JP2002303605A - Method for sample pretreatment and chip for electrophoresis - Google Patents

Abstract

PROBLEM TO BE SOLVED: To shorten the time required for refining a sample and introducing an aimed component contained in the sample to an analytical flow passage and, in addition, to make the handling of a very small quantity of sample easier. SOLUTION: After a refining column section 5 is filled up with a filler through gel-filtration and analytical flow passages S1 , S2 ,..., Sn are filled up with a matrix for separation, a buffer solution is injected into a sample introducing flow passage 3. An unrefined sample is introduced to the flow passage 3 from one end side 3a of the passage 3 and sent toward the other end 3b side. The unrefined sample is refined in the refining column section 5. The aimed component contained in the refined sample is detected by means of a photoluminescence detector D1 and, when the component reaches a branch section F1 , the component is electrophoretically introduced to the analytical flow passage S1 by producing a potential difference between electrodes respectively positioned in through holes A1 and G.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a sample pretreatment method in electrophoresis for analyzing a sample containing a very small amount of protein, nucleic acid, drug or the like at high speed and high resolution, and an electrophoresis chip used for electrophoresis. Things.

[0002]

2. Description of the Related Art Electrophoresis has been conventionally used for analyzing very small amounts of proteins and nucleic acids, and a typical example is capillary electrophoresis. In capillary electrophoresis, a glass capillary (hereinafter simply referred to as a capillary) having an inner diameter of 100 μm (micrometer) or less is filled with a separation matrix (electrophoresis medium), a sample is introduced at one end, and both ends are buffered. The sample is brought into contact with the solution, and a high voltage is applied between both ends via a buffer solution to develop the analyte in the capillary. Since 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 (deoxyribonucleic acid) can be analyzed at high speed and with high resolution.

[0003] The capillary has an outer diameter of 100 to 500.
There is a problem that it is not easy to handle when replacing the capillary, which is to be performed by the user, because it is as thin as about μm and easily broken. In addition, there is a problem that the heat release may not be sufficient, which may adversely affect the separation state. Further, since a voltage is applied to both ends of the capillary via the buffer solution, a length dimension necessary for at least contact with the buffer solution is required, and there is a problem that the length cannot be designed to be less than a certain length.

[0004] Therefore, instead of the capillary, the speed of analysis and the miniaturization of the apparatus can be expected as DJ
As shown in Harrison et al./Analyte. Chem. 1993, 283, 361-366, an electrophoresis chip formed by joining two substrates has been proposed. FIG. 4 shows an example of the electrophoresis chip.

[0005] The electrophoresis chip 11 is made of a pair of transparent base materials 11a and 11b made of a transparent plate-like inorganic material (eg, glass, quartz, silicon, etc.) or plastic, and is formed by semiconductor photolithography and etching, or micromachining. The electrophoresis capillary grooves 13, 15 crossing each other on the surface of one of the substrates 11b by the technique.
The other substrate 11a is provided with through-holes at positions corresponding to the ends of the grooves 13 and 15 as an anode reservoir 17a, a cathode reservoir 17c, a sample reservoir 17s, and a waste reservoir 17w. The electrophoresis chip 11 is used in a state where both base materials 11a and 11b are overlapped and joined as shown in FIG.

When performing electrophoresis using the electrophoresis chip 11, prior to analysis, for example, by pumping using a syringe, one of the reservoirs, for example, from the anode reservoir 17a to the inside of the grooves 13, 15 and the reservoir. The electrophoresis medium is filled in 17a, 17c, 17s, and 17w. Next, the reservoirs 17a, 17c, 17s, 1
7w is removed, the sample is injected into the sample reservoir 17s corresponding to one end of the shorter groove (sample injection channel) 13, and the other reservoir 1
Inject buffer solution into 7a, 17c, 17w.

The electrophoresis chip 11 into which the electrophoresis medium, the sample and the buffer solution have been injected is mounted on an electrophoresis apparatus. A predetermined voltage is applied to each of the reservoirs 17a, 17c, 17s, and 17w to cause the sample to migrate into the groove 13 and to guide the sample to the intersection 19 between the grooves 13 and 15. Each reservoir 17
a, 17c, 17s, and 17w are switched, and the sample present at the intersection 19 is moved into the groove 15 by the voltage between the reservoirs 17a and 17c at both ends of the longer groove (separation channel) 15. inject. After injecting the sample into the groove 15, the sample contained in the reservoir 17s is replaced with a buffer solution. Thereafter, a voltage for electrophoresis is applied to each of the reservoirs 17a, 17c, 17s, and 17w, and the sample injected into the groove 15 is separated in the groove 15. By disposing a detector at an appropriate position in the groove 15, a sample separated by electrophoresis is detected. Detection is performed by means such as an absorption spectrophotometry, a fluorescence photometry, an electrochemical or electric conductivity method.

