JP2000175683A - Formation method of chip and structured material used for isolation of nucleic acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a chip capable of isolating a nucleic acid from a specimen in a short period of time without a defect such as a cut and in a high recovery rate by including a structured material containing silicon oxides and having holes each with a diameter larger than the silicon oxides, in its flow passage. SOLUTION: This chip used for isolating a nucleic acid, including a structured material containing silicon oxides and having holes each with a diameter larger than the silicon oxides in its flow passage, is obtained by compounding particles 33 of silicon oxides on the surface of a resin particle 32a having a larger diameter than that of the silicon oxides, then putting the compounded particles into a mold 34, heat-treating them at a heat resisting temperature of the resin particles 32a or higher to melt and adhere the resin particles 32a each other for forming a structured material 31, taking out the structured material 31 from the above mold 34, immersing it into a sol.gel solution containing a silicon compound, then taking out the structured material 31 from the sol .gel solution, performing a condensation polymerization of the sol .gel solution thereon by a heat treatment to prepare a structured material 31 capable of capturing the nucleic acid from a liquid specimen and then sealing it in a chip case.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a nucleic acid capturing chip for separating nucleic acid components or plasmid DNA from a nucleic acid-containing sample such as a biological sample, a structure contained therein, and a method for forming the same.

[0002]

2. Description of the Related Art Conventional biological samples containing nucleic acids are
As a method for separating nucleic acids, first, a biological sample or the like is allowed to act on a surfactant in the presence of a protease to release nucleic acids. Thereafter, the mixture is mixed with phenol (and chloroform), and the aqueous and organic phases are separated several times by a centrifuge.
Thereafter, there is a method of recovering the nucleic acid in the form of a precipitate from the aqueous phase using alcohol. Further, there is a method by column chromatography using a structure in which a specific type of particles such as silicon oxide is packed in a container. In recent years, various methods and apparatuses have been studied to make these methods easier.

[0003] JP-A-9-47278 discloses DNA
That can fully and inexpensively extract DNA from culture solution
A extraction purification method and apparatus are described. The tip of this device has a structure in which a first filter tube including a trap filter and a membrane filter and a second filter tube including a glass fiber filter, a glass powder layer and a membrane filter are vertically stacked, and a vacuum device (pump) Filtration of impurities by
In this configuration, DNA is adsorbed, washed, and eluted sequentially to perform extraction.

[0004]

When nucleic acids are separated by centrifugation, the size of the apparatus becomes large and the nucleic acids themselves are damaged due to high-speed rotation.

[0005] In column chromatography and the chip described in the above-mentioned literature, when a specific type of particle is packed in a container, it has a fine structure due to the nature of the fine particle (particularly if the particle size is uniform), and when a particle size distribution is present. Since small particles aggregate and become dense between large particles, the liquid flow becomes poor, and it takes a very long time for the sample to pass. Therefore, if a sample is separated using a vacuum pump in order to reduce the time, there is a problem that the nucleic acid is cut as in the case of centrifugation.

SUMMARY OF THE INVENTION An object of the present invention is to solve the above problems and eliminate the need for a vacuum pump as a structure through which a biological sample can easily pass, thereby making it suitable for a structure capable of separating nucleic acids from a biological sample without defects such as cutting. It is to provide a material and a method for manufacturing the material.

[0007]

In order to achieve the above object, a chip according to the present invention includes a structure including silicon oxides and having a hole having a diameter larger than that of the silicon oxide in its flow path. Shall be. In addition, it is assumed that silicon oxide particles are composited on the surface of a particle serving as a nucleus, and a structure formed by three-dimensionally combining the composite particles is included in the flow path.

It is already known that nucleic acids adsorb to silicon oxides such as silica and glass. Generally, silica (S
Although there are many transformations even if iO ₂ ), there are three major ones: quartz, tridymite, and cristobalite, and there are high-temperature type and low-temperature type, respectively. Quartz is not a salt, but may be classified as a silicate mineral due to its condensation form. Ordinary glass is an amorphous substance which is stabilized by containing ions of alkali metal or alkaline earth metal in a three-dimensional irregular network of condensed silicic acid. In addition, there is a method in which silica is formed from a hydrolysis reaction and a dehydration condensation reaction using a silicon alkoxide compound. In this document, all of these condensates of silica, glass, silicate, silicate mineral, and silicic acid, which are crystalline and non-crystalline, are collectively referred to as silicon oxides.

[0009] The structure is a size that can be inserted into a chip and is used for adsorbing DNA.

