JP2007071569A - Manufacturing method of probe immobilizing carrier having fine pattern - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method capable of miniaturizing a spot size to a droplet amount, when spotting a droplet of a solution including a probe onto the base material (substrate) surface which has been subjected to application processing with an immobilization agent. <P>SOLUTION: When the probe is immobilized onto the base material through bonding formation by a reaction between a reactive group carried by the probe and a reactive group introduced onto the base material, a manufacturing process to be used includes: (1) a process for introducing the reactive group onto the base material; (2) a process for providing on the base material a coating film comprising a reaction inactive material showing no reactivity to both the reactive group in the probe and the reactive group introduced onto the base material; (3) a process for spot-contacting the droplet of the solution including the probe onto the base material covered with the coating film, and allowing the droplet to permeate the coating film, to be thereby brought into contact with the base material onto which the reactive group is introduced; and (4) a process for allowing the reactive group carried by the probe included in the droplet to be reacted with the reactive group introduced onto the base material. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for producing a probe-immobilized carrier, and more particularly to a method for producing an immobilized probe-immobilized carrier by spotting a probe on a substrate in a fine pattern.

  A technique for detecting a target substance using a probe molecule that can specifically bind to the target substance has been widely applied. For example, in the probe hybridization method, a nucleic acid probe having a base sequence complementary to a base sequence of a target nucleic acid molecule is used as a probe, and the target nucleic acid molecule is detected by a hybridization reaction. This technique is widely used for determining the base sequence of nucleic acid molecules, detecting target nucleic acid molecules with a specific base sequence contained in a specimen, and identifying bacterial species based on genetic information of various bacteria. Has been. When detecting target nucleic acid molecules based on this probe hybridization method, it has been proposed to use a probe-immobilized carrier (probe array) in which a large number of nucleic acid probes are arrayed and immobilized on a solid support. ing. In particular, the use of so-called high-density probe arrays in which spot points for immobilizing individual nucleic acid probes are arranged in a high-density array and a large number of nucleic acid probes are immobilized on a solid support at high density has recently been used. It is attracting attention rapidly. By using such a high-density probe array when performing a hybridization reaction on a large number of nucleic acid probes, the reaction between the target nucleic acid molecules contained in the investigation sample and a large number of nucleic acid probes can be performed in a single operation. Can be done. That is, in one experiment, it becomes possible to evaluate the difference in reactivity among many types of nucleic acid probes immobilized on one probe array. In addition, high-density probe arrays can be made with high reproducibility, and a large number of probe arrays with the same quality can be prepared to detect differences in reactivity between survey samples in a sample set. It can also be used.

  Various methods are known as methods for preparing a probe array by immobilizing a plurality of nucleic acid probes on a solid support. First, a method developed by the first Affimetrix can be mentioned. This method uses a protective group that is selectively removed by light irradiation and a photolithographic technique that is used in semiconductor manufacturing, and each rectangular region of several tens of μm that is arranged at high density on a substrate, Oligonucleotide synthesis is performed independently. The probe array obtained by this method is currently the only standardized standard in the world. However, since DNA probes are directly synthesized on a substrate, a large number of photolithographic steps are required to produce individual probe arrays, which makes the price very high. This high cost probe array can only be used in large quantities by some researchers with ample research costs. On the other hand, as another method for manufacturing a probe array, P.A. A technique developed by Brown et al. This method has a very simple manufacturing process as compared to the method of Affimetrix described above. A solution of many types of DNA probes prepared in advance is dispensed into a 96- or 384-well microplate whose surface is chemically treated. Next, using a robot capable of high-density spots, DNA probes are spotted at high density on a slide glass (substrate) and immobilized. This method uses a large number of DNA probes prepared in advance, instead of synthesizing DNA probes for each substrate as in the method of Affimetrix. Therefore, when a large number of probe arrays having a limited quantity are produced, if a method of spotting and immobilizing DNA probes at a high density is used, the manufacturing cost per probe array is lowered. In particular, many research institutes do not need a multi-step photolithographic process, and it is possible to create custom arrays with various combinations of DNA probes according to the needs of individual researchers. The method is being used.

  Also, as a probe spotting means, there is a probe array manufacturing method capable of forming a micro spot by spotting a probe-containing solution on the surface of a substrate (substrate) with an inkjet head capable of ejecting micro droplets. It has been proposed (Patent Document 1).

  In the probe array for immobilizing many kinds of pre-prepared probes on a substrate, an immobilizing agent for immobilizing DNA is usually applied on a glass substrate, and the immobilizing agent-coated surface is applied. It is produced by spotting a large number of target DNAs in a high-density array. However, when the surface treated with the immobilizing agent is hydrophilic, droplets of the solution containing the probe spotted on the surface spread on the surface and the droplets at adjacent spot points come into contact with each other. , There may be a problem that liquid mixing occurs between the two. In order to prevent the mixing of droplets between adjacent spot points, it is necessary to provide a constant interval between the spot points in consideration of the wetting and spreading of the droplets. It becomes a restriction. In addition, even when the spotted probe solution has little wetting and spreading on the surface of the base material (substrate) coated with the immobilizing agent, it may be desired to further reduce the spot diameter of each spot point. For example, in the case of a valuable research sample that originally has a small amount of sample, it is desired to further reduce the amount of sample liquid used for reaction with a large number of probes on the probe array. That is, it is desired to further reduce the spot diameter of each spot point and further reduce the area of the entire probe array. That is, the smaller the spot diameter, the higher the density of spot points. For example, by further increasing the density of spot points, even if the number of types of probes constituting the probe array is the same, the area for spotting these probes can be further reduced, so the amount of sample liquid in contact with this narrow area is reduced. It becomes possible. Alternatively, by further increasing the density of the spot points, it is possible to increase the total number of probe types that can be spotted for the same area. By utilizing this advantage, it is possible to increase the total number of types of probes that can be reacted with the sample solution, that is, to increase the amount of information obtained, even if the amount of sample solution used is the same.

  As a method of suppressing wetting and spreading on the surface of the spotted droplet, there is a method in which an area that can be a spot on the substrate is made hydrophilic and an area that can be a background around the surface is hydrophobic. As a means for this, a method has been proposed in which a fine hydrophilic pattern is formed in advance corresponding to a spot position on a hydrophobic substrate, and then a solution containing a probe is spotted on the hydrophilic area (Patent Document). 2, Patent Document 3, Patent Document 4). However, since these methods preliminarily form the hydrophilic / hydrophobic pattern on the substrate, a photomask for patterning the hydrophilic immobilizing agent application region is required, and this patterning step increases the cost for manufacturing the probe array. It can be a factor.

  In addition to these methods, attempts are made to make the tip of the spotting robot pin even thinner in order to reduce the amount of probe solution droplets spotted on the substrate and form a fine spot diameter. Attempts have been made to further reduce the discharge port of the inkjet head. The spot diameter is determined depending not only on the amount of droplets to be spotted, but also on the wettability of the probe solution with respect to the surface of the substrate (substrate) coated with the hydrophilic fixing agent, that is, the contact angle of the probe solution. . Therefore, even if the amount of spotted droplets is reduced, the spot diameter finally obtained does not decrease in proportion to the amount of droplets. Further, when the nozzle diameter of the ink jet head is reduced, minute particles are likely to be clogged in the nozzle. For this reason, as the nozzle diameter becomes thinner, there is another problem that the yield at the time of manufacturing the ink-jet head becomes inversely proportional. In order to minimize the occurrence of defects due to the clogging of fine particles within a narrow nozzle diameter, it is necessary to suppress the entry of fine particles as much as possible, and further strict management of the clean room environment is required. For example, with the introduction of a clean room facility that maintains a higher degree of cleanliness, it may be a factor in increasing manufacturing costs.

Theoretically, if the wettability of the spotted solution with respect to the substrate surface is lowered and the contact angle is increased, the spot diameter can be reduced in the same droplet amount. In order to reduce the wettability of the probe solution to the surface of the substrate (substrate) coated with the immobilizing agent, that is, to increase the contact angle of the probe solution, the chemical structure of the immobilizing agent applied to the substrate surface is changed. Or changing the composition of the spotting solution. For example, when the chemical structure of the immobilizing agent applied to the substrate surface is changed, the reaction mechanism between the probe and the immobilizing agent often changes. In that case, in order to make the contact angle of the desired probe solution compatible with the immobilization efficiency of the probe, the trial and error are often repeated, which may increase the development cost.
JP 2003-254969 A JP 2003-028864 A JP 2003-33177 A JP 2002-267667 A

  The present invention solves the above-mentioned problems, and the object of the present invention is to spot a liquid droplet containing a probe on the surface of a substrate (substrate) coated with a fixing agent. It is an object of the present invention to provide a novel technique that enables the spot diameter to be reduced with respect to the drop amount. That is, when manufacturing a probe-immobilized carrier used for detection of a target substance (target), in order to suppress the droplets of a solution containing a probe spotted on the substrate surface from spreading on the substrate surface, It is an object of the present invention to provide a novel technique that enables the reduction of the spot diameter with respect to the droplet amount without using a technique for applying hydrophilic / hydrophobic patterning having a function of limiting a spot area on the surface of a substrate. In particular, it does not change the immobilizing agent applied to the substrate surface, which is used for immobilizing the probe, and makes it possible to reduce the spot diameter of the droplets to be spotted. An object of the present invention is to provide a method for producing a probe-immobilized carrier that uses a novel spot means.

