JPH11183437A - Electrophoresis member and electrophoresis device using same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To enable the highly accurate measurement of a trace sample in a capillary part. SOLUTION: Potential differences are provided for both ends of a channel groove 53 for separation filled with a liquid for electrophoresis to cause a sample to migrate. An insulating film/semiconductor interface formed of a silicon nitriding film 29 and a silicon substrate 21b formed so as to cover the channel groove 53 for analysis is irradiated with light modulated into a predetermined frequency form the side of the silicon substrate 21b by a laser light source 47 and is, for example, impressed with a bias voltage V of 1 V by a voltage impressing power source 51, and a generated optical a.c. current I is measured by an optical a.c. current measuring device 49. As a result, it is possible to detect changes in the pH values of a capillary part irradiated with laser light as changes in the optical a.c. current I, and it is possible to detect even a trace sample without impairing detection sensitivity.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a capillary electrophoresis apparatus used for analyzing very small amounts of proteins and nucleic acids at high speed and high resolution, and an electrophoresis member used therefor.

[0002]

2. Description of the Related Art In the case of analyzing a very small amount of protein or nucleic acid, an electrophoresis apparatus has been conventionally used, and a typical example thereof is a slab gel electrophoresis apparatus.
In this apparatus, after a gel is filled between a pair of glass plates to form a gel electrophoresis section, a sample is injected into one end of the gel electrophoresis section, a voltage (up to 100 V) is applied to both ends, and the analyte is electrically charged. It is developed on a gel by electrophoresis. The developed analytes can be detected by irradiating the gel plate with laser light to detect the absorbance, staining with a stain, or preliminarily analyzing the analyte by RI (radioactive isotope).
, And the detection is performed by an autoradiograph.

However, in a slab gel electrophoresis apparatus, heat generation due to Joule heat in the gel poses a problem, so that analysis cannot be performed by applying a high voltage. for that reason,
It has a drawback that it takes a long time (several tens of hours) for the analysis time (migration time), and it cannot be said that it is a useful device in an application field requiring quick analysis such as DNA diagnosis.

Therefore, as an alternative device, an inner diameter of 1
A capillary electrophoresis apparatus that fills an electrophoresis buffer in a glass capillary of about 00 μm or less, introduces a sample into one end, and applies a high voltage to both ends of the capillary to develop an analyte in the capillary. Proposed. FIG. 1 shows a configuration example of the capillary electrophoresis apparatus.

[0005] Both ends of the glass capillary 20 are immersed in electrophoresis buffer reservoirs 11a and 11b filled with the electrophoresis buffer 13, respectively.
The electrodes 15a and 15b for applying a high voltage connected to the high voltage power supply 17 are immersed in 11b. Under such a configuration, a high voltage is applied to both ends of the glass capillary 20, whereby the analyte is developed in the capillary and detected by the detector 19. This device has a large surface area with respect to the volume in the glass capillary, that is, a high cooling effect, so that a high voltage can be applied.
A very small amount of sample such as A can be analyzed at high speed and with high resolution.

However, in the capillary electrophoresis apparatus, the outer diameter of the glass capillary used is 10 to several tens.
Since it is as thin as about 0 μm and easily broken, it is not easy to handle the user when replacing the capillary, which is not suitable for downsizing the apparatus. DJHarrison et
As described in al./Anal.Chim.Acta 283 (1993) 361-366, a plate-like capillary member formed by joining two substrates has been proposed. FIG. 2 shows an example of a conventional plate-like capillary member.

[0007] Using photo fabrication technology,
The analysis channel groove 5 and the sample injection channel groove 3 which intersect each other, and the substrate 1a which is located at both ends of the grooves 5 and 3 and on which the migration buffer reservoir 7 is formed, correspond to the migration buffer reservoir 7. A plate-like capillary member is formed by joining a substrate 1b having a through hole 9 formed at a position by ultrasonic processing. Since this capillary member has a plate shape, it is less susceptible to breakage than conventional glass capillaries, is easy to handle, and is suitable for miniaturization of the apparatus.

At the time of analysis using a plate-like capillary member, a needle-like electrode (not shown) is inserted into the electrophoresis buffer reservoir 7 through the through hole 9 and a high voltage is applied to both ends of the flow channel groove 5 for analysis. Then, the analysis target is developed in the flow channel for analysis 5. The developed analyte is irradiated with laser light from the outside and detected by a method of measuring the absorbance.