[0010] When passing through the inside of the structure included in the chip, the nucleic acid is captured by silicon oxides existing on the flow path surface inside the structure. Since the structure in the present invention is formed by three-dimensionally combining particles, a flow path through which a liquid passes always exists as a through hole. For this reason, the liquid sample has good flowability, and the liquid sample can be suctioned and discharged without suction using a special vacuum device.

[0011] Such a nucleic acid capturing structure comprises a step of compounding silicon oxide particles on the surface of a resin particle having a diameter larger than that of the silicon oxide, and a step of putting the composite particles into a mold to form a resin. A step of molding the structure by fusing the resin particles to each other by heat treatment at or above the heat resistance temperature of the particles, a step of immersing the structure taken out of the mold into a sol-gel solution containing a silicon compound, and After taking out from the sol-gel solution, it can be formed by performing a step of subjecting the sol-gel solution to polycondensation by heat treatment.

The nucleic acid-capturing structure comprises a step of combining silicon oxide particles on the surface of resin particles having a diameter larger than that of the silicon oxide, and a step of sol-gel containing the composite particles and a silicon compound. Mixing the solution and placing it in a mold;
The sol-gel solution can be formed by subjecting the sol-gel solution to polycondensation by heat treatment to form a mixed material into a molded body and removing it from the mold.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION The nucleic acid capturing chip of the present invention is a nucleic acid for a pretreatment device for separating nucleic acid from a fluid containing nucleic acid such as a biological sample in order to obtain genetic information from the nucleic acid in a clinical test or research field. Used for extraction. In this genetic test pretreatment device, nucleic acids are extracted in the following procedure. FIG. 1 is a plan view of the apparatus, and FIG.
FIG. 1 shows a flow chart for nucleic acid separation.

In FIG. 2, the portions into which the liquid sample, the cleaning liquid, and the like enter when suctioning and discharging are described later are chips 15, 19.
And are connected to the syringes 2 and 3 via the nozzles 8 and 9, the nozzle holders 6 and 7, and the pipes 4 and 5. The separation chip 19 includes a structure (not shown) for supplementing the nucleic acid in the chip. The tips of these tips are tapered on the inner diameter side. A structure for capturing nucleic acids in the liquid sample is sealed in the tapered portion. Further, the chip used for dispensing various liquid samples contains nothing. Here, in order to distinguish it from the separation chip 19 enclosing the structure, a chip containing nothing is referred to as a dispensing chip 15.

The configuration of the apparatus will be described below.

The syringes 2 and 3 can independently and automatically control the suction and discharge of the liquid sample. The syringes 2 and 3 are independently connected to nozzles 8 and 9 through pipes 4 and 5, respectively.
The nozzles 8 and 9 are fixed to nozzle holders 6 and 7. Nozzle holders 6 and 7 are arms 10 and 11, respectively.
Are held in a state where they can be moved in the Y-axis direction and the Z-axis direction. The arms 10 and 11 can be independently moved in the X-axis direction, and can be partially overlapped in the X-axis direction by providing a difference in the Z-axis direction. The movement of the arms 10 and 11 to a main part on the apparatus plane can be controlled by a combination of the operations of the arms 10 and 11.

The dispensing tip rack 14 can store dispensing tips 15 and can be provided with a total of three dispensing tips. The reaction vessel rack 16 can accommodate 48 reaction vessels 17. In the refined product rack 20, 48 refined product storage containers 21 can be installed. Also, the cleaning liquid bottle 2
2. One eluent bottle 23, one diluent bottle 24, and one binding promoter bottle 25 can be installed. The chip rack 18 can be provided with 48 separation chips 19.

Next, a rough operation of the apparatus will be described.

First, the nozzle 9 is moved above the target dispensing tip 15 on the dispensing tip rack 14 by the control of the arm 11 and the nozzle holder 7. Thereafter, the nozzle holder 7 is moved downward to bring the predetermined position of the dispensing tip 15 into contact with the nozzle 9, and the dispensing tip 15 can be automatically attached to the tip of the nozzle 9. By performing the same control with the nozzle 8, the nozzle holder 6, and the arm 10, the separation tip 19 can be attached to the tip of the nozzle 8.

Under the control of the arm 11 and the nozzle holder 7, the nozzle 8 is moved to a position just above the tip punch 27. Thereafter, the nozzle 9 is controlled by controlling the nozzle holder 7.
And the dispensing tip 15 is moved so that the junction is below the tip punch 27. Further, the nozzle 8 is moved in the direction of the chip punch 27. Thereafter, the nozzle holder 7 is moved upward while keeping a part of the nozzle 9 in contact with the chip punch 27. Thereby, the dispensing tip 15 can be automatically removed from the nozzle 9. By performing the same control with the nozzle 8, the nozzle holder 6, and the arm 10, the separation chip 19 can be removed from the nozzle 8.