  In order to solve the above-mentioned problems, the present inventor has conducted extensive research and has obtained the following knowledge. That is, the surface of the base material (substrate) subjected to the immobilizing agent application treatment has affinity for a solvent of the solution containing the probe, for example, an aqueous solvent. For this reason, when a droplet of a solution containing a probe is spotted, the final spot diameter is increased as the aqueous solvent wets and spreads. On the other hand, if a coating film made of another substance is formed in advance on the surface of the base material (substrate) subjected to the fixing agent coating treatment, the following phenomenon occurs. That is, when spotting a droplet of a solution containing a probe, if the droplet permeates through the coating film and comes into contact with the substrate (substrate) surface, the circumference of the dot diameter of the droplet is It will be in the state surrounded by a coating film. At that time, in order for the aqueous solvent of the solution containing the probe to cause further wetting and spreading on the surface of the base material (substrate), it is necessary to spread the coating film surrounding the periphery.

  If the surrounding coating material is a non-hydrophilic material that has a poor affinity for the solvent of the solution containing the probe, e.g., an aqueous solvent, and is immiscible with each other, the spotted droplet and its A clear interface is formed with the surrounding coating film. At this interface, an internal pressure due to the load of the droplet itself is applied from the spotted droplet side to the coating film side. On the other hand, an external pressure derived from the load of the non-hydrophilic substance that originally existed in the region pushed outward is applied to the droplet side from the coating film side with the spot. Furthermore, the adhesive force at the contact surface between the coating film and the substrate (substrate) surface and the stress required for plastic deformation of the coating film also act as repulsive forces that suppress the wetting and spreading of the droplets. It has been found that when they reach equilibrium, the wetting and spreading of the droplets does not proceed any further. Therefore, compared with the case where the coating film is not formed, by forming a coating film made of another substance in advance on the surface of the base material (substrate) coated with the fixing agent, there is a difference in degree. However, it was found that wetting and spreading of spotted droplets is suppressed.

  At that time, the substance constituting the coating film is used as a reactive group introduced to the surface of the substrate (substrate) or a reactive group provided in the probe, which is used for fixing the probe. On the other hand, when reactivity is shown, it causes the following problems. First, at the stage where a coating film is formed on the surface of the base material (substrate), the film that reacts with the reactive group introduced on the surface of the base material (substrate) and is immobilized on the surface of the base material (substrate) This causes a problem of forming a layer. Or it reacts with the reactive group provided in the probe contained in the spotted liquid droplet, and as a result, it becomes a factor of the problem of reducing the amount of probes immobilized on the substrate (substrate) surface. . In other words, a reactive substance that does not react with any of the reactive group included in the probe and the reactive group introduced onto the substrate is used as the material constituting the coating film. It has been found that the above-mentioned problems can be avoided.

  The present inventor completed the present invention based on the above findings.

That is, the method for producing a probe-immobilized carrier according to the present invention includes:
In a method for producing a probe-immobilized carrier in which a probe capable of specifically binding to a target substance is immobilized on a substrate,
The probe is immobilized on the substrate through bond formation by a reaction between a reactive group included in the probe and a reactive group introduced on the substrate.
The following steps (i) to (iv):
Step (i): introducing a reactive group capable of reacting with the reactive group included in the probe onto the substrate;
Step (ii): A coating film made of a reaction inert substance that is not reactive with any of the reactive groups provided in the probe and the reactive groups introduced onto the substrate. Providing on top;
Step (iii): The droplet of the solution containing the probe is spotted on the base material covered with the coating film made of the reaction inert substance by the spotting means, and the spotted droplet is Allowing the coating film made of a reaction inert substance to pass through and contacting with the base material into which the reactive group is introduced;
Step (iv): a step of reacting a reactive group included in a probe contained in a droplet to be deposited with a reactive group introduced on a substrate in contact with the droplet This is a method for producing a probe-immobilized carrier.

  The overall shape of the droplet spotted on the substrate (substrate) surface is governed by the liquid volume of the droplet and the contact angle of the droplet in contact with the substrate (substrate) surface. Considering this point, conventionally, as a technique for minimizing the dot diameter where the spotted droplet contacts the substrate (substrate) surface, contact the droplet volume with the substrate (substrate) surface. Means to increase the corner have been used. For example, when a droplet of a solution using an aqueous solvent is spotted, a fine hydrophilic pattern is formed at the center of the spot point, and the periphery is surrounded by a hydrophobic surface. In that state, in the liquid droplets that protrude from the hydrophilic pattern, the outer periphery of the contact surface is located on the hydrophobic surface, the contact angle of the liquid droplet is increased, and the effect of miniaturizing the dot diameter is achieved. It had been. Also, by applying the immobilizing agent used to immobilize the probe only to the minute hydrophilic pattern portion, the diameter of the dot on which the probe is immobilized on the substrate (substrate) surface is miniaturized. It was possible to do.

  In the step of forming a hydrophilic pattern on which the fixing agent coating process is performed on the surface of the base material (substrate), in order to perform patterning into a predetermined pattern shape, for example, a photomask used for photolithography, and A patterning device is used. The costs associated with their use can be a factor in unnecessary manufacturing cost increases in some cases. Or, to increase the contact angle of the solvent used in the solution to the surface of the base material (substrate) subjected to the fixing agent coating treatment, the fixing agent used or the solvent itself used in the solution is changed. Attempts have also been made. To change the immobilizing agent used, it is also necessary to create a probe with a reactive group that is compatible with the modified immobilizing agent. A lot of development time is required in the development process that repeats. Therefore, depending on the case, it may be a factor of unnecessary development cost increase.

  In the method for producing a probe-immobilizing carrier according to the present invention, the reactive group used for immobilizing the probe and the reactive group to be introduced on the surface of the substrate (substrate) can be used as they are. . Then, a coating film made of a substance that is inactive to the reaction is formed on the surface of the substrate (substrate) into which the reactive group is introduced, and then a droplet of the solution containing the probe is spotted and spotted. The droplets to be permeated through the coating film made of a reaction inert substance are brought into contact with the substrate into which the reactive group has been introduced. By adopting this method, it is possible to reduce the dot diameter at which the droplet contacts the surface of the substrate (substrate). This method only uses a coating film made of a reaction-inert substance, and there is no additional process requiring an apparatus that causes a significant increase in manufacturing cost. Further, selection of a coating film made of a suitable reaction-inert substance does not require an unnecessarily long development period. Since it has the above-mentioned two advantages, it also has an advantage that there is no factor for a large development cost increase.

A method for producing a probe-fixing carrier according to the present invention includes:
In a method for producing a probe-immobilized carrier in which a probe capable of specifically binding to a target substance is immobilized on a substrate,
The probe is immobilized on the substrate through bond formation by a reaction between a reactive group included in the probe and a reactive group introduced on the substrate.
The following steps (i) to (iv):
Step (i): introducing a reactive group capable of reacting with the reactive group included in the probe onto the substrate;
Step (ii): A coating film made of a reaction inert substance that is not reactive with any of the reactive groups provided in the probe and the reactive groups introduced onto the substrate. Providing on top;
Step (iii): The droplet of the solution containing the probe is spotted on the base material covered with the coating film made of the reaction inert substance by the spotting means, and the spotted droplet is Allowing the coating film made of a reaction inert substance to pass through and contacting with the base material into which the reactive group is introduced;
Step (iv): a step of reacting a reactive group included in a probe contained in a droplet to be deposited with a reactive group introduced on a substrate in contact with the droplet In this method, it is preferable to select the following form.

First, in the method for producing a probe fixing carrier according to the present invention,
After step (iv)
Step (v): It is preferable to further provide a step of removing the droplets to be spotted,
After step (iv)
Step (v ′): It is more preferable to further provide a step of removing the droplets to be deposited and the coating film made of the reaction inert substance.

