JP4045535B2 - Biochemical analysis method using beads - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
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 showed an active reaction based on electrophoresis after performing an interaction such as a hybridization reaction on the surface of the bead that functions as a carrier for immobilizing substances such as nucleotides. It relates to a biochemical analysis method.
[0002]
[Prior art]
Currently, molecular integration substrates for bioassays called so-called DNA chips or DNA microarrays (hereinafter collectively referred to as “DNA chips”), in which predetermined DNA is finely arranged by microarray technology, are used for gene mutation analysis, It is used for SNPs (single nucleotide polymorphism) analysis, gene expression frequency analysis, etc., and has begun to be widely used in fields such as drug discovery, clinical diagnosis, pharmacogenomics, forensic medicine and others.
[0003]
Since this DNA chip has a large number of DNA oligo chains and cDNA (complementary DNA) integrated on a glass substrate or silicon substrate, comprehensive analysis of intermolecular interactions such as hybridization becomes possible. It is characterized by dots.
[0004]
To briefly explain an analysis method using a DNA chip, a fluorescent probe dNTP is obtained by reverse transcription PCR reaction or the like by extracting mRNA extracted from cells, tissues, etc. with respect to a DNA probe solid-phased on a glass substrate or a silicon substrate. In this method, PCR amplification is carried out while incorporating, hybridization is performed on the substrate, and fluorescence is measured with a predetermined detector.
[0005]
Here, DNA chips can be classified into two types. The first type is one in which oligonucleotides are directly synthesized on a predetermined substrate using a photolithographic technique applying a semiconductor exposure technique, and a typical one is by Affymetrix (Affymetrix). (For example, refer to Japanese translations of PCT publication No. 4-505760). Although this type of chip has a high degree of integration, there is a limit to DNA synthesis on the substrate, and the length is about several tens of bases. The second type is also referred to as “Stanford method”, and is prepared by dispensing and solidifying a prepared DNA on a substrate using a tip-breaking pin ( For example, refer to Japanese Patent No. 3272365. This type of chip is less integrated than the former, but has the advantage that a DNA fragment of about 1 kb can be solid-phased.
[0006]
Currently, a predetermined detection substance is solid-phased on a minute detection surface portion provided on a thin flat plate called a biosensor chip including a protein chip, and a target substance is applied to the detection substance. Biosensor technology has been developed to pass a minute volume of the solution containing the solution and observe / analyze the interaction between the two substances using the principle of surface plasmon resonance, crystal oscillator, etc., such as antibody antigen reaction and hormone response reaction. It has become a useful technique in analyzing the interaction between substances.
[0007]
[Problems to be solved by the invention]
However, the above-described conventional DNA chip technology is a technology in which a nucleotide such as a DNA probe is immobilized (immobilized) in a narrow spot portion of a two-dimensional substrate and a hybridization reaction is performed. The degree of freedom of the nucleotide molecule is 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 low and the reaction time tends to be long.
[0008]
In addition, in the biosensor technology described above, it is difficult to handle a nanoliter level of a very small amount of solution, and there are technical issues such as the need for highly skilled technology when making high throughput. The apparatus or system for implementing the technique was very expensive.
[0009]
Therefore, the main object of the present invention is to make a reaction by making full use of a technique for performing an inter-substance reaction such as hybridization on an optically or electrically distinguishable bead surface and a bead fractionation technique by electrophoresis. The objective is to provide a biochemical analysis method that improves efficiency and simplifies the overall procedure.
[0010]
[Means for Solving the Problems]
In order to solve the above technical problem, first, in the present application, (1) a target substance is placed in a liquid phase with respect to a detection substance fixed on a bead surface that is optically or electrically distinguishable. (2) an electric field application procedure in which a DC electric field and / or an AC electric field is applied to an electrolyte containing the beads obtained through the reaction procedure, and the procedures of (1) and (2) A biochemical analysis method comprising at least
[0011]
Here, in the biochemical analysis method according to the present invention, the microbeads are caused to function as a carrier for a detection substance and a reaction substance (a substance in which a detection substance and a target substance are bound) and a counterweight, thereby allowing the group of beads. Is put into a predetermined electrolyte flow path, and electrophoresis is performed with a driving force in accordance with the amount of electric charge of the substance held on the beads, thereby separating the beads .
[0012]
That is, when the detection substance immobilized on the beads and the target substance in the sample solution show an active reaction, the magnitude of the Coulomb force is generated according to the total charge of the substance held on the bead surface. This difference in Coulomb force appears as a difference in moving speed. More specifically, when the detection substance immobilized on the beads and the target substance in the sample solution show an active reaction, the Coulomb force of the reactant held on the beads is obviously increased . .