The liquid receivers 29 and 30 can receive the liquid discharged from the nozzles 8 and 9. The liquid receivers 29 and 30
It functions as a home position of the nozzle, and the received liquid is sent to a waste liquid treatment process (not shown) as a waste liquid. The washing unit 26 can wash the dispensing tip 15 attached to the nozzle holder 7 via the nozzle 9 by discharging running water.

Hereinafter, the recovery of DNA added to serum using the automatic apparatus will be described in more detail.

A commercially available purified product of λ DNA (Ferm
Entas) was added, and SDS (sodium dodecyl sulfate) was added at a final concentration of 1% as a sample liquid 13 (liquid sample) to prevent nuclease.
The sample bottle was placed in a sample bottle, and the sample bottle was stored in the sample rack 12 on the apparatus shown in FIG. After setting the dispensing tip rack 18, the reagent bottles 22, 23, 24, 25, the reaction container 17, and the purified product container 21 at predetermined positions on the apparatus in FIG. 1, the apparatus performs the following operations.

First, in the first step, an operation of mixing the sample liquid 13 and the binding promoter using the dispensing tip 15 is performed.

The dispensing tip 15 is attached to the nozzle 9 by a predetermined operation under the control of the arm 11 and the nozzle holder 7. Thereafter, by controlling the arm 11, the nozzle holder 7, and the syringe 3, a predetermined amount of guanidine hydrochloride as a binding promoter is sucked from the binding promoter bottle 25. Further, 50 μl of air is sucked, and the nozzle 9 and the dispensing tip 15 are moved to the washing section 26 by control, where the outer wall of the dispensing tip 15 is washed with running water. After the cleaning, the nozzle holder 7 is moved under control to the predetermined sample liquid 1 on the sample rack 12.
3 and aspirates a predetermined amount of the sample liquid 13 by controlling the syringe 3. After the suction, the nozzle holder 7 is moved to a predetermined reaction container 17 on the reaction container rack 16 by control, and then the entire amount is discharged. After the discharge, the sample liquid 13 and guanidine hydrochloride are mixed by further performing suction and discharge. After mixing, the nozzle holder 7 is moved to the position of the tip punch 27 by control, and the dispensing tip 15 is removed from the nozzle 9 by a predetermined amount of operation.

In the second step, a nucleic acid (here, DNA) is bonded to a structure for capturing the nucleic acid in the chip 19.

By controlling the arm 10 and the nozzle holder 6,
The separation tip 19 is attached to the nozzle 8 by a predetermined operation. Thereafter, the nozzle holder 6 is moved to the reaction container 17 containing the mixture on the reaction container rack 16, and the mixture is sucked into the separation chip 19 by controlling the syringe 2. After the suction, the suction and discharge are repeated a predetermined number of times under the control of the syringe 2 to bring the structure into contact with the mixed liquid.

In the third step, the residue after the nucleic acid is captured in the structure is discharged.

After the suction and discharge are repeated a predetermined number of times by the separation chip 19, the liquid mixture in the reaction vessel 17 is sucked into the separation chip 19. Thereafter, the arm is moved to the waste liquid port 28 under the control of the nozzle holder 6 and the separation chip 19 is moved.
The mixed liquid in the nozzle 8 is discharged by controlling the syringe 2. After the ejection, the separation chip 19 moves to the liquid receiver 29 under the control of the arm 10 and the nozzle holder 6.

In the fourth step, the structure containing the nucleic acid in the separation chip 19 is washed.

The dispensing tip 15 is attached to the nozzle 9 by a predetermined operation under the control of the arm 11 and the nozzle holder 7. Thereafter, the washing bottle 22 is placed in the dispensing tip 15 under the control of the arm 11, the nozzle holder 7 and the syringe 3.
A predetermined amount of cleaning liquid is sucked from the apparatus. Thereafter, the nozzle holder 7 is moved to a predetermined reaction vessel 17 of the reaction vessel rack 16 by control, and the cleaning liquid is discharged. After discharging, arm 11
By controlling the nozzle holder 7 and the nozzle holder 7, the nozzle holder 7 is moved to the position of the tip punch 27, and the dispensing tip 15 is removed from the nozzle 9 by a predetermined operation.

