JP2004350613A - Apparatus for feeding or recovering sample substance and method for treating sample substance - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an apparatus and a method for feeding and recovering a sample substance, by which the sample substance is not dried and it is not required to carry out washing operation by using a washing liquid and loss of the sample substance for detection can remarkably be decreased. <P>SOLUTION: The apparatus is at least equipped with a solution tank 11 for storing a solution containing a chargeable sample substance S (T or D), a conductive gel storage tank 12 made to communicate with the solution tank 11 and a first electrode E<SB>1</SB>for forming a direct electric field X<SB>1</SB>(or X<SB>2</SB>). The apparatus or the method comprises feeding the sample substance S (T or D) in the solution tank 11 through a conductive gel G to a reaction region 21 on a substrate 2 by forming the direct electric field X<SB>1</SB>(or X<SB>2</SB>) by applying voltage to a space between the first electrode E<SB>1</SB>and a second electrode E<SB>2</SB>attached to the reaction region 21 of the outside or recovering the sample substance S (T or D) which exists in the reaction region 21 through the conductive gel G into the solution tank 11 of the apparatus 1. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention provides an apparatus for supplying a charged sample material to a reaction region formed on a substrate or the like by using the action of a DC electric field, or for recovering a sample material from the reaction region. About the method.
[0002]
[Prior art]
There is a technique of supplying a sample substance dropwise to a minute reaction area. Specifically, there is a method in which a minute droplet containing a sample substance such as a DNA probe is accurately dropped on a predetermined reaction region (also referred to as a “spot region”). As typical examples of this method, an ink jet printing method, a micro mechanical spotting method, and the like are known.
[0003]
The “ink-jet printing method” is a method in which a nozzle used in an ink-jet printer is applied, and is a method in which a sample material for detection is jetted from a printer head to a substrate using electricity, and fixed. This method includes a piezoelectric ink jet method, a bubble jet method, and an ultrasonic jet method. The piezoelectric ink jet method is a method in which a droplet is ejected by a displacement pressure generated by applying a pulse to a piezoelectric body. The bubble jet method is a thermal method in which droplets are ejected by the pressure of bubbles generated by heating a heater in a nozzle. The ultrasonic jet method is a method in which an ultrasonic beam is applied to a free surface of a liquid to apply a locally high pressure to discharge a microdroplet from the portion.
[0004]
The “micromechanical spotting method” is a method in which a microdroplet containing a sample substance for detection is spotted on a detection surface on a substrate using a printhead equipped with a microspotting pen, capillary (capillary tube), or tweezers. It is.
[0005]
Such a sample substance spotting technique is based on a so-called DNA chip or DNA microarray (hereinafter, referred to as a “microchip”) in which various types and a large number of DNA oligo chains and cDNA (complementary DNA) are arranged in a fine array on a glass substrate or a silicon substrate. This is an important technique for producing or manufacturing an integrated substrate for a bioassay called "DNA chip".
[0006]
The “DNA chip” is, for example, a sample solution containing a target DNA is supplied to a DNA probe or the like supplied and immobilized on a glass substrate or a silicon substrate, and hybridization is performed. After performing an operation of washing away DNAs not shown or mishybridized DNA (hereinafter, referred to as “washing operation”), fluorescence measurement or the like is performed with a predetermined detector to analyze the interaction between molecules and perform gene analysis. It is widely used for mutation analysis, SNPs (single nucleotide polymorphism) analysis, gene expression frequency analysis and the like.
[0007]
In addition, Patent Document 1 discloses a technique for creating a high-density microarray by electrospraying a sample solution. Patent Literature 2 discloses a technique of supplying a sample solution to the same spot a plurality of times by the above-described inkjet printing method. Patent Literature 3 discloses a technique in which a liquid containing a probe is attached to a solid phase surface by the above-described inkjet printing method. Patent Literature 4 discloses a technique of performing a detection experiment by washing with a buffer after hybridization.
[0008]
[Patent Document 1]
JP-A-2001-281252.
[Patent Document 2]
JP-A-2001-186880.
[Patent Document 3]
JP-A-11-187900.
[Patent Document 4]
JP-A-2003-300.
[0009]
[Problems to be solved by the invention]
However, in the conventional sample material (including solution) spotting technique, an operation of dropping the sample substance on the substrate (operation of supplying the sample substance) and an operation of immobilizing the dropped sample substance on a predetermined surface portion are performed. When performing, there is a problem that since the sample substance is exposed to a dry atmosphere, the molecular structure (for example, a three-dimensional structure such as a higher-order structure) may be adversely affected.
[0010]
In addition, in the above-mentioned washing operation performed after performing the interaction (for example, hybridization), in order to minimize false positive and false negative detection errors, selection of an appropriate washing solution and use of the washing solution are performed. The experimenter had to give due consideration to setting the conditions.
[0011]
Further, the above-mentioned washing operation causes the insufficiently immobilized or unimmobilized detection sample substance (eg, DNA probe) present in the reaction area to be washed away together with the sample substance to be washed. In addition, there is a problem that a loss occurs in a valuable experimental material (a sample substance for detection), which raises a manufacturing cost of a DNA chip or the like.
[0012]
Therefore, the present invention mainly (1) does not dry the sample substance, (2) does not need to perform a cleaning operation using a cleaning liquid, and (3) can greatly reduce the loss of the sample substance for detection. It is an object of the present invention to provide an apparatus and a method for supplying or recovering a sample substance, which have the above advantages (1) to (3).
[0013]
[Means for Solving the Problems]
In the present invention, first, the following “sample substance supply / recovery device” is provided. In this apparatus, a sample substance having a property of being charged to a negative charge or a positive charge is supplied to a predetermined reaction region by a driving force of a DC electric field, or is present in a free state or a weak binding force in the reaction region. This is a device devised so that a sample substance can be collected by the driving force. Specifically, the apparatus according to the present invention includes a “solution tank” for storing a solution containing a charged sample substance, an “conductive gel storage tank” connected to the solution tank, and the solution and the conductive material. And a "first electrode" for forming a DC electric field in the conductive gel. In this configuration, the interface between the solution stored in the solution tank and the conductive gel is in contact.
[0014]
In this device configuration, for example, the conductive gel is passed from the solution in the solution tank by applying a voltage to the first electrode disposed in the upper part of the solution tank and the second electrode attached to the external reaction area. To form a “DC electric field” (for example, about 10 V / cm) reaching the reaction region. This “DC electric field” plays a role of driving the charged sample substance in the direction of the electric field. That is, an effect of moving the negatively charged sample substance to the anode side and the positively charged sample substance to the cathode side is exhibited.
