JP4349002B2 - Sample substance supply and recovery method using apparatus for supplying or recovering sample substance - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an apparatus for supplying a charged sample material to a reaction region formed on a substrate or the like using the action of a direct current electric field, and for recovering the sample material from the reaction region, and Regarding the method.
[0002]
[Prior art]
There is a technique in which a sample substance is dropped and supplied to a minute reaction region. Specifically, there is a method in which a minute droplet containing a sample substance such as a DNA probe is accurately dropped onto a predetermined reaction region (also referred to as a “spot region”). As typical examples of this method, an inkjet printing method, a micromechanical spotting method, and the like are known.
[0003]
The “ink-jet printing method” is a method of applying nozzles used in an ink-jet printer, and is a method in which a sample material for detection is ejected from a printer head onto a substrate using electricity and fixed as in an ink-jet printer. 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 and a minute drop is emitted from the portion by applying a high pressure locally.
[0004]
The “micro mechanical spotting method” is a method of spotting microdrops containing a sample material for detection on a detection surface portion on a substrate by using a print head equipped with a micro spotting pen, a capillary (capillary tube), or tweezers. It is.
[0005]
Such a spotting technique for a sample substance is a so-called DNA chip or DNA microarray (hereinafter referred to as “DNA chip” or “DNA microarray”) in which a large number of DNA oligo chains and cDNA (complementary DNA) are finely arranged and integrated on a glass substrate or silicon substrate. This is an important technique for producing or manufacturing an integrated substrate for bioassay called “DNA chip”.
[0006]
For example, a “DNA chip” performs hybridization by supplying a sample solution containing a target DNA to a DNA probe or the like supplied and immobilized on a glass substrate or a silicon substrate, followed by hybridization. After performing the work of washing away DNA not shown or mishybridized DNA (hereinafter referred to as “washing work”), the fluorescence measurement is performed with a predetermined detector, thereby analyzing the interaction between molecules and the gene. 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 Document 2 discloses a technique for supplying a sample solution to the same spot a plurality of times by the inkjet printing method. Patent Document 3 discloses a technique in which a liquid containing a probe is attached to a solid surface by the inkjet printing method. Patent Document 4 discloses a technique for performing a detection experiment by washing with a buffer after hybridization.
[0008]
[Patent Document 1]
JP 2001-281252 A.
[Patent Document 2]
JP 2001-186880 A.
[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 spotting technique of sample material (including solution), the work of dropping the sample material on the substrate (work of supplying the sample material), the work of fixing the dropped sample material on a predetermined surface portion, etc. When performing, since the sample substance is exposed to a dry atmosphere, there is a problem that the molecular structure (for example, a three-dimensional structure such as a higher order structure) may be adversely affected.
[0010]
Further, in the above washing operation performed after the interaction (for example, hybridization) is performed, in order to prevent false positive and false negative detection errors as much as possible, the selection of an accurate washing solution and the washing solution The experimental workers had to give due consideration to the setting of conditions.
[0011]
Furthermore, because of the washing operation, the incompletely immobilized or non-immobilized detection sample substance (for example, DNA probe) existing in the reaction region is washed away together with the sample substance for the purpose of washing. However, there is a problem in that valuable experimental materials (detection sample substances) are lost, which increases the manufacturing cost of DNA chips and the like.
[0012]
Therefore, the present invention mainly (1) does not dry the sample material, (2) it is not necessary to perform a cleaning operation using the cleaning liquid, (3) can greatly reduce the loss of the sample material for detection, It is an object of the present invention to provide an apparatus and method for supplying or recovering a sample substance having the advantages (1) to (3).
[0013]
[Means for Solving the Problems]
In the present invention, first, the following “sample substance supply and recovery device” is provided. In this apparatus, a sample substance having a property of being charged negatively or positively is supplied to a predetermined reaction region by a driving force of a DC electric field, or is present in the reaction region in a free state or a weak binding force. It is an apparatus devised so that the sample substance can be collected by the driving force. Specifically, the apparatus according to the present invention includes a “solution tank” that stores a solution containing a charged sample substance, a “conducting gel storage tank” that communicates with the solution tank, the solution, and the conductive material. And a “first electrode” for forming a direct current electric field in the gel. In this configuration, the interface between the solution stored in the solution tank and the conductive gel is in contact.
[0014]
In this apparatus configuration, for example, the conductive gel is passed from the solution in the solution tank by applying to the first electrode arranged in the upper part of the solution tank and the second electrode attached to the external reaction region. Thus, a “DC electric field” (for example, about 10 V / cm) reaching the reaction region can be formed. This “DC electric field” serves to drive a charged sample substance along the direction of the electric field. That is, the negatively charged sample material is moved to the anode side, and the positively charged sample material is moved to the cathode side.
[0015]
For example, when supplying a negatively charged sample substance to the reaction region, a negative electric charge is formed by forming a DC electric field using the first electrode as a cathode and the second electrode attached to the reaction region as an anode. Is collected in the solution tank, a DC electric field may be formed by using the first electrode as an anode and the second electrode as a cathode. Similarly, when a positively charged sample substance is supplied 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 this positive charge 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]
Due to the driving action of the charged sample material by the direct current electric field, the sample material in the solution tank is supplied to the reaction region by moving through the electric gel, or the sample material existing in the reaction region is supplied to the electric motor. It is possible to freely move the gel and collect it in the solution tank. The sample material collected in the solution tank is, for example, a sample material in an unfixed or insufficiently fixed state at a predetermined surface portion of the reaction region.
