JP2007044033A - Method for analyzing interaction of biomolecule immobilized on carrier and apparatus therefor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a means for rapidly and efficiently treating a large amount of a carrier in an analysis of interaction between biomolecules using the carrier in which the biomolecules are immobilized. <P>SOLUTION: A reactor is used for making the biomolecules on the carrier interact with biomolecules in a sample. The reactor is equipped with a temperature controller 14 for controlling the temperature of the sample and the carrier 23, a carrier holder 11 having a plurality of carrier housing parts having discharge ports 22, a washing head 12 provided with a lid body having a plurality of feed holes for feeding a wash liquid or a gas to the carrier housing parts of the carrier holder and a holder housing container 13 having a plurality of recessed parts for housing the carrier housing parts of the carrier holder 11. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a reaction apparatus for interacting biomolecules immobilized on a carrier, a biomolecule analysis apparatus including the reaction apparatus, and a method for analyzing the interaction of biomolecules.

  Currently, a method is widely used in which biomolecules such as nucleic acids, sugar chains, and proteins are immobilized on a carrier, and the target biomolecules are reacted to study the interaction between the two molecules.

  A carrier on which DNA or protein is immobilized is called a DNA chip or protein chip, and the DNA or protein is aligned at a density of several hundred to several hundred thousand per square centimeter on a carrier such as a dispensing object or silicon. Created by immobilization. When all the gene sequences of a certain biological species have been elucidated, a technique has been developed in which a DNA chip is prepared based on the information to obtain information on the total expression of a sample by a single hybridization. In other words, DNA chips with thousands of different DNA probes are included by aligning and immobilizing DNA with a known sequence in an array state and adding an unknown sample to cause hybridization. At the same time, several thousand genes can be identified. In the field of basic research, DNA chips are used to estimate the function of newly discovered genes by comparing them with the expression patterns of known genes, to identify important substances in signal transduction pathways, and to identify new genes Used for research. It is also an important technology in fields that require high throughput, such as discovery of new target molecules for therapeutic drugs, disease diagnosis, genomic toxicology, and search for disease-related genes.

  The main type of protein chip is an antibody array on which an antibody is immobilized and an ELISA method is performed on the chip. This is because the ELISA method is a well-established technique and enables highly sensitive analysis. In the future, protein chips are expected to play a role in clinical diagnosis, health monitoring, environment, food inspection, and so on.

  The measurement work using a DNA chip or protein chip as described above requires repeated simple work due to the large number of samples, and requires a large installation area because the equipment used in each process is independent. The reaction temperature must be controlled and the reaction takes a long time to proceed, and each process is mechanized automatically because it is necessary to handle a very small amount of sample with high accuracy. Has long been anxious.

  An object of the present invention is to provide a means for rapidly and efficiently treating a large amount of carriers in an analysis of interactions between biomolecules using a carrier on which biomolecules are immobilized.

  As a result of intensive studies to solve the above problems, the present inventors have found that a temperature control device, a carrier holder having a plurality of carrier accommodating portions having discharge ports, and a lid having a plurality of supply holes are provided. By interacting with biomolecules using a reaction device equipped with a head and a holder container that can accommodate a carrier accommodating part, the interaction, washing and drying of biomolecules on the carrier can be carried out efficiently and quickly. The inventors have found what can be done and have completed the present invention.

That is, the present invention includes the following inventions.
(1) A reaction apparatus for interacting biomolecules on a carrier with biomolecules in a sample,
A temperature control device for controlling the temperature of the sample and the carrier, a carrier holder having a plurality of carrier accommodating portions having discharge ports, and a lid having a plurality of supply holes for supplying cleaning liquid or gas to the carrier accommodating portion of the carrier holder. The reaction apparatus comprising: a cleaning head provided; and a holder housing container having a plurality of recesses capable of housing the carrier housing portion of the carrier holder.

(2) The reactor according to (1),
A loading stage for arranging a plurality of carriers on which biomolecules are immobilized;
A transfer device for transferring a carrier from a loading stage to a carrier accommodating portion of a carrier holder in a reaction device, and a dispensing device for dispensing a plurality of samples containing biomolecules into a carrier accommodating portion of a carrier holder of a reaction device A biomolecule analyzer comprising:
(3) The apparatus according to (2), wherein the loading stage includes a cleaning device.

(4) The device according to (2) or (3), wherein the dispensing device includes a temperature control device for controlling the temperature of the sample containing the biomolecule.
(5) The device according to any one of (1) to (4), wherein the carrier housing portion of the carrier holder can be sealed with the lid of the cleaning head and the holder housing container.

(6) The apparatus according to any one of (1) to (5), wherein the temperature control device of the reaction apparatus is installed in the holder container and the holder container is movable.

(7) A method for analyzing the interaction of biomolecules,
Arranging a plurality of carriers on which biomolecules are immobilized on a loading stage;
A step of transferring a carrier from a loading stage to a carrier holder having a plurality of carrier accommodating portions having discharge ports, wherein the carrier accommodating portion of the carrier holder includes a plurality of carrier accommodating portions of the carrier holder. Are accommodated in the plurality of recesses of the holder housing container having the recesses of
A step of dispensing a plurality of samples containing biomolecules into a carrier accommodating portion of a carrier holder by a dispensing device;
By moving a cleaning head having a lid having a plurality of supply holes for supplying the cleaning liquid or gas to the carrier accommodating portion of the carrier holder and covering the carrier holder with the lid, the carrier accommodating portion of the carrier holder is Sealing,
The temperature control device changes the temperature of the sample and the carrier to allow the biomolecules immobilized on the carrier to interact with the biomolecules contained in the sample, and the carrier holder and the holder container are separated for cleaning. The method comprising the step of cleaning or drying the carrier by supplying a cleaning liquid or gas from the supply hole of the head to the carrier containing portion.

(8) The method according to (7), wherein the temperature control device is installed in the holder container, and the carrier holder is accommodated and separated from the holder container by moving the holder container.

(9) The load stage includes a cleaning device and a temperature control device, and at least the surface of the load stage or the load holder on which the carrier is arranged or the bottom of the carrier is made of a heat-peelable adhesive and is peeled off by the temperature control device. The method according to (7) or (8), wherein the biomolecule is immobilized on the support by changing the temperature to below the temperature, washing is performed, and the support is released by raising the temperature to the release temperature.

(10) A step of subjecting a nucleic acid in a sample to a PCR reaction by changing the temperature with a temperature control device provided in the dispensing device before dispensing the sample, since the biomolecule contained in the sample is a nucleic acid. The method according to any one of (7) to (9), further comprising:

(11) The biomolecule is a nucleic acid, and the PCR reaction is performed by changing the temperature with a temperature controller in at least a part of the step of allowing the nucleic acid immobilized on the carrier to interact with the nucleic acid contained in the sample. The method according to any one of (7) to (10).
(12) The method according to (10) or (11), wherein the PCR reaction is an asymmetric PCR reaction.
(13) The method according to any one of (7) to (12), wherein the carrier is a solid support having a carbon layer and a chemical modification group on a substrate surface made of a silicon material.

  According to the present invention, in the analysis of the interaction between biomolecules using the carrier on which the biomolecule is immobilized, the interaction, washing and drying of the biomolecule on the carrier can be carried out efficiently, and a large amount of the carrier can be rapidly produced. Can be processed.

  In one embodiment, the present invention relates to a reaction apparatus for interacting biomolecules on a carrier with biomolecules in a sample. The reaction apparatus includes a temperature control device for controlling the temperature of the sample and the carrier, a carrier holder having a plurality of carrier accommodating portions having discharge ports, and a plurality of supply holes for supplying cleaning liquid or gas to the carrier accommodating portion of the carrier holder. And a holder housing container having a plurality of recesses capable of housing the carrier housing portion of the carrier holder.

  In another embodiment, the present invention relates to a biomolecule analyzer comprising a loading stage, the reaction device, a transfer device, and a dispensing device.

  In the present invention, biomolecules include nucleic acids such as single-stranded and double-stranded DNA and RNA, polypeptides, sugars and the like. In the present invention, polypeptides include oligopeptides and proteins. Examples of the interaction between biomolecules include interactions between polypeptides, interactions between nucleic acids, and interactions between polypeptides and nucleic acids. Specifically, antigen-antibody reaction, avidin-biotin binding reaction, Examples include enzyme-substrate binding reactions, hybridization between nucleic acid complementary strands, ligand-receptor binding reactions, nucleic acid-transcription factor binding reactions, and cell adhesion factor binding reactions. The present invention is preferably used in analysis of nucleic acid interaction, particularly hybridization between nucleic acid complementary strands.