[0015]
For example, when supplying a negatively charged sample substance to the reaction region, a DC electric field is formed using the first electrode as a cathode and the second electrode attached to the reaction region as an anode, and the negatively charged sample material is formed. Is collected in a solution tank, a DC electric field may be formed using the first electrode as an anode and the second electrode as a cathode. Similarly, when supplying a positively charged sample substance to the reaction region, a DC electric field is formed using the first electrode as an anode and the second electrode attached to the reaction region as a cathode, and the positive electrode is charged. When the sample substance is collected in the solution tank, a DC electric field may be formed using the first electrode as a cathode and the second electrode as an anode.
[0016]
By the action of driving the charged sample material by the DC electric field, the sample material in the solution tank is supplied to the reaction region by moving and passing through the motorized gel, or the sample material present in the reaction region is It is possible to freely move the conductive gel and collect it in the solution tank. The sample substance collected in the solution tank is, for example, an unfixed or insufficiently fixed sample substance at a predetermined surface of the reaction region.
[0017]
The work relating to the supply or collection of the charged sample substance is performed in a state where the conductive gel is brought into close contact with the reaction area (the interface of the solution stored in the reaction area). No exposure to dry atmosphere. Therefore, there is an advantage that the molecular structure of the sample substance (for example, a three-dimensional structure such as a higher-order structure) is not adversely affected in the course of the work. That is, the sample substance can be supplied to an external reaction region or recovered from the reaction region while maintaining a molecular structure state capable of interacting with a predetermined partner molecule.
[0018]
In addition, in the apparatus according to the present invention, in addition to the above-described “DC electric field”, a device for forming a “high-frequency electric field” in the solution stored in the reaction region may be provided.
[0019]
For example, when a high-frequency electric field of about 1 MV / m is applied, a nucleotide molecule (a nucleotide molecule composed of a large number of polarization vectors composed of a negative charge of a nucleotide chain having phosphate ions and the like and a positive charge of an ionized hydrogen atom) becomes dielectric. Polarization occurs so that the nucleotide molecule can be stretched linearly parallel to the electric field.
[0020]
More specifically, a nucleotide molecule such as a DNA probe that functions as a detection sample substance for detecting hybridization is supplied to the reaction region, and one end (terminal) of the nucleotide molecule is placed on a predetermined surface portion (detection surface) of the reaction region. In the state where () is immobilized, the nucleotide molecules are rounded or layered in a random coil shape or the like. In such a three-dimensional structure, the bases are overlaid with each other, so that steric hindrance occurs during the reaction. When a nucleotide molecule is stretched linearly by the action of a high-frequency electric field, bases do not overlap each other, so steric hindrance during the reaction is reduced, and the hybridization reaction proceeds smoothly in a short time, so detection Efficiency and accuracy can be improved.
[0021]
Here, as a specific means for forming the “high-frequency electric field”, a “third electrode” other than the first electrode and a second electrode attached to the reaction region (for example, the bottom surface) may be used. It can be formed, or can be formed by a “counter electrode” opposed to the reaction region.
[0022]
The first electrode and the second electrode serve to form a “DC electric field” for supplying or recovering the sample substance, and the first electrode and the third electrode, or the counter electrode, for extending the sample substance. It plays a role in forming a “high-frequency electric field”. Note that the first electrode and the second electrode may be designed so that both a DC electric field and a high-frequency electric field can be formed.
[0023]
Here, as the third electrode used for forming the high-frequency electric field, a movable electrode configured to be movable to a position facing the second electrode provided in the reaction region and to be able to retreat from the position is employed. be able to. This “movable electrode” is waiting at another predetermined position during the supply operation or the recovery operation of the sample substance, and when these operations are completed, the position opposite to the second electrode (attached to the reaction area) (E.g., a position above the reaction area) to form a high-frequency electric field, and when the electric field forming operation (extending operation) is completed, returning to the standby position again can be performed by computer control or the like. Electrodes.
[0024]
Further, as the third electrode, an electrode provided at a lower surface (or a bottom surface) position of the above-mentioned conductive gel storage tank and having a shape through which the sample substance can pass can be adopted. For example, it is an electrode plate in which fine holes of a size that allows a sample substance to pass through are formed uniformly and in large numbers. These micropores serve to pass the sample substance moving by the action of the DC electric field formed between the first electrode and the second electrode in the direction of the reaction region or in the direction of the solution tank.
[0025]
The device according to the present invention is a device for supplying a nucleotide molecule (nucleotide chain) for detection such as a DNA probe to a reaction region on a substrate, or extending a nucleotide molecule for detection immobilized on a predetermined surface portion of the reaction region. It is suitable as a device for performing the above.
[0026]
As described above, the device according to the present invention is suitable as a device for efficiently and quickly supplying a charged sample substance contained in a solution in a solution tank to a reaction region. In addition, forcibly collect detection sample material that is not immobilized in the reaction area (at a predetermined surface area) and that is in a free state or inadequately solidified (weak immobilized bond). It is also suitable as a device for performing. Alternatively, the present invention is also suitable as a device for forcibly recovering a substance that does not show any interaction with the immobilized detection sample substance or a sample substance that has erroneously interacted (for example, mishybridized).
[0027]
In this apparatus, by adjusting the intensity of the DC electric field, it is possible to adjust the supply speed, the recovery speed, the supply amount, the recovery amount, the type of the recovery material, and the like of the sample substance. For example, in the collection operation of the sample substance, the intensity of the DC electric field is reduced to collect only the detection sample substance which is not immobilized and is in a free state (for example, the detection sample substance which is in an insufficiently solidified state). ), Or only free substances that did not interact with the immobilized sample substance for detection can be collected (so that sample substances that have erroneously interacted can be prevented).
[0028]
Secondly, the present invention provides the following “sample substance supply method”. Specifically, the present invention provides a method for supplying a sample substance, which allows a charged sample substance contained in a solution to move and pass through a conductive gel by the action of a DC electric field and supply the charged sample substance to a predetermined reaction region.
[0029]
In this method, a charged sample substance such as a nucleotide molecule is temporarily stored in a solution, and the sample substance is transferred from the solution to a conductive gel by forming a DC electric field. The gel is passed and supplied to a predetermined reaction area. That is, the present method is a method in which a charged sample substance is moved in a route of a solution → a conductive gel → a reaction region.