[0017]
Since the work related to the supply or recovery of the charged sample substance is performed in a state where the conductive gel is in close contact with the reaction region (the solution interface stored in the reaction area), the sample substance is removed during the work process. There is no exposure to a dry atmosphere. For this reason, 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 the external reaction region or recovered from the reaction region while maintaining the molecular structure state capable of interacting with a predetermined partner molecule.
[0018]
In addition to the above-described “DC electric field”, the apparatus according to the present invention can be devised to include means for forming a “high-frequency electric field” in the solution stored in the reaction region.
[0019]
For example, when a high-frequency electric field of about 1 MV / m is applied, a dielectric is applied to nucleotide molecules (nucleotide molecules composed of a number of polarization vectors composed of negative charges of nucleotide chains including phosphate ions and positive charges of ionized hydrogen atoms). Polarization occurs, so that the nucleotide molecules can be stretched linearly parallel to the electric field.
[0020]
More specifically, a nucleotide molecule such as a DNA probe that functions as a sample material for detection for detecting hybridization is supplied to the reaction region, and one end (terminal) of the nucleotide molecule is provided on a predetermined surface portion (detection surface) of the reaction region. ) Is immobilized, this nucleotide molecule is rounded or stacked in a random coil shape or the like. In such a three-dimensional structure state, bases overlap each other, resulting in steric hindrance during the reaction. When a nucleotide molecule is stretched in a straight line by the action of a high-frequency electric field, bases do not overlap each other, so there is less steric hindrance during the reaction, and the hybridization reaction proceeds smoothly in a short time. Efficiency and accuracy can be improved.
[0021]
Here, as a specific means for forming the “high-frequency electric field”, between the second electrode attached to the reaction region (for example, the bottom surface thereof) and the “third electrode” other than the first electrode, It can also be formed, or can be formed by “counter electrodes” that are arranged so as to sandwich the reaction region.
[0022]
The first electrode and the second electrode serve to form a “DC electric field” for supplying or recovering the sample material, and the first electrode and the third electrode, or the counter electrode, for extending the sample material. Plays the role of creating a “high frequency electric field”. In addition, you may devise so that both a direct current electric field and a high frequency electric field can be formed using a 1st electrode and a 2nd electrode.
[0023]
Here, as the third electrode used for the formation of the high-frequency electric field, a movable electrode configured to be movable to a position facing the second electrode attached to the reaction region and to be able to leave the position is adopted. be able to. This “movable electrode” stands by at another predetermined position at the time of supplying or recovering the sample substance, and when these operations are completed, the position facing the second electrode (attached to the reaction region) (For example, a position above the reaction region) to form a high-frequency electric field, and when this electric field forming work (extension work) is completed, the operation of returning to the standby position again can be performed by computer control or the like. Electrode.
[0024]
In addition, as the third electrode, an electrode provided at the position of the lower surface (or the bottom surface) of the above-described conductive gel container and having a shape through which the sample substance can pass can also be employed. For example, it is an electrode plate in which a large number of fine holes of a size through which a sample substance can pass are formed uniformly. These micropores play a role of allowing a sample substance that is moved by the action of a DC electric field formed between the first electrode and the second electrode to pass in the reaction region direction or the solution tank direction.
[0025]
The apparatus according to the present invention is an apparatus for supplying a detection nucleotide molecule (nucleotide chain) such as a DNA probe to a reaction region on a substrate, or extends a detection nucleotide molecule immobilized on a predetermined surface portion of the reaction region. It is suitable as a device to perform.
[0026]
As described above, the apparatus according to the present invention is suitable as an apparatus for efficiently and rapidly supplying the charged sample substance contained in the solution in the solution tank to the reaction region. In addition, the sample material for detection that is not immobilized in the reaction area (predetermined surface part) or the sample material for detection that is in a state of insufficient solidification (a state where the immobilized binding is weak) is forcibly recovered. It is also suitable as a device to perform. Alternatively, it is also suitable as a device for forcibly recovering a substance that has not interacted with the immobilized sample substance for detection or a sample substance that has been misinteracted (for example, mishybridization).
[0027]
In this apparatus, it is possible to adjust the supply rate, recovery rate, supply rate, recovery rate, type of recovery material, etc. of the sample substance by adjusting the strength of the DC electric field. For example, in the sample material recovery operation, by reducing the strength of the DC electric field, only the detection sample material that is not immobilized and recovered is recovered (the detection sample material that is not sufficiently solidified). It is possible to collect only the free substance that did not interact with the immobilized detection sample substance (to prevent collection of erroneously interacted sample substance).
[0028]
Secondly, the present invention provides the following “sample substance supply method”. Specifically, there is provided a sample substance supply method for supplying a charged sample substance contained in a solution to a predetermined reaction region by moving and passing through a conductive gel by the action of a direct current electric field.