  In the present invention, the loading stage is configured to arrange a plurality of carriers on which biomolecules are immobilized. The loading stage may be configured to arrange a loading holder on which a plurality of carriers are already arranged. The loading stage preferably includes a cleaning device, and the cleaning device can clean the carrier on which the biomolecule is immobilized before performing the interaction between the biomolecules in the reaction device. As the cleaning device, those usually used in this technical field can be used, and usually includes a water supply device, a stirring device, a drying device, and the like. The water supply apparatus usually includes a container containing a cleaning liquid and a container containing sterilizing water for rinsing, and the cleaning liquid that is usually used in the art can be used. For example, SSC / SDS can be used. Drying after washing the carrier can be performed by, for example, a blow drying head or a centrifuge that performs blow drying.

  The cleaning device preferably comprises a temperature control device. The temperature control device usually includes a heating device. At least the surface of the loading stage or the loading holder on which the carrier is arranged, or the bottom of the carrier, is preferably made of a heat-peeling adhesive. The heat-peeling adhesive is placed at a constant temperature by a temperature control device after the carrier is placed. By heating, the carrier can be brought into a peelable state. There is no limitation in particular as a heat-peelable adhesive, A well-known thing can be used in this technical field, for example, an ultraviolet curing adhesive and the adhesive which mix | blended them with the foaming agent can be used. The heat-peelable adhesive has a property of easily foaming and peeling off from the adhesive surface when heated, and can be easily peeled off by heating. Before washing, the carrier on which the biomolecule is spotted may be immobilized on the carrier by heating it using a temperature control device.

  Moreover, the sheet | seat (thermally peelable adhesive sheet) by which the said heat peelable adhesive was apply | coated to the film-form base materials, such as polyester, in layers can also be used, and a commercial item is utilized as such a sheet. Can do. The heat-peelable adhesive or the heat-peelable adhesive sheet can be brought into a state in which the carrier can be peeled by heating at 50 to 200 ° C. for 1 second to 20 minutes.

  By using a heat-peelable adhesive or a heat-peelable adhesive sheet, the carrier can be fixed by temperature control to wash the carrier, and after washing, the carrier can be transferred to the reactor in a state where the carrier can be peeled off. it can. Since the immobilization and peeling of the carrier can be performed by temperature control, automatic operation is possible, and the risk of contamination of the carrier on which the biomolecule is immobilized can be reduced.

  The carrier arranged on the loading stage is preferably transferred to the carrier accommodating part of the carrier holder in the reaction apparatus by the transfer device. The transfer device is not particularly limited as long as a plurality of carriers arranged on the loading stage can be transferred to the plurality of carrier accommodating portions in the carrier holder. The transfer device preferably includes a gripping tool and an arm unit that can freely move in a plane direction and a vertical direction. The transfer device grips the side surface of the carrier and moves the gripping tool to a biomolecule fixed on the carrier. The carrier is transported to the carrier holder without contact. It is preferable that the transfer device has a configuration in which the carrier after interaction and cleaning can be taken out from the carrier holder and transported to an arbitrary plane position.

  A reaction apparatus for causing a biomolecule on a carrier to interact with another biomolecule includes a temperature control device, a carrier holder, a cleaning head, and a holder receiving container. FIG. 1 shows a side view of an embodiment of a reaction apparatus. Reference numeral 11 denotes a carrier holder, 12 denotes a cleaning head, 13 denotes a holder container, and 14 denotes a temperature control device. Further, M represents a forward / backward movement motor for the cleaning head, and CY represents a vertical drive cylinder. 1 is a cylinder that drives the cleaning head 12 up and down, and CY described on the right side is a cylinder that drives the holder storage container 13 up and down.

  The carrier holder 11 has a plurality of carrier accommodating portions 21 having discharge ports 22. Each carrier accommodating portion is usually configured as a concave container having a bottom portion on which the carrier 23 can be arranged, and has a discharge port 22 for discharging a cleaning liquid and a drying gas. The discharge port 22 is not particularly limited as long as it has a structure capable of discharging the cleaning liquid and the drying gas, and may be plural or single. Moreover, you may have a structure where the carrier accommodating part 21 is not. FIG. 2 shows a cross-sectional view of one embodiment of the carrier holder 11. FIG. 2 shows two carrier accommodating portions 21, each carrier accommodating portion has a discharge port 22, and a carrier 23 is accommodated in each. FIG. 3 shows a schematic top view of one embodiment of the carrier holder 11 in which a plurality of carrier accommodating portions are arranged. The carrier accommodating portion 21 is open upward, and preferably has a groove 31 for a gripping tool for gripping the carrier 23 in the transfer device to enter and exit.

  The cleaning head 12 includes a lid having a plurality of supply holes for supplying a cleaning liquid or gas to the carrier accommodating portion 21 of the carrier holder 11 and is preferably movable. The lid of the cleaning head 12 is configured to cover the surface of the carrier holder 11 where the carrier accommodating portion 21 is open. The supply hole of the lid of the cleaning head 12 is preferably formed so as to correspond to each carrier accommodating portion of the carrier holder 11. The cleaning head 12 is connected to a cleaning liquid supply unit and a gas supply unit, and cleaning liquid and gas are supplied from these units to the space above the lid in the cleaning head 12 and circulate.

  FIG. 4 shows a plan view of one embodiment of the cleaning head 12. In FIG. 4, reference numeral 41 denotes a cleaning liquid or a drying gas, 42 denotes a lid, and 43 denotes a supply hole of the lid. The position in the planar direction of the supply hole of the lid and the opening of the carrier accommodating portion 21 of the carrier holder 11 are made to coincide with each other, preferably the lid and the carrier holder 11 are brought into close contact with each other. By circulating the drying gas, the cleaning liquid and the drying gas are discharged from the supply hole, and the carrier in the carrier accommodating portion can be washed or dried (see FIG. 5b).

  Further, the cleaning head 12 can be shifted in the plane direction to shift the opening position of the carrier accommodating portion 21 of the carrier holder 11 and the position of the supply hole of the lid. The lid 52 and the carrier holder 11 are brought into close contact with each other, whereby the opening 52 of the carrier accommodating portion can be blocked (see FIG. 5a).

  The holder housing container 13 has a plurality of recesses that can accommodate the carrier housing portion 21 of the carrier holder. Although the carrier accommodating part 21 of the carrier holder 11 has the discharge port 22 as described above, the discharge port 22 is blocked by being accommodated in the concave portion of the holder accommodating container (see FIG. 5a). The concave portion of the carrier holder is preferably formed so as to correspond to the position of the carrier accommodating portion 21 of the carrier holder 11 so that each carrier accommodating portion can be accommodated.

  A sample containing a biomolecule is normally dispensed by the dispensing device 63 into the concave portion 53 of the holder housing container. The sample is dispensed into the carrier accommodating portion 21 in which the carrier in which the biomolecule is immobilized is transferred, which is accommodated in the concave portion of the holder accommodating container, thereby bringing the biomolecule on the carrier into contact with the biomolecule in the sample. Can be made. Preferably, the lid of the cleaning head 12 is brought into close contact with the carrier holder accommodated in the holder accommodating container 13 with the opening 52 of the carrier accommodating portion and the supply hole of the cleaning head lid shifted. The carrier accommodating portion 21 can be sealed. FIG. 5a shows an embodiment in which the carrier accommodating part 21 is sealed as described above. In FIG. 5a, 13 is a holder container, 11 is a carrier holder, 21 is a carrier container, 51 is a supply hole for the lid, 52 is an opening in the carrier container, 12 is a cleaning head, and 42 is a lid for the cleaning head. Showing the body.

  In the reaction apparatus of the present invention, the temperature controller 14 for controlling the temperature of the carrier 23 and the sample is preferably installed in the holder accommodating container 13. By controlling the temperature of the sample and the carrier in the carrier container accommodated in the hermetically sealed holder container by the temperature control device 14, the PCR reaction can be carried out or the conditions of biomolecule interaction, for example, the hybridization conditions can be set. Can be controlled.

  After the interaction or reaction of biomolecules is performed, when the carrier holder 11 and the holder container are separated, the sample flows out from the discharge port 22 of the carrier container 21, so that the carrier 23 and the sample can be separated. Then, after the lid is moved so that the position of the supply hole of the lid of the cleaning head 12 and the opening 52 of the carrier accommodating portion coincides, preferably the lid of the cleaning head and the carrier holder 11 are brought into close contact with each other. By supplying the cleaning liquid from the holes, the carrier in the carrier accommodating portion can be efficiently washed. The carrier 23 can also be dried by supplying a gas, preferably an inert gas such as nitrogen, from the supply hole. FIG. 5b shows an embodiment in which the carrier holder 11 and the holder container 13 are separated. In FIG. 5b, 12 is a cleaning head, 11 is a carrier holder, 13 is a holder accommodating container, 21 is a carrier accommodating portion, 42 is a lid for the cleaning head, 51 is a supply hole for the lid, 52 is a carrier accommodating portion. The opening 53 is a recess of the holder housing container, and the arrow 54 indicates the direction in which the cleaning liquid or gas flows.