In such an electrophoresis, a sample introduced into an electrophoresis chip has been subjected to a pretreatment and purified. For example, a sample in which nucleic acid and impurities such as salts and unreacted primers are mixed, such as a sample prepared by the Sanger method or a sample prepared by the PCR (Polymerase chain Reaction) method, is subjected to gel filtration or ethanol precipitation in advance. After purification and removal of contaminants, it is introduced into an electrophoresis chip. In the gel filtration method and the ethanol precipitation method, a centrifugal operation is performed in order to shorten the treatment time or increase the nucleic acid recovery rate.

[0009]

The sample to be analyzed by electrophoresis needs to be purified and then introduced into an electrophoresis chip. However, since purification and introduction are separate steps, it takes a long time. there were. In particular, when a sample prepared by the Sanger method or the PCR method is purified by the gel filtration method or the ethanol precipitation method, the centrifugal operation is performed, so that the sample must be temporarily stored in a centrifuge, which takes a long time. Was. Further, in the case of a small amount of sample, there is a problem that it is difficult to collect the purified sample and introduce it into an electrophoresis chip.

A first object of the present invention is to purify a sample,
Another object of the present invention is to provide a pretreatment method capable of shortening the time required for introducing a target component in a sample into an analysis channel for performing electrophoresis and facilitating handling of a small amount of sample. A second object of the present invention is to provide a method for electrophoresis that can reduce the time required for purifying a sample and introducing a target component in the sample into an analysis channel for performing electrophoresis, and can easily handle a small amount of sample. Is to provide a chip for use.

[0011]

In the sample pretreatment method according to the first aspect of the present invention, a sample before purification is introduced into a purification column for purifying the sample, and the sample is purified. The target component in the purified sample is electrophoretically introduced into an analysis channel branched from the channel on the elution side.

After the sample before purification is introduced into the purification column and purified, the target component in the purified sample eluted from the purification column is directly electrophoretically transferred to the analysis channel without being separately collected in a container. To be introduced. As a result, the time required for purifying the sample and introducing the target component in the sample into the analysis channel for performing electrophoresis can be reduced,
In addition, handling of a small amount of sample can be facilitated.

An electrophoresis chip according to a second aspect of the present invention includes a sample introduction channel provided with a purification column for purifying a sample inside a plate-like substrate, and a sample introduction channel. An analysis channel connected downstream of the purification column is provided, and a hole reaching the sample introduction channel at a position corresponding to both ends of the sample introduction channel from the surface of the plate-like substrate, and a sample introduction channel. An electrode for generating a potential difference between the sample introduction channel and the analysis channel is provided at at least one location and at a position corresponding to at least one location of the analysis channel. However, the electrode includes a case where holes for arranging the electrodes are provided in the plate-shaped member, and the electrodes are arranged in those holes when the electrophoresis chip is used.

The purification column is filled with a gel filtration filler,
The analysis channel is filled with the separation matrix, and the buffer solution is injected into the sample introduction channel. A sample before purification is introduced from one end of the sample introduction channel, and is sent toward the other end. The sample before purification is purified in the purification column, and eluted from the elution side of the purification column. When the target component in the purified sample reaches the branch between the sample introduction channel and the analysis channel, a voltage is applied to the electrode to cause a predetermined potential difference between the sample introduction channel and the analysis channel. ,
The target component is electrophoretically introduced into the analysis channel. As a result, the time required for purifying the sample and introducing the target component in the sample into the analysis channel for performing electrophoresis can be shortened, and the handling of a small amount of sample can be facilitated.

[0015]

BEST MODE FOR CARRYING OUT THE INVENTION In the electrophoresis chip of the present invention, a plurality of analysis channels connected to individual positions downstream of a purification column portion of a sample introduction channel are provided inside a plate-like substrate. Preferably, the electrode is provided at a position corresponding to at least one of the analysis channels. as a result,
A plurality of types of samples can be sequentially sent to the sample introduction channel, and the target components of different samples can be sequentially introduced into the respective analysis channels, so that multi-analyte analysis can be supported. .