On the other hand, the reaction inert substance constituting the coating film made of the reaction inert substance is:
When a droplet of a solution containing the probe is spotted on a substrate into which the reactive group has been introduced in a state where it is not covered with the coating film, the reactive group is introduced. Compared with the dot diameter of the part where the droplet to be spotted on the substrate
When a droplet of a solution containing the probe is spotted on a substrate on which the reactive group has been introduced in a state covered with the coating film, the reactive group is introduced. It is desirable that the substance has the property of reducing the dot diameter at the site where the droplets to be deposited on the substrate are in contact.

Further, the reaction inert substance constituting the coating film made of the reaction inert substance is:
When a droplet of a solution containing the probe is spotted on a base material into which the reactive group has been introduced in a state covered with the coating film,
At the temperature at which the spotting is carried out, it is possible to allow the droplets to be spotted to pass through the coating film made of the reaction-inactive substance and to come into contact with the substrate into which the reactive group has been introduced. A substance exhibiting fluidity is preferable.

For example, as the reaction inert substance constituting the coating film made of the reaction inert substance,
A substance selected from the group consisting of naphthene, paraffin, aromatic hydrocarbon compounds, or derivatives obtained from these hydrocarbon compounds can be used.

  In addition, as the reaction inert substance, it is desirable to use a substance having poor affinity for the solvent constituting the solution containing the probe.

In the method for producing a probe fixing carrier according to the present invention,
The spotting means is
It is preferable that the droplet is spotted by an inkjet method.

For example, in the droplet spotting means by the inkjet method,
It is possible to use a system in which bubbles are generated in a solution containing the probe by thermal energy, and the solution is ejected as droplets by a pressure increase caused by the bubble generation, and droplets are spotted. it can. Alternatively, the droplet spotting means by the inkjet method includes:
It is also possible to use a system in which a pressure rise is caused by a piezoelectric element in a solution containing the probe, the solution is ejected as droplets, and droplets are spotted.

In addition, as the spotting means,
A solution containing the probe is attached to the tip of the protrusion,
A solution liquid containing the probe, which is attached to the tip of the protrusion, which is attached to the tip of the protrusion through the coating film made of a reaction-inactive substance and brought into contact with the substrate. You may employ | adopt the method of spotting a drop.

  On the other hand, a sulfanyl group can be used as a reactive group provided in the probe. Furthermore, an amino group can also be employed as a reactive group provided in the probe.

  On the other hand, a maleimide group can be used as a reactive group introduced on the substrate. Furthermore, introduction of the reactive group onto the substrate can be performed using N-hydroxysuccinimidyl ester. This N-hydroxysuccinimidyl ester is an activated ester of carboxylic acid, and as a result, corresponds to introduction of a carboxyl group.

  The method for producing a probe-immobilized carrier according to the present invention is a particularly useful technique when the probe is a nucleic acid probe.

  Hereinafter, the present invention will be described in more detail.

In the method for producing a probe fixing carrier according to the present invention,
When a probe that can specifically bind to a target substance is immobilized on the surface of a substrate to produce a probe-immobilized carrier, the probe is immobilized on the surface of the substrate by a reactive group included in the probe. And a reactive group introduced on the base material through bond formation by reaction. At that time, a probe containing a reactive group is dissolved in a solvent at a predetermined concentration to prepare a solution containing the probe in advance. On the other hand, a reactive group capable of reacting with a reactive group included in the probe is introduced on the surface of the substrate. A droplet of a solution containing the probe is spotted on the surface of the substrate into which the reactive group has been introduced, and the reactive group included in the probe at a site where the droplet contacts the surface of the substrate, A reaction between the reactive groups introduced on the substrate is caused. Therefore, the probe is fixed on the surface of the base material only at a portion where the droplet contacts the base material surface.

In the present invention, as a means for suppressing the expansion of the dot diameter at the site where the droplet and the substrate surface contact,
On the surface of the base material into which the reactive group has been introduced, a substrate made of a reaction inert substance that does not react with any of the reactive group provided in the probe and the reactive group to be introduced onto the base material. With the covering film provided, droplets of the solution containing the probe are spotted from the surface. The droplets to be spotted pass through the coating film made of a reaction-inactive substance, and the tip part comes into contact with the surface of the base material into which the reactive group has been introduced.

  In FIG. 1, in the method of the present invention, a droplet to be spotted passes through a coating film made of a reaction-inert substance, and the tip is in contact with the surface of the substrate into which a reactive group is introduced. A schematic cross-sectional view is shown. A reactive group 5 used for fixing the probe 1 is introduced on the surface of the substrate 4. The reactive group 5 introduced on the surface of the substrate 4 can react with the reactive group 2 included in the probe 1, and the droplet 3 of the solution containing the probe 1 is formed on the substrate 4. The two are reacted while in contact with the surface. Prior to the spotting step of the droplet 3 of the solution containing the probe 1, the surface of the substrate 4 into which the reactive group 5 has been introduced is in contact with both the reactive group 2 of the probe and the reactive group 5 of the substrate. Then, it is covered with a coating film made of the reaction-inactive substance 6.

  When the droplet 3 is spotted, the droplet 3 first passes through the coating film in such a form that the coating film made of the reaction-inactive substance 6 existing under the spot point is pushed away. The tip of the droplet 3 reaches the surface of the substrate 4 and comes into contact. That is, the substance 6 originally covering the surface of the base material 4 needs to be displaced around the spot point in the process of spotting the droplet 3, and the fluidity is as high as this plastic deformation is possible. It is necessary to have. When this spotting is made, near the surface of the substrate 4, the periphery of the droplet 3 is surrounded by the reaction-inactive substance 6, and an interface is formed in which both are in contact with each other. On the other hand, the surface of the portion of the droplet 3 that is higher than the upper surface of the coating film made of the reaction-inactive substance 6 is in contact with the atmosphere, and the surface shape of the portion is the droplet 3 itself. Is governed by the load and surface tension.

  The shape of the portion that permeates the coating film made of the reaction-inactive substance 6 is a force that travels from the droplet 3 side to the coating film side, and a force that travels from the coating film side to the droplet 3 side. Are in equilibrium. The force from the droplet 3 side toward the coating film side corresponds to the internal pressure resulting from the load of the droplet 3 itself. On the other hand, the force from the coating film side toward the liquid droplet 3 side corresponds to the external pressure derived from the partial load of the substance 6 pushed away around the droplet 3 when the liquid droplet 3 is spotted. If this internal pressure is greater than the external pressure, the interface between the two is further expanded, and the dot diameter in contact between the droplet 3 and the surface of the substrate 4 is increased. At that time, the adhesive force at the contact surface between the coating film and the substrate (substrate) surface, and the stress required for plastic deformation of the coating film act as repulsive forces that suppress the expansion of the dot diameter. That is, if the difference between the internal pressure and the external pressure cannot overcome the repulsive force, the dot diameter does not further increase.

  At that time, the adhesion force at the contact surface between the coating film and the substrate (substrate) surface, which functions as a repulsive force, and the stress required for plastic deformation of the coating film are the same as the surface of the droplet 3 and the substrate 4. It is proportional to the outer periphery of the dot which contacts, that is, it is proportional to the dot diameter (diameter). On the other hand, the difference between the internal pressure and the external pressure gradually decreases as the dot diameter (diameter) increases. Of the two components constituting the repulsive force, the adhesion force at the contact surface between the coating film and the base material (substrate) surface is insensitive to the surface of the base material (substrate) into which the reactive group 5 is introduced. This is due to the affinity (wetting property) of the active substance 6. In addition, the stress required for plastic deformation of the coating film is derived from the fluidity (viscosity) of the substance 6 that is reaction-inactive.

  In the present invention, as described above, the coating film composed of the reaction-inactive substance 6 surrounds the droplet 3 of the solution containing the probe 1 and surrounds the droplet 3 and the substrate. 4 is used to suppress the enlargement of the dot diameter in contact with the surface of No. 4. Therefore, the reaction-inactive substance 6 is prevented so that the solvent used in the solution containing the probe 1 and the reaction-inactive substance 6 constituting the coating film do not mix with each other. As described above, a substance having poor affinity for the solvent is used. For example, when the solvent used in the solution containing the probe 1 is an aqueous solvent, a hydrophobic substance is used as the reaction-inactive substance 6. For example, when the probe 1 is a nucleic acid molecule, the solvent used for preparing the solution containing the probe 1 is an aqueous solvent, and a hydrophobic substance is used as the substance 6 that is inactive to the reaction.

