JP2003302373A - Biochemical analysis method using beads - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a biochemical analysis method that is useful for the all- inclusive analysis of genes and the analysis of interaction between substances. <P>SOLUTION: The biochemical analysis method is provided, which includes a reaction procedure for performing the mutual reaction of a target substance 3 in a liquid phase to a substance 2 for detection that is immobilized to the surface of beads 1 that can be optically or electrically identified (1), and a beads-fractionating procedure for fractionating the beads 1 (1a, 1b, 1c) that are obtained through the reaction procedure in an electrolyte channel 4 filled with electrolyte R (2). <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a biochemical analysis method useful for gene mutation analysis, gene expression analysis, polymorphism analysis, kinetics of intermolecular interaction and the like. Specifically, it includes a method of separating beads that show an active reaction based on electrophoresis after performing mutual reaction such as hybridization reaction on the surface of beads that function as a carrier for immobilizing substances such as nucleotides. Biochemical analysis method.

[0002]

2. Description of the Related Art At present, a molecular integrated substrate for a bioassay called a so-called DNA chip or a DNA microarray (hereinafter, collectively referred to as "DNA chip"), in which predetermined DNAs are finely arrayed by a microarray technology, is available. Mutation analysis of genes, SNPs (single nucleotide polymorphism) analysis,
It has been used for gene expression frequency analysis, etc., and has begun to be widely used in the fields of drug discovery, clinical diagnosis, pharmacogenomics, forensic medicine and others.

This DNA chip has various types and many kinds of DNA oligo chains and cDNA (co
It is characterized by the fact that it is possible to comprehensively analyze intermolecular interactions such as hybridization because of the accumulation of complementary DNA).

Briefly explaining an example of an analysis method using a DNA chip, mRNA extracted from cells, tissues or the like is subjected to reverse transcription PCR reaction or the like with respect to a DNA probe immobilized on a glass substrate or a silicon substrate. This is a method of performing PCR amplification while incorporating a fluorescent probe dNTP, performing hybridization on the substrate, and performing fluorescence measurement with a predetermined detector.

Here, DNA chips can be classified into two types. The first type is a method of directly synthesizing an oligonucleotide on a predetermined substrate by using a photolithography technique to which a semiconductor exposure technique is applied, and is typically produced by Affymetrix. (For example, refer to Japanese Patent Publication No. 4-505760). Although this type of chip has a high degree of integration, it has a limit in DNA synthesis on a substrate and has a length of about several tens of bases. The second type is also called the "Stanford method", and is prepared by dispensing and immobilizing DNA prepared in advance on a substrate using a tip pin ( See, for example, Japanese Patent No. 3272365). Although this type of chip has a lower degree of integration than the former, it has the advantage that it can immobilize a DNA fragment of about 1 kb.

[0006] At present, a predetermined detection substance is immobilized on a minute detection surface portion provided on a thin flat plate, which is called a biosensor chip including a protein chip, and the detection substance is A biosensor technology is in progress to observe and analyze the mutual reaction of both substances by using the surface plasmon resonance principle and the principle of a crystal oscillator, etc., by passing a small amount of a solution containing the target substance, antibody-antigen reaction and hormone. It has become a useful technique in the analysis of interaction reactions between substances such as response reactions.

[0007]

However, in the above-mentioned conventional DNA chip technology, a nucleotide such as a DNA probe is solid-phased (immobilized) at a narrow spot site of a two-dimensional substrate to carry out a hybridization reaction. Since this technique is performed, the degree of freedom of nucleotide molecules is spatially limited. In addition, steric hindrance may occur during the hybridization reaction. For these reasons, the conventional DNA chip technology has a technical problem that the efficiency of the hybridization reaction is poor and the reaction time tends to be long.

Further, in the above-mentioned biosensor technology, there is a technical problem that it is difficult to handle an extremely small amount of solution on a nanoliter level, and a highly skilled technology is required for high throughput. In addition, the devices or systems for implementing the technology were very expensive.