After the movement of the nozzle holder 7, the arm 10,
By controlling the nozzle holder 6, the separation chip 19 is moved to the reaction vessel 17 on the reaction vessel rack 16 containing the cleaning liquid,
The cleaning liquid is sucked into the separation chip 19 by controlling the syringe 2. After the suction, under the control of the syringe 2, suction and discharge are repeated a predetermined number of times by the separation chip 19, and the structure supplemented with the nucleic acid is washed with the washing liquid. Separation chip 19
After repeating suction and discharge a predetermined number of times, the arm 1
0, the cleaning liquid is moved to the waste liquid port 28 by the control of the nozzle holder 6 and the cleaning liquid in the separation chip 19 is discharged by the control of the syringe 2. After the ejection, the separation chip 19 moves to the liquid receiver 29 under the control of the arm 10 and the nozzle holder 6.

If necessary, the fourth step is repeated a predetermined number of times.

In the fifth step, the nucleic acid captured by the structure in the separation chip 19 is eluted.

The dispensing tip 15 is attached to the nozzle 9 by a predetermined operation under the control of the arm 11 and the nozzle holder 7. Thereafter, suction is performed from the eluent bottle 23 to the dispensing tip 15 by controlling the arm 11, the nozzle holder 7, and the syringe 3, and the nozzle holder 7 is moved to a predetermined reaction vessel 17 on the reaction vessel rack 16 by control. Thereafter, the eluent in the dispensing tip 15 is discharged into the reaction vessel 17. After the discharge, the nozzle holder 7 is moved to the position of the tip punch 27 under the control of the arm 11 and the nozzle holder 7, and the dispensing tip 15 is removed from the nozzle 9 by a predetermined operation.

After the movement of the nozzle holder 7, the arm 10,
By controlling the nozzle holder 6, the separation chip 19 is moved to a predetermined reaction vessel 17 on the reaction vessel rack 16 containing the eluent. Therefore, the eluent is sucked into the separation chip 19 by controlling the syringe 2. After the suction, the syringe 2 is controlled, suction and discharge are repeated a predetermined number of times, and the structure and the eluent are brought into contact. Thereafter, the arm 10 and the nozzle holder 6 are controlled in a state where the eluent in the reaction container is sucked into the separation chip 19, and the separation chip 19 is moved to the predetermined purified product storage container 2.
Move to 1. After the movement is completed, the solution in the separation chip 19 is discharged into the purified product storage container 21 by the operation of the syringe 2. After the ejection, the arm 10 and the nozzle holder 6 are operated to move to the liquid receiver 29. The fifth step is repeated a predetermined number of times as necessary.

After the fifth step, the arm 10 and the nozzle holder 6 are operated, and the nozzle holder 6 is
And the tip 19 is removed from the nozzle 8 by a predetermined operation.

Next, as a first embodiment, a method of forming a nucleic acid capturing structure provided in a separation chip will be described. FIG. 3 (a)
FIG.

The silica particles * 1 and the organic resin particles 32a * 2 were mixed with the silica particles at a resin particle volume concentration of 10%.
And converted to a specific gravity so as to be 60% by volume.
Thereafter, a predetermined amount of an ethyl silicate solution * 3 is added, and the mixture is stirred until it becomes clay-like, and PTFE (polytetrafluoroethylene) is added.
Into a mold 34. In the figure, reference numeral 33 denotes a mixture of silica particles and an ethyl silicate solution.

The PTFE mold 34 remains 100 to 20
Heat treatment is performed at 0 ° C. for 30 minutes to cause polycondensation of the ethyl silicate in the mold 34. As a result, a molded product that does not collapse even when removed from the mold is obtained.

The molded article taken out of the mold is heat-treated at least at 400 ° C. or higher for 3 hours to completely burn the resin particles 32a. As a result, the resin particles 32a become voids, and the structure 31a has a flow path formed therein. Although not shown in the figure, fine pores of silica particles are formed in the pores where the resin particles 32a were present, so that the contact area with the liquid sample is increased and the nucleic acid is easily trapped. ing.

* 1 is a SIGMA APPPROX with a particle size of 8 to
It is 10 μm. * 2: CL manufactured by Sumitomo Seika Co., Ltd.
The series has a particle size of 180 to 1000 μm. *
Reference numeral 3 denotes an ethyl silicate solution (silicon compound-containing sol-gel solution: an alcohol solution of silicon alkoxide).
5 g, 17.28 g of water, 0.3 g of hydrochloric acid (12N), and 5.52 g of ethanol.

In this method, the silica particles and the resin particles 32a are not limited to the above products.
Also, the ethyl silicate concentration can be changed. The particles of silicon oxides used are desirably at least 0.001 μm and at most 20 μm. If it is less than 0.001 μm, it will be scattered during preparation and the concentration will not be stable. There is also a problem that dust and the like are generated during handling. When the thickness is 20 μm or more, the distance between the organic material and the organic material for forming the flow path is increased, and the structure is fragile and easily collapsed.

