JP2004170232A - Electrophoretic device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To restrain a trouble caused by a leak current flow in a thin film in a joined interface. <P>SOLUTION: An injection flow passage 4, a separation flow passage 5, and a groove 15 for increasing resistance in a current route between each reservoir 3 and the flow passage 4 or 5 are formed in a glass substrate 1, and a through hole for the reservoir 3 is formed in a glass substrate 2, and the both substrates 1, 2 are joined to constitute this electrophoretic device. The groove 15 is formed along a periphery of the reservoir 3 and the passages 4, 5 to patternize an Si thin film 13, thins a current route from the reservoir 3 arranged with an electrode to the flow passage 4 or 5, or separates the reservoir 3 from the flow passage 4 or 5. The resistance gets high thereby in the current route between the each reservoir 3 and the flow passage 4 or 5. <P>COPYRIGHT: (C)2004,JPO

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to an electrophoresis device for performing analysis by electrophoresis, and more particularly to an electrophoresis device capable of analyzing a very small amount of sample with high speed and high separation, and also referred to as a microchip. .
Such electrophoresis devices are used in biochemistry, molecular biology, clinical practice, for example, in the field of DNA (deoxyribonucleic acid) and protein analysis.
[0002]
[Prior art]
When a very small amount of DNA, protein, or the like is analyzed, an electrophoresis method has been conventionally used, and a capillary electrophoresis apparatus is an example of a technique for realizing the apparatus. A capillary electrophoresis device is a device in which a glass capillary having an inner diameter of 100 μm or less is filled with an electrophoresis buffer, a sample is introduced into one end side, and a high voltage is applied between both ends to develop an analyte in the capillary. is there. Since the inside of the capillary has a large surface area with respect to the volume, that is, high cooling efficiency, a high voltage can be applied, and a very small amount of sample such as DNA can be analyzed at high speed and with high resolution.
[0003]
In recent years, an electrophoretic device formed by joining two substrates has been proposed as a form in which high-speed analysis and miniaturization of the apparatus can be expected in place of a glass capillary which is complicated to handle (Non-Patent Document) 1).
[0004]
FIG. 4 shows an example of the electrophoresis device. An injection channel 4 and a separation channel 5 which intersect each other are formed on the surface of one substrate 2 as shown in FIG. As shown in (A), reservoirs 3 are provided as through holes at positions corresponding to both ends of the injection flow path 4 and the separation flow path 5, and the substrates 1 and 2 are overlapped and bonded as shown in (C). It was done.
In such a conventional electrophoretic device, the sample introduction is basically based on the use of a cross-injector design flow path composed of an injection flow path 4 and a separation flow path 5 that intersect each other.
[0005]
When using this electrophoresis device, the electrophoresis buffer is injected into the injection channel 4 and the separation channel 5 from any one of the reservoirs 3. Then, after injecting about 1 to 2 μl (microliter) of the sample into the reservoir 3 at one end of the injection channel 4, an electrode is inserted into each reservoir 3, or an electrode previously formed in each reservoir 3 is used. Then, a predetermined voltage is applied while applying a plurality of voltages to the injection flow path 4 such that the sample is uniformly applied to the injection flow path 4 so as not to spread to the separation flow path 5 at the intersection 6. To the intersection 6 of the separation channel 5.
[0006]
Next, the applied voltage is switched to the separation channel 5, and a voltage is applied also to the injection channel 4 so that the sample moves in the reverse direction from the intersection 6, and only the sample at the intersection 6 is applied to the separation channel 5. And perform electrophoretic separation. By arranging a detector at an appropriate position in the separation channel 5, the separation component is detected.
[0007]
Analysis using such an electrophoresis device can perform high-speed analysis, consumes very little solvent, requires a very small amount of sample, and can reduce the size of the device, compared to conventional capillary electrophoresis analysis. It has advantages such as. These features make it possible to perform on-site (on-site or bedside) analysis, which was difficult to achieve with conventional analyzers in the field of analytical chemistry described above, and high-speed analysis in fields such as DNA analysis. Promising as an advantage in screening from an analytical point of view.
[0008]
In the above-described electrophoretic device, generally, an optical detection method is often adopted. At this time, an S / N ratio can be improved by providing an optical slit for the purpose of preventing stray light.