Therefore, the main object of the present invention is to make full use of a technique of performing a mutual reaction between substances such as hybridization on a bead surface which is optically or electrically distinguishable, and a technique of separating beads by electrophoresis. Therefore, it is intended to improve the reaction efficiency and provide a biochemical analysis method in which the whole procedure is simple.

[0010]

In order to solve the above technical problems, first, in the present application, (1) a bead surface that is color-coded into a plurality of gradations and is optically or electrically distinguishable. A reaction procedure in which a target substance is allowed to interact in a liquid phase with respect to the detection substance immobilized on (2) the beads obtained through the reaction process are electrophoresed and separated in an electrolyte flow path. Bead sorting procedure, these (1),
Provided is a biochemical analysis method including at least the procedure of (2).

Here, in the biochemical analysis method according to the present invention, the minute beads are caused to function as a carrier and a counterweight for the detection substance and the reaction substance (the substance in which the detection substance and the target substance are bound), and It is characterized in that the bead group is put into a predetermined electrolyte flow path, and electrophoresis is performed by using the charge of the substance held by the beads as a driving force to perform separation. That is, the classification of these beads is
It is based on the principle that when the magnitude of the Coulomb force occurs depending on the total amount of charges of the substance bound to the bead surface, the difference in the Coulomb force appears as a difference in the speed of electrophoresis.

More specifically, when the detection substance immobilized on the bead and the target substance in the sample solution show an active reaction, the Coulomb force of the reaction substance retained on the bead is obvious. As it grows. As a result, the beads are attracted to the electrode side opposite to the charge of the reactant in the electrolytic solution flow path to which a DC (Direct current) electric field is applied, and move faster, and the electrode region is moved within a short time. Come to. For example, when the reactant is DNA, the reactant moves to the positive electrode side because it has a negative electrode. On the other hand, if no active reaction is shown,
The beads either stay without migrating or move slowly in the electrolyte flow path.

Here, the beads that can be used in the present invention are minute beads having a structure capable of being optically or electrically distinguished by any means. It is also necessary to record in advance the type of detection substance immobilized on each bead. In the present invention, since it is sufficient to detect the beads themselves which are optically or electrically distinguishable, the tendency labeling and RI labeling of the reaction substances, which have been essential in the past, are unnecessary.

As a method of optically identifying the beads, a method of changing the color tone of the beads can be adopted. For example, two colors of cyanine dyes Cy3 and Cy5 are mixed and dyed, and the level of the color is determined by the content ratio of each fluorescent dye. A group of beads with a tone,
It is possible to combine a plurality of fluorescent dyes without mixing them and to adopt a color-coded bead group or the like. Since the bead has a clear contour, it has an advantage that it can be optically read with high resolution and a low-cost optical system can be adopted.

As a method of electrically identifying the beads, for example, magnetic beads can be used to electrically identify the beads by detecting a current generated by electromagnetic induction.

Thus, using a predetermined read sensor capable of optically or electrically identifying the beads, which kind of beads rapidly moved to the positive electrode side, slowly moved, or did not move. Since it can be accurately grasped, it is possible to easily trace the presence or absence of the mutual reaction between the target substance and the detection substance, the reaction strength, the affinity, the complementarity of the base sequence, and the like. That is, the biochemical analysis method according to the present invention can be used for gene mutation analysis, gene expression analysis, polymorphism analysis, kinetics analysis of intermolecular interaction, analysis of antigen-antibody reaction, hormone response reaction and the like.

The surface of the beads is coated with a substance having a binding site for the detection substance so that various detection substances can be immobilized. The surface of the beads after the coating treatment is fixed with a detection substance such as a single-stranded or double-stranded full-length or fragment nucleotide, peptide, protein, lipid, low-molecular compound, sugar, liposome or other biological substance. Phase it. It is also possible to immobilize different detection substances for each bead that is optically or electrically identified as the same species.

Next, in the present invention, a bead group on which a predetermined substance for detection is immobilized is put into a predetermined buffer solution,
A mutual reaction such as hybridization with the target substance is allowed to proceed for a predetermined time.