[0013]
Here, the beads that can be used in the present invention are minute beads that are configured to be optically or electrically distinguishable regardless of the means. It is also necessary to record in advance the type of the detection substance immobilized on each bead. In the present invention, it is only necessary to detect the bead itself that is optically or electrically discriminable, so that the fluorescent label and RI label of the reactive substance that have been essential in the past are unnecessary.
[0014]
As a method for optically identifying the beads, a method of changing the color tone of the beads can be adopted. For example, two cyanine dyes Cy3 and Cy5 are mixed and dyed, and a color gradation is applied depending on the content ratio of each fluorescent dye. A group of beads or a group of beads that are combined and color-coded without mixing a plurality of fluorescent dyes can be employed. Since the bead has a clear outline, there is an advantage that it can be read optically with high resolution and a low-cost optical system can be adopted.
[0015]
As an electrical identification method for beads, for example, magnetic beads can be used, and electrical identification can be performed by detecting a current generated by electromagnetic induction. When magnetic beads are used, for example, by placing a magnet on the electrode side that is the starting point of electrophoresis, it is possible to temporarily hold the group of beads introduced into the electrolyte flow path to the electrode side. Thereby, the free diffusion of the bead group before the start of the bead migration can be prevented. In addition, at the same time as the start of the bead migration, the magnetic force of the magnet is devised so as not to reach the magnetic beads.
[0016]
This makes it possible to accurately grasp which kind of beads moved quickly, slowly moved, or did not move using a predetermined reading sensor capable of optically or electrically identifying the beads. Therefore, the presence / absence of interaction between the target substance and the detection substance, reaction intensity, affinity, complementarity of the base sequence, etc. can be easily traced. That is, the biochemical analysis method according to the present invention can be used for gene mutation analysis, gene expression analysis, polymorphism analysis, kinetic analysis of intermolecular interaction, antigen-antibody reaction, hormone response reaction, and the like.
[0017]
The bead surface is coated with a substance having a binding site with the detection substance so that various detection substances can be immobilized. Further, a single-stranded or double-stranded full-length or fragment nucleotide, peptide, protein, lipid, low-molecular compound, sugar, liposome or other biological substance is immobilized on the surface of the beads after the coating treatment. Keep in phase. Different detection substances may be immobilized for each bead that is optically or electrically identified as the same species.
[0018]
In the present invention, the electric field application procedure may be performed in an independent electrolyte flow path for each of the different detection substances.
[0019]
Alternatively, a procedure for dissociating a target substance with insufficient reaction from a detection substance by electrically vibrating a reactive substance bound to the bead surface may be employed. For example, an AC ( Alternating current ) electric field is applied to the electrolyte, and vibration is applied to the target substance remaining in an insufficiently bound state (for example, mishybrid) on the bead surface to physically dissociate from the detection substance. Can do. According to such a dissociation procedure, the bead group that has undergone the reaction procedure is broadly divided into “beads that hold a substance that has shown an active reaction” and “unreacted beads in which only a detection substance is fixed”. be able to. As a result, the bead group can be divided into one electrode side and the other electrode side in the electrolyte flow path.
[0020]
Further, by adopting the dissociation procedure, there is an advantage that the cleaning and drying procedures that are essential in the existing DNA chip, protein chip and other biosensor chips are completely unnecessary. That is, unnecessary consideration regarding the selection of the composition of the cleaning liquid and the cleaning conditions is not required, and the cleaning and drying procedures themselves are not required, so that the entire analysis operation can be simplified.
[0021]
When both the detection object and the target substance are single-stranded nucleotides and the reaction procedure is a hybridization reaction, the electrolyte channel has been migrated to one electrode side after the procedure (2). The base sequence of the target nucleotide chain can also be determined by collecting the beads and PCR-amplifying the target nucleotide chain of the double-stranded nucleotide bound to the bead.
[0022]
In this application, the term hybridization refers to all complementary strand-forming reactions between all natural or synthetic nucleic acids and nucleic acid derivatives including deoxyribonucleic acid (DNA), ribonucleic acid (RNA), polynucleotides and oligonucleotides. Includes.