On the other hand, as a method for detecting a local pH value of a solution, LAP from Molecular Devices, USA
S (Light Addressable Potentiometric Sensor) has been proposed (DGHafeman, JWParce and HMMcConn)
ell: Science 240 (1988) 1182). In LAPS, an insulating film is formed on a semiconductor substrate to form an insulating film / semiconductor interface, and a solution (sample: Electrolyte) and an insulating film (Insulato) are formed.
r), the semiconductor has an EIS structure. When light modulated to a specific frequency is irradiated from the semiconductor substrate side or the solution side, and a bias voltage V is applied to the solution, an optical alternating current I flows through the EIS structure. At this time, the IV curve representing the relationship between the applied bias voltage V and the flowing optical alternating current I shifts in the bias voltage axis direction depending on the pH of the solution. As a result, under a constant bias voltage V, a change in the pH value in a region irradiated with light can be detected as a change in the optical alternating current. Utilizing this feature of being able to detect the local pH value irradiated with light, it has been developed, for example, as a pH value image sensor that images a two-dimensional distribution of pH (M. Nakao et al: Jpn.JA)
ppl. Phys. 33 (1994) L394).

[0010]

The conventional plate-shaped capillary electrophoresis apparatus is easy to handle and suitable for miniaturization, but has a problem that the detection sensitivity is lowered as the amount of the sample becomes extremely small. Accordingly, an object of the present invention is to provide a plate-shaped capillary electrophoresis member capable of performing highly sensitive measurement on a very small amount of sample in the capillary, and an electrophoresis apparatus using the same.

[0011]

In order to solve the above-mentioned problems, an electrophoretic member according to the present invention applies a high voltage to a capillary portion filled with an electrophoresis running solution to cause electrophoresis of the sample in the capillary portion. In the electrophoresis member having a capillary portion for electrophoresis formed in the capillary electrophoresis apparatus for performing the separation analysis of the first member, the narrow groove of the first member having the narrow groove for separation analysis formed on the substrate was formed. A second member of a silicon substrate that covers the narrow groove to form a capillary portion on the surface is joined to form a plate-like capillary member, and at least one of the first member and the second member has a narrow groove end. A through hole is formed at a position corresponding to the portion, and at least a detecting portion of the inner wall of the capillary of the second member has an insulating film /
A semiconductor structure is formed.

The space for the capillary portion is formed by covering the separation analysis narrow groove of the first member with the second member. An insulating film is formed on at least the detection portion of the second member forming the capillary portion. When the electrophoresis liquid is injected into the capillary portion, the portion where the insulating film is formed has an EIS structure having a electrophoresis liquid / insulating film / semiconductor structure.

A capillary electrophoresis apparatus according to the present invention comprises: an electrophoresis member according to the present invention; means for applying a potential difference to both ends of a capillary portion filled with an electrophoresis running solution to electrophoretize a sample; A light source for irradiating the film / semiconductor structure with light, a detector for measuring an optical alternating current generated in the insulating film / semiconductor structure of the detection unit by the light from the light source, and a means for positioning the electrophoretic member I have.

EI formed at the detection part of the capillary part
A portion of the S structure is irradiated with light modulated to a specific frequency from a semiconductor substrate side or a liquid sample side by a light source.
The optical alternating current flowing through the S structure is measured. At this time, the IV curve due to the bias voltage V applied to the solution (sample) and the alternating current I flowing through the solution shifts in the direction of the bias voltage axis depending on the pH of the solution. A change in the pH value of the irradiated area can be detected as a change in the photo-current. The present invention
The basic principle of LAPS is applied to an electrophoresis apparatus. If a detection method for detecting such a surface state is used, in a plate-shaped capillary electrophoresis apparatus, despite the extremely small amount of the sample, the detection sensitivity is maintained by securing the surface area of the insulating film in contact with the liquid sample without impairing the detection sensitivity. It becomes possible to detect.

[0015]

FIG. 3 is a structural view showing an embodiment of the electrophoretic member, wherein FIG. 3A is a perspective view, and FIG. 3B is a sectional view taken along line AA of FIG. For example, on one surface of a substrate 21a made of glass, an analysis channel groove 53 for separating a sample by electrophoresis and a sample channel groove 55 for guiding the sample to the analysis channel groove 53 are formed. . At both ends of the flow channel groove 53 for analysis and the flow channel groove 55 for sample, a migration buffer reservoir 57 for storing a migration solution is formed. A through-hole 37 is formed at the position of the detection section of the analysis channel 53, and the platinum electrode 35 is inserted therein. The platinum electrode 35 is made of, for example, a low melting point SiO ₂ material 3.
9 to fix the through hole 37 back. Although not shown, an electrophoresis solution or a sample is injected into the analysis channel groove 53 and the sample channel groove 55 on each of the electrophoresis buffer reservoirs 57, and a migration voltage is applied to the electrophoresis solution. Through holes for guiding electrodes for the substrate 21a
Is formed.