According to this method, the substance for detection and the target substance can be diffused in the buffer solution by Brownian motion or the like, which has the advantage of increasing the frequency of contact between the substances. Further, the bead surface has a large surface area and a large reaction space as compared with the conventional spot portion on the substrate of the DNA chip and the detection surface portion on the thin plate of the sensor chip. Steric hindrance during the reaction is not a big problem. Therefore, hybridization and other mutual reactions can proceed efficiently and the reaction can be completed in a short time.

In the present application, the term "hybridization" refers to deoxyribonucleic acid (DNA), ribonucleic acid (RNA), all natural or synthetic nucleic acids including polynucleotides and oligonucleotides, and all complementary strands between nucleic acid derivatives. The formation reaction is included.

In the present invention, when the substance for detection is a nucleotide, the Tm (melting Temperature) of the nucleotide or G
By grouping the beads according to the C content and distinguishing the reaction field for each group, it is possible to devise to carry out the hybridization reaction under the optimal composition or temperature condition of the buffer solution. According to this device, it is possible to carry out the hybridization reaction with high accuracy as compared with the existing DNA chip which has to carry out the hybridization under a uniform reaction condition.

Next, the "sorting procedure" of the bead group is performed by introducing the bead group that has undergone the washing procedure into a predetermined electrolyte solution flow path and performing electrophoresis based on the principle described above. Here, by devising the use of magnetic beads as the beads, and by arranging a magnet on the negative electrode side that is the migration start point, the beads that have been introduced into the electrolyte solution flow path are once placed on the negative electrode side. It is possible to keep it. As a result, free diffusion of the bead group before the start of electrophoresis can be prevented, and accurate electrophoresis measurement can be performed. At the same time as the start of electrophoresis, a device is devised so that the magnetic force of the magnet does not reach the magnetic beads.

In the separation procedure, the substance retained on the bead surface is electrically vibrated to detect the substance immobilized on the bead surface, for example, in the form of mishybridization. A procedure for dissociating the bound target substance can also be performed.

Specifically, the electrolytic solution contains AC of low and medium frequencies.
(Alternating current) An electric field is applied before or at the same time as a DC electric field for electrophoretic separation, and vibration is applied to the target substance remaining in an insufficiently bound state (mishybridization) on the bead surface so that the target substance is physically removed from the detection substance. Detach.

According to this dissociation procedure, beads that have undergone the reaction procedure are roughly classified into "beads that retain a substance that exhibits an active reaction" and "beads in an unreacted state in which only a substance for detection is immobilized". can do. As a result, the bead group can be divided into the negative electrode side and the positive electrode side in the electrolytic solution flow path.

Further, by adopting the above-mentioned dissociation procedure, there is an advantage that the washing and drying procedures which are indispensable in the existing DNA chip, protein chip and other biosensor chips are completely unnecessary. That is, it is possible to simplify the entire analysis work, since extra consideration regarding the selection of the composition of the cleaning liquid and the cleaning conditions is unnecessary, and the cleaning and drying procedure itself is unnecessary.

Next, in the present invention, when the detection substance and the target substance are both single-stranded nucleotides, that is, when the reaction procedure is a hybridization reaction, an electrolyte solution flow path is provided after the fractionation procedure. The beads that have moved to the positive electrode side of the inside are collected, the beads that have migrated to the positive electrode side in the electrolyte flow path are collected, and the target nucleotide chain of the double-stranded nucleotide bound to the beads is subjected to PCR amplification treatment. Then, the base sequence of the target nucleotide chain can be easily known by sequencing. In addition, for the purpose of facilitating the recovery operation, the electrolyte solution flow path to be used may be provided with a structure capable of easily discharging and recovering the beads collected on the positive electrode side to the outside of the tank.

As described above, according to the present invention, an exhaustive gene analysis means having a completely different structure from that of a DNA chip, which is a structure for performing hybridization at a narrow spot portion on a substrate, and a narrow detection surface of a thin plate-shaped sensor chip. It has the technical significance of providing an exhaustive mutual reaction analysis means having a completely different structure from the biosensor chip that promotes mutual reaction between substances.