[0030]
Further, this method can be a method including a step of immobilizing the sample substance on a surface (detection surface) formed so as to face the reaction region. That is, the method may be provided with both a procedure for supplying the sample substance and a procedure for immobilizing the sample substance.
[0031]
In the immobilization procedure, the DC electric field is turned off, so that the solidification reaction of the sample substance on the surface is performed exclusively by Brownian motion of the substance. This allows the immobilization reaction to proceed smoothly without being affected by the electric field.
[0032]
Furthermore, the present method is developed into a method including a procedure of extending an immobilized nucleotide molecule by the action of a high-frequency electric field so that the immobilized nucleotide molecule can be arranged in a linear structure that is easy to hybridize. I do.
[0033]
The present method is directed to a method of supplying another charged sample substance that interacts with the sample substance already immobilized in the reaction area to the reaction area by moving the conductive gel by the action of a DC electric field. Can be developed. That is, it is possible to provide a method of sequentially supplying at least two types of sample substances that interact with each other to a predetermined reaction region by the action of a DC electric field. In addition, by turning off the DC electric field when performing this interaction, by devising that the interaction is performed exclusively by Brownian motion, the interaction can proceed smoothly under the influence of the electric field. it can.
[0034]
Thirdly, the present invention provides the following “sample substance recovery method”.
[0035]
First, the present invention provides a sample substance recovery method in which a charged sample substance existing in a solution in a reaction region is moved in a conductive gel by the action of a DC electric field and collected in a predetermined solution tank.
[0036]
The sample substance recovered from the reaction area by the present method is a substance that has not shown an interaction with the detection sample substance immobilized on a predetermined surface portion (detection surface) of the reaction area, or a substance of the reaction area. A sample substance in an unfixed state or in an insufficiently fixed state at a predetermined surface site can be exemplified.
[0037]
Next, in the present invention, (1) a procedure of supplying a charged detection sample substance contained in a solution to a predetermined reaction region by moving a conductive gel by the action of a DC electric field; A) immobilizing the sample substance for detection on a predetermined surface site facing the reaction area; and (3) applying a DC electric field to the sample substance for detection in an unfixed or insufficiently immobilized state on the surface site. And (4) transferring the charged target sample substance contained in the solution to the conductive gel by the action of a DC electric field. (5) a procedure for supplying an interaction between the immobilized detection sample substance and the target sample substance, (6) a procedure for performing the interaction between the immobilized detection sample substance and the target sample substance, Target sample substance that did not show an interaction between Beauty erroneous interactions with target sample substance, provides a procedure for recovering the solution vessel is moved passed through the conductive gel by the action of the DC electric field, the sample material supply recovery method comprising.
[0038]
This method is called supply of detection sample material → fixation of detection sample material → recovery of detection sample material in an unfixed state → supply of target sample material → interaction → collection of target sample material that showed no interaction. It is a comprehensive method that includes a whole series of interaction detection procedures.
[0039]
In this method, when the immobilized detection sample substance is a nucleotide molecule, the immobilization and / or the interaction is performed by devising a procedure for extending the nucleotide molecule by the action of a high-frequency electric field. In such a case, a method devised to turn off the DC electric field can be provided.
[0040]
Here, the main technical terms in the present application are defined.
[0041]
A "reaction zone" is a sample solution storage area that can provide a place for hybridization and other interactions between substances in the liquid phase. For example, a small concave structure or a well structure portion formed on a substrate can be cited. “Surface” or “surface site” is a detection surface that has been subjected to a surface treatment necessary to immobilize a sample substance for detection in order to detect an interaction between substances.
[0042]
"Interaction" broadly refers to chemical bonding or dissociation between substances. For example, in addition to the interaction between single-stranded nucleotide molecules, that is, hybridization, the interaction between a double-stranded nucleotide molecule and a peptide (or protein), the enzymatic response reaction and other intermolecular interactions, the antigen-antibody reaction, the interprotein etc. Include the interaction between small molecules, the interaction between small molecules and large molecules, and the interaction between small molecules, and are not interpreted narrowly.
[0043]
The “detection sample substance” is a substance that is directly or indirectly immobilized on the surface of the reaction region, and the “target sample substance” is a substance that exhibits a specific interaction with the detection sample substance. In some cases, it is labeled with a fluorescent substance or the like.
[0044]
The term “nucleotide molecule” means a polymer of a phosphate ester of a nucleoside in which a purine or pyrimidine base and a sugar are glycoside-linked, and an oligonucleotide including a DNA probe, a polynucleotide, a DNA obtained by polymerizing a purine nucleotide and a pyrimidine nucleotide ( (Full length or fragment thereof), cDNA (cDNA probe), RNA, polyamide nucleotide derivative (PNA), etc. obtained by reverse transcription.
[0045]
“Hybridization” means a reaction of forming a complementary strand (duplex) between nucleotide chains having a complementary base sequence structure.
[0046]
"Steric hindrance" means that the presence of a bulky substituent in the vicinity of a reaction center or the like in a molecule or the posture or tertiary structure (higher-order structure) of a reaction molecule makes it difficult for a molecule of a reaction partner to approach. This means a phenomenon in which a desired reaction (in the present application, hybridization) hardly occurs.
[0047]
“Counter electrode” means a pair of electrodes to which a voltage can be applied.
[0048]
"Conductive gel" means a conductive gel such as agarose gel.
[0049]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, preferred embodiments of the present invention will be described with reference to the accompanying drawings. The present invention is not narrowly limited to the embodiments described below.
[0050]
First, reference numeral 1 shown in FIG. 1 indicates a preferred embodiment of the sample substance supply / recovery device according to the present invention. This sample material supply / recovery device 1 (hereinafter abbreviated as “device 1”) is a device that supplies a chargeable sample material S to a predetermined region of a substrate 2 disposed below, or from the substrate 2 It functions as an apparatus for collecting the predetermined sample material S.
[0051]
The present apparatus 1 has a container-shaped solution tank 11 for storing the solution L containing the sample substance S, a conductive gel storage tank 12 for storing the conductive gel G, and a second tank attached to the upper part of the dissolution tank 11. One electrode E ₁ It is composed of
[0052]
The conductive gel storage tank 12 is connected to the solution tank 11, and the interface between the solution L and the conductive gel G stored in the solution tank 11 is in contact with each other. The conductive gel G also serves to seal the solution L in the solution tank 1 so as not to leak downward.