[0029]
In this method, a charged sample material such as a nucleotide molecule is accommodated in a temporary solution, and the sample material is moved from the solution to a conductive gel by the formation of a direct current electric field. The gel is passed through and supplied to the predetermined reaction area. That is, this method is a method in which a charged sample substance is moved along a route of solution → conductive gel → reaction region.
[0030]
Further, this method can include a procedure for immobilizing the sample substance on a surface (detection surface) formed so as to face the reaction region. That is, a method including both a sample substance supply procedure and an immobilization procedure can be adopted.
[0031]
In addition, the DC electric field is turned off during the immobilization procedure, so that the solidification reaction of the sample substance on the surface is performed exclusively by the Brownian motion of the substance. Thereby, an immobilization reaction can be smoothly advanced in a state without the influence of an electric field.
[0032]
Furthermore, the present method can be developed into a method including a procedure for extending a nucleotide molecule immobilized by the action of a high-frequency electric field, so that the immobilized nucleotide molecule can be arranged into a linear structure that is easy to hybridize. To do.
[0033]
This method is a method in which another charged sample substance that interacts with the sample substance already immobilized in the reaction region is supplied to the reaction region by moving the conductive gel by the action of a direct current electric field. It can be developed. That is, it is possible to provide a method of sequentially supplying at least two kinds 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, it is possible to make the interaction proceed smoothly under the influence of the electric field by devising the interaction to be performed exclusively by the Brownian motion. it can.
[0034]
Thirdly, the present invention provides the following “sample substance recovery method”.
[0035]
First, there is provided a sample material recovery method for recovering a charged sample material present in a solution in a reaction region in a predetermined solution tank by moving the inside of a conductive gel by the action of a direct current electric field.
[0036]
The sample substance recovered from the reaction region by this method is a substance that has not interacted with the detection sample substance immobilized on the predetermined surface portion (detection surface) of the reaction region, or the reaction region. The sample substance which is in the non-fixed state or the immobilization state in a predetermined surface part can be mentioned.
[0037]
Next, in the present invention, (1) a procedure for supplying the charged detection sample material contained in the solution to a predetermined reaction region by moving the conductive gel by the action of a direct current electric field; ) A procedure for immobilizing the detection sample substance on a predetermined surface part facing the reaction region; and (3) a DC electric field for the detection sample substance in an unfixed or insufficiently immobilized state on the surface part. (4) The charged target sample substance contained in the solution is moved by the action of the direct current electric field to move the conductive gel through the conductive gel. A procedure for supplying to a predetermined reaction region; (5) a procedure for performing an interaction between the immobilized sample material for detection and a target sample material; and (6) an immobilized sample material for detection. Target sample material that did not show any 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 consists of supplying the sample material for detection → immobilizing the sample material for detection → collecting the sample material for detection in an unfixed state, etc. → supplying the target sample material → interaction → collecting the target sample material that did not show any interaction. It is a comprehensive method that includes a whole series of interaction detection procedures.
[0039]
In this method, in the case where the immobilized sample material for detection is a nucleotide molecule, it is devised to include a procedure for extending the nucleotide molecule by the action of a high-frequency electric field, and the immobilization and / or the interaction is performed. In some cases, a method devised to turn off the DC electric field can be provided.
[0040]
Here, main technical terms in the present application are defined.
[0041]
The “reaction area” is a sample solution storage area that can provide a field of interaction between hybridization and other substances in the liquid phase. For example, a minute concave structure or well structure portion formed on the substrate can be mentioned. The “surface” or “surface part” is a detection surface that has been subjected to a surface treatment necessary for immobilizing a sample material for detection in order to detect an interaction between the materials.
[0042]
“Interaction” broadly means a chemical bond or dissociation between substances. For example, in addition to the interaction between single-stranded nucleotide molecules, that is, hybridization, interaction between double-stranded nucleotide molecule and peptide (or protein), enzyme response reaction, other intermolecular interactions, antigen-antibody reaction, protein-to-protein, etc. It includes a wide range of interactions between macromolecules, interactions between small molecules and macromolecules, and interactions between small molecules and is not interpreted narrowly.
[0043]
The “detection sample substance” is a substance that is directly or indirectly immobilized on the surface region 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]
“Nucleotide molecule” means a polymer of a phosphate ester of a nucleoside in which a purine or pyrimidine base and a sugar are glycosidically bonded, and includes an oligonucleotide, a polynucleotide containing a DNA probe, a DNA in which a purine nucleotide and a pyrimidine nucleotide are polymerized ( Full length or a fragment thereof), cDNA obtained by reverse transcription (cDNA probe), RNA, polyamide nucleotide derivative (PNA) and the like are widely included.
[0045]
“Hybridization” means a complementary strand (duplex) formation reaction between nucleotide strands having a complementary base sequence structure.
[0046]
“Steric hindrance” makes it difficult to access the reaction partner molecule due to the presence of bulky substituents near the reaction center in the molecule, the posture of the reaction molecule, and the three-dimensional structure (higher order structure). This means a phenomenon in which a desired reaction (hybridization in the present application) hardly occurs.
