80 sampler base 85 Y-axis drive 90 Z-axis drive 95 X-axis drive 100 sample plates 101 first deepening 102 second deepening 115 Electrode 150 automatic sampling unit 160 Irradiation detection / thermostat unit

Claims

[1] Capillary electrophoresis device comprising: a thermostat (1) with a heat source, a first heat conduction layer and a detection window (41) for detecting a sample, wherein the thermostat (1) is designed to keep a capillary (6) at a predetermined temperature; a capillary holder (5) with a second heat conduction layer to sandwich the capillary (6) between the second heat conduction layer and the first heat conduction layer, wherein the capillary holder (5) is designed to hold the capillary (6); and a detection unit designed to detect the sample to be electrophoresed in the capillary (6), wherein in the heat source, a heat generation amount of a periphery of the detection window (41) and / or an end of the capillary (6) is higher than a heat generation amount of another portion, and wherein the capillary electrophoresis device characterized by is that the capillary holder (5) has recesses (55) arranged along a section in which the capillary (6) is held. [2] A capillary electrophoresis apparatus according to claim 1, wherein the heat generation amount of the heat source is set to decrease with increasing distance from the periphery of the detection window (41). [3] A capillary electrophoresis apparatus according to claim 1, wherein the heat generation amount of the heat source is set to decrease with increasing distance from the end of the capillary (6). [4] Capillary electrophoresis device according to claim 1, wherein the heat source is a foil heater (70) and has series-connected resistance heating wires, and a width of the resistance heating wires of the periphery of the detection window (41) and / or the end of the capillary (6) in the capillary holder (5) is narrower than a width of the resistance heating wires at other locations. [5] Capillary electrophoresis device according to claim 1, wherein the heat source is a foil heater (70) and has series-connected resistance heating wires, and the resistance heating wires of the periphery of the detection window (41) and / or the end of the capillary (6) are arranged more densely in the capillary holder (5) than the resistance heating wires at other locations. [6] Capillary electrophoresis device according to claim 1, wherein the recess (55) is provided for each of the capillaries (6). [7] Capillary electrophoresis device according to claim 1, wherein a height of the recess (55) is smaller than an outer diameter of the capillary (6). [8] Capillary electrophoresis device according to claim 1, wherein the recess (55) has at least two different heights which are smaller or larger than an outer diameter of the capillary (6) along the capillary (6). [9] The capillary electrophoresis device according to claim 1, wherein the second heat conduction layer has flexibility. [10] A capillary electrophoresis apparatus according to claim 1, wherein a heat diffusion plate (45) is provided between the first heat conduction layer and the heat source. [11] A capillary electrophoresis apparatus according to claim 1, wherein the end of the capillary (6) is an end on a cathode side. [12] A capillary electrophoresis device according to claim 1, wherein both ends of the capillary (6) protrude from the capillary holder (5), one end being fixed to the capillary holder (5) and another end being fixed to an electrode holder (57). [13] A thermostat (1) used in a capillary electrophoresis apparatus according to claim 1, wherein the thermostat (1) comprises a heat source, a heat conduction layer, and a detection window (41) for detecting a sample, and accommodates a capillary (6) used for electrophoresis of a sample to be detected, and wherein, in the heat source, a heat generation amount of a periphery of the detection window (41) and / or an end of the capillary (6) is higher than a heat generation amount of another portion.

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