[0053]
The charged sample substance S contained in the solution L of the solution tank 11 can be widely used as long as it has a negative charge or a positive charge in the solution L, and examples thereof include nucleotide molecules, peptides, and proteins. Can be. As the solution L, a solution obtained by dissolving nucleotide molecules such as DNA with a buffer solution or the like can be used.
[0054]
The conductive gel G stored in the conductive gel storage tank 12 cannot pass through the solvent (such as a buffer) of the solution L, but has a property that the charged sample material S can be moved and passed by the action of the DC electric field. Any gel can be appropriately used as long as it has a gel.
[0055]
The substrate 2 on the lower side of the device 1 is formed of quartz glass, synthetic resin, or the like, and is, for example, a substrate constituting a DNA chip, a protein chip, or another sensor chip. On the upper surface of the substrate 2, there is provided a reaction region (spot region) 21 which is a concave structure portion having a small volume of a well shape or the like and capable of storing a solution such as a buffer solution.
[0056]
The sample substance S is supplied to the reaction region 21 from the solution tank 11 of the apparatus 1 by a predetermined procedure (described later). Since the present apparatus 1 is configured to supply or recover the sample material S in a state of being in close contact with the reaction region 21, the sample material S can be spotted on the substrate 2 without exposing the sample material S to a dry atmosphere.
[0057]
In any part of the reaction region 21, a surface portion or a metal thin film surface portion 3 (hereinafter, referred to as a metal thin film surface portion 3) which has been subjected to a surface treatment so that the sample material S (for example, a sample material D for detection such as a DNA probe) can be immobilized. "Detection surface 3") is provided.
[0058]
The detection surface 3 is appropriately provided on a bottom surface, a side wall surface, or the like of the reaction region 21 and functions as a portion that chemically bonds to a part of the sample material S supplied from the apparatus 1 and fixes the sample material S. Surface. For example, when the sample substance S is a nucleotide molecule for detection such as a DNA probe, one end (terminal) of the nucleotide molecule is fixed to the detection surface 3. In the present invention, the configuration of the detection surface 3 is not narrowly limited.
[0059]
On the back side of the detection surface 3, reference numeral E in FIG. ₂ A second electrode indicated by. This second electrode E ₂ The first electrode E of the device 1 when the device 1 is positioned above the reaction area 21; ₁ And a pair of opposing electrodes can be formed, and a voltage can be applied via a power supply V by turning on / off the switch 4.
[0060]
With this configuration, the electrode E ₁ -E ₂ Plays a role in forming a DC electric field (described later) passing through the solution L, the conductive gel G, and the reaction region 21 (the solution therein). The first electrode E ₁ And the second electrode E ₂ Is configured so that the cathode and anode can be freely switched.
[0061]
Specifically, when the sample material S has a negative charge, the first electrode E ₁ Is the cathode, the second electrode E ₂ When a DC electric field is formed using the sample as a positive electrode, the sample material S becomes a second electrode E as an anode. ₂ Move towards the side. First electrode E ₁ To the anode and the second electrode E ₂ When a direct current electric field is formed with the cathode as the cathode, the sample material S is the first electrode E as the anode. ₂ Move towards the side.
[0062]
On the other hand, when the sample material S is positively charged, the first electrode E ₁ Is the cathode, the second electrode E ₂ When a direct current electric field is formed using the sample electrode as an anode, the sample material S becomes a first electrode E as a cathode. ₁ Move towards the side. First electrode E ₁ To the anode and the second electrode E ₂ Is used as a cathode to form a DC electric field, the sample material S becomes a second electrode E as a cathode. ₂ Move towards the side.
[0063]
Hereinafter, a procedure for supplying and immobilizing the detection sample substance D, which is one of the sample substances S, to the detection surface 3 of the reaction region 21 of the substrate 2 will be described with reference to FIGS. In the following description, a case where the sample substance S is a negatively charged detection sample substance D such as a DNA probe will be described as a representative example, but the present invention is not limited to this.
[0064]
First, FIG. 2A shows a state where the apparatus 1 is located above the substrate 2 at a distance. In this state, the electrode E ₁ -E ₂ , The detection sample substance D is still in the solution L in the solution tank 11.
[0065]
Subsequently, in FIG. 2B, the apparatus 1 arrives at the reaction area 21 of the substrate 2 and the first electrode E ₁ -Second electrode E ₂ A voltage is applied between them (in this case, the first electrode E ₁ Is the cathode, the second electrode E ₂ Is the anode). Thereby, the solution L → the conductive gel G → the DC electric field X passing through the reaction region 21 ₁ Is formed. This DC electric field X ₁ The sample substance D for detection moves from the solution tank 11 to the electric gel storage tank 12 by the action of and is supplied to the reaction area 21. Note that D in FIG. ₁ Indicates a detection sample substance immobilized on the detection surface 3.
[0066]
Next, FIG. 2 (C) shows a detection sample substance (symbol D) immobilized in an unfixed state or an incomplete state existing in the reaction region 21. ₂ Indicated by FIG. 3 is a diagram showing a state in which the liquid is collected in the solution tank 11 of the apparatus 1. At this stage of the procedure, the first electrode E ₁ Is the anode, the second electrode E ₂ Has been switched to the cathode, and the DC electric field X ₁ DC electric field X opposite to ₂ Is formed.
[0067]
When the detection sample substance D is supplied to the reaction region 21 through the procedure shown in FIG. 2B, a specific site of the detection sample substance D is chemically bonded to the detection surface D and is immobilized. Immobilization may be performed via a linker molecule or a spacer molecule in some cases.
[0068]
In this immobilization, due to steric hindrance, etc., the immobilized or incompletely immobilized detection sample substance D ₂ Are mixed in the reaction region 21. Therefore, the detection sample substance D securely immobilized in the reaction region 21 ₁ Select an appropriate voltage strength that can leave only the DC electric field X ₁ DC electric field X opposite to ₂ To form a sample substance D for detection. ₂ Is passed through the conductive gel storage tank 12 and collected in the upper solution tank 11 (see FIG. 2C). As a result, the reaction sample 21 contains the immobilized detection sample substance D. ₁ Only can be present.