Further, it is preferable that a plurality of purification column sections are provided in parallel in the sample introduction channel inside the plate-like base material. As a result, a plurality of types of samples can be respectively introduced into different purification column sections for purification, and problems such as mixed contamination between samples can be prevented. Further, if each purification column is filled with a different gel filtration filler, it becomes possible to perform purification in accordance with the target component contained in the sample.

[0017]

FIG. 1 is a plan view showing an embodiment of an electrophoresis chip. FIG. 2 is an exploded perspective view showing the embodiment. This example is applied to a DNA sequencer. The electrophoresis chip 1 is formed by bonding a pair of transparent plate-like inorganic members (eg, glass, quartz, silicon, etc.) or plate-like substrates 1a and 1b made of plastic.

The lower plate-like substrate 1b will be described. On the surface of the lower plate-shaped substrate 1b on the side to be bonded to the upper plate-shaped substrate 1a, for example, by photoengraving technology used in a semiconductor manufacturing process, micro-machining technology, ordinary machining technology, laser processing technology, etc. Width is 1
The sample introduction channel 3 is formed of a groove having a depth of about 00 μm and a depth of about 50 μm. On one end 3a side of the sample introduction channel 3, for example, a purification column portion 5 is formed which is formed of a concave portion having a width of about 250 μm, a length of about 5 cm, and a depth of about 50 μm, and containing a gel filtration filler. ing. Sample to the other end 3b side of the inlet flow path 3, a width dimension of 90μm approximately, analysis flow path S ₁ in which the depth is made of a groove of about 50 [mu] m, S _2, ..., in the order S _n from the purification column unit 5 side The branch is formed. Sample introduction channel 3 analysis flow path from S _1, S _2, ..., bifurcation portions S _n is branched F _1, F _2, ..., and F _n.

The upper plate-like substrate 1a will be described. A sample is introduced into the sample introduction channel 3 from one end 3a at positions corresponding to both ends 3a and 3b of the sample introduction channel 3,
Through holes 7a and 7b for discharging from the other end 3b are formed. Analysis passage S _1, S _2, ..., a position corresponding to a position slightly apart from the sample introduction channel 3 of S _n, analysis flow path S _1, S _2, ..., to place the cathode electrode S _n through-hole C _1, C ₂ of, ..., C _n are formed. Analysis passage S _1, S _2, ..., arranged in a position corresponding to the opposite end, the analysis flow path S _1, S _2, ..., an anode electrode S _n is the sample introduction channel 3 S _n A ₁ , A ₂ ,..., A
_n is formed. A purification column unit 5 of the sample introduction channel 3 to a position corresponding to between the analysis flow path S _1, the through-hole G for arranging the ground electrode is formed.

The chip 1 for electrophoresis is composed of the plate-shaped bases 1a and 1a.
b is used in a state of being bonded. This embodiment is called D
When applied to an NA sequencer, for example, a gel filtration filler such as Sephadex (a product of Amersham Pharmacia Biotech Co., Ltd.) is packed in the purification column section 5,
Analysis passage S _1, S _2, ..., through the S _n hole _{C 1, C 2, ...,} C n
Through holes A _1, A _2, and ..., and filling the separation matrix such linear polyacrylamide at a position corresponding to between the A _n, the sample introduction channel 3 and analysis channel S _1, S _2, ..., S
TE other than the part filled with the separation matrix of _n
(Tris EDTA) A liquid feeding medium such as a buffer is filled.

When the electrophoresis chip 1 is used,
Through-hole _{C 1, C 2, ...,} C n, the through hole A _1, A _2, ..., to A _n and the through-hole G, the sample introduction channel 3 and analysis channel S _1,
S _2, ..., arranged electrodes so immersed in each filled liquid S _n, the through-hole _{C 1, C 2, ...,} C n, the through hole A _1,
A ₂ ,..., _An and the through-hole G are hermetically sealed with the electrodes arranged inside. Further, each branch portion F of the sample introduction channel 3
_1, F _2, ..., fluorescence detectors D _1, D ₂ at a position corresponding to the downstream of F _n, ..., place the D _n. The fluorescence detectors D ₁ , D ₂ ,
, D _n are arranged at positions slightly downstream from the branch portions F ₁ , F ₂ ,..., F _n in consideration of the distribution of the purified sample eluted from the purification column 5.