[0047]
“Counter electrodes” means a pair of electrodes to which a voltage can be applied.
[0048]
“Conductive gel” means a conductive gel such as agarose gel.
[0049]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, preferred embodiments of the present invention will be described with reference to the accompanying drawings. The present invention is not limited to the embodiments described below.
[0050]
First, reference numeral 1 shown in FIG. 1 shows a preferred embodiment of a sample substance supply / recovery device according to the present invention. This sample substance supply and recovery apparatus 1 (hereinafter abbreviated as “apparatus 1”) is an apparatus that supplies a charged sample substance S to a predetermined region of a substrate 2 disposed on the lower side, or from the substrate 2. It functions as an apparatus for collecting the predetermined sample material S.
[0051]
The apparatus 1 includes a container-like solution tank 11 that stores a solution L containing the sample substance S, a conductive gel container 12 that contains a conductive gel G, and a first part attached to the upper part of the dissolution tank 11. One electrode E ₁ It consists of and.
[0052]
The conductive gel storage tank 12 is connected to the solution tank 11, and the interface between the solution L stored in the solution tank 11 and the conductive gel G is in contact. The conductive gel G also plays a role of sealing so that the solution L in the solution tank 1 does not 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 is a negatively charged or positively charged substance in the solution L. Examples thereof include nucleotide molecules, peptides, and proteins. Can do. In addition, as the solution L, what melt | dissolved nucleotide molecules, such as DNA, with a buffer solution etc. can be used.
[0054]
The conductive gel G stored in the conductive gel storage tank 12 cannot pass the solvent (buffer solution or the like) of the solution L, but has the property that the charged sample substance S can be moved and passed by the action of the DC electric field. Any gel can be used as appropriate.
[0055]
The lower substrate 2 of the apparatus 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 other sensor chip. On the upper surface of the substrate 2, there is provided a reaction region (spot region) 21, which is a recess structure portion having a minute volume well shape or the like and can store a solution such as a buffer solution.
[0056]
The sample material 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 apparatus 1 is configured to supply or recover the sample material S in close contact with the reaction region 21, the sample material S can be spotted on the substrate 2 without being exposed to a dry atmosphere.
[0057]
The surface region or the metal thin film surface region 3 (hereinafter, referred to as surface treatment) that can immobilize the sample material S (for example, the sample material D for detection such as a DNA probe) can be fixed to any part of the reaction region 21. "Detection surface 3") is provided.
[0058]
The detection surface 3 is appropriately provided on the bottom surface, the side wall surface, and the like of the reaction region 21 and functions as a portion that chemically bonds with a part of the sample material S supplied from the apparatus 1 and fixes the sample material S. It is the surface to do. For example, when the sample substance S is a detection nucleotide molecule 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 itself is not narrowly limited.
[0059]
The back surface side portion of the detection surface 3 has a symbol E in FIG. ₂ The second electrode indicated by is attached. This second electrode E ₂ Is the first electrode E of the device 1 when the device 1 is positioned above the reaction zone 21. ₁ And a pair of counter electrodes can be formed, and voltage can be applied via the power source V by turning on / off the switch 4.
[0060]
With this configuration, the electrode E ₁ -E ₂ Serves to form a direct current 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 second electrode E ₂ In this configuration, the cathode and the anode can be freely switched.
[0061]
More specifically, when the sample material S is negatively charged, the first electrode E ₁ The cathode, second electrode E ₂ When the DC electric field is formed with the anode as the anode, the sample material S is the second electrode E as the anode. ₂ Move towards the side. First electrode E ₁ Anode, second electrode E ₂ When a direct current electric field is formed using as a cathode, the sample material S is the first electrode E which is an anode. ₂ Move towards the side.
[0062]
On the other hand, when the sample material S is positively charged, the first electrode E ₁ The cathode, second electrode E ₂ When the direct current electric field is formed with the anode as the anode, the sample material S is the first electrode E as the cathode. ₁ Move towards the side. First electrode E ₁ Anode, second electrode E ₂ When the direct current electric field is formed using as a cathode, the sample material S is a second electrode E which is a cathode. ₂ Move towards the side.
[0063]
Hereinafter, a procedure for supplying and immobilizing the detection sample material D, which is one of the sample materials 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 material S is a detection sample material D having a negative charge like a DNA probe will be described as a representative example. However, the present invention is not limited to this.
[0064]
First, FIG. 2A shows a state in which the apparatus 1 is located above the substrate 2. In this state, the electrode E is still ₁ -E ₂ Since no voltage is applied to the sample material D for detection, the detection sample substance D still remains in the solution L of the solution tank 11.
[0065]
Subsequently, in FIG. 2B, the apparatus 1 arrives at the reaction region 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, second electrode E ₂ Is the anode). Thereby, the direct current electric field X passing through the solution L → the conductive gel G → the reaction region 21. ₁ Is formed. This DC electric field X ₁ As a result, the sample substance D for detection moves from the solution tank 11 through the electric gel storage tank 12 and is supplied to the reaction region 21. In addition, D in FIG. 2 (B) ₁ Indicates the sample material for detection immobilized on the detection surface 3.