[0069]
FIG. 3 is a diagram schematically showing the procedure of FIGS. 2A to 2C in the form of an electric field forming procedure. First, the first electrode E ₁ Is the cathode, the second electrode E ₂ DC electric field X ₁ To supply the detection sample substance D to the reaction area 21. Subsequently, the electric field is temporarily turned off to allow the immobilization reaction of the detection sample substance D to proceed, and after a predetermined time, the first electrode E ₁ To the anode and the second electrode E ₂ To the cathode, the DC electric field X ₁ DC electric field X opposite to ₂ To form
[0070]
Here, when the DC electric field is turned off at the time of the immobilization reaction, the chemical bond (immobilization reaction) between the detection surface 3 and the detection sample substance D is exclusively changed to the natural Brownian motion of the detection sample substance D. This is preferable because it can be advanced in a state where it is entrusted.
[0071]
Based on the above device configuration or procedure, the substrate 2 in which the detection sample substance D is immobilized in the reaction region 21 can be formed. This can be widely used for the production or production of a DNA chip on which a nucleotide molecule for detection such as a DNA probe is immobilized, a protein chip on which a protein for detection is immobilized, or other sensor chips.
[0072]
Next, based on FIGS. 4 and 5, the detection sample substance D is placed on the detection surface 3 in the reaction area 21. ₁ A procedure for supplying a target sample substance T, which is a sample substance S, to the substrate 2 on which is fixed is described.
[0073]
First, FIG. 4A shows a state where the apparatus 1 is positioned above the substrate 2 so as to be separated therefrom. In this state, the electrode E ₁ -E ₂ Since no voltage is applied to the target sample substance T, the target sample substance T still remains in the solution L in the solution tank 11.
[0074]
Subsequently, in FIG. 4 (B), the apparatus 1 arrives at the reaction region 21 of the substrate 2 in a sealed state, and the first electrode E ₁ -Second electrode E ₂ A voltage is applied between them (in this case, the first electrode E ₁ Is the cathode, the second electrode E ₂ Is the anode). Thus, the solution L → the conductive gel G → the DC electric field X passing through the reaction region 21 ₁ Is formed. This DC electric field X ₁ The negatively charged target sample substance T moves from the solution tank 11 through the motorized gel storage tank 12 and is supplied to the reaction area 21 by the action of.
[0075]
Next, FIG. 4C shows the detection sample substance D immobilized in the reaction area 21. ₁ And a target sample substance that did not show an interaction (here, hybridization) ₂ FIG. 3 is a diagram showing a state in which the liquid crystal is collected into the solution tank 11 of the apparatus 1. At this stage of the procedure, the first electrode E ₁ Is the anode, the second electrode E ₂ Is switched to the cathode and the DC electric field X ₁ DC electric field X opposite to ₂ Is formed.
[0076]
When the target sample substance T is supplied to the reaction region 21 through the procedure shown in FIG. 4B, the specific site (specific base sequence portion) of the target sample substance T is immobilized on the detection surface 3. Detection sample substance D ₁ And the interaction (here, hybridization).
[0077]
At this time, the detection sample substance D ₁ Since the target sample substance T and the target sample substance T are rounded in a random coil shape, steric hindrance and the like occur. Thereby, the sample substance D for detection is contained in the reaction area 21. ₁ Sample substance T that interacts with and binds (forms a duplex) ₁ In addition to the above, the target sample substance and the sample substance D for detection which are released into the reaction region 12 without interaction. ₁ And a target sample substance in a state of misinteraction (here, mishybridization) (hereinafter collectively referred to as “target sample substance T”). ₂ ". ) Exists.
[0078]
These target sample substances T ₂ Leaving in the reaction region 21 may cause false positives or false negatives in the operation stage for detecting the presence or absence of the interaction. Therefore, before the detection operation stage, the target sample substance T ₂ It is desirable to remove.
[0079]
Therefore, in the reaction area 21, the detection sample substance D ₁ Target substance T that interacted with ₁ Select an appropriate voltage intensity that can leave only the DC electric field X ₁ DC electric field X opposite to ₂ , The target sample substance T that did not show an interaction or showed a false interaction ₂ Is passed through the conductive gel storage tank 12 and collected in the upper solution tank 1 (see FIG. 4C). As a result, the target sample substance T that has shown an interaction ₁ (And D ₁ ) Can be present.
[0080]
FIG. 5 is a diagram schematically showing a series of method procedures from FIGS. 2A to 2C to FIGS. 4A to 4C according to the format of an electric field forming procedure.
[0081]
First, the first electrode E ₁ Is the cathode, the second electrode E ₂ DC electric field X ₁ The negatively charged detection sample substance D is supplied to the reaction region 21 (see FIG. 2B), and then the electric field is temporarily turned off to fix the detection sample substance D to the detection surface 3. Reaction, and after a predetermined time, the first electrode E ₁ To the anode and the second electrode E ₂ To the cathode, the DC electric field X ₁ DC electric field X opposite to ₂ And the sample substance D for detection in an unfixed state or insufficiently fixed ₂ Is collected in the solution tank 11 (see FIG. 2C). The recovered sample material D for detection ₂ Can be reused.
[0082]
Next, the device 1 containing the target sample substance T is moved to the substrate 2 (see FIG. 4A). ₁ Is the cathode, the second electrode E ₂ DC electric field X ₁ To supply the negatively charged target sample substance T to the reaction region 21 (see FIG. 4B).
[0083]
Subsequently, the electric field is temporarily turned off, and the immobilized detection sample substance D is detected. ₁ The interaction between the target sample material T and the target sample material T is allowed to proceed efficiently by Brownian motion between the materials, and after a predetermined time, the first electrode E ₁ To the anode and the second electrode E ₂ Is switched to a cathode and the DC electric field X ₁ DC electric field X opposite to ₂ To form
[0084]
This DC electric field X ₂ The detection sample substance D ₁ Is released into the reaction region 12 without interacting with ₁ Sample substance T that interacts incorrectly with ₂ Is passed through the conductive gel G and collected in the solution tank 11 (see FIG. 4C).
[0085]
If the above-described apparatus configuration or procedure is used, the detection sample substance D is supplied into the reaction region 21 of the substrate 2, the immobilization reaction of the detection sample substance D proceeds efficiently, and then the detection accuracy is adversely affected. Sample substance D in an unfixed state or the like that gives ₂ Is recovered from the reaction area 21, and then the target sample substance T is supplied to the reaction area 21, and the target sample substance T is immobilized (on the detection surface 3). ₁ (Eg, hybridization) between the target sample substance T and the target sample substance T, which has a false interaction that adversely affects the detection accuracy. ₂ Is recovered from the reaction region 21 in a series of methods (assays).