  In the cleaning head 12, the operation of aligning or shifting the position of the supply hole of the lid with the opening of the carrier accommodating portion 21 once separated the lid of the cleaning head 12 and the carrier holder 11 to the upper limit. Thereafter, it is preferable that the cleaning head 12 is moved in the plane direction.

  Accommodation of the carrier holder 11 in the holder accommodation container and separation of the carrier holder 11 from the holder accommodation container 13 may be performed by moving either the carrier holder 11 or the holder accommodation container 13, but preferably the holder accommodation. This is done by moving the container 13 up and down.

  The dispensing device 63 dispenses a plurality of samples containing biomolecules to the carrier accommodating portion 21 accommodated in the concave portion 53 of the holder accommodating container in the reaction device. As the dispensing device 63, those commonly used in the art can be used. Usually, a container (for example, a 96-well or 384-well plate) that accommodates a plurality of samples, and a lid-closable holder that accommodates the container; Dispensing comprising a plurality of tip nozzles, a mechanism for attaching and detaching the tip to and from the tip nozzle, a mechanism for aspirating or injecting the sample by the attached tip, and an arm unit that can move freely in the plane and vertical directions Has a head.

  The holder portion of the dispensing device 63 preferably includes a temperature control device. The temperature control device usually includes a heating device and a cooling device. When the biomolecule contained in the sample is a nucleic acid, the temperature inside the container is changed by changing the temperature of the sample contained in the container of the dispensing device 63 by the temperature control device with the holder portion sealed with a lid. PCR reaction can be carried out. By adopting a configuration in which the PCR reaction can be performed in the dispensing device 63, when the nucleic acids are allowed to interact, it is not necessary to amplify the nucleic acid contained in the sample in advance with another device, and the sample can be used as it is in the dispensing device 63. Just sampling into a container, it can be used for interaction as it is.

  In addition to the reaction apparatus, loading stage 61, transfer apparatus, and dispensing apparatus 63, the biomolecule analyzer of the present invention includes an unloading stage 62 for taking out and arranging the carrier 23 after interaction and washing. It is preferable to provide.

  FIG. 6 shows a schematic top view of an embodiment of a biomolecule analyzer including a loading stage 61, a reaction device, a transfer device, and a dispensing device 63. In FIG. 6, 11 is a carrier holder, 13 is a holder container, 12 is a cleaning head, 61 is a loading stage, 62 is an unloading stage, and 63 is a dispensing device. In FIG. 6, the carrier placed on the loading stage is optionally washed and transferred to the carrier holder 11 by a transfer device (not shown). Here, the carrier accommodating portions of the carrier holder 11 are respectively accommodated in the concave portions of the holder accommodating container, and as a result, the discharge port of the carrier accommodating portion is blocked. The dispensing device 63 descends after reaching the upper part of the carrier holder 11 by moving the head part that sucks the sample into the plurality of chips attached to the chip nozzle in the XY direction, Dispense the sample into Subsequently, the cleaning head 12 moves in the XY direction, moves to the upper part of the carrier holder 11, and further moves up and down (Z direction), whereby the lid of the cleaning head 12 is moved to the carrier holder 11. Close contact with. Then, interaction or reaction of biomolecules is carried out in the carrier accommodating part. At this time, the inside of the carrier accommodating portion is sealed by the lid of the cleaning head 12 being in close contact with the carrier holder 11 with the position of the supply hole being shifted from the opening of the carrier holder 11. At this time, the temperature control device controls the temperature in the carrier accommodating portion. After completion of the reaction, the holder housing container 13 and the carrier holder 11 are separated, whereby the sample flows out from the outlet of the carrier holder 11 and the carrier and the sample are separated. The cleaning head 12 once moves away from the carrier holder 11 and moves slightly in the XY direction, aligns the position of the supply hole of the lid and the position of the opening of the carrier accommodating portion, and the lid and the carrier holder 11 again. In close contact. Then, the cleaning liquid or gas is supplied from the supply hole, whereby the carrier is cleaned and dried. Thereafter, the cleaning head 12 is separated from the upper part of the carrier holder 11, and the carrier is taken out from the carrier holder 11 by the transfer device.

The invention also relates to a method for analyzing biomolecular interactions. The method of the present invention comprises:
a) arranging a plurality of carriers 23 on which biomolecules are immobilized on a loading stage 61;
b) a step of transferring the carrier 23 from the loading stage 61 to the carrier holder 11 having a plurality of carrier containing portions 21 having discharge ports, preferably by a transfer device, wherein the carrier containing portion of the carrier holder 11 21 is housed in the plurality of recesses of the holder housing container 13 having a plurality of recesses capable of housing the carrier housing portion 21 of the carrier holder 11,
c) Dispensing a plurality of samples containing biomolecules into the carrier accommodating part of the carrier holder 11 by the dispensing device 63;
d) The carrier holder 11 is covered with the lid by moving the washing head 12 provided with a lid having a plurality of supply holes for supplying the cleaning liquid or gas to the carrier accommodating portion 21 of the carrier holder. A step of sealing the eleven carrier accommodating portions 21;
e) The biomolecules immobilized on the carrier and the biomolecules contained in the sample by changing the temperature of the sample and the carrier by the temperature control device 14, preferably the temperature control device 14 installed in the holder container 13. And f) separating the carrier holder 11 and the holder housing container 13, and preferably lowering the holder housing container 13 to separate the carrier housing portion 21 of the carrier holder 11 from the holder housing container 13. And a step of cleaning or drying the carrier 23 by supplying a cleaning liquid or gas from the supply hole of the lid of the cleaning head 12 to the carrier accommodating portion 21.

  In step f, the biomolecules that did not interact in step e are washed away, and only the biomolecules that interact with the biomolecules on the carrier remain on the carrier. The carrier is dried by blowing an inert gas such as nitrogen from the supply hole. The carrier treated as described above is subjected to a step of detecting an interaction. The method for detecting the interaction can be appropriately selected by those skilled in the art based on the biomolecule used and the interaction.

  Samples containing biomolecules used in the present invention are not particularly limited, but include cell extracts, bacterial cell extracts, cell-free synthetic products, PCR (Polymerase chain Reaction) products, enzyme-treated products, synthetic DNAs, synthetic RNAs, Examples include synthetic peptides. Moreover, it is preferable to install in the dispensing device 63 a sample from which a target biomolecule has been extracted in advance. Extraction of biomolecules can be performed by a method usually used in the art. For example, when DNA is extracted, a method of phenol extraction and ethanol precipitation, a method of using cetyltrimethylammonium bromide, a method of using glass beads, or the like can be used. When RNA is extracted, a guanidine-cesium chloride ultracentrifugation method, a hot phenol method, a guanidinium thiocyanate-phenol-chloroform (AGPC) method, or the like can be used.

  When nucleic acid is allowed to interact as a biomolecule contained in the sample, the nucleic acid in the sample is subjected to PCR reaction by controlling the temperature with a temperature control device provided in the dispensing device 63 before dispensing the sample. Preferably, it can be amplified by an asymmetric PCR reaction. Amplification is preferably performed until the amplified fragment is at a detectable level.

  The PCR reaction conditions are usually (1) heat denaturation at 94 to 98 ° C. for 0 second to 5 minutes, (2) heat denaturation at 95 to 98 ° C. for 0 second to 5 minutes, and (3) 37 to 68 ° C. (2) Annealing reaction of 0 to 2 minutes, (4) An extension reaction at 70 to 75 ° C for 5 seconds to 5 minutes, (5) An extension reaction at 70 to 75 ° C for about 5 minutes, Nucleic acids are amplified by repeating the process for 20-40 cycles.

  Conditions for the PCR reaction can be appropriately set by those skilled in the art based on the type of primer, the type of DNA polymerase, the length of the nucleic acid to be amplified, and the like. In the steps (2) and (3), the nucleic acid is sufficiently changed even if the temperature is changed to a predetermined temperature but is not maintained at that temperature (that is, 0 seconds) and the change to the temperature of the next step is started. PCR reaction time can be shortened.

  Asymmetric PCR is a method in which two primers for amplification are used in equal amounts in normal PCR, whereas the primer to be obtained as a single strand is put in a larger amount than the other primer and PCR is performed. is there. By doing so, only two strands are created at the beginning of the PCR cycle, but once the smaller primer is used up, single-stranded DNA will be created each time. Become. When double-stranded DNA is used as a target, the DNAs hybridize with each other and cannot be hybridized with the probe nucleic acid, resulting in low sensitivity. By using the prepared single-stranded DNA, high-sensitivity detection is possible without such problems.