[0009]
The present inventors have proposed a method of forming an optical slit efficiently by interposing a light-shielding thin film at a bonding interface between two substrates and using the thin film as an optical slit (Patent Document 1) 1).
Light hitting the light-shielding film of the optical slit is blocked by the light-shielding film and does not enter the detector, thereby improving detection sensitivity.
[0010]
[Non-patent document 1]
D. J. Harrison et al. , Science 261 (1993) pp. 895-897
[Patent Document 1]
Japanese Patent Application Laid-Open No. 2000-121547
[Problems to be solved by the invention]
In general, in the above-described electrophoresis device, a method of controlling the flow of a solution by using two crossed flow paths and four electrodes for introducing and separating a sample is often used. In the electrophoresis, a high voltage is applied to each electrode. For example, a small leak current may flow through the thin film at the junction interface due to the voltage applied when the sample is introduced, and a problem such as generation of bubbles in the separation channel may occur.
[0012]
An object of the present invention is to suppress the occurrence of problems caused by leakage current flowing through a thin film when a thin film is provided at the junction interface of the electrophoretic device for light shielding or the like. .
[0013]
[Means for Solving the Problems]
In the electrophoretic device of the present invention, the two substrates are joined so that a flow path including a separation flow path is formed therein, and a reservoir is formed at an end of each flow path. An electrophoresis device in which a migration buffer is filled, a voltage is applied to the flow channel via the reservoir, and a sample is introduced into the separation flow channel and sample components are separated in the separation flow channel. A conductive thin film is present at the bonding interface, and the thin film is subjected to processing for increasing the electrical resistance of the thin film between the reservoir and the flow path at least in a portion where a high voltage is applied. It is characterized by the following.
[0014]
The resistivity of the thin film existing at the bonding interface changes depending on the material, the film formation method, the heat treatment, and the like. However, even if the conditions are optimized, a sufficient resistance value is not always obtained. Therefore, in the present invention, the resistance of the current path of the target portion is increased by processing the thin film existing at the interface.
[0015]
An electrophoresis device uses two crossed flow paths, generally called an injection flow path and a separation flow path, and applies a sample placed in a reservoir (sample inlet) at one end of the injection flow path to a voltage (or electroosmotic flow). ) Is controlled so that only the plugs near the intersection with the separation flow path are cut out and introduced into the separation flow path. Actually, by switching the voltage applied to the electrodes at both ends of the injection flow path and the separation flow path, the potential gradient in the flow path is changed, but the current accompanying the voltage applied to each electrode is changed. When a certain amount or more of current flows through the thin film at the bonding interface in addition to the flow path, the liquid in the flow path exchanges electrons with the electrons where the electrons reach the flow path, resulting in bubbles Problems such as occurrence occur. According to the present invention, the current path from the electrode to the flow path is made thinner or cut in accordance with the flow path design, thereby increasing the resistance of the current path, so that problems such as bubble generation can be suppressed. .
[0016]
The processing can be performed at the same time as, for example, the processing of the flow channel by utilizing the fine processing technology. In that case, desired characteristics can be obtained without complicating the manufacturing process.
[0017]
BEST MODE FOR CARRYING OUT THE INVENTION
As a material of the substrate, a resin plate can be used in addition to a glass plate such as quartz glass or Pyrex (registered trademark).
The thin film existing at the bonding interface can be a light-shielding film. As the light-shielding film, any film having a large absorption coefficient with respect to the measurement light, such as a metal thin film such as Cr (chromium) in addition to a Si (silicon) thin film, can be used.
[0018]
In addition, the thin film present at the bonding interface has a large selection between the substrate and the etching so that it can be used as a mask when etching the substrate in a microfabrication technique for forming a flow path in the substrate. It is even more advantageous if it has a ratio.
[0019]
FIG. 1 shows an electrophoresis device according to one embodiment. FIG. 2 is a sectional view taken along the line AA ′ in FIG.