[0086]
Hereinafter, inventions relating to a device configuration or procedure further improved so as to use a means or procedure for forming a “high-frequency electric field” in addition to a DC electric field will be described with reference to FIGS. 6 to 10.
[0087]
These improved inventions are characterized in that when a high-frequency electric field of about 1 MV / m is applied, a nucleotide molecule (a nucleotide comprising a large number of polarization vectors composed of a negative charge of a nucleotide chain having phosphate ions and the like and a positive charge of ionized hydrogen atoms) This phenomenon utilizes the phenomenon or principle in which dielectric polarization occurs in a molecule), and as a result, nucleotide molecules are linearly stretched in parallel with an electric field.
[0088]
As a specific means or method for forming a high-frequency electric field, a method using a movable electrode (third electrode) and a second electrode (FIG. 6), a third electrode and a second electrode (FIG. 7), and a method of installing a counter electrode opposed to a solution stored in the reaction region 4 of the substrate B (FIG. 9). The device or method including the means or procedure for forming a high-frequency electric field is particularly effective when the sample substance D for detection is a nucleotide molecule such as a DNA probe.
[0089]
First, reference symbol E in FIG. ₃₁ Is the first electrode E described above. ₁ And the second electrode E ₂ The third electrode is a “movable electrode” prepared for forming a high-frequency electric field. According to the method shown in FIGS. 2A to 2C and FIG. 3 and other methods, the detection sample substance D ₁ A substrate 2 (DNA chip or the like) on which (a DNA probe or the like) is immobilized is prepared and used.
[0090]
Movable electrode E ₃₁ Are all controlled by a computer, and are defined to perform a predetermined positioning operation according to a predetermined computer program. Specifically, first, an operation of moving from the standby position onto the substrate 2 is performed, and then, an operation of arranging to close the reaction region 21 is performed (see FIG. 6A).
[0091]
Subsequently, the movable electrode E ₃₁ And the second electrode E ₂ A high-frequency electric field Y of about 1 MV / m is formed in a solution (buffer solution or the like) in the reaction region 12. Thereby, the detection sample substance D immobilized in the reaction area 21 ₁ (A nucleotide molecule such as a DNA probe) can be linearly stretched by the action of the high-frequency electric field Y (stretching operation, symbol D in FIG. 6B). ₃ reference).
[0092]
Subsequently, when the extension operation is completed, the movable electrode E ₃₁ Is moved from above the reaction region 12 (FIG. 6C), and instead, the device 1 is moved to above the reaction region 12 (see FIG. 6D).
[0093]
Next, the first electrode E ₁ And the second electrode E ₂ To the second electrode E ₂ DC electric field X toward ₁ To form This DC electric field X ₁ Thereby, the target sample substance T in the solution L accommodated in the solution tank 1 of the apparatus 1 is supplied to the reaction region 21 (see FIG. 6E).
[0094]
Subsequently, after the electric field is turned off for a predetermined time and the interaction proceeds efficiently, the first electrode E ₁ And the second electrode E ₂ Between the first electrode E ₁ DC electric field X toward ₂ To form This DC electric field X ₂ As a result, the target sample substance T in the reaction region 21 ₂ Is collected in the solution L in the solution tank 1 of the apparatus 1 (see FIG. 6F). When this collection operation is completed, the apparatus 1 is pulled up from the substrate 2 (see FIG. 6 (G)).
[0095]
By performing the above procedure shown in FIG. 6, the detection sample substance D in the extended state is obtained. ₃ Interaction between the target substance T and the target substance T can be performed under the condition that the influence of the steric hindrance is small.
[0096]
Next, in FIG. 7 and FIG. 8, in order to form a “high-frequency electric field” indicated by the reference character Y, the third electrode E is placed at the lower surface (or the bottom surface) of the conductive gel container 12. ₃₂ Is shown in the embodiment.
[0097]
In this embodiment, the device 1 includes the third electrode E ₃₂ Are integrated, so that the movable electrode E ₃₁ There is an operational advantage that there is no need to move. In addition, the third electrode E ₃₂ This also has a function of making it difficult for the gel G to fall out of the storage tank 12, and is also preferable in this respect.
[0098]
This third electrode E ₃₂ As shown in FIG. 7, is an electrode plate which is disposed by being fitted to the lower surface (or the bottom surface) of the conductive gel storage tank 12, and a large number of micro holes 4 penetrating the electrode plate are regularly arranged. It is provided with the form. The diameter or the size of the micropores 4 is set to such an extent that at least the sample material S (T, D) can pass smoothly.
[0099]
Hereinafter, with reference to FIG. ₃₂ A method using the device 1 in which is integrated will be described. First, in FIG. 8A, the detection sample substance D ₁ The third electrode E is placed on the substrate 2 on which the fixation of ₃₂ Indicates the procedure for moving the integrated device 1. Then, the device 1 is attached to the reaction region 21 and the second electrode E ₂ And the third electrode E ₃₂ And a high frequency electric field Y is formed in the reaction region 21 (see FIG. 8B). Due to the action of the high-frequency electric field Y, the detection sample substance D fixed to the detection surface 3 in an extended state. ₃ Are arranged in large numbers.
[0100]
Next, in the procedure shown in FIG. ₁ And the second electrode E ₂ And a DC electric field X from the solution tank 11 to the reaction area 12 via the conductive gel containing tank 12. ₁ To form This DC electric field X ₁ Supplies the target sample substance T to the reaction region 21 of the substrate 2.
[0101]
In the procedure shown in FIG. 8D, the immobilized detection sample substance D ₃ The target sample substance T that has not shown an interaction or has erroneously interacted with each other due to the specific interaction between the target sample substance T and the target sample substance T ₂ With the DC electric field X ₁ DC electric field X from reaction region 12 in the opposite direction to solution bath 11 through conductive gel storage bath 12 ₂ Is collected in the solution tank 1 of the apparatus 1.
[0102]
Next, another embodiment for forming the high-frequency electric field Y will be described with reference to FIG. In this embodiment, the detection surface 3 is formed on one of the side walls of the reaction region 21 of the substrate 2, and the counter electrode E is sandwiched between the reaction regions 21. ₄ , E ₅ Is arranged.