  When the solvent used in the solution containing the probe 1 is an aqueous solvent, if the surface of the base material (substrate) into which the reactive group 5 is introduced has poor affinity for the aqueous solvent, On the surface of the substrate (substrate) into which the reactive group 5 has been introduced, the contact angle exhibited by the solution containing the probe 1 is large. In that case, the effect of reducing the spot diameter according to the present invention is limited. Specifically, the factor that restricts the distance between the spot points of the probe is not the dot diameter at which the droplet 3 and the surface of the substrate 4 are in contact, but the maximum diameter of the dropped droplet 3 itself. ing. Originally, when the contact angle is large, when the dot diameter of contact between the droplet 3 and the surface of the substrate 4 is narrowed, the maximum diameter of the droplet 3 that is spotted does not substantially change, or It will be slightly enlarged. In that case, it is difficult to reduce the distance between the spot points of the probe, which should be set in order to avoid contact between adjacent droplets 3.

  On the other hand, when the solvent used in the solution containing the probe 1 is an aqueous solvent, the surface of the base material (substrate) into which the reactive group 5 is introduced has a high affinity for the aqueous solvent. It becomes the state of. That is, the contact angle exhibited by the solution containing the probe 1 is originally reduced on the surface of the base material (substrate) into which the reactive group 5 has been introduced. It becomes more prominent. Specifically, the factor that limits the distance between the spot points of the probe is the maximum diameter of the droplet 3 that is spotted, but the maximum diameter is substantially the same as the droplet 3 and the substrate 4. The dot diameter is in contact with the surface. Originally, when the contact angle is small, when the dot diameter between the droplet 3 and the surface of the substrate 4 is reduced, the maximum diameter of the droplet 3 that is spotted is also reduced. . In that case, it becomes possible to reduce the interval between the spot points of the probe, which should be set in order to avoid contact between the adjacent droplets 3 in proportion to the decrease in the dot diameter.

  For example, when the solvent used in the solution containing the probe 1 is an aqueous solvent, if the surface of the base material (substrate) into which the reactive group 5 is introduced is hydrophilic, the reaction-inactive substance 6 is obtained. When using a hydrophobic substance, the following state occurs. That is, the adhesion force at the contact surface between the coating film and the substrate (substrate) surface is small. Therefore, the stress required for plastic deformation of the coating film is mainly used as a repulsive force that suppresses the expansion of the dot diameter. The lower the fluidity of the reaction-inactive substance 6 (the higher the viscosity), the greater the stress required for plastic deformation of the coating film. In order to enhance the effect of suppressing the increase in the diameter of the contacted droplet 3 and the surface of the substrate 4, it is more preferable that the reaction-inactive substance 6 has low fluidity (high viscosity).

  If the substance constituting the coating film has reactivity with the reactive group 5 introduced on the surface of the substrate, the reaction between the two in the stage of coating the surface of the substrate. May progress. For example, when the substance constituting the coating film is immobilized on the surface of the base material due to the reaction with the reactive group introduced on the surface of the base material 4, the coating of the immobilized substance is applied. The layer may impede contact between the droplet 3 of the solution containing the probe 1 and the surface of the substrate 4. Further, when the reactive group introduced on the surface of the substrate 4 is consumed by the reaction with this substance, the droplet 3 of the solution containing the probe 1 and the surface of the substrate 4 are brought into contact with each other. At this time, the surface density of the reactive group 5 of the substrate that can be used for the reaction with the reactive group of the probe decreases. In order to avoid this problem, the substance constituting the coating film uses a substance that does not show reactivity with the reactive group 5 of the substrate.

  In addition, the droplet 3 of the solution containing the probe 1 and the substance constituting the coating film are in contact with each other at the interface. If the substance constituting the coating film has reactivity with the reactive group 2 of the probe, the reaction between the two may proceed at the interface. When the probe 1 existing in the vicinity of the surface of the base material 4 is consumed in the droplet 3 along with this reaction, the probe can react with the reactive group 5 introduced on the surface of the base material 4. The concentration of 1 decreases. In order to avoid this problem, the substance constituting the coating film uses a substance that does not show reactivity with the reactive group 2 of the probe. Accordingly, in the present invention, the substance 6 constituting the coating film is a reaction-inactive substance that does not react with either the reactive group 2 of the probe or the reactive group 5 of the substrate. Is used.

  A coating film composed of a reaction-inactive substance 6 surrounds the droplet 3 of the solution containing the probe 1 and has a dot diameter that makes contact with the surface of the droplet 3 and the substrate 4. A state in which expansion is suppressed is assumed. In this state, the reactive group 2 of the probe contained in the droplet 3 is reacted with the reactive group 5 introduced on the surface of the substrate 4. Due to this reaction on the surface, the probe 1 is fixed to the surface of the substrate 4 only within the dot diameter where the droplet 3 and the surface of the substrate 4 are in contact with each other. That is, for the droplet 3 to be spotted, a coating film made of a reaction-inactive substance 6 surrounds the periphery of the droplet 3 and the dot diameter at which the droplet 3 and the surface of the substrate 4 are in contact with each other is enlarged. Therefore, the fixed region (spot diameter) of the probe 1 on the surface of the substrate 4 is miniaturized.

  As described above, in the present invention, the reaction-inactive substance 6 constituting the coating film covering the surface of the substrate 4 exhibits a proper fluidity (viscosity) and contains a probe 1. It is necessary to select a substance having poor affinity for the solvent used in the above. At the same time, it is more preferable that the substance has a poor affinity for the surface of the substrate 4 into which a reactive group has been previously introduced.

For example, as an example of a hydrophobic substance that can be used as the reaction-inactive substance 6 that constitutes the coating film that covers the surface of the base material 4, it has a reactive functional group or a partial structure rich in reactivity. Non-hydrocarbons can be mentioned. In addition, a hydrogen atom present in the form of CH ₃ —, —CH ₂ —,> CH— in a hydrocarbon that does not have a reactive functional group or a partial structure rich in reactivity is substituted with a fluorine atom. The fluorine-substituted hydrocarbon also becomes a hydrophobic substance that can be used as the substance 6 that is inactive to the reaction. Instead of the forms of CH ₃ —, —CH ₂ —,> CH—, fluorine-substituted hydrocarbons containing the corresponding CF ₃ —, —CF ₂ —,> CF— form are compared with the base hydrocarbon. Higher hydrophobicity. At the same time, the fluidity is lower (the viscosity is higher), so it is generally more suitable as a reaction-inert hydrophobic substance. Examples of the hydrocarbon not having a reactive functional group or a partial structure rich in reactivity include naphthene, paraffin, and aromatic hydrocarbon compounds. In addition, derivatives of these hydrocarbon compounds that do not have reactivity can be used as well.

  The reaction-inactive substance 6 constituting the coating film covering the surface of the substrate 4 is configured to push away the coating film made up of the reaction-inactive substance 6 when the droplet 3 is spotted. It is necessary to permeate the coating film and to exhibit fluidity. However, a substance that can achieve a desired fluidity by forming a mixture with a plurality of kinds of substances can be used even if the substance is not a liquid by itself. That is, a coating film that covers the surface of the substrate 4 can be formed by adjusting the target fluidity (viscosity) as a mixture obtained by adding an appropriate amount of a liquid substance to a substance that is not liquid alone. .

  As described above, the reaction-inactive substance 6 constituting the coating film covering the surface of the substrate 4 is generally more preferably low in fluidity (high in viscosity). At that time, those having a viscosity higher than necessary are difficult to permeate the coating film in such a form that when the droplet 3 is spotted, the coating film made of the substance 6 which is inactive to the reaction is pushed away. Therefore, it is not suitable for the purpose of use of the present invention.

  For example, when the ink jet method is used as a means for spotting the droplet 3, the droplet ejected from the ejection port of the inkjet head flies at a constant speed and is coated with the substance 6. Reach the substrate covered with a membrane. After that, it collides with the coating film with an impulse proportional to the momentum, plastically deforms the reaction-inactive substance 6 constituting the coating film, pushes it away, and brings its tip into contact with the substrate surface. At this time, the kinetic energy consumed in the process of plastically deforming and pushing away the reaction-inactive substance 6 is proportional to the viscosity of the reaction-inactive substance 6 and the film thickness of the coating film. That is, when the viscosity of the reaction-inactive substance 6 is excessively high, the reaction-inactive substance 6 that forms the coating film is consumed even if all of the kinetic energy of the flying droplets is consumed. It becomes impossible to make the tip reach the surface of the base material by being plastically deformed and pushed away. Further, even when the film thickness of the coating film is excessively large, the reaction-inactive substance 6 constituting the coating film is plastically deformed even if all the kinetic energy of the flying droplets is consumed. It is impossible to make the tip reach the surface of the substrate.