The silicon substrate 21b is bonded to the surface of the substrate 21a on which the analysis channel groove 53, the sample channel groove 55, and the migration buffer reservoir 57 are formed, and the silicon substrate 21b is bonded between the substrate 21a and the substrate 21b. An insulating film 22 composed of a silicon nitride film 29 and a silicon oxide film 31 is formed on the substrate 21b. The silicon oxide film 31 on the analysis flow channel 53, the sample flow channel 55, and the migration buffer reservoir 57 has been removed, and the analysis flow channel 53 and the sample flow channel 55 are covered with the silicon nitride film 29. And a capillary portion. The inner wall surface of the capillary has an insulating film / semiconductor structure. An ohmic contact electrode 33 is formed on a part of the silicon substrate 21b.

An example of a process for producing a plate-shaped capillary member according to the present invention by photofabrication technology will be described with reference to FIGS. 3, 4 and 5. 4 and 5 are process sectional views of the same embodiment.
The photofabrication technique refers to a technique of transferring a pattern of a photomask to make a copy, and generally applies a photoresist or a photosensitive material called a resist to a substrate surface and transfers the pattern with light. Then, the transferred planar pattern is processed into a three-dimensional shape by etching or the like. The photoresist used is not particularly limited as long as it can withstand the subsequent etching step. Further, the thickness needs to be thick enough to withstand the subsequent etching step, but is generally about several μm. Further, the exposure of the photoresist can be performed using an exposure apparatus (aligner or stepper) generally used in semiconductor manufacturing.

(A) An etching protection film 25 (for example, a silicon film having a thickness of several thousand degrees) is formed on a substrate 21a by, for example, a sputter deposition apparatus, and a patterning resist 23 ( For example, A
Z4620) is applied using a spinner. Here, quartz is used as the material of the substrate 21a in addition to various types of glass. Here, glass is used. The thickness of the substrate 21a is desirably, for example, about 0.2 to 1 mm. The material and thickness of the etching protection film 25 are not particularly limited as long as they can withstand a subsequent etching step.
When the substrate 21a is glass, Au / Cr (approximately 2000 °) in which an Au film is laminated on a Cr film can be used.

(B) Then, the resist 23 is exposed to UV light from an exposure device through a photolithographic mask 27, and then developed and patterned into a desired shape. (C) Next, the etching protective film 25 is patterned using the patterned resist 23 as an etching mask. (D) Subsequently, using the patterned resist 23 and the etching protection film 25 as a mask, the glass substrate 21
is etched at room temperature with, for example, a hydrofluoric acid aqueous solution of about 46% to form an analysis channel groove 53, a sample channel groove 55, and a migration buffer reservoir 57. (E) Thereafter, the resist 23 and the protective film 25 are removed by etching.

(F) On the other hand, an oxide film (SiO ₂ : not shown) is formed on the silicon substrate 21b, and a silicon nitride film (Si ₃ N ₄ ) is formed thereon using, for example, a CVD method.
29 to form a semiconductor / insulating film structure. The thickness of the silicon nitride film 29 is, for example, about 100 nm, but is not particularly limited. Further, a silicon oxide film (SiO ₂ ) 31 is formed on the silicon nitride film 29. Thereafter, gold antimony is deposited to a thickness of about 50 nm on the back surface of the silicon substrate 21b (the surface opposite to the surface on which the silicon nitride film 29 is formed) to make ohmic contact. Gold antimony is formed on the entire back surface of the substrate 21b and then patterned by a photofabrication technique, or is patterned into an electrode shape by vapor deposition through a mask. Thereafter, alloying is performed at 500 ° C. to form an electrode 33. This condition is not particularly limited as long as the formed electrode 33 can make ohmic contact with the silicon substrate 21b.

Subsequently, the analysis flow channel 5 is formed in the silicon substrate 21b, the silicon nitride film 29 and the silicon oxide film 31.
3. For example, a tapered through hole for injecting an electrophoresis running solution or a sample is formed in the sample channel groove 55 and the electrophoresis buffer reservoir 57. Here, means for forming the through hole is not particularly limited, but ultrasonic processing or sand blast processing can be used. Although the size of the through hole is not particularly limited, for example, the opening diameter is desirably about several 100 μm to several mm.