When the organic substance used is the resin particles 32a as in this embodiment, it is desirable that the thickness be 50 μm or more and 1000 μm or less. If it is 50 μm or less, water permeability is poor and pressure is applied at the time of suction and discharge of the liquid, and the nucleic acid may be cut. On the other hand, if the thickness is 1000 μm or more, the pores become large, and the structure cannot be formed and collapse.

Here, the ethyl silicate solution (sol-gel solution containing a silicon compound) functions as a binder for binding particles of silicon oxides. In this example, an ethyl silicate solution was used, but various metal sol-gel solutions may be used as the binder, but by using a sol-gel solution containing a silicon compound, the binder portion can also bind to nucleic acids. Therefore, the efficiency of nucleic acid capture is improved.

The composition of the sol-gel solution is mainly composed of silicon alkoxide, and water (for hydrolysis), acid or alkali (for catalyst) and solvent (for preparation of homogeneous solution; Use)
Is used. In addition, the sol-gel liquid is not limited to the above composition, and a carboxylate or an inorganic compound can be used instead of the alkoxide. It is also possible to use ethylene glycol, ethylene oxide, triethanolamine, xylene and the like as the solvent. If necessary, it is also possible to add an additive so that, for example, cracks hardly occur.

In the present invention, since the volume concentration of the resin particles 32 a with respect to the silica particles affects the flow of the sample liquid 13,
It is desirable that the concentration be as high as possible so that the nucleic acid capturing structure does not collapse after the heat treatment. The resin particle volume concentration varies depending on the particle diameter of the resin particles 32a used,
It is preferable to reduce the volume concentration as the structure becomes larger. Further, since the specific gravity varies depending on the particles, the resin particle concentration also varies depending on the specific gravity.

In addition, since the heat treatment is intended for molding, the temperature is higher than the temperature at which the ethyl silicate solution is solidified.
Any temperature may be used as long as it is lower than the melting point of PTFE used in the mold. The time is used as a guide until the solvent (ethanol and water in this embodiment) is consumed. The heat treatment is performed until the resin particles 32a burn and disappear. The heat treatment temperature may be changed depending on the used resin particles 32a, and the time can be changed depending on the type and amount of the resin particles 32a.

Next, another embodiment of the method for forming a nucleic acid capturing structure according to the second embodiment will be described.

FIG. 3B shows a forming process. less than,
Each step will be described.

Silica Particles and Organic Fiber 32b
With an organic substance volume concentration of 10 to 60 Vo
It is converted to a specific gravity and weighed so as to be 1%. The fibers 32b are put in a PTFE mold 34, and then a predetermined amount of a sol-gel solution is added. In the figure, 33a indicates a mixture of silica particles and a sol-gel liquid.

While keeping the PTFE mold 34, 100-2
Heat treatment is performed at 00 ° C. for 30 minutes to cause polycondensation of the ethyl silicate in the mold 34.

The molded product taken out of the mold 34 is heat-treated at least at 400 ° C. for 3 hours to completely burn the fiber 32b. As a result, the structure 31 in which the flow path to which the silica particles adhere is formed by forming a hole in the fiber 32b portion.
b is formed.

As in the present embodiment, the fiber 32b is used as an organic substance.
Is used, it is desirable to increase the amount of the ethyl silicate solution used as a binder as compared with the case of the resin particles 32a as in the previous embodiment. Since the fibers 32b are continuous unlike the resin particles 32a, in order to form the structure 31, the amount of the binder is increased and the stronger structure 3
Must be 1. Specifically, after the step, an appropriate amount of an ethyl silicate solution is infiltrated into the molded body, and then heat-treated to polycondensate the ethyl silicate. Repeat this step several times if necessary. Note that any of synthetic fibers and natural fibers may be used as the fibers 32b, and plants such as trees and bamboos can be used as the natural fibers.

Next, as a third embodiment, another method for forming a nucleic acid separating structure will be described with reference to FIG.

The hollow resin particles 36 * 4 and ethanol were
Approximately 10 wt% hollow resin particle weight concentration with respect to ethanol
And stirred with a spoonful. An appropriate amount of ethanol dispersion liquid * 5 of SiO2 ultrafine particles is added, and the mixture is further stirred well with a spoonful of scrub and put into a mold 34 made of PTFE.

The PTFE mold 34 is heat-treated at 170 ° C. for 20 minutes and taken out of the mold 34. As a result, a flow path is formed by the hollow resin particles 36, and SiO2 ultrafine particles 37, which are silicon oxide, are present on the flow path surface, and the nucleic acid is supplemented here.