  In the present invention, the concentration ratio of the other primer to the one primer concentration in asymmetric PCR is usually less than 1, preferably 1/2 or less, more preferably 1/100 to 1/10.

A label such as a radioisotope, a fluorescent substance, or a luminescent substance is allowed to act on dNTP (containing equal amounts of dATP, dCTP, dGTP, dTTP) or a primer for PCR that is incorporated during the amplification reaction. By detecting this, the interaction of nucleic acids can be analyzed. As a radioactive isotope, ³² P, ¹²⁵ I, ³⁵ S, or the like can be used. As the fluorescent substance, for example, fluorescein (FITC), sulforhodamine (TR), tetramethylrhodamine (TRITC) and the like can be used. As the luminescent substance, luciferin or the like can be used. There are no particular restrictions on the type of the labeled body and the method for introducing the labeled body, and various conventionally known means can be used. For example, as a method for introducing a label, a random prime method using a radioisotope can be mentioned.

  When the sample-derived nucleic acid interacts with the nucleic acid on the carrier, the hybridization buffer containing SSC and SDS in the nucleic acid extraction sample or PCR product (for example, 1 to 5 × SSC / 0.1 to 1% SDS) Is preferably added and allowed to interact with the nucleic acid on the support. By using the hybridization buffer, the hybridization time can be shortened.

  Further, in the embodiment in which the nucleic acid immobilized on the carrier 23 in step e interacts with the nucleic acid in the sample, that is, in the hybridizing mode, the temperature controller 14 controls the temperature in at least a part of step e. It is preferred to perform PCR, preferably asymmetric PCR. The term “at least part” means that the asymmetric PCR reaction and the hybridization reaction are performed in parallel during at least a part of the time, and more preferably the PCR reaction and the hybridization reaction are performed in parallel. In addition, a polynucleotide probe can be present in a sample containing nucleic acid to perform hybridization with the probe extension reaction. Furthermore, the reaction rate can be increased by stirring by vibration or applying an electric field.

  In asymmetric PCR, the nucleic acid probe immobilized on the carrier 23 is preferably one that binds to a specific site of a single-stranded amplified fragment amplified by asymmetric PCR and does not bind to an unreacted primer. The reason for this is that the efficiency of hybridization is markedly improved when targeting amplified fragments that are amplified in large amounts by asymmetric PCR and present as single strands, and that complementary strands of amplified single strands are difficult to produce. is there. In addition, if a nucleic acid having the same base sequence as a primer used for asymmetric PCR is used as a probe, unreacted primers in the PCR solution will bind preferentially to the amplified fragment, and sufficient hybridization efficiency with the probe cannot be obtained. Because.

  The hybridization reaction needs to be performed under stringent conditions. Stringent conditions are well known in the art and are not particularly limited. For example, stringent hybridization conditions indicate, for example, conditions in which the sodium concentration is 15 to 40 mM, preferably 15 to 20 mM, and the temperature is 40 to 70 ° C., preferably 60 to 65 ° C. In particular, the case where the sodium concentration is 16 to 17 mM and the temperature is 65 ° C. is most preferable.

  In one embodiment, at least the surface of the loading stage 61 or the loading holder on which the carrier 23 is disposed, or the bottom of the carrier is made of a heat-peelable adhesive or a heat-peelable adhesive sheet. In this case, the loading stage 61 includes a cleaning device, and the cleaning device includes a temperature control device. After the carrier 23 is placed on the loading stage 61, the carrier 23 can be made peelable by heating the heat peelable adhesive or the heat peelable adhesive sheet to a certain temperature at which the carrier 23 can be peeled off. .

  In the present invention, the carrier 23 on which a biomolecule to be treated is immobilized can be produced by a method known in the art. For example, the biomolecule is immobilized on the carrier by dissolving the biomolecule in a spotting buffer and spotting it on the carrier. As the spotting buffer, PEG (polyethylene glycol) solution, glycerin solution, PBS (phosphate buffered saline), DMSO (dimethyl sulfoxide), 3 × SSC (saline sodium citrate), pure water, etc. may be used. it can. The prepared spotting solution can be dispensed into a 96-well or 384-well plate, and the dispensed solution can be spotted on a carrier by a spotter device or the like. Alternatively, spotting may be performed manually with a micropipette. At this time, a plurality of types of biomolecules can be rapidly analyzed by arranging a plurality of types of biomolecules in an array as independent spots.

  When the biomolecule to be immobilized is a nucleic acid, it is spotted on a carrier surface that has been surface-treated with a polycation (polylysine, polyethyleneimine, etc.) using a spotter device, and the DNA is charged on the carrier. Generally, a method of electrostatically coupling to the substrate is used. In addition, a method using a silane coupling agent having an amino group, an aldehyde group, an epoxy group or the like is also used as a method for treating the carrier surface. In this case, since an amino group, an aldehyde group, and the like are introduced to the support surface by a covalent bond, they are stably present on the support surface as compared with the case of using a polycation.

  In the present invention, as a carrier for immobilizing a biomolecule, those known in the art can be used and are not particularly limited. For example, noble metals such as platinum, platinum black, gold, palladium, rhodium, silver, mercury, tungsten and their compounds, and conductor materials such as graphite and carbon typified by carbon fiber; single crystal silicon, amorphous Silicon materials represented by silicon, silicon carbide, silicon oxide, silicon nitride, etc., composite materials of these silicon materials represented by SOI (silicon on insulator), etc .; glass, quartz glass, alumina, sapphire, ceramics, Inorganic materials such as stellite and photosensitive glass; polyethylene, ethylene, polypropylene, cyclic polyolefin, polyisobutylene, polyethylene terephthalate, unsaturated polyester, fluorine-containing resin, polyvinyl chloride, polyvinylidene chloride, polyvinyl acetate, polyvinyl alcohol , Polyvinyl acetal, acrylic resin, polyacrylonitrile, polystyrene, acetal resin, polycarbonate, polyamide, phenol resin, urea resin, epoxy resin, melamine resin, styrene / acrylonitrile copolymer, acrylonitrile / butadiene styrene copolymer, polyphenylene oxide And organic materials such as polysulfone.

  The present invention is suitably used for the carrier 23 having a fine flat plate structure. The shape is not limited to a rectangle, a square, or a round shape, but a shape of 1 to 75 mm square, preferably 1 to 10 mm square, more preferably 3 to 5 mm square is usually used. Since it is easy to produce a carrier having a fine plate-like structure, silicon materials and resin materials can be mentioned. The present invention is preferably used particularly for the carrier 23 made of single crystal silicon.

  In the present invention, it is preferable to use a solid support having a carbon layer on the surface of the substrate. Furthermore, what introduce | transduced the specific chemical modification group into the carbon layer is preferable. This is because by introducing a specific chemical modification, it becomes easier to hold the biomolecule to be analyzed and it can be stably immobilized. Here, the substrate means a base material from which the carbon layer is formed. Such a base material is not particularly limited, and those made of the above materials can be used. Similarly, the base material in the solid support as described above is preferably made of a silicon material, particularly single crystal silicon.

  The carbon layer formed on the substrate in the present invention is not particularly limited, but diamond, diamond-like carbon, amorphous carbon, graphite, hafnium carbide, niobium carbide, silicon carbide, tantalum carbide, thorium carbide, titanium carbide, uranium carbide. , Tungsten carbide, zirconium carbide, molybdenum carbide, chromium carbide, vanadium carbide and the like, and a diamond-like carbon (DLC) layer is preferable. The carbon layer has excellent chemical stability, can withstand subsequent reactions in the introduction of chemical modification groups and binding to the analyte, and the binding is flexible because of the electrostatic binding to the analyte. It is advantageous in that it has the property of being transparent, it is transparent to the detection system UV because it has no UV absorption, and it can be energized during electroblotting. Further, it is advantageous in that nonspecific adsorption is small in the binding reaction with the analyte.

  In the present invention, the carbon layer can be formed by a known method. For example, a microwave plasma CVD (Chemical vapor deposition) method, an ECRCVD (Electrical cyclotron resonance) method, an ICP (Inductive coupled plasma) method, a direct current sputtering method, an ICP (Inductive coupled plasma method) Examples thereof include a vapor deposition method, a laser vapor deposition method, an EB (Electron beam) vapor deposition method, and a resistance heating vapor deposition method.