[0023]
Next, in addition to the above-described method invention, the present invention provides an electrolyte flow path for moving the beads by an electric field according to the amount of charge of the substance held on the optically or electrically distinguishable beads, There is provided a biochemical analyzer comprising an electric field applying means for applying a direct current electric field and / or an alternating electric field to an electrolyte flow path. In addition, for the purpose of facilitating the recovery operation of the beads, the electrolyte flow path to be used may be provided with a structure that allows the beads collected on the electrode side to be easily discharged and recovered outside the tank.
[0024]
As described above, the present invention provides a comprehensive gene analyzing means having a configuration completely different from a DNA chip that is configured to perform hybridization at a narrow spot site on a substrate, and a substance on a narrow detection surface of a thin plate-shaped sensor chip. It has the technical significance of providing a comprehensive interaction analysis means having a completely different structure from the biosensor chip that promotes the interaction between the two.
[0025]
The bead surface has a larger surface area and wider reaction space than the spot sites on the substrate of the conventional DNA chip and the detection surface sites on the thin plate of the sensor chip. The steric hindrance is not a big problem. For this reason, hybridization and other interactions can proceed efficiently, and the reaction can be completed in a short time.
[0029]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, preferred embodiments of the present invention will be described 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 for explaining the coating process of the beads.
[0030]
Reference numeral 1 in FIGS. 1 and 2 denotes a spherical polystyrene bead suitable for the biochemical analysis method according to the present invention. The beads 1 are stained by stepwise changing the content ratio of two kinds 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 of 10 × 10 = 100 gradations is produced. Note that the number of gradations of the beads 1 can be appropriately determined as necessary, and is not limited to 100 gradations.
[0031]
These beads 1 can be identified by their color tones read by an optical sensor configured to emit laser light having two wavelengths (657 nm and 720 nm). The bead 1 can be adopted as long as it has an optically distinguishable configuration. In the present invention, it is also possible to use an identification method by detecting a current generated by electromagnetic induction using magnetic beads instead of the optically distinguishable beads 1.
[0032]
Next, as shown in FIG. 2, the beads 1 are subjected to a coating process suitable for fixing various detection substances. For example, the surface film 101 of the beads 1 is formed by coating with an amino group, a carboxyl group, a thiol group, an aldehyde group, streptavidin, NTA (nitrilotriacetic acid), etc., and further binding a linker or a capture. . Amino groups, carboxyl groups, thiol groups, and aldehyde groups are suitable for immobilizing molecules with binding sites for these functional groups, and streptavidin is suitable for immobilizing biotinylated DNA fragments and biotinylated ligands. NTA is suitable for fixing His-Tag proteins.
[0033]
Next, a detection substance is fixed to the beads 1 in a predetermined buffer solution. Substances for detection include single-stranded or double-stranded nucleotides (full length or fragments, including both DNA and RNA), amino acids, peptides, proteins, lipids, low molecular weight compounds, liposomes and other biological materials. Select according to the purpose of analysis.
[0034]
When the detection substance is a single-stranded nucleotide, it can be used for analysis of the hybridization reaction with the target nucleotide. When the detection substance is a double-stranded nucleotide, specific binding between the predetermined sequence site of the nucleotide and the receptor molecule, etc. It can be used for analysis of hormone response reaction composed of The hormone response is useful for analysis of endocrine disrupting substances. In addition, when the detection substance is a nucleotide, the beads are grouped according to the Tm ( melting temperature) or GC content of the nucleotide, and the reaction field is distinguished for each group. , It can be devised to perform a hybridization reaction. According to this device, the hybridization reaction can be performed with higher accuracy than the existing DNA chip that must be hybridized under uniform reaction conditions.
[0035]
FIG. 3A schematically shows a bead in a state where a single-stranded nucleotide functioning as the detection substance 2 is fixed to the bead 1. Although a large number of single-stranded nucleotides are actually immobilized on the surface of the bead 1, in FIG. 3, it is omitted for convenience of explanation. The beads on which the detection substance 2 is thus fixed may be referred to as probe beads.
[0036]
FIG. 3B schematically shows a state in which the single-stranded nucleotide that is the target substance 3 is actively hybridized with the single-stranded nucleotide that functions as the detection substance 2.
[0037]
In the example shown in FIG. 3 (B), the case where the detection substance 2 and the target substance 3 showed interaction active, the negative charge amount to which the bead 1 is held is increased, the beads 1, for example, in the electrolyte flow path multiplied by the DC electric field will move quickly to the positive electrode side.
[0038]
FIG. 4 is a diagram schematically showing how the beads 1 move in the electrolyte flow path.