[0029]

Preferred embodiments of the present invention will be described below with reference to the accompanying drawings. First, FIG. 1 is a diagram showing an embodiment of beads used in the biochemical analysis method according to the present invention, and FIG. 2 is a diagram illustrating coating treatment of the beads.

Reference numeral 1 in FIGS. 1 and 2 denotes spherical polystyrene beads suitable for the biochemical analysis method according to the present invention. The beads 1 are dyed by gradually changing the content ratios of the two types of fluorescent dyes Cy3 (fluorescence wavelength 570 nm, excitation wavelength 543 nm) and Cy5 (fluorescence wavelength 670 nm, excitation wavelength 633 nm). A group of beads having 10 × 10 = 100 gradations is manufactured. The number of gradations of the beads 1 can be appropriately determined as necessary and is not limited to 100 gradations.

These beads 1 have two wavelengths (657n
The color tone is read and identifiable by an optical sensor configured to emit laser light of m, 720 nm).
Note that the beads 1 can be adopted as long as they are optically distinguishable. In the present invention, it is possible to use an identification method by using magnetic beads instead of the beads 1 that can be optically identified, and detecting a current generated by electromagnetic induction.

Next, as shown in FIG. 2, the beads 1 are subjected to a coating treatment suitable for fixing various substances for detection. For example, amino group, carboxyl group, thiol group, aldehyde group, streptavidin, NTA
(Nitrilotriacetic acid), etc. are coated, and then a linker or capture is attached.
The surface layer film 101 of the beads 1 is formed. In addition, an amino group,
Carboxyl groups, thiol groups, and aldehyde groups are suitable for immobilizing molecules having binding sites for these functional groups, streptavidin is suitable for immobilizing biotinylated DNA fragments and biotinylated ligands, and NTA Is H
It is suitable for immobilization of is-Tag protein.

Next, the detection substance is fixed to the beads 1 in a predetermined buffer solution. The substance for detection is a single-stranded or double-stranded nucleotide (full length or fragment, DNA
And both RNA. ), Amino acids, peptides, proteins, lipids, low molecular weight compounds, liposomes and other biological substances are selected according to the purpose of analysis.

When the substance to be detected is a single-stranded nucleotide, it can be used for analysis of hybridization reaction with a target nucleotide, and when it is a double-stranded nucleotide, the specificity between the predetermined sequence site of the nucleotide and the receptor molecule can be used. It can be used for analysis of hormone-responsive reactions composed of dynamic binding. The hormone response reaction is useful for analysis of endocrine disrupting substances and the like.

In FIG. 3A, the beads 1 and the detection substance 2 are attached.
The beads in which the single-stranded nucleotides functioning as are immobilized are schematically shown. Although many single-stranded nucleotides are actually immobilized on the surface of the bead 1, they are omitted in FIG. 3 for convenience of explanation. The beads to which the substance 2 for detection is thus immobilized may be called probe beads.

FIG. 3B schematically shows a state in which the single-stranded nucleotide which is the target substance 3 is actively hybridized with the single-stranded nucleotide which functions as the substance for detection 2.

In the example shown in FIG. 3B, when the detection substance 2 and the target substance 3 actively interact with each other, the amount of negative charges held by the beads 1 increases,
The beads 1 rapidly move to the positive electrode side in the electrolytic solution flow path to which the DC electric field is applied.

FIG. 4 is a diagram schematically showing the movement of the beads 1 in the electrolytic solution flow path.

The electrolytic solution flow path indicated by reference numeral 4 may be formed in a tank shape, may be adopted in a structure in which it is formed in an elongated capillary shape on the substrate, and may be formed in multiple channels. The shape of the flow channel is not limited to a narrow one. When different detection substances are immobilized on beads that are optically (or electrically) identified as the same species, electrophoresis is performed in independent electrolytic solution channels. A negative electrode 41 is formed at one end and a positive electrode 42 is formed at the other end of the electrolyte flow path 4 so that they can be conducted. The arrangement of the positive and negative electrodes is appropriately determined according to the charge of the substance to be electrophoresed.