The substrates 1 and 2 are glass substrates, for example, quartz glass substrates. On one surface of the glass substrate 1, minute flow paths 4 and 5 (injection flow path 4 and separation flow path 5) used as liquid sample flow paths, and current between each reservoir (electrode) 3 and flow paths 4 and 5 are formed. A groove 15 for increasing the resistance of the path is formed by etching. A through hole is formed in the glass substrate 2 by, for example, a method such as sand plasting, and this serves as a reservoir ((1) to (4)) 3 for storing a sample or an electrophoresis buffer. The reservoir 3 corresponds to an electrode for applying a voltage. By joining these two glass substrates 1 and 2 by, for example, a hydrofluoric acid joining method or the like, the electrophoretic device of the present invention can be realized.
[0020]
The injection flow path 4 and the separation flow path 5 are formed so as to intersect with each other, and reservoirs (1) to (4) 3 are arranged at both ends of both flow paths 4 and 5. The channels 4 and 5 have a width of several hundreds μm or less, for example, 10 to 200 μm, and a depth of several hundreds μm or less, for example, 2 to 90 μm. The injection channel 4 has a length of several mm to several tens of mm, and the separation channel 5 has a length of 10 to 100 mm.
[0021]
The etching of the glass substrate 1 is performed using the Si thin film 13 as a mask, and the Si thin film 13 of the etching mask is left as an optical slit.
[0022]
The groove 15 for increasing the resistance of the current path is formed along the periphery of the reservoir 3 and along the flow paths 4 and 5, thereby patterning the Si thin film 13. From the reservoir 3 where the electrodes are arranged. The current path to the flow path 4 or 5 is narrowed, or the space between the reservoir 3 and the flow path 4 or 5 is divided. Thus, the resistance of the current path between the reservoir 3 and the flow path 4 or 5 is increased.
[0023]
When this electrophoretic device is used, the electrophoresis buffer is injected from one of the reservoirs 3 into the injection channel 4 and the separation channel 5, and about 1 to 2 μl (micrometer) of the sample is injected into one reservoir, for example, the reservoir (3). Liter). After the injection, a predetermined voltage is applied to each of the reservoirs 3 so as to electrophoretically spread the sample into the injection flow path 4 so as not to spread to the separation flow path 5 at the crossing point. And guided to the intersection of the injection channel 4 and the separation channel 5. Then, at an appropriate timing, the applied voltage is switched to the separation channel 5, and a voltage is applied also to the injection channel 4 so that the sample moves in the opposite direction from the intersection, and only the sample at the intersection is separated into the separation channel 5. And perform electrophoretic separation.
[0024]
By arranging a detector at an appropriate position in the separation channel 5, the separation component is detected. Detection of a separation peak can be performed by various methods. For example, optical fluorescence detection at the detection position, UV (ultraviolet) absorption detection, chemiluminescence detection, electrochemical detection with a working electrode and detection electrode, or mass spectrometry by ionizing from the end of the separation channel by the electrospray method There is a method such as detection with a meter.
[0025]
Here, for example, when a high voltage is applied to the reservoir (3) for sample introduction and electrophoretic separation, if there is no groove 15 for increasing the resistance, a portion of the separation channel 5 near the reservoir (3) Although there is a possibility that air bubbles may be generated in the portion (immediately below in FIG. 1), in the pattern of the silicon thin film 13 of this embodiment, the current in this portion is formed due to the groove 5 formed so as to surround the reservoir (3). Since the resistance of the path is increased, for example, ten times or more, it is possible to suppress the generation of bubbles.
[0026]
Next, a method of manufacturing the electrophoretic device of this embodiment will be described with reference to FIG. Here, the groove 15 and the flow paths 4 and 5 are collectively expressed as a groove 8.
(A) First, after cleaning the glass substrate 1, a thin film forming apparatus, for example, a sputtering film forming apparatus, forms an Si thin film 13 to a thickness of 3000 mm as an etching protection film also serving as a light shielding film, and further etches the film. As the photoresist 16 for patterning the protective film, for example, AZ4620 is spin-coated at a rotation speed of 3000 rpm for 40 seconds. At this time, the thickness of the resist 16 is about 7 μm. The material and thickness of the photoresist used are not particularly limited, and may be any material and thickness that can withstand the subsequent etching process. Also, the material and thickness of the etching protection film are not particularly limited, and may be any material and thickness that can withstand the subsequent substrate etching step. Here, a Si thin film is used according to the embodiment.