[0039]
The electrolyte flow path indicated by reference numeral 4 may be formed in a tank shape, may be adopted in a configuration in which it is formed in a narrow capillary shape on a substrate, and may be multi-channeled. The form is not limited narrowly. When a different detection substance is immobilized for each bead that is optically (or electrically) identified as the same species, electrophoresis by an electric field is performed in each independent electrolyte flow path. When a DC electric field is applied to the electrolyte flow path 4, an electrode 41 that functions as a negative electrode is formed at one end , and an electrode 42 that functions as a positive electrode is formed at the other end, thereby enabling conduction . The arrangement of the positive and negative electrodes is appropriately determined depending on the charge of the substance to be electrophoresed.
[0040]
The electrolyte channel 4 is filled with an electrolyte R such as a low melting point agarose gel. In the vicinity of the 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 channel 4. A group of beads in the sample solution S that has been added to the electrolyte flow path 4 is allowed to primarily stay in the movement start region 43 in the vicinity of the electrode 41.
[0041]
It is also possible to employ magnetic beads as the beads 1. In this case, the magnet M is disposed in the movement start area 43, and the magnetic beads can be reliably retained in the movement start area 43 by the magnetic force of the magnet N. Then, when applying an electric field E in the electrolyte solution R, to release the magnetic force, it is possible to perform the migration due to the electric field. As a result, it is possible to prevent the group of beads from moving due to diffusion, Brownian motion, or the like before the start of electrophoresis, so that it is easy to observe the movement caused only by the action of the electric field E.
[0042]
Here, for example, when a DC electric field is applied to the electrolytic solution R, the group of beads 1 is on the side of the electrode 42 that functions as a positive electrode at a speed corresponding to the charge amount (Coulomb force) of the substance held on the beads 1. Will be moved to. The symbol 1a shown in FIG. 4 schematically shows a bead group showing an active reaction, the symbol 1b is a bead group in which the reaction is insufficient, and the symbol 1c is a bead that shows no active reaction at all. Shows the group .
[0043]
The beads 1a group is attracted and moved quickly to the electrode 42 within a short time, and the one beads 1b group moves slowly. That is, the bead 1a and the beads 1b and 1c have a remarkably different migration speed depending on the amount of charge of the substance being held, and the band based on the color tone of the bead 1 depends on the speed difference. It is formed in the flow path 4. By confirming which kind of beads move fast and gather on the positive electrode part 42 side and which kind of beads move slowly, the reaction state between the detection substance 2 and the target substance 3 can be confirmed.
[0044]
Here, before or at the same time as applying a DC electric field to the electrolyte R, by applying a low / medium frequency AC electric field, the substance held in the group of beads 1 was electrically vibrated and showed no active reaction. The target substance 3 may be devised so as to be dissociated from the detection substance 2.
[0045]
By this dissociation procedure, the target substance 3 can be dissociated from the bead 1b group that has been insufficiently reacted (mishybridization etc.), so that the bead group 1 in the electrolyte R is replaced with the bead 1a that has shown an active reaction. It becomes possible to bisect the group and the bead 1c group that did not show any active reaction. As a result, since it is not necessary to perform a troublesome washing / drying procedure such as an existing DNA chip or biosensor chip, work efficiency is very good.
[0046]
Next, by collecting the beads 1a group moved to the positive electrode part 42 side and analyzing the color tone of the beads 1a by a reading device, it is easy to determine which kind of detection substance and target substance showed an active reaction. I can grasp.
[0047]
The collection of the beads 1 group from the electrolyte flow path 4 may be automatically taken out from the openable / closable opening 45 provided at a suitable position on the electrode 42 side where the beads gather .
[0048]
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, electrophoretic migration is performed. After the separation procedure based on the above, the group of beads 1a moved to the electrode 42 side in the electrolyte channel 4 is collected, the target nucleotide chain of double-stranded nucleotides held in the group of beads 1a is subjected to PCR amplification treatment, and the sequence By sequencing, the base sequence of the target nucleotide chain can be easily known.
[0049]
【Example】
In order to verify the effectiveness of the biochemical analysis method according to the present invention, probe beads were prepared by immobilizing single-stranded 25-base DNA probes on beads. Both the cDNA probe of this probe bead and a bead that had been hybridized with a predetermined mRNA and a bead that had not been hybridized were added to an electrolyte 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.