  In the high-frequency plasma CVD method, a source gas (methane) is decomposed by glow discharge generated between electrodes by a high frequency to synthesize a DLC (diamond-like carbon) layer on a substrate. In the ionization vapor deposition method, the source gas (benzene) is decomposed and ionized using thermoelectrons generated by a tungsten filament, and a carbon layer is formed on the substrate by a bias voltage. The DLC layer may be formed by ionized vapor deposition in a mixed gas composed of 1 to 99% by volume of hydrogen gas and 99 to 1% by volume of remaining methane gas.

  In the arc evaporation method, an arc discharge is generated in a vacuum by applying a DC voltage between a solid graphite material (cathode evaporation source) and a vacuum vessel (anode), and a plasma of carbon atoms is generated from the cathode to generate an evaporation source. Further, by applying a negative bias voltage to the substrate, carbon ions in the plasma can be accelerated toward the substrate to form a carbon layer.

  In the laser vapor deposition method, for example, a carbon layer can be formed by irradiating a graphite target plate with Nd: YAG laser (pulse oscillation) light and melting it, and depositing carbon atoms on a glass substrate.

  The thickness of the carbon layer on the surface of the solid support of the present invention is usually from a monomolecular layer to about 100 μm. If it is too thin, the surface of the base substrate may be locally exposed. Therefore, the thickness is preferably 2 nm to 1 μm, more preferably 5 nm to 500 nm. Note that all of the solid support may be made of a carbon material.

  In order to immobilize the biomolecule, it is preferable to introduce a chemical modifying group into the surface of the substrate on which the carbon layer is formed. The introduction of such a chemical modification group can be appropriately selected by those skilled in the art and is not particularly limited. For example, an amino group, a carboxyl group, an epoxy group, a formyl group, a hydroxyl group, and an activated ester group are introduced. To do. It is also effective to introduce metal chelates such as nickel chelates and cobalt chelates.

  Introduction of amino groups can be carried out, for example, by irradiating the carbon layer with ultraviolet rays in ammonia gas. Alternatively, the carbon layer can be chlorinated by irradiation with ultraviolet rays in chlorine gas, and further irradiated with ultraviolet rays in ammonia gas. Alternatively, it can also be carried out by reacting a polyvalent amine gas such as methylenediamine or ethylenediamine with a chlorinated carbon layer.

  The introduction of the carboxyl group can be carried out, for example, by reacting a suitable polycarboxylic acid with the carbon layer aminated as described above.

  Introduction of an epoxy group can be carried out, for example, by reacting a suitable polyvalent epoxy compound with the carbon layer aminated as described above. Or it can obtain by making an organic peracid react with the carbon = carbon double bond which a carbon layer contains. Examples of the organic peracid include peracetic acid, perbenzoic acid, diperoxyphthalic acid, performic acid, and trifluoroperacetic acid.

  Introduction of the formyl group can be carried out, for example, by reacting glutaraldehyde with the carbon layer aminated as described above.

  Introduction of hydroxyl groups can be carried out, for example, by reacting water with the carbon layer chlorinated as described above.

  The introduction of the activated ester group is, for example, by irradiating the carbon layer with ultraviolet rays in chlorine gas to chlorinate the surface, and then irradiating with ultraviolet rays in ammonia gas and aminating, and then using an appropriate acid chloride or dicarboxylic acid. Carboxylation with an anhydride can be carried out by dehydration condensation of the terminal carboxyl group with carbodiimide or dicyclohexylcarbodiimide and N-hydroxysuccinimide. By this treatment, a group in which an activated ester group such as an N-hydroxysuccinimide ester group is bonded to the end of the hydrocarbon group via an amide bond can be formed (Japanese Patent Laid-Open No. 2001-139532).

  When immobilizing nucleic acids such as DNA and RNA, it is preferable to introduce an amino group, a carbodiimide group, an epoxy group, a formyl group, or an activated ester group. When immobilizing a polypeptide, it is preferable to introduce an amino group, a carbodiimide group, an epoxy group, a formyl group, a metal chelate or an activated ester group. When a solid support into which a metal chelate is introduced is used, a polypeptide having a label having an affinity for a metal ion such as a polyhistidine sequence can be immobilized effectively and stably. The metal chelate can be introduced, for example, by chlorinating the substrate on which the carbon layer is formed and then aminating it, and then adding a halocarboxylic acid such as chloroacetic acid to introduce a chelate ligand. Labels such as polyhistidine sequences can be introduced by methods known to those skilled in the art.

  The chemical modification group may be introduced by forming an electrostatic layer on the surface carbon layer. The electrostatic layer can be formed using a positively charged compound such as an amino group-containing compound.

  A carbon layer obtained by kneading and molding a polymer material and a carbon-based material can also be used. Examples of the carbon-based material include carbon black and graphite. Accordingly, in the present invention, a carrier having a chemically modifying group on the surface of a material obtained by kneading a polymer material and carbon black and / or graphite can be used as the carrier. A carbonaceous material may be used independently and may be used in mixture of multiple types.

  Examples of the polymer material include polytetrafluoroethylene (PTFE), tetrafluoroethylene-perfluoroalkyl vinyl ether copolymer (PFA), tetrafluoroethylene / hexafluoropropylene copolymer (4.6 fluoride, EFP), Fluororesin such as tetrafluoroethylene / ethylene copolymer (ETFE), polyvinylidene fluoride (difluoride, PVDF), polychlorotrifluoroethylene (trifluoride, PCTFE), polypropylene, polyethylene, polybutene, polystyrene, poly Polyolefins such as vinyl chloride, polyvinyl alcohol and polyvinyl acetate, polyamides such as nylon (for example, nylon 6, nylon 66, nylon 11, nylon 12, nylon MXD6), polybutylene terephthalate, polyethylene Polyesters such as terephthalate and polytrimethylene terephthalate, polycarbonate, polyurethane, polylactic acid, ABS resin (Acrylonitrile Butadiene Styrene resin), acrylic resins, methylpentene resins, phenol resins, melamine resins and epoxy resins. As the carbon black, known carbon black can be used, and examples thereof include channel black, thermal black, furnace black, acetylene black, ketjen black, and graphitized carbon black. As graphite, known graphite can be used, and examples thereof include artificial graphite, scaly graphite, scaly graphite, and soil graphite. The kneading ratio between the polymer material and the carbon black and / or the graphite is not particularly limited. An adhesive may be added during kneading and molding may be performed. The kneaded material can not only suppress nonspecific adsorption but also suppress autofluorescence, and can immobilize not only peptides but also nucleic acids such as DNA.

  In one embodiment of the present invention, genes that have been narrowed down so as to obtain sufficient information for the target analysis among all gene information of animals and plants are immobilized on a carrier. For example, for the purpose of investigating the influence of chemical substances on animal and plant genes, they are narrowed down to genes related to estrogen and immobilized on a carrier. For example, an estrogen-related gene such as Estrogen receptor 1, Trefoil factor 1, Tumor protein D52-like 1 is immobilized on a carrier, and DNA is extracted from a tissue or cell of an animal or plant on which estrogen has acted, and the carrier is used as a target. React with genes. By examining the behavior of estrogen-related genes, it is possible to know the effects of estrogen on animals and plants. For example, if the expression level of the Estrogen receptor 1 gene is increased, it indicates that the cell has taken up a large amount of estrogen, indicating the possibility of being affected in some way. The effect of more genes can be determined by examining the increase or decrease in the expression level.

  In another embodiment of the present invention, all gene information of animals and plants is appropriately classified and immobilized on a carrier. For example, among genes involved in cancer, genes involved in organs are classified and immobilized on a carrier. For example, in the case of liver cancer, α-fetoprotein, tyrosine aminotransferase, Cyp7a1, etc. are immobilized on a carrier as liver-specific genes, and ICDH, BFP, CA125, etc. are immobilized as liver-specific genes. Then, DNA is extracted from the patient's liver and reacted with the gene on the carrier as a target. By examining the reacted target, it is possible to examine whether the liver is cancerous or, if it is cancerous, the degree of progression. Here, the gene includes cDNA, mRNA, aRNA and fragments thereof. By using this device, a disease-related gene can be searched quickly and accurately, and a target molecule for a therapeutic agent can be found.

  In the following, DNA is extracted from cultured cells to prepare a sample, subjected to PCR reaction, and then dispensed into a carrier holder of a reaction apparatus on which a carrier on which DNA is immobilized is transferred. One embodiment of the present invention that interacts will be described.

Sample pretreatment:
A sample is prepared by culturing cells and adjusting the number of cells. The number of cells is 1 × 10 ⁶ or less. The cells are dissolved in 180 μl PBS and dispersed well. Next, 20 μl of cell lysate A (EDT) and 180 μl of cell lysate B (LDT) are added, mixed well, and heated at 70 ° C. for 10 minutes. Add 240 μl of 100% ethanol to make a pretreated sample.