FIG. 3 is a flowchart showing one embodiment of the operation and pre-processing method of this embodiment. Here, a solution before purification prepared by the Sanger method was used as a sample.
The sample before purification contains a fluorescently labeled Sanger reaction product, salt, unreacted substances, and the like. A TE buffer was used as a liquid sending medium. In FIG. 3, n indicates a sample number, and m indicates the number of samples to be subjected to preprocessing. The operation will be described with reference to FIGS.

In order to preprocess the sample of sample number 1, the sample number n is set to "1" (step S1).
For example, using a liquid pump for liquid chromatography, etc.
The sample before purification is passed through the sample introduction channel 3 through the through hole 7a.
To the other end 3a, and sends the liquid inside the sample flow path 3 toward the other end 3b (step S2). The sample before purification is introduced into the purification column unit 5 and is eluted and purified in order from the Sanger reaction product having a higher molecular weight by the action of the gel filtration filler packed in the purification column unit 5. At this time,
Analysis passage S _1, S _2, ..., in order to prevent contamination of the unwanted Sanger reaction products and contaminants to S _n, the through-hole C _1, C _2, ..., a cathode electrode disposed on C _n the potential charged slightly negatively, through holes a _1, a _2, ..., leaving the potential of arranged anode electrode a _n to float.

The Sanger reaction products of sample number n of arrival of the Sanger reaction products of the branch portion F _n corresponding to the analysis flow path S _n of introducing (target component) is detected by a fluorescence detector D _n (step S3 ). Based on the detection signal of the fluorescence detector D _n, Sanger reaction products to determine whether the host vehicle has reached the branch unit F _n (step S4). When Sanger reaction products has not been detected by the fluorescence detector D _n (N
o), continue detection of Sanger reaction products (step S)
3).

[0025] The fluorescence detector D _n when detecting the Sanger reaction products (Yes), float the potential of the through-hole C _n to arranged cathode electrodes corresponding to the analysis flow path S _n introducing the Sanger reaction products to the potential of arranged anode electrode in the through hole a _n positively charged, introduced electrophoretically Sanger reaction product analysis flow path S _n with negative charge (step S5). After introducing the analytical flow path S _n Sanger reaction products, in order to prevent contamination of the analytical channel unwanted Sanger reaction products and contaminants to S _n, the cathode electrode disposed in the through-hole C _n charged potential to the negative, to float the potential of arranged anode electrode in the through hole a _n.

The TE buffer is further supplied to the sample introduction channel 3 to wash the inside of the sample introduction channel 3 and the inside of the purification column section 5 (step S6). It is determined whether or not the pre-processing has been performed for all of the samples of the number m (step S7). When n ≠ m (No), “1” is added to n (step S8), and the process returns to step S2 to perform preprocessing for the next sample number “n + 1”. When n = m, and ends the preprocessing, analysis flow path S _1, S _2, ..., the process proceeds to the step of analyzing the Sanger reaction products introduced into S _n.

As described above, in the embodiment of the electrophoresis chip and the pretreatment method, it is not necessary to separately collect the purified sample in the container, and the target component in the sample is continuously transferred to the analysis channel by the electrophoresis. Can be introduced to
The time required for purifying the sample and introducing the target component in the sample can be reduced. Furthermore, in the examples of the electrophoresis chip and the pretreatment method, handling of a small amount of sample is facilitated.

In the above embodiment, the pretreatment method and the electrophoresis chip according to the present invention are applied to a DNA sequencer, but the present invention is not limited to this.
The pretreatment method and the electrophoresis chip according to the present invention can also be applied to electrophoresis for analyzing the analysis of substances other than NA. Further, the flow path configuration of the electrophoresis chip of the present invention is not limited to the above embodiment, and various changes can be made within the scope of the present invention described in the appended claims. For example, the number of analysis channels may be one, or a plurality of purification columns may be provided. Also,
In the above-described embodiment of the electrophoresis chip, the analysis channel is formed by one groove, but the analysis channel constituting the electrophoresis chip of the present invention is not limited to this. For example, it may be formed by a plurality of intersecting flow paths, such as by forming two intersecting flow paths as shown in FIG.

Further, through for the embodiment of the electrophoretic chip disposing the electrode apertures _{A 1, A 2, ...,} A n, the through-hole _{C 1, C 2, ...,} C n and the through-hole G Are provided, and the electrodes are arranged in the through holes when the electrophoresis chip 1 is used. However, the electrophoresis chip of the present invention is not limited to this, and the electrodes are previously provided on the electrophoresis chip 1. It may be arranged. For example, a metal pattern is formed inside the above-described through hole and on the surface of the upper substrate 1a to use the metal pattern inside the through hole as an electrode, or a desired metal pattern is formed between the upper substrate 1a and the lower substrate 1b. to form a sample introduction channel 3 and analysis channel S _1, S _2, ..., or can the desired electrode metal pattern end in contact with the position of S _n.