[0050]
<Bead pretreatment>
First, a 1 mL tube of Dynabeads Oligo dt25 (referred to as “▲ 1 ▼ tube”) of DynaDirect's mRNADirectKit (product numbers: No. 610.11 and 610.12) is shaken lightly and sinks to the bottom. The bead group (specific gravity 1.6, diameter 2.8 μm) was uniformly suspended. Next, a 1.5 μL microcentrifugation tube (referred to as “▲ 2 ▼ tube”) is placed on a Dynal MPC magnet stand, and 10 μL of the suspension is collected from the tube and dispensed. did. After 30 seconds, when the beads are attracted to the magnet and the suspension is clear, pipette off the liquid and discard. {Circle around (2)} The tube was taken out of the MPC magnet stand, injected with a volume of 10 μL of Lysis / Binding buffer, and the tube was shaken lightly again to be resuspended and stored.
[0051]
<Extraction of mRNA>
10 μL of a cryopreserved mouse brain tissue preservation solution was collected, dispensed into a 1.5 mL microcentrifugation tube, 20 μL of chloroform was added, and the mixture was stirred with a vortex mixer for 30 seconds. Subsequently, the tube was set in a centrifuge and centrifuged at 10,000 rpm for 30 seconds. After completion of the centrifugation, only the aqueous layer on the upper side of the tube was carefully sucked with a pipette, dispensed into an RNase-free microcentrifugation, 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, the mixture was centrifuged and rotated at 10,000 rpm for 1 minute to precipitate foreign matter, and the supernatant was taken out into another tube.
[0052]
<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 3-5 minutes. Then, it was placed on a Dynal MPC magnet stand and waited for 30 seconds to clear. Next, all the supernatant was discarded, the tube was removed from the stand, and 10 μl of mRNA extract was dispensed. The tube was placed on a rotator or rocker and incubated at room temperature for 3-5 minutes. Subsequently, the tube was placed on the stand again, waited for 1 minute, and the supernatant was removed by suction with a pipette. Add 20 μl of wash buffer solution A from the above kit, shake it lightly on the stand, repeat the procedure of removing the supernatant twice, add 20 μl of wash buffer solution B from the above kit, and wash once with the same procedure. did. This sample solution was stored at ice temperature.
[0053]
<Separation procedure by electrophoresis>
The sample solution obtained in paragraph 0043 and the beads prepared in the procedure of paragraph 0049, that is, beads that were not hybridized, were separately added to the agarose gel solution, and these were set on separate glass slides, and each microscope was applied while applying an electric field. I examined it. Specifically, 50 μl of 2% low melting point agarose gel TAE solution was added to the sample solution and suspended by shaking lightly. 10 μl of the suspension was sampled and dropped onto the center of the slide glass. The cover glass was carefully covered so that bubbles did not enter the suspension, and the periphery was closed with a high melting point 1% agarose gel. Then, a slide glass was set in a microscope, a DC electric field was applied at 20 v / cm and 10 mA, and the moving speed of the bead group was observed at × 200 magnification.
[0054]
<Result>
As a result of the above experiment, beads that showed active hybridization rapidly moved to the positive electrode side. On the other hand, the electrophoresis speed of beads that did not show active hybridization was clearly slow, and rapid electrophoresis could not be observed even when the electric field was increased to 30 v / cm. This proves the effectiveness of the biochemical analysis method according to the present invention.
[0055]
As described above, the embodiments and examples of the present invention have been described with the reaction system that performs hybridization on beads as a representative example, but the biochemical analysis method according to the present invention is not limited to hybridization, a useful method for analysis of interaction between low-high molecular charge quantity of material retained on the beads I row electrophoresis by an electric field based on the difference (Coulomb force), optically or electrically Widely includes those having the technical idea of sorting identifiable beads.
[0056]
【The invention's effect】
According to the biochemical analysis method of the present invention, an interreaction between substances is performed on a bead surface having an optically or electrically distinguishable configuration, and the beads are separated by electrophoresis using an electric field . Using the reading sensor, it is possible to accurately grasp which kind of beads move quickly to the electrode side, or slowly or not, so that the target substance and the detection substance can be detected. Presence / absence of reaction, reaction intensity, affinity, complementarity of base sequences and the like can be easily traced. In addition, since the bead has a clear outline, it can be easily read with high optical resolution, so that an inexpensive optical system can be used.
[0057]
Therefore, the biochemical analysis method according to the present invention can be used for gene mutation analysis, gene expression analysis, polymorphism analysis, kinetic analysis of intermolecular interaction, antigen-antibody reaction, enzyme response reaction, and the like.