[0103]
The detection sample substance D supplied from the apparatus 1 is immobilized on the detection surface 3 on the side wall (D in FIG. 9A). ₁ reference). This immobilized detection sample substance D ₁ To the counter electrode E ₄ , E ₅ Are extended in the horizontal direction by a high-frequency electric field Y formed by applying a voltage to (see FIG. 9B).
[0104]
Next, the device 1 is brought into close contact with the reaction region 12 so that the first electrode E ₁ And the second electrode E ₂ And a DC electric field X from the solution tank 11 to the reaction area 12 through the conductive gel containing tank 12. ₁ To form This DC electric field X ₁ The target sample substance T is supplied to the reaction region 21 of the substrate 2 by the action of (see FIG. 9C).
[0105]
Subsequently, although not specifically shown in FIG. ₁ Is turned off to allow the interaction to proceed, followed by the target sample substance T that did not show an interaction or showed a false interaction. ₂ To the DC electric field X ₂ And start detection.
[0106]
FIG. 10 is a diagram illustrating a method of supplying and recovering the sample substance S (T, D) using the high-frequency electric field Y according to an electric field forming procedure.
[0107]
First, the first electrode E ₁ And the second electrode E ₂ DC electric field X formed between ₁ Thus, the detection sample substance D is supplied from the solution tank 1 to the reaction region 21 via the conductive gel G. Next, this DC electric field X ₁ Turn off to allow the immobilization reaction to proceed.
[0108]
And the DC electric field X ₁ DC electric field X in the opposite direction to ₂ To the first electrode E ₁ And the second electrode E ₂ And the immobilized or incompletely immobilized detection sample substance D ₂ Is collected in the solution tank 11 from the reaction region 21 via the conductive gel G.
[0109]
Subsequently, the high-frequency electric field Y is applied to the third electrode E ₃₁ (Movable electrode) or E ₃₂ And the second electrode E ₂ And the counter electrode E ₄ -E ₅ Or the like, thereby forming the detection sample substance D immobilized on the detection surface 3. ₁ Elongate.
[0110]
Then again the DC electric field X ₁ Thus, the target sample substance T is supplied from the solution tank 1 to the reaction region 21 via the conductive gel G. Next, the DC electric field X ₁ Is turned off, and the detection sample substance D in the extended state is ₃ With the target sample substance T.
[0111]
Then, the DC electric field X ₁ DC electric field X in the opposite direction to ₂ To the first electrode E ₁ And the second electrode E ₂ Between the target sample substance T and the target sample substance T that did not interact with each other or showed a false interaction (mishybridization). ₂ Is collected from the reaction region 21 into the solution tank 11 via the conductive gel G.
[0112]
FIG. 11 shows an example of the substrate 2. The substrate 2 may be any substrate as long as it can accumulate many and many kinds of detection sample substances D on a glass substrate, a silicon substrate, or the like. In FIG. 11, a substrate on a disk is used, but the shape, configuration, and the like of the substrate are not limited thereto.
[0113]
In FIG. 11, a large number of reaction regions 21 are provided on the surface of the substrate 2. The apparatus 1 is attached to the reaction region 21 on the substrate 2 to supply or collect the sample substance S (the sample substance D for detection or the target sample substance T). The apparatus 1 includes one reaction region (for example, R ₁ ) Can be supplied or recovered with the aim of only the sample substance S, or a plurality of reaction regions (for example, R ₂ Or R ₃ ) Or the sample material S can be simultaneously supplied to and collected from all the reaction regions 21 on the substrate 2.
[0114]
Generally, in many cases, many kinds of detection sample substances D are integrated on the substrate 2. In such a case, the apparatus 1 corresponding to one reaction area 21 is prepared, and a different sample substance D for detection is supplied to each reaction area 21 and the substrate 2 for detection (for example, DNA, Chip).
[0115]
On the other hand, in general, the target sample substance T is often supplied to a plurality of reaction regions in one kind. In such a case, the apparatus 1 capable of simultaneously supplying the target sample substance T to a plurality of reaction regions 21 in a predetermined range is used.
[0116]
As described above, the apparatus 1 according to the present invention corresponds to one reaction region 21 and one row of reaction regions 21 (R ₂ ) Corresponding, multiple rows of reaction regions 21 (R ₃ ), Those corresponding to all the reaction regions 21 on the substrate 2, and the like can be selectively used according to the purpose.
[0117]
In the case of the apparatus 1 corresponding to a large number (or all) of the reaction regions 21, the procedure can be efficiently performed by appropriately changing the sample substances D and T to be put in the solution tank 1. A series of steps such as supply, immobilization, and recovery can be performed at the same time by placing a separate substance of the sample substance D for detection in each of the solution tanks 1 and then performing the subsequent procedures.
[0118]
In the case of the target sample substance T, the same kind of substance is put into all the solution tanks 1 (of the apparatus 1), and then the subsequent steps are performed, so that the interaction can be detected in one operation. . In addition, for example, several experiments, tests, and the like can be performed in a single operation by putting different substances in every several columns. Note that a series of steps, in particular, the movement and combination of the apparatus 1 and the substrate 2 and the procedure for forming an electric field can be executed by computer control or the like.
[0119]
【The invention's effect】
The present invention has the following effects.
[0120]
By using the “sample material supply / recovery device” according to the present invention, the sample material can be spotted on the substrate without exposing the sample material to a dry atmosphere.
[0121]
This apparatus can not only spot a sample substance for detection on a substrate but also supply a target sample substance to a substrate on which the sample substance for detection is immobilized, and the sample substance for detection interacts with the target sample substance (for example, hybridization). Can be used in experiments for detecting whether or not
[0122]
In addition, when performing an experiment to detect whether or not an interaction is exhibited by using the apparatus or the method according to the present invention, a cleaning step using a conventional cleaning solution is not required, and therefore, the experimenter needs to perform an appropriate operation. A simple and effective interaction experiment can be performed without having to consider the selection of a suitable washing solution and setting the conditions of the washing solution. Further, loss of the sample substance due to the washing operation is prevented, and an efficient and economical experiment can be performed.
[0123]
In the apparatus or method according to the present invention, in addition to the DC electric field, since a high-frequency electric field can be used, especially when a nucleotide molecule such as a DNA probe is used as a sample substance for detection, it can be extended. It is possible to improve the efficiency of the interaction (such as hybridization) with the target sample substance.