DNA extraction:
The pretreated sample is added to a cartridge of an extraction apparatus (for example, Quick Gene 800 manufactured by Fuji Photo Film). The following is done automatically. The sample passes through the column filter and only the DNA is adsorbed to the column. The DNA adsorption column is washed with a washing solution. Subsequently, the DNA is separated from the column by passing the collection buffer. This extract is used as a DNA sample.

Preparation before device:
A 3 mm square silicon carrier on which probe DNA is spotted is prepared by various spotter devices. The silicon carrier is prepared by coating a 3 mm square silicon substrate with a diamond-like carbon layer, modifying with a carboxyl group, and then active esterifying with N-hydroxysuccinimide. For example, 32 silicon carriers are arranged on a glass slide as a load holder. In the case of a 5 mm square silicon carrier, for example, 8 sheets are arranged per glass slide. Then, the loading holder on which the carriers are arranged is arranged on the loading stage.

  The apparatus is turned on and the DNA sample is placed in the 96-well plate of the dispensing apparatus 63, respectively. Enter work data. (Thickness of carrier, pitch, number of slides, loading holder reference position, unloading holder reference position, coordinates of first carrier in loading holder, etc.). The carrier immobilization conditions (for example, temperature 80 ° C., heating for 1 hour) and the carrier peeling conditions (for example, temperature 100 ° C., heating for 1 minute) are input to the cleaning device installed in the load holder. Enter the carrier cleaning conditions in the loading stage (for example, number of cleaning steps, designation of cleaning liquid supply bottle, cleaning time, rinse time, number of rinses, number of times of dry air spray, reciprocating speed, temperature, etc.). Enter the hybridization conditions in the reactor (eg, temperature 45 ° C., 3 hours). The cleaning conditions in the reactor are input (for example, the number of cleaning steps, the specification of the cleaning liquid supply bottle, the cleaning liquid discharge time, the drying air blowing time, etc.). A cleaning solution, for example, 2 × SSC / 0.2% SDS or pure water, is put into various cleaning solution bottles.

A tip (pipette tip, for example, 96 tip) is placed on the dispensing device 63. If 96 chips are not needed, attach a blind jig (the tip of the tip is blocked). When the tip mounting button is pressed, the tip nozzle is lowered, and the tip is inserted and returned to the initial position. Determine the dispensing volume.
The carrier holder 11 is placed on the stage of the reactor while being accommodated in the holder accommodating container 13.
A solution of the following components for performing PCR is dispensed into each well of a 96-well plate containing the DNA sample of the dispensing device 63.

  Create a temperature program for PCR. For example, (1) 95 ° C. 2 minutes → (2) 95 ° C. 30 seconds → (3) 55 ° C. 30 seconds → (4) 72 ° C. 30 seconds → (5) 72 ° C. 5 minutes. Repeat (2) to (4) 25 times. Press the PCR start button to execute PCR.

Device operation:
The loading stage 61 is heated. When the immobilization temperature (80 ° C.) is reached, the immobilization time (1 hour) is maintained. When the immobilization time ends, the cleaning liquid is introduced by a pump and supplied until the carrier is completely immersed in the cleaning liquid. Washing is carried out for about a washing time (15 minutes) in a running water state by rotation with a stirrer or circulation of washing liquid by a pump. At this time, when the heated cleaning liquid is used, the stage is heated to the set temperature when heated. When the cleaning time comes, the cleaning liquid is discharged. Subsequently, pure water is supplied until the carrier is completely immersed. Wash water with running water. The cleaning time is the time set by the rinse time, and then discharged. Gas is ejected from the blow drying head to dry the carrier. The blow drying head moves back and forth to cover the carrier.

  After the immobilization process and the washing and drying steps are completed, heating for peeling the carrier is performed. After the peeling heating is completed, the transfer device is operated to take the carrier to the loading holder on the loading stage. The transfer device holds and conveys the side surface of the carrier. At this time, if there is a deviation in the position of the carrier, the carrier cannot be accurately conveyed, and means for searching for the position of the carrier is provided. As a means for this search, a full search and a reference search are used. In the case of full search, position detection by a sensor is performed for each carrier. In the case of the reference search, only the carrier to be transported first is searched at the start of transporting, and then the transport is performed with a shortened search time by transferring at the input pitch. The carrier taken by the transfer device is stored in the carrier accommodating portion 21 of the carrier holder 11 set in the reaction device. All carriers are transferred based on the work data.

  If the PCR reaction is completed in the dispensing device 63, the dispensing operation is started as it is. The tip nozzle equipped with the tip of the dispensing device 63 is lowered to a position where the sample can be sucked from the 96 well plate. Simultaneously in 96 wells, the DNA sample after the PCR reaction is sucked in a set volume. The dispensing head ascends, moves to the upper part of the reaction apparatus and descends, and dispenses the sample into the carrier accommodating portion of the carrier holder 11 in which the carrier is accommodated. The dispensing head rises and retracts. The cleaning head is lowered, and the lid is in close contact with the carrier holder to seal the carrier accommodating portion 21.

  The sample and the carrier are heated by the temperature controller 14 to carry out the hybridization reaction (45 ° C. × 3 hours). Here, the PCR reaction may be performed by changing the temperature by the temperature control device 14 during at least a part of the time when the hybridization reaction is performed.

  When the hybridization is completed, the cleaning head 12 is raised and the lid is removed. The supply hole of the lid of the cleaning head 12 moves so as to be aligned with the opening 52 of the carrier accommodating portion. Again, the cleaning head 12 descends. Further, the holder accommodating container 13 is also lowered. The cleaning liquid is supplied from the supply hole. By separating the carrier holder and the holder housing container and supplying the cleaning liquid or gas from the supply hole of the cleaning head to the carrier storage portion, it is possible to prevent cross-contamination between biomolecules dispensed on the carrier. After the completion of cleaning, gas is fed from the supply hole to dry the carrier. The cleaning head 12 is raised and retracted.

  The transferred carrier is immediately measured by a known detection method. Examples of the detection method include a detection method using a fluorescence measuring device, a radioisotope measuring device, a chemiluminescence measuring device, or the like. If measurement is not performed immediately, it must be stored according to each detection method. In particular, when performing fluorescence measurement, in order to prevent discoloration of the fluorescent material, it is preferable to store the light at a low temperature of −20 ° C. or lower and light shielding. When chemiluminescence is measured, it is often carried out in a solution, and it can be measured with a chemiluminescence measuring instrument after a predetermined luminescence time, excluding the cleaning process and the transfer process to the unloading holder.

(Example 1) DNA interaction
Preparation of silicon support A 3 mm square silicon substrate was coated with a diamond-like carbon layer, modified with a carboxyl group, and then active esterified with N-hydroxysuccinimide to prepare a silicon support.

Preparation of DNA-immobilized carrier Oligonucleotide (manufactured by Hokkaido System Science) was adjusted to 100 μM with sterile water. 4 times concentrated Sol. 6 (manufactured by Toyo Kohan Co., Ltd.) to obtain a DNA solution having a final concentration of 5 μM. A heat-releasable double-sided tape (manufactured by Nitto Denko) was applied as a sheet coated with a heat-releasable adhesive on a slide glass, and a silicon carrier was arranged as shown in FIG. 7 using a transfer device (manufactured by Hitachi High-Technologies Corporation). . The DNA solution was spotted 3200 points (100 points / chip) with SPBIO2000 (manufactured by Hitachi Soft Engineering).

Washing of DNA Immobilization Carrier A slide glass on which a DNA immobilization carrier was placed was placed in a baking chamber at 80 ° C. and allowed to stand for 1 hour for immobilization. Subsequently, the substrate was washed with a room temperature washing solution (2 × SSC / 0.2% SDS) for 15 minutes. Subsequently, cleaning was performed for 5 minutes with a boiling (about 90 ° C. to 100 ° C.) cleaning solution (2 × SSC / 0.2% SDS). Rinsing was performed three times with ultrapure water. Water was blown away with a centrifuge.

Preparation of DNA Sample DNA was extracted using an automatic nucleic acid extraction system QuickGene800 (manufactured by Fuji Photo Film Co., Ltd.). A cell lysate was added to various cells to dissolve the cells, and the lysate was added to the apparatus cartridge to extract DNA. PCR was performed using TaKaRa Ex Taq ^™ (manufactured by Takara Bio Inc.) to obtain a DNA sample. At this time, the concentration of one primer was reduced to 1/10 to amplify more DNA strands extending than the other primer. CyDye was incorporated into the PCR product by using CyDye-labeled dCTP.