[Brief description of the drawings]
FIG. 1 is a view showing an embodiment of a bead (1) used in the biochemical analysis method according to the present invention. FIG. 2 is a view for explaining a coating treatment of the bead (1). The figure which shows typically the bead (1) of the state by which the single-stranded nucleotide which functions as a substance for a detection (2) was fixed (B) Single strand which is a target substance (3) with respect to the same single-stranded nucleotide Fig. 4 schematically shows the state where nucleotides are actively hybridized. Fig. 4 schematically shows how the beads (1) move in the electrolyte flow path (4).
1 Bead 2 Detection substance 3 Target substance 4 Electrolyte flow path
41, 42 Electrode R Electrolyte

Claims (8)

A biochemical analysis method including at least the following procedures (1), (2), and (3).
(1) A reaction procedure in which a target single- stranded nucleotide is subjected to a hybridization reaction with a single-stranded nucleotide for detection immobilized on a bead surface optically or electrically distinguishable.
(2) An electric field application procedure in which a DC electric field and / or an AC electric field is applied to the electrolytic solution containing the beads obtained through the reaction procedure.
(3) An interaction analysis procedure for analyzing the interaction based on a migration state of the beads obtained through the electric field application procedure. A biochemical analysis method including at least the following procedures (1), (2), and (3).
(1) optical or electrical detection substance different for each bead identified in the same species were fixed, reacted procedure for interaction with a target substance with respect to the detection substance.
(2) An electric field application procedure in which a DC electric field and / or an AC electric field is applied to an electrolyte solution containing the beads obtained through the reaction procedure in an independent electrolyte channel for each of the different detection substances .
(3) An interaction analysis procedure for analyzing the interaction based on a migration state of the beads obtained through the electric field application procedure. The detection substance is any one of single-stranded or double-stranded nucleotides, amino acids, peptides, proteins, lipids, low molecular compounds, sugars, liposomes, and other biological substances, and has a charge in the electrolyte. The biochemical analysis method according to claim 2 , wherein the substance is a substance that can be migrated by the driving force of the electric charge. A biochemical analysis method including at least the following procedures (1), (2), (3), and (4) .
(1) A reaction procedure in which a target substance interacts with a detection substance immobilized on a bead surface that is optically or electrically distinguishable.
(2) A dissociation procedure for dissociating a target substance with insufficient reaction from a substance for detection by electrically vibrating a reactive substance bound to the bead surface.
(3) An electric field application procedure in which a DC electric field and / or an AC electric field is applied to an electrolytic solution containing the beads obtained through the dissociation procedure.
(4) An interaction analysis procedure for analyzing the interaction based on a migration state of the beads obtained through the electric field application procedure. A biochemical analysis method including at least the following procedures (1), (2), and (3).
(1) A reaction procedure in which a target substance interacts with a detection substance fixed on the surface of a magnetic bead that is optically or electrically distinguishable.
(2) An electric field application procedure in which a DC electric field and / or an AC electric field is applied to an electrolytic solution containing the magnetic beads obtained through the reaction procedure.
(3) the magnetic beads obtained by the above electric field application step, by electrically identified by detecting the current generated by electromagnetic induction to detect the electrophoretic state of the magnetic beads, on the basis of the electrophoretic state An interaction analysis procedure for analyzing the interaction. A biochemical analysis method including at least the following procedures (1), (2), (3), and (4) .
(1) A reaction procedure in which a target single- stranded nucleotide is subjected to a hybridization reaction with a single-stranded nucleotide for detection immobilized on a bead surface optically or electrically distinguishable.
(2) An electric field application procedure in which a DC electric field and / or an AC electric field is applied to the electrolytic solution containing the beads obtained through the reaction procedure.
(3) An interaction analysis procedure for analyzing the interaction based on a migration state of the beads obtained through the electric field application procedure.
(4) Collect the beads that have migrated to the electrode side in the electrolyte flow path, PCR amplify the target nucleotide chain of double-stranded nucleotides bound to the beads, and determine the base sequence of the target nucleotide chain Procedure for determining the nucleotide sequence to be determined.   The biochemical analysis method according to claim 1, wherein the electric field application procedure of (2) is a procedure of separating beads based on a difference in moving speed according to a charge amount of a substance held on the beads. A biochemical analyzer that detects the interaction between a detection substance and a target substance,
Beads said detection substance different for each same type optically or electrically discernible the same species have been fixed, according to the charge amount of material retained in the beads, each bead of the same type by an electric field Each independent electrolyte flow path to be moved;
Electric field applying means for applying a DC electric field and / or an AC electric field to the electrolyte flow path;
A reading sensor capable of optically or electrically discriminating the migration state of the beads;
A biochemical analyzer comprising:

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