  On the other hand, the flying speed of the liquid droplets discharged from the discharge port of the inkjet head, that is, the discharge speed is selected within a predetermined range in consideration of the liquid volume of the liquid droplets and the liquid viscosity of the solution containing the probe 1. Is done. Therefore, there is an upper limit for the kinetic energy of the flying droplets. That is, it is difficult to increase the discharge speed of the liquid droplets discharged from the discharge port of the inkjet head. Considering this point, the viscosity of the reaction-inactive substance 6 and the film thickness of the coating film are within appropriate ranges according to the discharge speed of the droplets discharged from the discharge port of the inkjet head to be used. select. If the viscosity of the specific substance 6 is mentioned, it is desirable to select the viscosity within the range of the penetration of 30 ° or less. On the other hand, if the viscosity of the substance 6 is excessively small, the action of the present invention cannot be exerted. Therefore, it is desirable to select the viscosity so that the penetration does not fall below 10 °. At that time, the viscosity of the single substance cannot be changed, but as the reaction-inactive substance 6 constituting the coating film, plural kinds of substances are mixed at an appropriate ratio, and the mixture shows A technique for adjusting the viscosity may be used.

  The film thickness of the coating film composed of the reaction-inactive substance 6 depends on the size (liquid amount) and the flying speed of the droplet 3 and the viscosity of the substance 6, and when the target is spotted. Depending on the dot diameter, the diameter is appropriately selected. Usually, the droplet 3 is selected in a range not exceeding the diameter when the droplet 3 is formed into a spherical shape. That is, when the film thickness of the coating film exceeds the diameter when the droplet 3 is formed into a spherical shape, the flow is increased when the upper surface of the coating film is higher than the upper surface of the deposited droplet 3. The upper surface of the deposited droplet 3 is completely covered by the substance 6 exhibiting the property. When the droplet 3 sinks inside the coating film, the droplet 3 receives buoyancy, so that the additional load that makes the droplet 3 contact the surface of the substrate 4 becomes extremely small. The dot diameter at the time of spotting may become unnecessarily small. In addition, the outermost surface including the upper surface of the droplet 3 that has been spotted is covered with the reaction-inactive substance 6, and as it is, it is immobilized on the target substance and the substrate surface. It becomes difficult to contact the probe. In order to avoid such a state, it is necessary to select the film thickness of the coating film so that the upper surface of the coating film is at a lower position than the upper surface of the dropped droplet 3. There is. Specifically, it is desirable that the film thickness of the coating film made of the reaction-inactive substance 6 is in a range of 10 to 300,000 nm (a range of 0.01 to 300 μm).

  In general, the film thickness of the coating film composed of the reaction-inactive substance 6 is preferably uniform on the surface of the substrate 4. At this time, it is preferable that the coating film formed of the reaction-inactive substance 6 is formed so as to cover the entire surface of the substrate 4 because the coating film forming process is usually simplified. For example, the following method can be used as means for forming a coating film composed of the reaction-inactive substance 6 so as to cover the entire surface of the substrate 4. That is, using a solution in which the substance 6 is dissolved in a low-boiling solvent, it is applied to a uniform thickness by a known coating method such as spin coating, slit coating, dip coating, and then the low-boiling solvent is removed by evaporation. Thus, a method for forming a coating film having a desired film thickness can be used. Specifically, since the boiling point of the substance 6 is high and the viscosity in the melted state is high, even when the desired thin film cannot be coated, a solution using a low boiling point solvent as a diluent solvent is suitable for thin film coating. The liquid viscosity can be made. Thereafter, when the low boiling point solvent used for the diluting solvent is evaporated, the substance 6 having a high boiling point is concentrated, and a coating film having a predetermined film thickness is formed on the substrate 4. Moreover, the following method can be utilized as a method of performing thin film coating of the substance 6 without using a dilution solvent. That is, the base material 4 into which the reactive group 5 is introduced and the substance 6 are sealed in a vacuum or normal pressure chamber, and the gas of the substance 6 itself is used as the surface of the base material 4 into which the reactive group 5 is introduced. A method of forming a coating film by agglomerating on the surface can be mentioned. At this time, the gas of the substance 6 itself can be generated by means for evaporating the substance 6 by the saturated vapor pressure of the substance 6 itself or by evaporation means such as heating and sputtering.

  Instead of forming a coating film made of the reaction-inactive substance 6 on the entire surface of the substrate 4, the substance 6 may be coated only on the region where the probe is fixed. The following method can be used as a means for specifying the part and coating the substance 6. That is, by using a spotting means such as an ink jet method or a pin method, a solution in which the substance 6 is dissolved in a predetermined solvent is spotted only on a region where the probe is fixed, and the solvent is removed by evaporation. A method of forming a coating film by concentrating can be mentioned.

  In the present invention, a reactive group used for immobilizing the probe is introduced on the surface of the substrate. The material of the base material itself is not particularly limited as long as it does not hinder the detection substance (target substance) using the obtained probe fixing base material. However, for example, it is preferable to use a stable material for a diluting solvent used for forming a coating film composed of the reaction-inactive substance 6 among inorganic materials and polymer materials. When the material constituting the base material itself has a reactive group used for immobilizing the probe, a step of newly introducing the reactive group can be omitted. A desired reactive group is introduced onto the surface of a substrate formed of a material not having a reactive group, using a known method. Examples of reactive groups that can be used for probe immobilization include amino groups, maleimide groups, acrylamide groups, N-hydrosuccinimidyl esters (an active ester of carboxyl groups), formyl groups, carboxyl groups, and epoxy groups. Can do. However, it is not limited to the reactive group illustrated.

  In addition, according to the present invention, probes immobilized on the surface of a substrate include biologically high proteins such as proteins (including complex proteins), nucleic acids, sugar chains (including complex carbohydrates), and lipids (including complex lipids). Includes molecules. Specifically, examples of probes immobilized on the surface of a substrate include enzymes, hormones, pheromones, antibodies, antigens, haptens, peptides, synthetic peptides, DNA, synthetic DNA, RNA, synthetic RNA, PNA, synthetic PNA , Ganglioside, lectin and the like.

  The probe is immobilized on the surface of the substrate through bond formation by a reaction between a reactive group included in the probe and a reactive group introduced on the substrate. That is, it is preferable to select a reactive group capable of reacting with the reactive group included in the probe and introduce it onto the substrate.

  On the other hand, examples of the reactive group included in the probe include a sulfanyl group (—SH) and an amino group. When a sulfanyl group (-SH) is introduced into a single-stranded DNA used as a probe, for example, when a single-stranded DNA is automatically synthesized, it is introduced at the time of synthesis with an automatic DNA synthesizer. That is, a sulfanyl group (—SH) can be introduced at the end of a DNA chain using a thiol-modifier (manufactured by Glen Research). In addition, the introduction means is not particularly limited as long as the sulfanyl group (—SH) can be selectively introduced into a desired site efficiently. Alternatively, when an amino group is introduced into a single-stranded DNA used as a probe, for example, when a single-stranded DNA is automatically synthesized, it is introduced at the time of synthesis with an automatic DNA synthesizer. That is, an amino group can be introduced at the end of a DNA strand using an amino-Modifier (manufactured by Glen Research). In addition, the introduction means is not particularly limited as long as the amino group can be efficiently and selectively introduced into a desired site.

  In the present invention, a droplet of a solution containing a probe is spotted on a base material covered with a coating film made of a reaction inert substance. As the spotting means, a droplet of a predetermined liquid amount is spotted (spotted) at a target spot position by using an inkjet method, a pin method, a pin & ring method, or the like. In the present invention, when the target is to reduce the dot diameter of a droplet to be spotted at each spot position, the liquid amount of the droplet to be spotted at each spot position is also reduced proportionally. In addition, if the goal is to reduce the distance between spot points and increase the spot point density per unit area, select a spotting means that can achieve spot position accuracy that can accommodate spotting with a high density. It is preferable to do.

  In the spotting means applying the pin method or the pin & ring method, for example, depending on the tip diameter of the pin to be used, the amount of liquid that can be attached to the tip, the amount of liquid to be spotted on each spot position, the spot position The accuracy and spot diameter are adjusted. Also in the present invention, spotting means applying this pin method or pin and ring method can be used.

  On the other hand, the spotting means to which the ink jet method is applied is excellent in the amount of liquid droplets spotted at each spot position, the accuracy of the spot position, and the spot position interval control, and can be suitably used in the present invention. In the inkjet method, a solution containing a probe is placed in a very thin nozzle, and the vicinity of the tip of the nozzle is momentarily pressurized or heated, so that a droplet of the solution containing a very small amount of probe is accurately ejected from the tip of the nozzle. , Fly through the space and spot it on the substrate surface.

  In the spotting method to which the inkjet method is applied, a solvent that satisfies the following requirements is used as a solvent used for preparing a solution containing a probe. First, when ejected from an inkjet head, the contained probe is not substantially affected. At the same time, it is a solvent that can eject droplets with high reproducibility at a desired minute liquid amount and ejection speed using an inkjet head. As long as these two requirements are satisfied, the composition of the solvent is not particularly limited. For example, when the probe immobilized on the surface of the substrate is a water-soluble substance, an aqueous solvent is used as the solvent used for preparing the solution containing the probe. This example uses a water-soluble substance such as an enzyme, hormone, pheromone, antibody, antigen, hapten, peptide, synthetic peptide, DNA, synthetic DNA, RNA, synthetic RNA, PNA, synthetic PNA, ganglioside, lectin as a probe Corresponds to the case.