Note that * 4 is F-80ED manufactured by Matsumoto Resin Pharmaceutical Co., Ltd. and has an average particle size of 80 to 90 μm, and * 5 is OSCAL manufactured by Catalyst Chemicals Co., Ltd. and has a particle size of 40 to 300 μm.
nm. Also in this method, the hollow resin particles 36 and the ultrafine SiO2 particles 34 are not limited to the products described above.

In the present forming method, an ethyl silicate solution (sol-gel solution containing a silicon compound) can be used as a binder. However, as shown in this embodiment, a temperature slightly exceeding the heat resistance temperature of the hollow resin particles 34 is used. , The hollow resin particles 34 were melt-adhered to each other, and a sponge-like molded body was obtained. However, if the heat treatment temperature is too low, the hollow resin particles 34
Since there is no fusion adhesion between them, they do not become a molded body but become powdery. On the other hand, if the heat treatment temperature is too high, the hollow resin particles 34 rupture, shrink and shrink, and are not molded into an elastic sponge. Further, if the heat treatment time is too long, the heat treatment time is reduced as in the case where the temperature is too high. In the case of solid resin particles, if the heat treatment temperature is too high and the melting of the particles progresses, it becomes impossible to secure a flow path. Therefore, in this forming method, it is necessary to select a heat treatment temperature and time suitable for the material.

In the method of forming a nucleic acid separating structure of each of the above embodiments, a metal can be used for the mold 34, but there is a possibility that the metal surface and the components of the sol-gel solution may bind.
When the bonding occurs, the structure 31 is broken or broken when the structure 31 is removed from the mold. When using a metal mold, it is desirable to treat the surface with a substance that does not react with the sol-gel solution so that bonding does not easily occur.

Next, as a fourth embodiment, another method for forming a nucleic acid capturing structure will be described.

The porous body made of metal is immersed in an ethyl silicate solution.

The porous body taken out of the solution
A heat treatment is performed at 200 ° C. for 30 minutes to cause polycondensation of the ethyl silicate to form a silicon alkoxide film on the porous portion of the porous body.

If necessary, this process is repeated a plurality of times.

In the structure of this embodiment, the surface area of the flow channel is increased by using the porous portion of the porous material as the flow channel, and the nucleic acid is supplemented with silicon alkoxide. That is, silicon alkoxide is used not as a binder but as a nucleic acid capturing material.

The structure of the first embodiment may be used instead of the metal porous body, or silicon oxide (eg, silica particles) may be mixed with ethyl silicate. In that case, the first
Unlike the examples, the silica particles are used to increase the surface area of the flow channel by providing irregularities in the flow channel, so any material may be used as long as the material can provide the unevenness to the flow channel without using silica particles. .

Alternatively, the porous body of the metal is first immersed in a sol-gel solution that can be used as a binder containing some particles, and then subjected to heat treatment to form irregularities in the porous portion, and then immersed in ethyl silicate. Heat treated,
A coating may be formed on the uneven surface.

The heat treatment conditions in this case are not particularly limited as long as they are lower than the heat resistance temperature of the porous body. Ethyl silicate becomes crystalline silica as it is heat-treated at a high temperature, and becomes a desirable state for nucleic acid recovery.

Next, another method for forming the nucleic acid capturing structure of the fifth embodiment will be described.

FIG. 6 shows a forming process. Hereinafter, each step will be described.

The resin particles 32a * 2 and the silica particles 37
* 6 (0.2 to 10 wt% of resin particle weight) is weighed. These two particles are combined by a particle combining device.

The composite particles (composite particles 40)
A predetermined amount is weighed and put into a mold 34 made of PTFE.

Resin particles 3 in PTFE mold 34
Heat treatment for about 30 minutes at or above the heat resistance temperature of 2a;
a) After bonding 41 between the particles by fusing together, the particles are taken out of the mold.

The structure taken out from the mold is taken out after being immersed in * 3 in an ethyl silicate solution in which silica particles 37 * 5 are monodispersed.

The excess liquid is lightly sucked or blown off or blown off to dry naturally or dried at 40 to 50 ° C., and then heat-treated at 100 ° C.

The operation as needed is repeated several times.

Here, * 2 indicates the aforementioned Sumitomo Seika Co., Ltd.
CL series with a particle size of 180-1000 μm. *
6 is flaky particles made by Asahi Glass Co., Ltd., having an average particle size of 4 to 5 μm.
(Thickness: about 0.05 μm). * 5 is an ethanol solution having a particle size of 40 to 300 nm, OSCAL manufactured by Catalyst Chemicals, Inc. * 3: Ethyl silicate solution (silicon compound-containing sol-gel solution: alcohol solution of silicon alkoxide) 25 g of ethyl silicate, 17.28 g of water, 0.3 g of hydrochloric acid (12N), 5.42 g of ethanol
It is adjusted by.