[0066]
Next, FIG. 2C shows a sample material for detection (reference symbol D) immobilized in an unfixed state or an incomplete state existing in the reaction region 21. ₂ It shows with. FIG. 3 is a diagram showing a state in which the material is collected in the solution tank 11 of the apparatus 1. In this procedural step, the first electrode E ₁ Is the anode, 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.
[0067]
When the detection sample substance D is supplied to the reaction region 21 through the procedure shown in FIG. 2B, a specific part of the detection sample substance D is chemically bonded to the detection surface D and immobilized. Immobilization may be performed via a linker molecule or a spacer molecule.
[0068]
At the time of this immobilization, due to steric hindrance or the like, the sample material D for detection which has not been immobilized or has been imperfectly immobilized ₂ Are mixed in the reaction region 21. Therefore, the sample material D for detection that is reliably immobilized in the reaction region 21 ₁ Select the appropriate voltage strength that can leave only the DC electric field X ₁ DC electric field X opposite to ₂ Sample material D for detection ₂ Is moved through the conductive gel container 12 and collected in the upper solution tank 11 (see FIG. 2C). As a result, the detection sample substance D immobilized in the reaction region 21 is obtained. ₁ Can only exist.
[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 The cathode, second electrode E ₂ DC electric field X ₁ To supply the detection sample substance D to the reaction region 21. Subsequently, the electric field is turned off once to proceed with the immobilization reaction of the sample material D for detection, and after a predetermined time, the first electrode E ₁ Anode, second electrode E ₂ To the cathode, the DC electric field X ₁ DC electric field X opposite to ₂ Form.
[0070]
Here, when the DC electric field is turned off during the immobilization reaction, the chemical bond (immobilization reaction) between the detection surface 3 and the detection sample substance D is exclusively converted to the natural Brownian motion of the detection sample substance D. Since it can be made to proceed in the entrusted state, it is preferable.
[0071]
Based on the above apparatus configuration or procedure, the substrate 2 in a state where the detection sample substance D is immobilized in the reaction region 21 can be formed. This can be widely used for the production or manufacture of DNA chips on which detection nucleotide molecules such as DNA probes are immobilized, protein chips on which detection proteins are immobilized, and other sensor chips.
[0072]
Next, based on FIGS. 4 and 5, the sample material D for detection is applied to the detection surface 3 in the reaction region 21. ₁ A procedure for supplying the target sample material T, which is the sample material S, to the substrate 2 in a state where is fixed is described.
[0073]
First, FIG. 4A shows a state in which the apparatus 1 is positioned away above the substrate 2. In this state, the electrode E is still ₁ -E ₂ Since the voltage is not applied to the target sample substance T, the target sample substance T still remains in the solution L of 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, second electrode E ₂ Is the anode). Thereby, the direct current electric field X passing through the solution L → the conductive gel G → the reaction region 21. ₁ Is formed. This DC electric field X ₁ As a result, the target sample substance T having a negative charge moves from the solution tank 11 through the electric gel storage tank 12 and is supplied to the reaction region 21.
[0075]
Next, FIG. 4C shows a detection sample substance D immobilized on the reaction region 21. ₁ Target sample material that did not show any interaction (hybridization in this case) (symbol T ₂ FIG. 2 is a diagram illustrating a state in which the solution is collected into the solution tank 11 of the apparatus 1. In this procedural step, the first electrode E ₁ Is the anode, 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. Sample material D for detection ₁ And interaction (here, hybridization).
[0077]
At this time, the sample material D for detection ₁ Since the target sample substance T is rounded in a random coil shape, steric hindrance or the like occurs. As a result, the sample material D for detection is present in the reaction region 21. ₁ Target sample substance T that interacts and binds (forms a double chain) ₁ In addition to the target sample material and the detection sample material D that are released in the reaction region 12 without interaction. ₁ And the target sample substance in a state of erroneous interaction (here, mishybridization) (hereinafter these are collectively referred to as “target sample substance T ₂ ". ) Exists.
[0078]
These target sample substances T ₂ Is left in the reaction region 21, which 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 is extracted from the reaction region 21. ₂ It is desirable to exclude.
[0079]
Therefore, in the reaction region 21, the sample material D for detection ₁ Sample substance T that interacted with ₁ Select the 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 which has formed an interaction and does not show an interaction or shows an erroneous interaction ₂ Is moved through the conductive gel storage tank 12 and collected in the upper solution tank 1 (see FIG. 4C). Thereby, in the reaction region 21, the target sample substance T that has shown an interaction is shown. ₁ (And D ₁ Only) 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 in accordance with the format of the electric field forming procedure.
[0081]
First, the first electrode E ₁ The cathode, second electrode E ₂ DC electric field X ₁ And the negatively charged detection sample substance D is supplied to the reaction region 21 (see FIG. 2B), and then the electric field is turned off to fix the detection sample substance D to the detection surface 3. The first electrode E is allowed to proceed after a predetermined time. ₁ Anode, second electrode E ₂ To the cathode, the DC electric field X ₁ DC electric field X opposite to ₂ Sample substance D for detection in an unfixed state or insufficiently fixed ₂ Is recovered in the solution tank 11 (see FIG. 2C). The recovered sample material D for detection ₂ Can be reused.