For example, when the ink jet head is a bubble jet head provided with a mechanism for applying a thermal energy to the solvent and discharging it, the following solvent is preferably used. That is, an aqueous solvent containing glycerin, thiodiglycol, isopropyl alcohol, and acetylene alcohol is preferable as a solvent for the solution containing the water-soluble substance. Furthermore, the following aqueous solvent can be specifically shown as an aqueous solvent suitably used as the solvent of the solution containing the water-soluble substance. That is, glycerin 5 to 10 wt%, thiodiglycol (HO—CH ₂ —CH ₂ —S—CH ₂ —CH ₂ —H) 5 to 10 wt%, acetylene alcohol (HC≡C—CH ₂ —OH) 0.5 An aqueous solvent containing ˜1 wt% is preferably used. In the case where the inkjet head is a piezo jet head that discharges a solution using a piezoelectric element, an aqueous solvent containing ethylene glycol and isopropyl alcohol is preferable as a solvent for the solution containing the water-soluble substance. Furthermore, specifically, an aqueous solvent containing 5 to 10 wt% of ethylene glycol (HO—CH ₂ —CH ₂ —OH) and 0.5 to ₂ wt% of isopropyl alcohol (CH ₃ —CH (CH ₃ ) —OH) is used. And preferably used as a solvent for a solution containing the water-soluble substance. In addition, in the spotting means applying the ink jet method, the liquid amount of one discharged droplet can be selected in the range of 1 pl to 100 pl. In this case, when an aqueous solvent is used for preparing a solution containing a probe, assuming that the droplet has a spherical shape, the diameter (spot diameter) corresponds to a range of 12 μm to 1200 μm.

  By reacting the reactive group included in the probe contained in the droplet to be deposited with the reactive group introduced on the substrate in contact with the droplet, the surface of the substrate is reacted. Immobilization of the probe is performed. The reaction between the reactive group provided in the probe and the reactive group introduced on the substrate is the same as the conventional reaction, and the reaction conditions are also the same. For example, when an aqueous solvent is used to prepare a solution containing the probe, the reaction is carried out at a temperature of 15-25 ° C. and a relative humidity of 70-99 RH% to prevent rapid transpiration of water contained in the droplets. Conditions such as standing in a constant temperature and humidity chamber are used.

  If a droplet containing an unreacted probe is left, when the target substance is detected using the prepared probe fixing carrier, the target substance contained in the detection sample is immobilized on the probe. In addition, specific binding occurs with unfixed probes. Therefore, it becomes a factor that reduces the quantitativeness of detection. Therefore, usually, after the reaction for immobilizing the probe on the surface of the substrate is completed, the droplet containing the unreacted probe is removed. Removal of droplets containing unreacted probes can be removed by washing by diluting the droplets using a solvent capable of dissolving the probes. For example, when the probe is an enzyme, hormone, pheromone, antibody, antigen, hapten, peptide, synthetic peptide, DNA, synthetic DNA, RNA, synthetic RNA, PNA, synthetic PNA, ganglioside, lectin, etc., the following method is used Is possible. That is, it can wash | clean using the washing | cleaning solution which melt | dissolved surfactant which shows a washing | cleaning action in the buffer solution which can melt | dissolve these. For example, when the probe is a DNA probe, the following buffer solution can be used. That is, phosphate buffer, Tris-HCl buffer, Tris-acetate buffer, HEPES buffer, MOPS buffer, sodium acetate buffer, and sodium citrate buffer can be used. Alternatively, these buffers can be washed as necessary using a washing solution in which a salt such as sodium chloride, a chelating agent such as EDTA, or the following surfactant is dissolved. That is, you may wash | clean with the buffer solution which melt | dissolved nonionic surfactant, such as anionic surfactants, such as SDS, BriJ58, Nonidet P-40, Triton X-100, Tween 20, and Tween 80.

  On the other hand, the coating film composed of the reaction-inactive substance 6 does not need to be removed unless it becomes an inhibiting factor when detecting the target substance using the produced probe-immobilized carrier. Since the coating film composed of the reaction-inactive substance 6 has fluidity, the spot film where the probe is immobilized is often removed after removing the droplet containing the unreacted probe. The surface is again covered. Therefore, it is usually preferable to remove the coating film composed of the substance 6 that is inactive to the reaction, in addition to the removal of the droplets containing the unreacted probe.

  In addition, when the coating film is composed of a mixture of a plurality of reaction-inactive substances, and the fluidity is lost due to the evaporation of the low-boiling components contained therein, the probe was immobilized. The surface of the spot part is not covered again. In such a state, it is not always necessary to remove the coating film. In particular, when the target substance is detected, this solid coating film covers the probe non-fixed region. In this state, if the reaction-inactive substance constituting the coating film existing in the background area does not exhibit the action of adsorbing the target substance, the coating film exhibits a blocking effect. . That is, in order to prevent non-specific adsorption of the target substance in the background area, the effect of increasing the S / N ratio between the probe fixing region and the background area may be exhibited.

  When removing the coating film, any means can be used as long as the reaction-inactive substance 6 can be removed, but a cleaning means that does not affect the probe immobilized on the substrate surface should be used. Is preferred. For example, when the probe is an enzyme, hormone, pheromone, antibody, antigen, hapten, peptide, synthetic peptide, DNA, synthetic DNA, RNA, synthetic RNA, PNA, synthetic PNA, ganglioside, lectin, etc., the following means are used Is possible. That is, a liquid droplet containing an unreacted probe is washed by using a washing solution in which a surfactant exhibiting a washing action for a reaction-inactive substance is dissolved in a buffer solution capable of dissolving these probes. The coating film can be removed simultaneously with the removal. For example, when the probe is a DNA probe, a phosphate buffer, Tris-HCl buffer, Tris-acetate buffer, HEPES buffer, MOPS can be applied to a droplet containing a hydrophobic, reaction-inactive substance and an unreacted probe. Buffer, sodium acetate buffer, sodium citrate buffer, or a salt such as sodium chloride, a chelating agent such as EDTA, an anionic surfactant such as SDS, BriJ58, Nonidet P-40, if necessary. Washing can be performed on a buffer solution in which a nonionic surfactant such as Triton X-100, Tween 20, or Tween 80 is dissolved.

  Hereinafter, the present invention will be described in more detail with reference to examples.

Example 1
(I) Probe synthesis and synthesis of a target substance (target) with a fluorescent label A single-stranded DNA probe is used as a probe that can specifically bind to a single-stranded nucleic acid molecule of the target substance. A single-stranded nucleic acid molecule of SEQ ID NO: 1 is synthesized using an automatic DNA synthesizer. In addition, the single-stranded nucleic acid molecule of SEQ ID NO: 1 was synthesized at the 5 ′ end of the single-stranded DNA at the time of synthesis using an automatic DNA synthesizer (Thiol-Modifier) (manufactured by Glen Research). ) To introduce a sulfanyl (—SH) group as a reactive group. After completion of the synthesis, normal deprotection is performed, the DNA is recovered, purified by high performance liquid chromatography, and the one having the target DNA strand is collected. In the following experiment, a single-stranded DNA molecule having a reactive group introduced at the 5 ′ end is used as a probe to be immobilized on a substrate.
SEQ ID NO: 1
5 ′ HS— (CH ₂ ) ₆ —O—PO ₂ —O-ACTGGCCGTCGTTTACA 3 ′
In addition, a DNA strand part of the single-stranded DNA probe of SEQ ID NO: 1 and a single-stranded DNA molecule having a complementary base sequence are synthesized by an automatic DNA synthesizer, and Cy3 is bound to its 5 ′ end. It is used as a target substance (target) with a fluorescent label.

(Ii) Preparation of probe fixing carrier-Substrate cleaning As a base material of the probe fixing carrier, a 1 inch x 3 inch square synthetic quartz glass substrate is used. The quartz glass substrate is cleaned by the following procedure. That is, according to the steps of pure water brush cleaning, pure water rinse, alkaline detergent ultrasonic cleaning, pure water rinse, pure water ultrasonic cleaning, pure water rinse, and nitrogen blow drying, fine dust and organic substances adhering to the surface are removed. A quartz glass substrate having a clean surface is prepared. In the step of ultrasonic cleaning with alkaline detergent, an aqueous solution of semi-clean KG (manufactured by Yokohama Oil & Fats Co., Ltd.) having a concentration of 1% is used as the alkaline detergent, and ultrasonic cleaning is performed at a liquid temperature of 25 ° C. for 2 minutes.