In this method, the composite particles 40 are formed by mechanically combining the silica particles 37 with the resin particles 32a. In this example, the particles were compounded using Theta Composer (manufactured by Tokuju Corp.). Other methods of compounding include mechanomill (manufactured by Okada Seiko Co., Ltd.) and mechanofusion (manufactured by Hosokawa Micron Co., Ltd.). Of course, all of these methods can be used and are limited to the present embodiment. It is not something to be done.

Since the resin particles 32a are softer than the silica particles 37, the silica particles 37 are embedded in the surface of the resin particles 32a by the compounding. That is, a film-like material is formed by the silica particles 37, and thereby the properties of the resin itself are changed. As one of them, the apparent heat-resistant temperature has been improved. Therefore, it is desirable that the heat treatment temperature is set to be higher than the heat resistant temperature of the untreated resin particles 32a.

In this method, after the structure is molded, the surface of the flow path of the structure is subjected to a surface treatment by immersion treatment with an ethyl silicate solution containing silica particles 37. At this time, the silica particles that have been treated later bind to the silica particles that have been previously composited using the ethyl silicate solution as a binder. As a result, a structure is obtained in which the silica particles 37 are firmly bonded to the resin particles 32a and the silica particles 37 do not peel off from the resin particles 32a.

By repeating the above operations, the surface of the resin particles 32a can be modified with the silica particles 37, and the resin particles 32a for binding to nucleic acids can be modified.
Can be reduced. However, in order to ensure water permeability, a gap formed by the formed resins is not filled.

In the above embodiments, resin particles are used as cores, but silica particles (glass beads) can be used as cores. In this case, since the base is silicon oxide, it is effective for nucleic acid recovery.

Next, another method for forming the nucleic acid capturing structure of the sixth embodiment will be described. FIG. 7 shows a forming process. Hereinafter, each step will be described.

The resin particles 32a * 2 and the silica particles 37
* 1 (0.2 to 10 wt% of resin particle weight) is weighed. These two particles are combined by a particle combining device.

The composite particles (composite particles 40)
Add a predetermined amount of ethyl silicate solution to
Place in FE mold 34.

100-200 ° C. in PTFE mold 34
And heat-treated for 30 minutes at a time to condense the ethyl silicate in the mold to bind the particles together, and then take out the mold.

Note that * 1 is the SIGMA APPRO described above.
X is one having a particle size of 8 to 10 μm.

In the forming methods of the fifth and sixth embodiments, the silica particles 37 and the resin particles 32a are not limited to the above products. Also, the ethyl silicate concentration can be changed. The particles 37 of silicon oxides used are 0.001.
It is desirable that the thickness be in the range from μm to 100 μm. 0.001μm
In the following cases, the particles are scattered at the time of blending, the concentration is not stable, and there is a problem of dust in handling. In addition, compounding of particles
It is said that it is better to use particles having a size less than one tenth of the diameter of the resin particles 32a serving as base particles as child particles.
The following is assumed.

As the resin particles 32a to be used, generally used plastic resins are used. Saturated hydrocarbon resins hardly reacting with nucleic acids and various reagents used, for example, polyethylene, polypropylene and the like, and fluorine-based resins such as PTFE are used. Resins are preferred. However, it is also possible to eliminate the influence of the underlayer by sufficiently treating the surface of the resin with silicon oxide as in the fifth embodiment. In this case, the resin is not limited to the resin.

In the fifth and sixth embodiments, the filling rate of the particles of the structure is improved by heat-treating the mold in a pressurized state with the displacement of the structure being constant during the heat treatment in the process. Can be. Thereby, the porosity can be made constant. However, in this case, as the pressure increases, the resin particles 32a are also deformed. Note that it is also possible to perform heat treatment by releasing the pressurization after pressurizing and holding for a predetermined time before the heat treatment. In this case, the filling rate is lower than in the case where the heat treatment is performed in a pressurized state, but the resin particles 32a are not deformed. The heat treatment temperature may be changed depending on the resin particles used, and the time can be changed depending on the type and amount of the resin particles.

Further, in the case where the PTFE mold is processed in a mold capable of controlling the temperature on the side surface and the upper and lower surfaces, the temperature of the side surface is set higher than the upper and lower surfaces. Thereby, the resin particles are melted on the side surface, and the irregularities due to the particles are eliminated. When using the structure enclosed in the chip housing, the problem is that the sample sample flows through the gap formed between the chip housing inner wall and the structure,
That is, the structure cannot efficiently contact the specimen sample. By melting the particles only on the side surface by controlling the temperature of the mold to eliminate irregularities and make the surface smooth, the gap between the inner wall of the chip housing and the structure can be reduced. As a result, the structure is such that the sample liquid easily comes into contact with the structure, and the nucleic acid can be collected more effectively.