[0082]
Next, the apparatus 1 containing the target sample substance T is moved to the substrate 2 (see FIG. 4A), and then the first electrode E ₁ The cathode, second electrode E ₂ DC electric field X ₁ And the negatively charged target sample substance T is supplied to the reaction region 21 (see FIG. 4B).
[0083]
Subsequently, the electric field is turned off and the detection sample substance D is immobilized. ₁ The target electrode material T interacts efficiently with the Brownian motion between the materials, and after a predetermined time, the first electrode E ₁ Anode, second electrode E ₂ To the cathode and the DC electric field X ₁ DC electric field X opposite to ₂ Form.
[0084]
This DC electric field X ₂ By means of the sample material D for detection ₁ Is not released in the reaction region 12 without interacting with the sample material D or the sample material D for detection ₁ Sample material T that interacts with the ₂ Is moved through the conductive gel G and collected in the solution tank 11 (see FIG. 4C).
[0085]
If the above apparatus configuration or procedure is used, the sample material D for detection is supplied into the reaction region 21 of the substrate 2, and the immobilization reaction of the sample material D for detection proceeds efficiently, and thereafter the detection accuracy is adversely affected. Sample material D for detection in an unfixed state etc. ₂ Is recovered from the reaction region 21, and subsequently, the target sample material T is supplied to the reaction region 21, and the target sample material T is immobilized (on the detection surface 3). ₁ The target sample substance T that has caused an interaction (e.g., hybridization) between the target sample substance T and a false interaction that adversely affects the detection accuracy. ₂ A series of methods (assay) for recovering the product from the reaction region 21 can be carried out smoothly.
[0086]
Hereinafter, an invention related to an apparatus configuration or a procedure that is further improved so as to use means or a 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]
In these improved inventions, when a high frequency electric field of about 1 MV / m is applied, nucleotide molecules (nucleotides comprising a large number of polarization vectors composed of negative charges of nucleotide chains including phosphate ions and positive charges of ionized hydrogen atoms) This utilizes the phenomenon or principle in which a dielectric polarization occurs in the molecule, and as a result, the nucleotide molecule is stretched linearly in parallel with the 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 disposed on the lower surface of the gel container 2 And the like (FIG. 7), and a method of installing a counter electrode (FIG. 9) opposite to each other with the solution stored in the reaction region 4 of the substrate B interposed therebetween. The apparatus or method provided with the means or procedure for forming the high-frequency electric field is particularly effective when the detection sample substance D is a nucleotide molecule such as a DNA probe.
[0089]
First, the symbol E in FIG. ₃₁ Is the first electrode E described above. ₁ And second electrode E ₂ 3 shows a “movable electrode” prepared for forming a high-frequency electric field. In accordance with the method shown in FIGS. 2 (A) to 2 (C) and FIG. ₁ A substrate 2 (DNA chip or the like) on which (DNA probe or the like) is immobilized is prepared and used.
[0090]
Movable electrode E ₃₁ All the operations are controlled by a computer or the like, and are determined to perform a predetermined positioning operation in accordance with 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 arriving so as to close the reaction region 21 is performed (see FIG. 6A).
[0091]
Subsequently, this movable electrode E ₃₁ And second electrode E ₂ A high frequency electric field Y of about 1 MV / m is formed in the solution (buffer solution or the like) in the reaction region 12. Thereby, the sample substance D for detection immobilized in the reaction region 21 is obtained. ₁ (A nucleotide molecule such as a DNA probe) can be stretched in a straight line by the action of the high-frequency electric field Y (elongation work, symbol D in FIG. 6B). ₃ reference).
[0092]
Subsequently, when this extension work is completed, the movable electrode E ₃₁ Is moved from above the reaction region 12 (FIG. 6C), and instead, the apparatus 1 is moved above the reaction region 12 (see FIG. 6D).
[0093]
Next, the first electrode E ₁ And second electrode E ₂ To the second electrode E ₂ DC electric field X toward ₁ Form. This DC electric field X ₁ Thus, 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 is efficiently advanced, the first electrode E ₁ And second electrode E ₂ A voltage is applied between the first electrode E ₁ DC electric field X toward ₂ Form. This DC electric field X ₂ By the target sample substance T in the reaction region 21 ₂ Is recovered in the solution L of 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. 6G).
[0095]
By performing the above-described procedure shown in FIG. ₃ And target substance T can be interacted under conditions that are less affected by steric hindrance.
[0096]
Next, in FIG. 7 and FIG. 8, the third electrode E is formed at the lower surface (or bottom surface) position of the conductive gel container 12 in order to form the “high frequency electric field” indicated by the symbol Y. ₃₂ The embodiment which provided is shown.
[0097]
In this embodiment, the device 1 has a third electrode E ₃₂ In addition to the device 1, the movable electrode E is further integrated. ₃₁ There is a work advantage that there is no need to move. The third electrode E ₃₂ Is also suitable in this respect because it also exhibits the function of making it difficult to remove the gel G from the storage tank 12.