The electrolytic solution flow path 4 is filled with an electrolytic solution R such as low melting point agarose gel. In the vicinity of the negative electrode 41,
A sample solution S containing a group of beads for which a reaction procedure such as hybridization has been completed is added. This sample solution S can be devised so that it can be injected from an opening / closing opening 44 provided in the electrolyte solution flow path 4. The group of beads 1 in the sample solution S that has been added to the electrolytic solution channel 4 is primarily retained in the movement start region 43 near the negative electrode 41.

It is also possible to employ magnetic beads as the beads 1. In this case, the magnet M is arranged in the movement start region 43, and the magnetic bead group can surely keep the magnetic bead group in the movement start region 43 by the magnetic force of the magnet N. Then, when the DC electric field E is applied to the electrolytic solution R, the magnetic force can be released and the electrophoresis can be performed. As a result, it is possible to prevent the beads 1 group from moving due to diffusion, Brownian motion, etc. before the start of electrophoresis, and thus it becomes easy to observe the movement caused by the action of the DC electric field E alone.

When a DC electric field E is applied to the electrolytic solution R, the group of beads 1 moves to the positive electrode 42 side at a speed according to the charge amount (Coulomb force) of the substance held by the beads 1. It will be. Reference numeral 1a shown in FIG. 4 schematically shows a bead group showing an active reaction, and reference numeral 1b is
Bead group that showed no active reaction, code 1
c shows a bead group in which the reaction was insufficient.

The bead 1a group is quickly attracted to and moved by the positive electrode 42 in a short time, and the one bead 1b group is slowly moved. That is, beads 1a and beads 1
b, 1c, depending on the amount of charge of the substance held,
A remarkable difference occurs in the speed of electrophoresis, and a band based on the color tone of the beads 1 is formed in the electrolyte flow path 4 according to the difference in speed. Which kind of beads move fast and positive electrode part 4
The reaction status of the detection substance 2 and the target substance 3 can be confirmed by confirming which kind of beads gather slowly on the second side and move slowly.

Here, before or at the same time as the DC electric field E is applied to the electrolytic solution R, an AC electric field of low / medium frequency is applied to electrically vibrate the substance retained in the group of beads 1 to activate the active reaction. Target substance 3 which did not show
You may devise so that it may dissociate from the detection substance 2.

By this dissociation procedure, it is possible to dissociate the target substance 3 from the beads 1b that have undergone insufficient reaction (mishybridization, etc.), so that the beads 1 in the electrolytic solution R show an active reaction. Beads 1a
Group and beads 1c that showed no active reaction
It becomes possible to divide into two groups. As a result, existing DN
Since it is not necessary to perform troublesome washing and drying procedures such as A chip and biosensor chip, work efficiency is very good.

Next, the beads 1 moved to the positive electrode part 42 side.
By collecting the group a and analyzing the color tone of the beads 1a by the reading device, it is possible to easily grasp which kind of the detection substance and the target substance showed an active reaction.

The bead 1 group may be recovered from the electrolytic solution flow path 4 automatically through an openable / closable opening 45 provided at a suitable position on the positive electrode portion 42 side.

Here, in the biochemical analysis method according to the present invention, when the detection substance 2 and the target substance 3 are both single-stranded nucleotides, that is, when the above reaction procedure is a hybridization reaction, an electric reaction is performed. After the above-described fractionation procedure by electrophoresis, the beads 1a group that has moved to the positive electrode 41 side in the electrolytic solution flow path 4 is collected, and the target nucleotide chain of the double-stranded nucleotide retained in the beads 1a group is subjected to PCR amplification treatment. , The base sequence of the target nucleotide chain can be easily known by sequencing.

[0049]

EXAMPLE In order to verify the effectiveness of the biochemical analysis method according to the present invention, a single-stranded 25-base DNA probe was immobilized on beads to prepare probe beads. Both the cDNA probe of this probe bead, the beads hybridized with a predetermined mRNA and the beads not hybridized were added to an electrolytic solution under the same conditions and electrophoresed, and the difference in the migration speed was examined. If the difference in the moving speed of both beads can be observed, the effectiveness of the method of the present invention will be demonstrated.