[0027]
(B) Next, the photoresist 16 is exposed using the photomask 7 and then developed. The exposure of the photoresist 16 can be performed using an aligner generally used in semiconductor manufacturing. The developer is not particularly limited as long as it can be used for developing the photoresist to be used.
[0028]
(C) Next, the Si thin film 13 is patterned by dry etching using high-frequency plasma in SF ₆ gas. Again, the etching gas is not particularly limited, and may be any gas that can etch Si without any problem.
[0029]
(D) Using the patterned Si thin film 13 and the photoresist 16 as a mask, the substrate 1 is etched with, for example, a 46% hydrofluoric acid aqueous solution to form a groove 8. Here, the etchant for the substrate 1 is not particularly limited, and may be any solution that can etch quartz glass.
[0030]
(E) After removing the photoresist 16, the surface of the Si thin film 13 is oxidized to form a silicon oxide film 4a on the surface of the Si film.
[0031]
(F) On the other hand, as shown in (f), a through hole for the reservoir 3 is formed in the glass substrate 2 by a processing method such as a sand blast method.
[0032]
(G) Finally, the glass substrate 1 on which the grooves 8 and the silicon thin film 13 are formed and the glass substrate 2 on which the through holes 3 are formed are overlapped, and a 1% hydrofluoric acid aqueous solution is interposed at the interface, for example, if necessary. By discarding at room temperature for 24 hours while applying a load of about 1 MPa, the glass substrates 1 and 2 are joined to form the electrophoretic device of the embodiment shown in FIG. Note that the etching of the glass substrate 1 in the step (d) may be performed by dry etching.
[0033]
The materials and manufacturing methods described in the above embodiments are merely examples, and do not limit the materials and processing methods of the present invention.
Further, in the embodiment, the groove 15 is etched to the substrate, but the same effect can be obtained only by removing the thin film 13.
[0034]
【The invention's effect】
According to the electrophoretic device of the present invention, a conductive thin film exists at the bonding interface of the substrate on which the flow path including the separation flow path is formed, and the thin film has a reservoir and a flow path. Since the processing to increase the electrical resistance of the thin film is performed, the electrical resistance of the thin film existing between each reservoir and the flow path at the substrate interface can be greatly increased, and the sample injection and analysis can be performed. Problems such as generation of bubbles at the time can be avoided.
In addition, the processing is as simple as forming a groove or patterning the thin film. In particular, if it is formed simultaneously with the flow path including the separation flow path, the number of manufacturing steps does not increase.
[Brief description of the drawings]
FIG. 1 is a schematic plan view showing an electrophoretic device according to one embodiment.
FIG. 2 is a cross-sectional view taken along a line AA ′ in FIG.
FIG. 3 is a process cross-sectional view schematically showing a method of manufacturing the electrophoretic device of the embodiment.
4A and 4B are diagrams showing a conventional electrophoretic device, wherein FIG. 4A is a plan view of one substrate, FIG. 4B is a plan view of the other substrate, and FIG. FIG.
[Explanation of symbols]
1, 2 glass substrate 3 reservoir 4 injection channel 5 separation channel 13 Si thin film 15 groove for increasing resistance

Claims (6)

The two substrates are joined so that a flow path including a separation flow path is formed therein, a reservoir is formed at an end of each flow path, and the flow path and the reservoir are filled with an electrophoresis buffer. An electrophoretic device in which a voltage is applied to the flow path via a reservoir to introduce a sample into the separation flow path and separate a sample component in the separation flow path,
A conductive thin film is present at the bonding interface between the two substrates, and at least in a portion where a high voltage is applied to the thin film, processing for increasing the electrical resistance of the thin film between the reservoir and the flow path is performed. An electrophoretic device characterized by being applied. The electrophoretic device according to claim 1, wherein the processed thin film is patterned by a groove so that a current path from each reservoir to a flow path becomes narrow. The electrophoretic device according to claim 1, wherein the processed thin film is patterned by grooves so as to separate each reservoir from a flow path. The electrophoretic device according to claim 2, wherein the groove has the same depth as the flow channel. The electrophoretic device according to claim 1, wherein the thin film is a light shielding film. The electrophoretic device according to claim 5, wherein the thin film is a silicon thin film.

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