[0098]
This third electrode E ₃₂ 7 is an electrode plate that is fitted and arranged at the lower surface (or bottom surface) position of the conductive gel container 12 as shown in FIG. 7, and a large number of micro holes 4 that penetrate the electrode plate are regularly arranged. It is equipped with the form. The minute holes 4 are set to have a diameter or a size that allows at least the sample substance S (T, D) to pass smoothly.
[0099]
Hereinafter, with reference to FIG. ₃₂ A method using the apparatus 1 in which is integrated will be described. First, in FIG. 8A, the sample material for detection D ₁ On the substrate 2 on which the fixation of the third electrode E is already completed. ₃₂ Represents the procedure of moving the integrated device 1. Then, the apparatus 1 is put on the reaction region 21 and the second electrode E ₂ And third electrode E ₃₂ Is applied to form a high-frequency electric field Y in the reaction region 21 (see FIG. 8B). Due to the action of the high-frequency electric field Y, the detection sample substance D immobilized in an expanded state on the detection surface 3 ₃ Will be arranged in large numbers.
[0100]
Next, in the procedure shown in FIG. ₁ And second electrode E ₂ A direct current electric field X extending from the solution tank 11 to the reaction region 12 through the conductive gel containing tank 12 ₁ 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.
[0101]
In the procedure shown in FIG. 8D, the immobilized detection sample substance D ₃ The target sample material T that does not show any interaction or has misinteracted while the two are bound by a specific interaction between the target sample material T and the target sample material T. ₂ DC electric field X ₁ DC electric field X extending from the reaction region 12 in the opposite direction to the solution tank 11 through the conductive gel container 12 ₂ To collect 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 wall surfaces 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 disposed.
[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 sample material D for detection ₁ Counter electrode E ₄ , E ₅ It is extended in the lateral direction by a high-frequency electric field Y formed by applying a voltage to (see FIG. 9B).
[0104]
Next, the apparatus 1 is brought into close contact with the reaction region 12, and the first electrode E ₁ And second electrode E ₂ And a direct current electric field X extending from the solution tank 11 to the reaction region 12 through the conductive gel container 12. ₁ Form. This DC electric field X ₁ By this action, the target sample substance T is supplied to the reaction region 21 of the substrate 2 (see FIG. 9C).
[0105]
Subsequently, although not particularly shown in FIG. ₁ , The target sample material T that has not been interacted with or has been misinteracted is allowed to proceed. ₂ DC electric field X ₂ To collect and begin detection.
[0106]
FIG. 10 is a diagram for explaining a method for supplying and recovering the sample substance S (T, D) using the high-frequency electric field Y according to the electric field forming procedure.
[0107]
First, the first electrode E ₁ And 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 The immobilization reaction is allowed to proceed with OFF.
[0108]
And the DC electric field X ₁ DC electric field X in the opposite direction ₂ The first electrode E ₁ And second electrode E ₂ Sample material D for detection that is not immobilized or is not sufficiently immobilized ₂ Is recovered in the solution tank 11 from the reaction region 21 through the conductive gel G.
[0109]
Subsequently, the high frequency electric field Y is changed to E which is the third electrode. ₃₁ (Movable electrode) or E ₃₂ And second electrode E ₂ Or the counter electrode E ₄ -E ₅ Sample material D for detection immobilized on the detection surface 3 by forming between ₁ Elongate.
[0110]
And 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 sample material D for detection in the extended state ₃ And the interaction of the target sample substance T is advanced.
[0111]
Subsequently, DC electric field X ₁ DC electric field X in the opposite direction ₂ The first electrode E ₁ And second electrode E ₂ The target sample substance T that has not interacted with or has shown an erroneous interaction (mishybrid). ₂ From the reaction region 21 through the conductive gel G to the solution tank 11.
[0112]
FIG. 11 shows an example of the substrate 2. The substrate 2 may be any substrate as long as it can accumulate a wide variety and many sample materials D for detection on a glass substrate, a silicon substrate, or the like. In FIG. 11, a substrate on a circular plate is used, but the shape, configuration, etc. of the substrate are not limited to this.
[0113]
In FIG. 11, a large number of reaction regions 21 are arranged on the surface of the substrate 2. The apparatus 1 is attached to the reaction region 21 on the substrate 2 to supply or recover the sample material S (the detection sample material D or the target sample material T). The apparatus 1 has a single reaction zone (for example R ₁ ) May be used to supply or recover the sample substance S, or a plurality of reaction regions (for example, R ₂ Or R ₃ Or the sample substance S may be supplied to all the reaction regions 21 on the substrate 2 or recovered at the same time.
[0114]
In general, many kinds of detection sample materials D are often accumulated on the substrate 2. In such a case, the apparatus 1 corresponding to one reaction region 21 is prepared, and each detection region D is supplied with a different sample material D for detection. Chip).
[0115]
On the other hand, in general, the target sample substance T is often supplied to a plurality of reaction regions. In such a case, the apparatus 1 that can supply the target sample substance T simultaneously 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, one row of reaction regions 21 (R ₂ ) Corresponding, multiple rows of reaction regions 21 (R _Three ), And those corresponding to all the reaction regions 21 on the substrate 2 can be used properly 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. The sample material D for detection can be subjected to a series of steps such as supply, immobilization, and recovery at the same time by carrying out subsequent procedures after putting different materials in the respective solution tanks 1.