(G) Finally, the glass substrate 21a and the silicon substrate 21b on which the silicon nitride film 29 and the silicon oxide film 31 are formed are replaced with the glass substrate 21a and the silicon oxide film 3.
1 are superimposed so as to join, for example, about 400 ° C.
Bonding is performed using an anodic bonding technique performed by applying a high voltage while heating to a certain degree.

Electrodes for electrophoresis are provided on the front surface, the back surface, and the inner wall of the through hole of the substrate 21a via a metal mask before bonding the glass substrate 21a and the silicon substrate 21b in the step (G). Is formed by sputtering to form electrodes and contact pads. Here, the material for the electrodes and the contact pads is the glass substrate 21
The conductive film is not particularly limited as long as it is a conductive film that can withstand the heat of the bonding process between the silicon substrate 21a and the silicon substrate 21b.
Wiring materials (Al, A) generally used in semiconductor manufacturing
u, Cu, Cr) and the like, and these materials can be formed by means such as vacuum evaporation or sputtering. The thickness of the conductor film at this time is not particularly limited, but can be about several hundreds to several thousand degrees. In the step of forming an electrode for electrophoresis, the bonding of the glass substrate 21a and the silicon substrate 21b in the step (G) is performed by forming a film by sputtering on the surface of the silicon substrate 21a and the inner wall of the through hole via a metal mask. Is also good.

(H) Glass substrate 21a and silicon substrate 2
After bonding 1b, a hydrofluoric acid aqueous solution is poured into a space surrounded by the analysis flow channel 53 or the sample flow channel 55 and the silicon oxide film 31, and only the silicon oxide film 31 in contact with the space is removed. As a result, the flow channel groove 53 for analysis and the flow channel groove 55 for sample are covered with the silicon nitride film 29, into which the electrophoresis liquid is injected, to form a capillary portion on which the sample migrates.

(I) Analysis channel groove 5 of glass substrate 21a
The through holes 37 are formed at the positions of the detection units in the three regions, the platinum electrodes 35 are inserted, and backfilling is performed using, for example, a low melting point SiO ₂ material 39 or the like. Thus, the plate-like capillary member (tip) 50 according to the present invention is formed.

Next, a detection method using the plate-like capillary member according to the present invention will be described with reference to FIG. FIG.
In (A), an upper plate (glass substrate) 21a,
Insulating film (silicon nitride film 29 / silicon oxide film 31) 2
2. A chip 50 made of the silicon substrate 21b is formed. A voltage application power supply 51 is provided to the ohmic contact electrode 33 formed on the silicon substrate 21b via an AC current measuring device 49, and a voltage application power supply 51 is provided to the platinum electrode 35 to form a circuit. In order to generate hole electron pairs at the insulating film / semiconductor interface formed by the bonding surface of the silicon substrate 21b and the insulating film 29, the platinum electrode 3 in the capillary portion is formed.
A laser light source 47 is provided to irradiate the position where 5 is formed with laser light, and a modulator 45 that changes the laser light from the laser light source 47 is connected to the laser light source 47. The laser light is applied to the modulator 45 by, for example, 5 to 10
It is modulated to about kHz. In this embodiment, the laser light is irradiated from the silicon substrate 21b side.
Irradiation may be performed from the 1a side.

Here, when the applied voltage V from the voltage applying power source 51 is changed, the depletion layer at the interface between the insulating film and the semiconductor changes accordingly, and the optical AC measured by the AC current measuring device 49 is thereby changed. The magnitude of the current I changes. This I
The −V curve is as shown in FIG. 6 according to the change in the pH value of the solution in the capillary portion at the portion irradiated with light. FIG. 6 is a diagram showing the relationship between the applied voltage V and the flowing AC current I when the EIS structure is irradiated with laser light at various pHs, wherein the horizontal axis represents the applied voltage (V) and the vertical axis represents the AC current. (Arbitrary unit au). If the measurement of the photo-AC current is performed with the applied voltage kept constant at a voltage value at which the pH transition is large, for example, 1 V in FIG. 6, the magnitude of the flowing photo-AC current changes with respect to the pH value of the solution. By measuring the magnitude of the alternating current, the pH value of the solution can be measured.