<Pretreatment of Beads> First, the dynal (DY
NAL's mRNA DirectKit (product number:
No. Dynabea (610.11 and 610.12)
A 1 mL tube of ds Oligo dt25 (referred to as “tube”) was shaken lightly to uniformly suspend the beads (specific gravity 1.6, diameter 2.8 μm) that had sunk at the bottom. Next, a 1.5 μL microcentrifuge tube (referred to as “tube”) was placed on an MPC magnet stand manufactured by Dynal, and 10 μL of the suspension was collected from the tube and dispensed. After 30 seconds, when the beads have been attracted to the magnet and the suspension is clear, the liquid is pipetted off and discarded. Remove the tube from the MPC magnet stand and place a 10 μL volume of Lysis / Bindi.
The ng buffer solution was injected, and the tube was gently shaken again to be resuspended and stored.

<Extraction of mRNA> 10 μL of a cryopreserved mouse brain tissue preservation solution was sampled to obtain 1.5 mL.
Dispense into microcentrifuge tube of
L chloroform was added, and the mixture was stirred for 30 seconds with a vortex mixer. Then, the tube was set in a centrifuge and centrifuged at 10,000 rpm for 30 seconds. After completion of the centrifugation, only the water layer on the upper side of the tube was carefully sucked with a pipette, dispensed into an RNase-free microcentrifuge, and 20 μL of Lysis / Binding buffer solution was further added. Next, the tube was attached to a rotary mixer and rotated for 2 minutes to dissolve the cell wall. Next, it was centrifuged and spun at 10,000 rpm for 1 minute to settle foreign matters, and the supernatant was taken out into another tube.

<Reaction Procedure (Hybridization)>
The beads prepared in paragraph 0049 and the mRNA extract prepared in the procedure of paragraph 0050 were mixed and reacted at room temperature for 3 to 5 minutes. And MP of Dynal
It was set on the C magnet stand and waited for 30 seconds to clarify. Next, all the supernatant was discarded, the tube was removed from the stand, and 10 μl of the mRNA extract was dispensed. The tube was placed on a rotator or a rocker and incubated at room temperature for 3 to 5 minutes. Subsequently, the tube was placed again on the stand, waited for 1 minute, and the supernatant was removed by suction with a pipette. 20 μl of washing buffer solution A from the above kit
In addition, the operation of gently shaking and standing on the stand and removing the supernatant was repeated twice, and then 20 μl of the washing buffer solution B of the above kit was added, followed by washing once by the same operation. This sample solution was stored on ice.

<Separation Procedure by Electrophoresis> Paragraph 0043
The sample solution obtained in 1. and the beads prepared in the procedure of Paragraph No. 0049, that is, the beads that have not been hybridized, are separately added to the agarose gel solution, and these are set on separate slide glasses, and examined under a microscope while applying an electric field. It was Specifically, 5
0 μl of a 2% low melting point agarose gel TAE solution was added and gently shaken to suspend. Take 10 μl of the suspension, drop it onto the center of the slide glass, carefully cover the cover glass to prevent bubbles from entering the suspension, and set the surroundings to a high melting point of 1%.
Blocked with agarose gel. Then, the slide glass was placed on a microscope, a DC electric field was applied under the conditions of 20 v / cm and 10 mA, and the moving speed of the bead group was observed at × 200 magnification.

<Results> As a result of the above experiment, the beads showing active hybridization rapidly moved to the positive electrode side. On the other hand, the electrophoresis speed of the beads which did not show active hybridization was obviously slow, and rapid electrophoresis could not be observed even when the electric field was raised to 30 v / cm. From this, the effectiveness of the biochemical analysis method according to the present invention was verified.

The embodiments and examples of the present invention are as described above.
Although a reaction system in which hybridization is performed on beads has been described as a typical example, the biochemical analysis method according to the present invention is not limited to hybridization and is useful for analysis of interaction between low and high molecules. Method, the amount of electric charge (Coulomb force) of the substance retained by the beads
Those which have a technical idea of separating beads that are optically or electrically distinguishable from each other by electrophoresis based on the difference of are widely included.