Hybridization reaction The CyDye-labeled PCR product prepared above was dissolved in a hybridization solution (5 × SSC / 0.5% SDS) to obtain a 20 μl hybridization solution. This hybridizing solution was added to the DNA-immobilized carrier prepared above and reacted at 60 ° C. for 1 hour in the chamber. After the reaction, a room temperature washing solution (2 × SSC / 0.2% SDS) was fed at a flow rate of 10 ml / min. 2 × SSC was fed at a flow rate of 10 ml / min. Dry with N ₂ blow.

(Example 2) DNA-protein interaction
Preparation of silicon support A 3 mm square silicon substrate was coated with a diamond-like carbon layer, modified with a carboxyl group, and then active esterified with N-hydroxysuccinimide to prepare a silicon support.

Preparation of DNA-immobilized carrier Oligonucleotide (manufactured by Hokkaido System Science) was adjusted to 100 μM with sterile water. 4 times concentrated Sol. 6 (manufactured by Toyo Kohan Co., Ltd.) to obtain a DNA solution having a final concentration of 5 μM. A heat-releasable double-sided tape (manufactured by Nitto Denko) was applied as a sheet coated with a heat-releasable adhesive on a slide glass, and a silicon carrier was arranged as shown in FIG. 7 using a transfer device (manufactured by Hitachi High-Technologies Corporation). . The DNA solution was spotted 3200 points (100 points / chip) with SPBIO2000 (manufactured by Hitachi Soft Engineering).

Washing of DNA Immobilization Carrier A slide glass on which a DNA immobilization carrier was placed was placed in a baking chamber at 80 ° C. and allowed to stand for 1 hour for immobilization. Subsequently, the substrate was washed with a room temperature washing solution (2 × SSC / 0.2% SDS) for 15 minutes. Subsequently, cleaning was performed for 5 minutes with a boiling (about 90 ° C. to 100 ° C.) cleaning solution (2 × SSC / 0.2% SDS). Rinsing was performed three times with ultrapure water. Water was blown away with a centrifuge.

Preparation of Protein Sample Budding yeast (S. cerevisiae) was cultured overnight in YPD liquid medium, and collected by centrifugation at 4 ° C. and 3000 rpm. After rinsing with physiological saline, the cells recovered by centrifugation were stored at -80 ° C. 0.4 g of cells were added to Lysis Buffer (50 mM HEPES-KOH, pH 7.5, 10 mM MgCl ₂ , 150 mM KCl, 0.1 mM EDTA, 10% glycerin, 0.1% NP-40, 1 mM dithiothreitol, 1 mM metathiol. It was dissolved in 1 mL of sodium bisulfite, 0.2 mM PMSF, 1 mM benzamide, 1 μg / mL pepstatin) and transferred to a tube containing 0.5 mm diameter glass beads. The mixture was crushed for 1 minute using a bead beater (manufactured by BIOSPEC) and ice-cooled for 2 minutes. This crushing was repeated 4 times. The supernatant was collected and centrifuged at 12000 rpm, 4 ° C. for 10 minutes to obtain the supernatant as a protein extract.

  One pack of Cy3 Mono-reactive Dye (manufactured by Amersham Biosciences) was allowed to react with 5 mg of protein for 1 hour. Protein column purification was performed with Micro Bio-Spin (manufactured by BIO RAD) to obtain a protein sample.

The DNA-protein interaction protein sample was added to the DNA-immobilized carrier prepared above, and the mixture was reacted at 37 ° C. for 1 hour in the chamber. After the reaction, a room temperature washing solution (TE buffer solution) was fed at a flow rate of 10 ml / min. Dry with N ₂ blow.

(Example 3) Protein-protein interaction
Preparation of silicon support A 3 mm square silicon substrate was coated with a diamond-like carbon layer, modified with a carboxyl group, and then active esterified with N-hydroxysuccinimide to prepare a silicon support.

Preparation of protein-immobilized carrier Proteins were dissolved in PBS and used as spot samples. A heat-releasable double-sided tape (manufactured by Nitto Denko) was applied as a sheet coated with a heat-releasable adhesive on a slide glass, and a silicon carrier was arranged as shown in FIG. 7 using a transfer device (manufactured by Hitachi High-Technologies Corporation). . SPBIO2000 (manufactured by Hitachi Soft Engineering) spotted 3200 spots (100 spots / chip).

Washing of the protein-immobilized carrier The protein-immobilized carrier was placed in a baking chamber at 37 ° C and allowed to stand for 1 hour for immobilization. Subsequently, the substrate was washed with a room temperature washing solution (TE buffer solution) for 15 minutes. Water was blown away with a centrifuge.

Preparation of Protein Sample Budding yeast (S. cerevisiae) was cultured overnight in YPD liquid medium, and collected by centrifugation at 4 ° C. and 3000 rpm. After rinsing with physiological saline, the cells recovered by centrifugation were stored at -80 ° C. 0.4 g of cells were added to Lysis Buffer (50 mM HEPES-KOH, pH 7.5, 10 mM MgCl ₂ , 150 mM KCl, 0.1 mM EDTA, 10% glycerin, 0.1% NP-40, 1 mM dithiothreitol, 1 mM metathiol. It was dissolved in 1 mL of sodium bisulfite, 0.2 mM PMSF, 1 mM benzamide, 1 μg / mL pepstatin) and transferred to a tube containing 0.5 mm diameter glass beads. Using a bead beater (manufactured by BIOSPEC), the mixture was crushed for 1 minute and cooled on ice for 2 minutes. This crushing was repeated 4 times. The supernatant was collected and centrifuged at 12000 rpm, 4 ° C. for 10 minutes to obtain the supernatant as a protein extract.

  One pack of Cy3 Mono-reactive Dye (manufactured by Amersham Biosciences) was allowed to react with 5 mg of protein for 1 hour. Protein column purification was performed with Micro Bio-Spin (manufactured by BIO RAD) to obtain a protein sample.

Protein-Protein Interaction A protein sample was dissolved in a prehybridization solution for protein (1 × TE buffer), added to the protein-immobilized carrier prepared above, and reacted at 37 ° C. for 14 hours in a chamber. After the reaction, a room temperature washing solution (TE buffer solution) was fed at a flow rate of 10 ml / min. Dry with N ₂ blow.

(Example 4) Reduction of PCR reaction time In a dispensing apparatus, a PCR solution of the following components was dispensed in order to amplify a sample containing DNA (DNA extracted from λ phage).

  PCR reaction is usually performed by (1) heat denaturation at 94-98 ° C. for 0 seconds to 5 minutes, (2) heat denaturation at 95-98 ° C. for 0 seconds to 5 minutes, and (3) 0 seconds at 37-68 ° C. It consists of annealing for 2 minutes, (4) extension reaction at 70-75 ° C. for 5 seconds to 5 minutes, and (5) extension reaction at 70-75 ° C. for about 5 minutes. By repeating, the nucleic acid is amplified.

  In this example, the conditions in the steps (2) to (4) among the above steps (1) to (5) are set as PCR conditions 1 to 3 in Table 3 below, and this is repeated for 30 cycles. It was. In Table 3 below, for example, 95 ° C. 0 second means that the temperature is changed to 95 ° C., but is not maintained at that temperature, and the change to 55 ° C. is started as soon as 95 ° C. is reached. .

  1 μl of the PCR product after the reaction was subjected to electrophoresis to confirm DNA amplification. As a result, DNA amplification was sufficiently observed even under PCR condition 3 (FIG. 8), indicating that the PCR reaction time could be shortened to 40 minutes.

(Example 5) Reduction of hybridization time
Preparation of silicon carrier A diamond-like carbon layer was coated on a 3 mm square silicon substrate. The diamond-like carbon layer was formed by ionized vapor deposition. First, the chamber was evacuated by a turbo molecular pump until the inside of the chamber became 8 × 10 ⁻³ Pa or less. Next, the silicon substrate surface was previously cleaned with hydrogen plasma. The conditions were room temperature, hydrogen gas flow rate of 40 sccm, and high frequency power of 100 W. The diamond-like carbon layer was formed under the conditions of methane gas: hydrogen gas = 47.5 sccm: 2.5 sccm gas flow rate, 3 Pa pressure, 200 W high frequency power, and room temperature. The surface of the diamond-like carbon layer was aminated with ammonia plasma. The ammonia gas flow rate was 18 sccm, and the pressure was 3 Pa. In order to introduce a carboxyl group into the surface of the aminated diamond-like carbon layer, this substrate was placed in a solution of 0.14M succinic anhydride and 0.1M sodium borate (pH 8.0) dissolved in pyrrolidone for 10 minutes. After washing with water, it was vacuum dried. Subsequently, in order to active esterify the carboxyl group on the substrate surface, the substrate was activated by immersing in an activating solution at 25 ° C. for 20 minutes. The activation solution was prepared to be 0.2M carbodiimidide and 0.2M N-hydroxysuccinimide in 0.1M phosphate buffer. Thereafter, the activated substrate was washed with pure water and vacuum-dried at 100 ° C.