-Surface treatment Aminosilane coupling agent (trade name: KBM-603; manufactured by Shin-Etsu Chemical Co., Ltd.) is dissolved in pure water to a concentration of 1 wt%, and stirred for 30 minutes to hydrolyze the methoxy group. Let While being heated to 80 ° C. in a warm bath, the washed quartz glass substrate is immersed in this aminosilane coupling agent aqueous solution for 30 minutes. Thereafter, the quartz glass substrate taken out is washed with pure water and baked in an oven at 120 ° C. for 1 hour. By this aminosilane coupling agent treatment, amino groups are introduced onto the surface of the quartz glass substrate.

  Next, 2.7 mg of N-maleimidocaproyloxysuccinimide (manufactured by Dojin; hereinafter abbreviated as EMCS) was weighed and dissolved in 9 ml of a 1: 1 (volume ratio) mixed solvent of dimethyl sulfoxide (DMSO) / ethanol, Prepare an EMCS solution. That is, an EMCS solution having a final concentration of 0.3 mg / ml is prepared. An amino group-introduced quartz glass substrate is immersed in the EMCS solution at room temperature for 2 hours to allow EMCS to react with amino groups and to introduce maleimide groups on the surface. After the immersion treatment in the EMCS solution, the substrate is sequentially washed with a DMSO / ethanol mixed solvent and ethanol to remove unreacted EMCS. Then, it is dried under a nitrogen atmosphere.

-Coating of reaction inactive substance In this example, a sulfanyl group (-SH), which is a reactive group introduced at the 5 'end of the probe DNA, and a reactive group introduced on the surface of the quartz glass substrate The probe is allowed to react with the maleimide group to immobilize the probe DNA on the substrate surface. The following four types of saturated hydrocarbons are selected as substances that are inactive to both the sulfanyl group (—SH) and the maleimide group, and are examined as surface coating agents:
N-dodecane having 12 carbon atoms (manufactured by n-Dodecane Kishida Chemical Co .; melting point -9.6 ° C, boiling point 216.3 ° C);
N-hexadecane having 16 carbon atoms (manufactured by n-Hexadekane Kishida Chemical Co., Ltd .; melting point 18.4 ° C., boiling point 286.5 ° C.);
N-heptadecane having 17 carbon atoms (manufactured by n-Heptadecane Kishida Chemical Co., Ltd .; melting point 22 ° C., boiling point 303 ° C.);
N-octadecane having 18 carbon atoms (manufactured by Kanto Chemical Co., Inc .; melting point 27.5 ° C., boiling point 318 ° C.).

  These four types of saturated hydrocarbons all have high boiling points, but have different melting points, and have different fluidity and viscosity at ambient temperatures selected in the range of 20 ° C to 30 ° C. Yes. The four types of saturated hydrocarbons are dissolved in acetone, which is a low boiling point aprotic solvent, to prepare four types of coating solutions.

  These coating solutions are applied to the surface of the substrate into which the maleimide group has been introduced using a spin coater, and the low boiling point solvent acetone contained in the coating film is volatilized to form a coating film of each saturated hydrocarbon on the substrate surface. . In the step of volatilizing acetone, which is a low boiling point solvent, and the spotting step of the probe solution described below, the environmental temperature is set to 22.3 ° C. Therefore, the coating film of n-dodecane and n-hexadecane having a melting point lower than the environmental temperature is a liquid coating film having fluidity. On the other hand, the coating film of n-octadecane is mainly composed of n-octadecane, although the freezing point depression is observed due to a slight residual acetone. More significantly inferior. In addition, the coating film of n-heptadecane also has a freezing point depression due to a slight residual acetone, and is a liquid coating film, but its fluidity is more than that of the two coating films. Significantly inferior.

-Immobilization of probe DNA The single-stranded DNA probe fragment (SEQ ID NO: 1) chemically synthesized in the above step (i) is dissolved in the following aqueous solvent to prepare a single-stranded DNA probe aqueous solution. That is, an aqueous solvent containing glycerin 7.5 wt%, urea 7.5 wt%, thiodiglycol 7.5 wt%, acetylene alcohol (trade name: acetylenol E100; manufactured by Kawaken Fine Chemical Co., Ltd.) 1.0 wt% is used. is doing. The probe DNA concentration contained in this aqueous solution is 8.75 μM. The probe-containing solution is spotted by an ink jet method on the surface of the substrate provided with the reaction inactive substance coating film at a droplet amount of 5 pL per spot point. The spotted quartz glass substrate was then allowed to stand in a constant temperature and humidity chamber at a temperature of 25 ° C. and a relative humidity of 99 RH for 30 minutes, and the sulfanyl group (—SH) at the 5 ′ end of the DNA probe was placed on the maleimide on the substrate surface. The DNA probe is immobilized by reacting with a group.

(Iii) Removal of probe-containing droplet and reaction-inert substance The surface of the probe-immobilized carrier prepared in step (ii) includes a droplet containing an unreacted DNA probe and a substrate surface after the reaction is completed. Saturated hydrocarbons, which are reaction inactivating substances used for the coating film, remain. In this example, in order to remove these, in a cleaning agent in which SDS (Sodium dodecylsulfate manufactured by Kishida Chemical Co., Ltd.) was dissolved in 1M-NaCl / 50 mM phosphate buffer solution (pH 7.0) at a concentration of 1.0 wt%, The probe fixing carrier is immersed for 5 minutes. Saturated hydrocarbons are eluted in the buffer solution by the action of the surfactant SDS, and droplets containing unreacted DNA probes are also diluted and removed by the buffer solution. Thereafter, rinsing with pure water is performed to remove the buffer solution containing the surfactant SDS. After rinsing and washing, a DNA probe immobilization carrier for probe hybridization is obtained by drying in a nitrogen atmosphere.

(Iv) Preparation of DNA probe immobilization carrier for comparison As a DNA probe immobilization carrier for comparison, a probe-containing solution was spotted without forming a coating film of a reaction inert substance on the surface of a quartz glass substrate into which a maleimide group was introduced. The prepared probe immobilization carrier is prepared. A probe-containing solution is directly spotted on the surface of the quartz glass substrate into which the maleimide group has been introduced, by an inkjet method, with a droplet amount of 5 pL per spot point. The spotted quartz glass substrate was then allowed to stand in a constant temperature and humidity chamber at a temperature of 25 ° C. and a relative humidity of 99 RH for 30 minutes, and the sulfanyl group (—SH) at the 5 ′ end of the DNA probe was placed on the maleimide on the substrate surface. The DNA probe is immobilized by reacting with a group.

  After the reaction is completed, droplets containing unreacted DNA probes remain on the substrate surface. In order to remove this, 5 mL of the probe-immobilized carrier was added to a detergent in which SDS (Sodium dodecylsulfate Kishida Chemical Co., Ltd.) was dissolved at a concentration of 1.0 wt% in a 1M NaCl / 50 mM phosphate buffer solution (pH 7.0). Immerse for a minute. Droplets containing unreacted DNA probe are diluted and removed with a buffer solution. Thereafter, rinsing with pure water is performed to remove the buffer solution containing the surfactant SDS. A DNA probe immobilization carrier for comparison is obtained by rinsing and drying in a nitrogen atmosphere.

(V) Hybridization reaction and fluorescence evaluation of hybrid on spot spot The fluorescently labeled target substance (complementary strand DNA) synthesized in the step (i) is a 1M-NaCl / 50 mM phosphate buffer solution ( The target substance (complementary strand DNA) solution is prepared by dissolving in pH 7.0) to a final concentration of 5 nM. In this target substance (complementary strand DNA) solution, after rinsing and washing, a probe-immobilized carrier that has been dried in a nitrogen atmosphere is immersed, and a hybridization reaction is performed at a temperature of 45 ° C. for 2 hours. After the hybridization reaction, the probe-immobilized carrier is washed with a 1M NaCl / 50 mM phosphate buffer solution (pH 7.0), and the non-hybridized target substance (complementary strand DNA) remaining on the surface is washed away. Next, after washing lightly with pure water and removing the buffer solution components used for washing, drying is performed by nitrogen blowing. The above-described hybridization reaction, subsequent washing, and nitrogen blow drying are performed in the same manner for both the DNA probe fixing carrier and the comparative DNA probe fixing carrier of this example.

  A hybrid of the probe DNA and the target substance (complementary strand DNA) formed by the hybridization reaction is detected using a fluorescent label attached to the target substance (complementary strand DNA). In each probe immobilization carrier, the fluorescence intensity of the spot point where the probe DNA is immobilized is measured using a fluorescence scanner (trade name: GenePix4000B / Axon Instruments, Inc.). In addition, the measurement conditions of fluorescence intensity measure fluorescence intensity from fluorescent label Cy3 under measurement light: 532 nm under excitation light irradiation. Further, the fluorescence intensity observed at the spot point is measured two-dimensionally by the fluorescence scanner.