[0030]

According to the sample pretreatment method of the present invention, a sample is introduced into a purification column for purifying the sample, the sample is purified, and the sample is separated from a flow channel on the elution side of the purification column. Since the target component in the purified sample is electrophoretically introduced into the flow channel, the time required for purifying the sample and introducing the target component in the sample into the analysis flow channel for performing electrophoresis is reduced. It can be shortened and can easily handle a small amount of sample.

The electrophoresis chip of the present invention has a sample introduction flow path in which a purification column is provided inside a plate-shaped substrate, and an analysis flow path connected downstream of the purification column. A hole that reaches the sample introduction channel at a position corresponding to both ends of the sample introduction channel from the surface of the plate-shaped substrate, and an electrode for generating a potential difference between the sample introduction channel and the analysis channel are provided. As a result, the sample purified by the gel filtration packing material packed in the purification column can be successively electrophoretically introduced into the analysis channel, thereby purifying the sample and purifying the sample into the analysis channel. The time required for introducing the components can be reduced, and the handling of a small amount of sample can be facilitated.

In the electrophoresis chip of the present invention, a plurality of analysis flow paths connected to individual positions on the sample introduction flow path downstream of the purification column section are provided inside the plate-like base material. If electrodes are provided at positions corresponding to at least one position of the flow path, a plurality of types of samples are sequentially sent to the sample introduction flow path, and target components of different samples are sequentially sent to each analysis flow path. It can be introduced and can respond to multi-sample analysis.

In the electrophoresis chip of the present invention, if a plurality of purification column sections are provided in parallel in the sample introduction channel inside the plate-like base material, a plurality of types of samples can be used in different purification columns. It can be introduced into the section and purified, and problems such as mixed contamination between samples can be prevented. Further, if each purification column is filled with a different gel filtration filler, it becomes possible to perform purification in accordance with the target component contained in the sample.

[Brief description of the drawings]

FIG. 1 is a plan view showing an embodiment of an electrophoresis chip.

FIG. 2 is an exploded perspective view showing the same embodiment.

FIG. 3 is a flowchart showing one embodiment of the operation and preprocessing method of the embodiment.

FIG. 4 is a diagram showing a conventional electrophoresis chip;
(A) is a top view of one base material, (B) is a top view of the other base material, and (C) is a side view in a state where both base materials are overlapped.

[Explanation of symbols]

DESCRIPTION OF SYMBOLS 1 Electrophoresis chip 1a Upper plate-like substrate 1b Lower plate-like substrate 3 Sample introduction channel 3a One end of sample introduction channel 3b The other end of sample introduction channel 5 Purification column section 7a, 7b Penetration for sending sample holes _{a 1, a 2, ...,} a n through holes C _1, C ₂ for an anode electrode disposed, ..., C _n through hole D _1, D ₂ for the cathode electrode placement, ..., D _n fluorescence detector F ₁ , F _2, ..., a through hole S _1, S ₂ of F _n bifurcation G grounding electrodes arranged, ..., S _n analysis channels

Claims (4)

[Claims] 1. A sample before purification is introduced into a purification column for purifying the sample, the sample is purified, and the purified sample is supplied to an analysis channel branched from a channel on the elution side of the purification column. A pretreatment method for a sample, which comprises introducing a target component therein electrophoretically. 2. A sample introduction channel provided with a purification column for purifying a sample inside a plate-shaped substrate,
An analysis flow path connected downstream of the purification column portion of the sample introduction flow path, wherein the sample introduction flow path is located at a position corresponding to both ends of the sample introduction flow path from the surface of the plate-shaped substrate. And an electrode for generating a potential difference between the sample introduction channel and the analysis channel at a position corresponding to at least one location of the sample introduction channel and at least one location of the analysis channel. An electrophoresis chip, comprising: 3. A plurality of analysis channels connected to individual positions of the sample introduction channel on the downstream side of the purification column portion in the plate-shaped substrate, and at least one of the analysis channels is provided. The electrophoresis chip according to claim 2, wherein the electrode is provided at a position corresponding to one location. 4. The electrophoresis chip according to claim 3, wherein a plurality of purification column sections are provided in parallel in the sample introduction channel inside the plate-shaped substrate.