[0124]
In the present invention, a method of supplying a sample substance onto (a reaction area of) a substrate, a method of recovering a sample substance that has not been immobilized on (a detection surface of) a substrate, and a method of allowing a sample substance for detection to interact with a target sample substance A method capable of reliably detecting the action can be provided.
[0125]
In addition, the present invention is particularly effective for nucleotide molecules such as DNA probes.
[Brief description of the drawings]
FIG. 1 is a view showing a preferred embodiment of a sample substance supply / recovery device (1) according to the present invention.
FIG. 2 is a view showing a series of methods for supplying, immobilizing, and recovering a detection sample substance (D) using the same apparatus (1).
FIG. 3 is a diagram illustrating the same method according to an electric field forming procedure.
FIG. 4 is a view showing a series of methods for supplying, immobilizing, and recovering a target sample substance (T) using the same device (1).
FIG. 5 is a diagram illustrating the same method according to an electric field forming procedure.
FIG. 6 shows a movable electrode (E) as a third electrode. ₃₁ FIG. 4 is a diagram showing a series of methods using ()).
FIG. 7 shows a third electrode (E) arranged on the lower surface of the conductive gel container (12). ₃₂ FIG.
FIG. 8 shows a third electrode (E ₃₂ The figure which showed the series of methods using the device (1) provided with ().
FIG. 9 shows a counter electrode (E) in a reaction area (21). ₃ , E ₄ The figure which showed the series of methods in case of providing.
FIG. 10 is a diagram illustrating a series of methods including a procedure for forming a high-frequency electric field (Y) according to an electric field forming procedure.
FIG. 11 is a view showing an example of a region of a substrate 2 to which a sample substance (S) is supplied from the apparatus 1;
[Explanation of symbols]
1 Sample material supply and recovery device (device)
2 substrate
3 Detection surface (surface part where sample substance is immobilized)
11 Solution tank
12 Conductive gel storage tank
21 Reaction zone
D Sample substance for detection
D ₁ Immobilized detection sample substance
D ₂ Unfixed or insufficiently immobilized detection sample substance
D ₃ Extended sample material for detection
E ₁ First electrode
E ₂ Second electrode
E ₃₁ , E ₃₂ Third electrode
E ₄ , E ₅ Counter electrode
G Conductive gel
L solution
S sample substance
T Target sample substance
T ₁ Interacting target sample material
T ₂ Target sample material that did not interact or showed false interaction
X ₁ DC electric field for supply
X ₂ DC electric field for recovery
Y high frequency electric field

Claims (21)

A solution tank for storing a solution containing a chargeable sample substance, a conductive gel storage tank connected to the solution tank, and a first electrode for forming a DC electric field, at least comprising:
By applying a voltage to the first electrode and a second electrode provided in an external reaction region to form the DC electric field, one of the following means (1) and (2) can be used. Sample material supply and recovery device to be performed.
(1) A means for supplying the sample substance in the solution tank to the reaction region by moving and passing the electric gel.
(2) Means for collecting the sample substance present in the reaction region in the solution tank by moving and passing the electric gel. 2. The apparatus according to claim 1, further comprising means for forming a high-frequency electric field in the solution stored in the reaction region in addition to the DC electric field. The sample substance supply / recovery device according to claim 2, wherein the high-frequency electric field is formed between the second electrode and a third electrode other than the first electrode. The sample substance supply / recovery device according to claim 3, wherein the third electrode is a movable electrode that can move to a position facing the second electrode and can move away from the position. The sample material supply / recovery device according to claim 3, wherein the third electrode is provided at a lower surface position of the conductive gel storage tank, and has a shape through which the sample material can pass. 3. The sample substance supply / recovery device according to claim 2, wherein the high-frequency electric field is formed by a counter electrode opposed to the reaction region. The sample substance supply / recovery device according to claim 1, wherein the sample substance is a nucleotide molecule. The sample substance supply / recovery device according to claim 1, wherein the sample substance collected in the solution tank is a sample substance that is not fixed or insufficiently fixed to a predetermined surface portion of a reaction region. A sample substance supply method in which a charged sample substance contained in a solution is moved and passed through a conductive gel by the action of a DC electric field and supplied to a predetermined reaction region. The method according to claim 9, further comprising a step of immobilizing the sample substance on a surface facing the reaction region. 11. The method according to claim 10, wherein the DC electric field is turned off during the immobilization procedure. 10. The method according to claim 9, wherein the sample substance is a nucleotide molecule. 13. The method according to claim 12, further comprising the step of extending the immobilized nucleotide molecules by the action of a high-frequency electric field. The other charged sample material interacting with the sample material immobilized in the reaction region is supplied to the reaction region by moving the conductive gel by the action of a DC electric field. 11. The method for supplying a sample substance according to item 10. 15. The method according to claim 14, wherein the DC electric field is turned off when the interaction is performed. A sample substance collection method in which a charged sample substance present in a solution in a reaction region is moved and passed through a conductive gel by the action of a DC electric field and collected in a solution tank. 17. The sample substance recovery method according to claim 16, wherein the sample substance to be recovered is a sample substance which is unfixed and / or insufficiently fixed to a predetermined surface portion of the reaction region. The sample substance to be recovered is a substance that has not shown an interaction with a detection sample substance immobilized on a predetermined surface portion of the reaction region or / and a substance that has erroneously interacted. Item 17. The method for recovering a sample substance according to Item 16. A procedure for supplying a charged detection sample substance contained in a solution to a predetermined reaction region by moving a conductive gel by the action of a DC electric field,
For a predetermined surface site facing the reaction region, a procedure of immobilizing the detection sample material,
A procedure in which the detection sample substance in an unfixed state or an insufficiently fixed state is moved and passed through a conductive gel by the action of a DC electric field and collected in a solution tank on the surface site,
A procedure in which the charged target sample substance contained in the solution is supplied to a predetermined reaction region by moving the conductive gel by the action of a DC electric field,
Performing an interaction between the immobilized detection sample substance and the target sample substance,
The solution sample is moved by passing a target sample substance that has not shown an interaction with the immobilized detection sample substance or / and a target sample substance that has erroneously interacted through the conductive gel by the action of a DC electric field. And recovering the sample substance. 20. The method according to claim 19, further comprising the step of, when the immobilized detection sample substance is a nucleotide molecule, extending the nucleotide molecule by the action of a high-frequency electric field. 20. The method for supplying and recovering a sample substance according to claim 19, wherein the DC electric field is turned off when performing the immobilization and / or the interaction.

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