(Vi) Evaluation results As a result of two-dimensional imaging measurement of the fluorescence intensity derived from the hybrid formed by the above hybridization reaction, the average of the fluorescent spot diameters observed on the above five types of probe-immobilized carriers Values are shown in Table 1. According to the results shown in Table 1, the average value of the fluorescent spot diameter is 50.1 μm in the comparative DNA probe immobilization carrier on which the reaction inactive substance coating film was not formed but spotted. Further, after forming the n-dodecane-coated film, the spot-fixed probe-immobilized carrier and after forming the n-hexadecane-coated film, the average of the fluorescent spot diameters observed on the spot-implanted probe-immobilized carrier The values are 50.9 μm and 49.7 μm, respectively. That is, the formation of the n-dodecane coating film or the formation of the n-hexadecane coating film has no significant effect on the miniaturization of spots (dots). On the other hand, after forming the n-heptadecane-coated film, the spot-fixed probe-immobilized carrier and after forming the n-octadecane-coated film, the average of the fluorescent spot diameters observed on the spot-implanted probe-immobilized carrier The values are 32.5 μm and 23.1 μm, respectively. That is, the formation of the n-heptadecane coating film and the formation of the n-octadecane coating film exert a remarkable effect on the miniaturization of spots (dots).

By the way, the fluorescence intensity of each spot point observed on the DNA probe immobilization carrier for comparison and the four types of probe immobilization carriers that are spotted after forming the reaction inactive substance coating film are observed. The fluorescence intensity of each spot point is compared. In the comparison, it was confirmed that the fluorescence intensity values per unit area of the fluorescent spot were substantially equal. That is, by forming a coating film of each saturated hydrocarbon on the substrate surface, the contact area between the droplet containing the DNA probe to be spotted and the substrate surface into which the maleimide group is introduced changes. However, the reaction between the reactive group of the DNA probe; the sulfanyl group (—SH) and the reactive group of the substrate surface; the maleimide group in the contact region is substantially uninhibited. . Further, these four kinds of reaction inert substances are linear saturated hydrocarbons having a structure of CH ₃ — (CH ₂ ) _n —CH ₃ , and only the carbon chain length is different. However, due to the difference in carbon chain length, the flow in the coating film is caused at the environmental temperature of 22.3 ° C. in which the low boiling solvent acetone is volatilized and the probe solution spotting step is performed. Properties (viscosity) are different.

  That is, since the melting points of n-dodecane and n-hexadecane are significantly lower than 22.3 ° C., they are liquid at this temperature and their viscosity is not so high. On the other hand, n-heptadecane has a melting point of 22 ° C. and is liquid at this temperature, but its viscosity is considerably higher than the viscosity exhibited by the two kinds of saturated hydrocarbons. Furthermore, the melting point of n-octadecane itself is higher than 22.3 ° C., and in a state in which a small amount of acetone as a diluent solvent remains, the liquid state showing fluidity is maintained, but the viscosity thereof is n-heptadecane. It is in a higher state than the viscosity indicated by. When spotting droplets of a solution containing a probe due to the difference in viscosity exhibited by the flowable substances constituting the coating film, the higher the viscosity, the smaller the spot diameter formed away from the coating film. ing. If the flowable material constituting the coating film has a low viscosity, when spotting a droplet of the solution containing the probe, the force required to eliminate the material present in the spot region is small, and the spot It can be seen that the effect of suppressing the expansion of the diameter is small.

  INDUSTRIAL APPLICABILITY The present invention can be suitably used for manufacturing a probe array type probe fixing carrier in which individual spot diameters are miniaturized and spot points are arranged at a higher density per unit area.

It is sectional drawing which shows typically the state of the probe solution droplet spotted on the base-material surface when the spotting process of a probe solution in the manufacturing method of the probe fixed carrier concerning this invention was implemented.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Probe 2 Reactive group of probe 3 Droplet 4 Base material 5 Reactive group of base material 6 Substance which is reaction inactive with respect to reactive group of probe and reactive group of base material

Claims (15)

In a method for producing a probe-immobilized carrier in which a probe capable of specifically binding to a target substance is immobilized on a substrate,
The probe is immobilized on the substrate through bond formation by a reaction between a reactive group included in the probe and a reactive group introduced on the substrate.
The following steps (i) to (iv):
Step (i): introducing a reactive group capable of reacting with the reactive group included in the probe onto the substrate;
Step (ii): A coating film made of a reaction inert substance that is not reactive with any of the reactive groups provided in the probe and the reactive groups introduced onto the substrate. Providing on top;
Step (iii): The droplet of the solution containing the probe is spotted on the base material covered with the coating film made of the reaction inert substance by the spotting means, and the spotted droplet is Allowing the coating film made of a reaction inert substance to pass through and contacting with the base material into which the reactive group is introduced;
Step (iv): a step of reacting a reactive group included in a probe contained in a droplet to be deposited with a reactive group introduced on a substrate in contact with the droplet A method for producing a probe-immobilized carrier. After step (iv)
Step (v): The method for producing a probe-fixing carrier according to claim 1, further comprising a step of removing the droplets to be spotted. After step (iv)
Step (v '): The method for producing a probe-immobilized carrier according to claim 1, further comprising a step of removing a droplet to be deposited and a coating film made of a reaction inert substance. The reaction inert substance constituting the coating film made of the reaction inert substance is:
When a droplet of a solution containing the probe is spotted on a substrate into which the reactive group has been introduced in a state where it is not covered with the coating film, the reactive group is introduced. Compared with the dot diameter of the part where the droplet to be spotted on the substrate
When a droplet of a solution containing the probe is spotted on a substrate on which the reactive group has been introduced in a state covered with the coating film, the reactive group is introduced. The probe-immobilized carrier production according to any one of claims 1 to 3, wherein the probe-immobilized carrier is a substance having a property of reducing a dot diameter at a portion where a droplet to be spotted on a substrate is contacted. Method. The reaction inert substance constituting the coating film made of the reaction inert substance is:
When a droplet of a solution containing the probe is spotted on a base material into which the reactive group has been introduced in a state covered with the coating film,
At the temperature at which the spotting is carried out, it is possible to allow the droplets to be spotted to pass through the coating film made of the reaction-inactive substance and to come into contact with the substrate into which the reactive group has been introduced. It is a substance which shows fluidity | liquidity, The manufacturing method of the probe fixed support | carrier as described in any one of Claims 1-4 characterized by the above-mentioned. The reaction inert substance constituting the coating film made of the reaction inert substance is:
The probe immobilization according to any one of claims 1 to 5, wherein the probe immobilization is a substance selected from the group consisting of naphthene, paraffin, aromatic hydrocarbon compounds, or derivatives obtained from these hydrocarbon compounds. A method for producing a carrier. The spotting means is
The method for producing a probe-fixing carrier according to any one of claims 1 to 5, wherein the droplet fixing means is an ink jet method. The droplet spotting means by the inkjet method is:
It is a method in which bubbles are generated in a solution containing the probe by thermal energy, and the solution is ejected as droplets by a pressure increase caused by the bubble generation, and droplets are spotted. A method for producing a probe-immobilized carrier according to claim 7. The droplet spotting means by the inkjet method is:
8. The probe fixing carrier according to claim 7, wherein a pressure rise is caused by a piezoelectric element in the solution containing the probe, the solution is ejected as a droplet, and the droplet is spotted. Manufacturing method. The spotting means is
A solution containing the probe is attached to the tip of the protrusion,
A solution liquid containing the probe, which is attached to the tip of the protrusion, which is attached to the tip of the protrusion through the coating film made of a reaction-inactive substance and brought into contact with the substrate. The method for producing a probe-fixing carrier according to any one of claims 1 to 6, wherein the method is a method of spotting drops. The reactive group provided in this probe is a sulfanyl group, The manufacturing method of the probe fixed carrier as described in any one of Claims 1-10 characterized by the above-mentioned. The method for producing a probe-immobilized carrier according to any one of claims 1 to 10, wherein the reactive group provided on the probe is an amino group. The method for producing a probe-immobilized carrier according to any one of claims 1 to 11, wherein the reactive group introduced onto the substrate is a maleimide group. The probe-immobilized carrier production according to any one of claims 1 to 10, wherein the introduction of the reactive group onto the substrate is performed using N-hydroxysuccinimidyl ester. Method. The method for producing a probe-immobilized carrier according to any one of claims 1 to 14, wherein the probe is a nucleic acid probe.

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