[0056]

EFFECTS OF THE INVENTION According to the biochemical analysis method of the present invention, a mutual reaction between substances is caused to occur on the surface of beads having a structure capable of being optically or electrically discriminated, and the beads are separated by electrophoresis to obtain a predetermined amount. It is possible to accurately grasp which kind of beads moves rapidly to the positive electrode side, slowly moves, or does not move by using the reading sensor of. Presence / absence of mutual reaction of substances, reaction intensity, affinity, complementarity of base sequences, etc. can be easily traced. Also, because the beads have a well-defined contour, they can be easily read optically at high resolution, which also has the advantage of using a low-cost optical system.

Therefore, the biochemical analysis method according to the present invention can be applied to gene mutation analysis, gene expression analysis, polymorphism analysis, kinetics analysis of intermolecular interaction, analysis of antigen-antibody reaction, enzyme response reaction and the like. Available.

[Brief description of drawings]

FIG. 1 is a view showing an embodiment of beads (1) used in the biochemical analysis method according to the present invention.

FIG. 2 is a view explaining a coating treatment of the beads (1).

FIG. 3 (A) is a diagram schematically showing a bead (1) on which single-stranded nucleotides functioning as a substance for detection (2) are immobilized, (B) a target substance ( 3)
A diagram schematically showing a state in which single-stranded nucleotides that are actively hybridized

FIG. 4 is a diagram schematically showing the movement of beads (1) in an electrolyte flow path (4).

[Explanation of symbols]

1 beads 2 substances for detection 3 Target substance 4 Electrolyte flow path 41 Negative electrode 42 Positive electrode R electrolyte

Front page continuation (51) Int.Cl. ⁷ Identification code FI theme code (reference) G01N 33/543 541 G01N 33/566 33/566 33/66 Z 33/66 33/68 33/68 33/92 Z 33 / 92 27/26 315Z 301A F term (reference) 2G045 BB10 BB14 BB51 DA13 DA36 DA60 DA80 FB02 FB05 GC20 JA01 4B063 QA01 QA12 QA13 QQ43 QR32 QR55 QS34

Claims (8)

[Claims] 1. A biochemical analysis method including at least the following steps (1) and (2). (1) A reaction procedure in which a target substance is allowed to interact with each other in a liquid phase with respect to a detection substance immobilized on the surface of beads that is optically or electrically distinguishable. (2) A bead fractionation procedure in which the beads obtained through the reaction procedure are electrophoresed in an electrolyte channel to be fractionated. 2. The detection substance is any one of single-stranded or double-stranded nucleotides, amino acids, peptides, proteins, lipids, low molecular weight compounds, sugars, liposomes and other biological substances, The biochemical analysis method according to claim 1, wherein the biochemical analysis method is a substance that has an electric charge and is electrophoretic. 3. The biochemical analysis method according to claim 1, wherein different detection substances are immobilized for each bead that is optically or electrically identified as the same species. 4. The biochemical analysis method according to claim 3, wherein electrophoresis is performed for each of the different substances for detection in independent electrolyte flow paths. 5. The biochemical analysis method according to claim 1, further comprising a step of dissociating the insufficiently reacted target substance from the detection substance by electrically vibrating the reaction substance bound to the bead surface. . 6. The biochemical analysis method according to claim 1, wherein the beads are magnetic beads. 7. The biochemical analysis method according to claim 6, wherein the magnetic beads are electrically identified by detecting a current generated by electromagnetic induction. 8. When both the substance to be detected and the target substance are single-stranded nucleotides and the reaction procedure is a hybridization reaction, after the step (2), the inside of the electrolytic solution flow path is moved to the positive electrode side. The biochemical analysis method according to claim 1, wherein the electrophoresed beads are collected, and the target nucleotide chain of the double-stranded nucleotide bound to the beads is amplified by PCR to determine the base sequence of the target nucleotide chain. .