FIG. 7 is a schematic diagram showing a capillary electrophoresis apparatus using an electrophoresis member according to the present invention. 59
Denotes a stage, in which a concave portion 61 capable of positioning the electrophoretic member 50 is formed. 63a, 63b, 6
Reference numerals 3c and 63d denote electrodes for applying a potential difference to both ends of the flow path of the electrophoresis member and causing the sample to electrophorese,
The electrophoresis member 50 is provided at a position corresponding to a predetermined electrophoresis liquid inlet and a predetermined electrophoresis liquid outlet. Wiring 65 from AC current measuring device and voltage application power supply
Reference numerals a and 65b are respectively connected to a platinum electrode and an ohmic contact electrode.

The electrophoretic member 50 filled with the electrophoresis running liquid is inserted into the concave portion 61 and positioned on the stage 59, and the automatically positioned electrodes 63a, 63b, 63c and 63d.
A potential difference is applied to both ends of the path groove of the electrophoresis member 50 to guide the sample from the sample introduction channel 55 to the intersection of the analysis channel 53 and the sample introduction channel 55. The sample is electrophoresed with. When light is emitted from the laser light source 47, a change in the pH value of the electrophoresed sample can be detected by measuring the optical alternating current flowing through 65a and 65b. Thus, separation and measurement by electrophoresis can be performed only by mounting the electrophoretic member 50 in which the flow path is filled with the electrophoresis running solution in the concave portion 61 of the stage 59 and injecting the sample to be analyzed into the sample inlet.

[0030]

According to the present invention, the insulating film / semiconductor interface is formed on the inner wall of the capillary portion, a bias voltage is applied to the capillary portion, and the capillary portion is irradiated with light, and the light generated at the insulating film / semiconductor interface. Since AC current is measured, L
It is possible to measure the change in pH value using APS, and it is possible to measure the state, speed, amount, concentration, temperature, etc. of various reactions on a very small amount of sample in the capillary.

[Brief description of the drawings]

FIG. 1 is a diagram illustrating a configuration example of a conventional capillary electrophoresis apparatus.

FIG. 2 is a diagram illustrating an example of a conventional plate-like capillary member.

FIG. 3 is a configuration diagram of one embodiment, (A) is a perspective view,
(B) is a sectional view taken along line AA of (A).

FIG. 4 is a process sectional view of a first half of the embodiment.

FIG. 5 is a sectional view showing a step in the latter half of the embodiment.

FIG. 6 is a diagram showing the relationship between an applied voltage V and an alternating current I flowing when an EIS structure is irradiated with laser light at various pH values.

FIG. 7 is a schematic diagram illustrating a measurement unit of a capillary electrophoresis apparatus using the same example.

[Explanation of symbols]

 Reference Signs List 21a Glass substrate 21b Silicon substrate 22 Insulating film 29 Silicon nitride film 31 Silicon oxide film 33 Ohmic contact electrode 35 Platinum electrode 45 Modulator 47 Laser light source 49 Optical alternating current measuring instrument 50 Chip 51 Voltage application power supply 53 Analysis channel groove 55 Sample channel groove 57 Migration buffer reservoir

Claims (2)

[Claims] 1. A capillary electrophoresis apparatus which applies a high voltage to a capillary portion filled with an electrophoresis running solution to cause electrophoresis of a sample in the capillary portion and performs sample separation and analysis. In the electrophoresis member having a capillary portion formed thereon, a silicon substrate that forms a capillary portion on a surface of the first member having a narrow groove for separation analysis formed on a substrate on which the narrow groove is formed, Are joined to form a plate-like capillary member, and at least one of the first member and the second member has a through hole formed at a position corresponding to the end of the narrow groove. At the same time, at least a detecting portion of the inner wall of the capillary of the second member includes:
An electrophoretic member comprising an insulating film / semiconductor structure for detecting an optical alternating current. 2. A capillary electrophoresis apparatus for applying a high voltage to a capillary portion to electrophorese a sample in the capillary portion and separating and analyzing the sample, wherein a first groove for separation and analysis is formed on a substrate. On a surface of the member on which the narrow groove is formed, a second member of a silicon substrate that covers the narrow groove to form a capillary portion is joined to form a plate-like capillary member, and the first member or the first member At least one of the second members has a through hole formed at a position corresponding to the end of the narrow groove, and at least a detection portion of the inner wall of the capillary of the second member has an insulating member for detecting an optical AC current. An electrophoresis member on which a membrane / semiconductor structure is formed; means for applying a potential difference to both ends of the capillary portion filled with an electrophoresis liquid to electrophoretize a sample; A light source that irradiates light to the insulating film / semiconductor structure of the detection unit of the main unit; a detector that measures a photo-current generated in the insulating film / semiconductor structure of the detection unit by light from the light source; An electrophoresis apparatus comprising: means for positioning a member.