Preparation of Immobilization Carrier An oligonucleotide complementary to viral DNA (manufactured by Hokkaido System Science Co., Ltd.) was adjusted to 100 μM with sterile water. It was dissolved in a 20 vol% polyethylene glycol solution to obtain an oligonucleotide solution complementary to the final concentration of 5 μM viral DNA. A heat-releasable double-sided tape (manufactured by Nitto Denko Corporation) was applied as a sheet in which a heat-releasable adhesive was applied to a silicon carrier, and the silicon carrier was arranged as shown in FIG. 7 using a transfer device (manufactured by Hitachi High-Technologies Corporation). . SPBIO2000 (manufactured by Hitachi Soft Engineering) spotted 28 oligonucleotide solutions complementary to viral DNA on a silicon carrier.

Washing of DNA Immobilization Carrier The DNA immobilization carrier was placed in a baking chamber at 80 ° C. for 1 hour to be immobilized. Subsequently, the substrate was washed with a room temperature washing solution (2 × SSC / 0.2% SDS) for 15 minutes. Subsequently, cleaning was performed for 5 minutes with a boiling (about 90 ° C. to 100 ° C.) cleaning solution (2 × SSC / 0.2% SDS). Rinsing was performed three times with ultrapure water. Water was blown away with a centrifuge.

Preparation of DNA sample DNA extracted from human tissue was incorporated into a plasmid vector, and PCR was performed using this plasmid DNA as a template using TaKaRa Ex Taq ^™ (Takara Bio Inc.) to obtain a DNA sample. PCR was performed using the PCR solution shown in Table 4 below. At this time, the PCR product was Cy3 labeled by using a Cy3 labeled FW primer. In the PCR reaction, 30 cycles of 95 ° C. for 0 second, 55 ° C. for 0 second, and 72 ° C. for 5 seconds were performed (same as PCR condition 3 in Example 4).

Hybridization Reaction The Cy3-labeled PCR product prepared above was dissolved in a hybridization buffer (5 × SSC / 0.5% SDS) to obtain a 20 μl hybridization solution. This hybridizing solution was added to the DNA-immobilized carrier prepared above, and reacted in a chamber at 50 ° C. for 5 minutes, 10 minutes, 30 minutes, 1 hour and 4 hours. After the reaction, rinsing was performed in order, once with 2 × SSC / 0.2% SDS, twice with 2 × SSC, and twice with 80% ethanol. Thereafter, water was blown off by a centrifuge. As a result of measuring the fluorescence signal derived from the hybridized Cy3-labeled PCR product with a fluorescent scanner FLA8000 (Fuji Photo Film Co., Ltd.), the hybridization time reached almost a peak in 10 minutes. Minutes were found to be sufficient (Figure 9).

  On the other hand, when the hybridization buffer (SSC / SDS buffer) was not used, a sufficient fluorescence signal was not obtained with a hybridization time of 10 minutes (FIG. 10). Therefore, it was found that the hybridization time can be shortened by adding SSC / SDS.

The side view of one Embodiment of the reaction apparatus of this invention is shown. FIG. 3 shows a cross-sectional view of one embodiment of the carrier holder of the present invention. The top view schematic of one embodiment of the carrier holder of this invention with which the some carrier accommodating part was arranged is shown. The top view of one embodiment of the washing head of the present invention is shown. FIG. 5a shows an embodiment in which the carrier housing is sealed. FIG. 5b shows an embodiment in which the carrier holder and the holder container are separated. 1 shows a schematic top view of one embodiment of the apparatus of the present invention comprising a loading stage, a reaction apparatus, a transfer apparatus and a dispensing apparatus. The mode which arrange | positions a support | carrier to a slide glass is shown. The result of having applied the sample amplified in various PCR conditions in Example 4 to electrophoresis is shown. The result of having plotted the measurement result of the fluorescence signal obtained by hybridization with and without using the hybridization buffer with respect to the hybridization time is shown. Fluorescence images at a hybridization time of 10 minutes are shown with and without a hybridization buffer.

Explanation of symbols

DESCRIPTION OF SYMBOLS 11 ... Carrier holder, 12 ... Cleaning head, 13 ... Holder accommodating container, 14 ... Temperature control device, 21 ... Carrier accommodating part, 22 ... Discharge port, 23 ... Carrier, 31 ... groove, 41 ... cleaning liquid or gas for drying, 42 ... lid body, 43 ... supply hole for lid body, 51 ... supply hole for lid body, 52 ... Opening of carrier accommodating portion, 53... Recessed portion of holder accommodating container, 54... Direction of flow of cleaning liquid or gas, 61... Loading stage, 62. Equipment, M ... Motor head moving motor, CY ... Cylinder for vertical drive

Claims (13)

A reaction device for interacting biomolecules on a carrier with biomolecules in a sample,
A temperature control device for controlling the temperature of the sample and the carrier, a carrier holder having a plurality of carrier accommodating portions having discharge ports, and a lid having a plurality of supply holes for supplying cleaning liquid or gas to the carrier accommodating portion of the carrier holder. The reaction apparatus comprising: a cleaning head provided; and a holder housing container having a plurality of recesses capable of housing the carrier housing portion of the carrier holder. The reactor according to claim 1,
A loading stage for arranging a plurality of carriers on which biomolecules are immobilized;
A transfer device for transferring a carrier from a loading stage to a carrier accommodating portion of a carrier holder in the reaction device, and a dispensing device for dispensing a plurality of samples containing biomolecules into the carrier accommodating portion of the carrier holder of the reaction device A biomolecule analyzer comprising:   The apparatus of claim 2, wherein the loading stage comprises a cleaning device.   The apparatus of Claim 2 or 3 with which the dispensing apparatus is equipped with the temperature control apparatus for controlling the temperature of the sample containing a biomolecule.   The apparatus according to any one of claims 1 to 4, wherein the carrier accommodating portion of the carrier holder can be sealed by the lid of the cleaning head and the holder accommodating container.   The apparatus of any one of Claims 1-5 by which the temperature control apparatus of a reaction apparatus is installed in a holder storage container, and a holder storage container is movable. A method for analyzing the interaction of biomolecules,
Arranging a plurality of carriers on which biomolecules are immobilized on a loading stage;
A step of transferring a carrier from a loading stage to a carrier holder having a plurality of carrier accommodating portions having discharge ports, wherein the carrier accommodating portion of the carrier holder includes a plurality of carrier accommodating portions of the carrier holder. Are accommodated in the plurality of recesses of the holder housing container having the recesses of
A step of dispensing a plurality of samples containing biomolecules into a carrier accommodating portion of a carrier holder by a dispensing device;
By moving a cleaning head having a lid having a plurality of supply holes for supplying the cleaning liquid or gas to the carrier accommodating portion of the carrier holder and covering the carrier holder with the lid, the carrier accommodating portion of the carrier holder is Sealing,
The temperature control device changes the temperature of the sample and the carrier to allow the biomolecules immobilized on the carrier to interact with the biomolecules contained in the sample, and the carrier holder and the holder container are separated for cleaning. The method comprising the step of cleaning or drying the carrier by supplying a cleaning liquid or gas from the supply hole of the head to the carrier containing portion.   The method according to claim 7, wherein the temperature control device is installed in the holder receiving container, and the holder receiving container moves and carries out the receiving and separating of the carrier holder in the holder receiving container.   The loading stage is equipped with a cleaning device and a temperature control device, and at least the surface of the loading stage or loading holder on which the carrier is arranged or the bottom of the carrier is made of a heat-peelable adhesive and is below the temperature at which the temperature control device peels The method according to claim 7 or 8, wherein the biomolecule is immobilized on the carrier by washing to perform washing, and the carrier is peeled by raising the temperature to the peeling temperature.   The biomolecule contained in the sample is a nucleic acid, and further includes a step of subjecting the nucleic acid in the sample to a PCR reaction by changing the temperature with a temperature control device provided in the dispensing device before dispensing the sample. The method of any one of Claims 7-9.   The biomolecule is a nucleic acid, and at least a part of the step of interacting the nucleic acid immobilized on the carrier with the nucleic acid contained in the sample, the PCR reaction is performed by changing the temperature with a temperature controller. Item 11. The method according to any one of Items 7 to 10.   The method according to claim 10 or 11, wherein the PCR reaction is an asymmetric PCR reaction.   The method according to any one of claims 7 to 12, wherein the carrier is a solid support having a carbon layer and a chemically modifying group on a substrate surface made of a silicon material.

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