[0118]
In the case of the target sample substance T, the same kind of substance is put in 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, etc. can be performed in a single operation by inserting different substances for each sequence. A series of steps, in particular, movement and combination of the apparatus 1 and the substrate 2, an electric field formation procedure, and the like can be executed by computer control or the like.
[0119]
【The invention's effect】
The present invention has the following effects.
[0120]
According to the present invention, Sample substance supply and recovery method The sample material can be spotted on the substrate without exposing the sample material to a dry atmosphere.
[0121]
this Method In addition to spotting the detection sample substance on the substrate, the target sample substance is supplied to the substrate on which the detection sample substance is fixed, and the detection sample substance and the target sample substance exhibit an interaction (for example, hybridization). It can be useful for experiments to detect whether or not.
[0122]
In addition, when performing an experiment for detecting whether or not an interaction is exhibited using the method according to the present invention, since a cleaning step using a conventional cleaning liquid is not required, an experimental worker must perform an accurate cleaning. There is no need to consider the selection of the solution and the condition setting of the cleaning solution, and a simple and effective interaction experiment can be performed. Also, loss of sample material due to the cleaning operation can be prevented, and an efficient and economical experiment can be performed.
[0123]
In the method according to the present invention, a high-frequency electric field can be used in addition to a direct-current electric field. Therefore, in particular, when a nucleotide molecule such as a DNA probe is used as a sample material for detection, this can be extended. The efficiency of interaction (hybridization etc.) with a substance can be improved.
[0124]
In the present invention, a method of supplying a sample material onto a substrate (reaction area), a method of recovering a sample material not immobilized on the substrate (detection surface thereof), a mutual relationship between a sample material for detection and a target sample material. It is possible to provide a method capable of reliably detecting the action.
[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 diagram showing a preferred embodiment of a sample substance supply / recovery device (1) according to the present invention.
FIG. 2 is a diagram showing a series of methods for supplying, immobilizing and recovering a sample material for detection (D) using the apparatus (1).
FIG. 3 is a view for explaining the method according to an electric field forming procedure;
FIG. 4 is a diagram showing a series of methods for supplying, immobilizing and recovering a target sample substance (T) using the apparatus (1).
FIG. 5 is a diagram illustrating the method according to an electric field forming procedure.
FIG. 6 shows a movable electrode (E ₃₁ The figure which showed a series of methods at the time of using.
FIG. 7 shows a third electrode (E) disposed on the lower surface of the conductive gel container (12). ₃₂ FIG.
FIG. 8 shows the third electrode (E ₃₂ The figure which showed a series of methods using the apparatus (1) provided with).
FIG. 9 shows a counter electrode (E ₃ , E ₄ The figure which showed a series of methods at the time 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 formation procedure.
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. FIG.
[Explanation of symbols]
1 Sample substance supply and recovery device (device)
2 Substrate
3 Detection surface (surface part where sample substance is immobilized)
11 Solution tank
12 Conductive gel container
21 Reaction area
D Sample material for detection
D ₁ Immobilized sample material for detection
D ₂ Unfixed or improperly immobilized sample material for detection
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 material
T Target sample material
T ₁ Interacted target sample material
T ₂ Target sample material that did not interact or showed misinteraction
X ₁ DC electric field for supply
X ₂ DC electric field for recovery
Y high frequency electric field

Claims (3)

Using a solution tank for storing a solution containing the nucleotide molecules, and conductive gel containing tank communicating with the said solution chamber, a first electrode for forming a DC electric field, at least comprising Ru sample material supply and recovery apparatus,
By applying a voltage to the first electrode and a second electrode attached to an external reaction region to form the DC electric field,
(1) A procedure for supplying the nucleotide molecules for detection in the solution tank to the reaction region by moving and passing through an electric gel ;
(2) a procedure for immobilizing the detection nucleotide molecule to a predetermined surface portion facing the reaction region;
(3) A procedure for recovering the detection nucleotide molecule in an unimmobilized state or immobilization insufficient state on the surface site by moving the conductive nucleotide molecule through the conductive gel and collecting it in the solution tank;
(4) A procedure for supplying the target nucleotide molecule in the solution tank to a predetermined reaction region by moving the conductive gel;
(5) a procedure for performing an interaction between the immobilized detection nucleotide molecule and the target nucleotide molecule;
(6) A target nucleotide molecule that has not interacted with the immobilized detection nucleotide molecule or / and a target nucleotide molecule that has misinteracted are moved through the conductive gel and collected in a solution tank. And the steps to
And (7) a step of extending the immobilized detection nucleotide molecule by forming a high-frequency electric field with a counter electrode provided across the reaction region,
A sample substance supply and recovery method in which the procedure of (7) is performed between the procedure (3) and the procedure (4) . 2. The sample substance supply and recovery method according to claim 1, wherein the DC electric field is turned off during the step (2) . 2. The sample substance supply and recovery method according to claim 1, wherein the DC electric field is turned off during the step (5) .

Images