As a method for forming the structure, there is a method using ultrasonic waves other than the heat treatment. In this case, PT
A horn that generates ultrasonic waves is inserted in place of the FE mold pins, and ultrasonic bonding is performed. The ultrasonic wave is transmitted to the portion where the particles are in contact, the temperature of the contact portion rises, and the resin melts and joins. In this method, the processing time can be set to several seconds to several tens of seconds by setting a frequency and an output suitable for each resin.

Next, another method for forming the nucleic acid capturing structure of the seventh embodiment will be described. FIG. 9 shows a forming process. Hereinafter, each step will be described.

A glass tube 42 having an outer diameter smaller than the inner diameter of the chip and glass particles 43 softening at a lower temperature than the glass tube 42 are prepared. (In this embodiment, tempered glass is used for the glass tube 42, and ordinary glass (soda glass) is used for the glass particles 43.) The glass particles 43 are put in the glass tube 42, and heat treatment is performed at a temperature at which the glass particles 43 soften. The glass particles are fused together. (In this example, at 720-730 ° C. for 30 minutes) The structure cooled to room temperature is immersed in an ethyl silicate solution (sol-gel solution containing a silicon compound) and then dried.
At this time, the excess is removed by blowing with nitrogen gas or the like so that the ethyl silicate solution does not fill the gap formed by the glass particles 43. Repeat this step several times if necessary.

This structure is heat-treated at 200 to 400 ° C. to cause polycondensation of the ethyl silicate to form a silicon alkoxide film on the glass surface.

The silicon alkoxide coating on the glass surface formed by the above operation makes it possible to eliminate the influence of impurities such as sodium contained in the glass. The heat treatment temperature in the number of steps and
It can be changed as needed depending on the concentration of impurities in the glass.

In the structure of this embodiment, the particles are deformed and fused due to the temperature difference at which the glass tube and the glass particles are softened, but the glass tube is not deformed and thus can maintain a certain shape. I can do it.

The structure composed only of glass particles is brittle and easily broken, but the structure of the present embodiment has high strength because it is installed in the tube. When press-fitting into the chip, the tube can be sealed by applying pressure, and there is no fear that the structure will be collapsed.

When an ethyl silicate solution in which silica particles are monodispersed is used instead of the ethyl silicate solution, the influence of impurities in the glass can be reduced, and small irregularities are formed on the surface, which is advantageous for nucleic acid recovery. become.

It is also possible to use silica particles or the like instead of glass particles. In that case, it is possible to create a structure by using a quartz glass tube as the outer tube and changing the heat treatment temperature. If there is a difference between the softening temperature of the tube and the softening temperature of the particles to be put into the tube, a structure can be formed, and the material constituting the structure is not limited to this embodiment.

Further, since this structure is entirely made of glass, it can be sterilized by an autoclave.

Next, a description will be given of a method of manufacturing a chip using any one of the structures of the first to sixth embodiments. FIG. 5 shows a chip of the present invention.

The chip 31 of the present invention was manufactured by enclosing the structure 31 described in the above embodiment in a chip housing (for convenience, a chip not enclosing (including) the structure is referred to as such) 19a. . As a manufacturing method, the chip housing 19a
There is a method of press-fitting the structure 31 into the hole. When an adhesive is used to fix the chip housing 19a to the structure 31, a non-permeable adhesive 38 is used. This is because in the case of a permeable adhesive, it is difficult to secure a flow path due to penetration into the inside of the structure. Alternatively, there is a method in which after inserting the structure 31 into the chip housing 19a, the chip housing 19a is deformed and sealed. In addition, there is a method in which the structure 31 is sealed in the chip housing by using a mesh, a filter, or the like that does not hinder the passage of water as the holding member 39.

In each case, the sample liquid 1 containing the nucleic acid
It is desirable that the structure 31 be present over the entire cross section of the chip so that the structure 3 can efficiently pass through the inside of the structure 31. Note that a plurality of structures 31 can be placed in the chip 19 in order to increase the amount of nucleic acid captured.

Since the chip of the present invention has a simple structure, it is possible to separate the chip housing from the structure when discarding after use, wash the chip housing, disinfect the chip housing, and reuse the chip housing. .

Next, the results of an experiment for confirming nucleic acid capture using the chip of the present invention will be described.

An experiment for confirming nucleic acid capture was performed using the chip 19 manufactured by press-fitting the formed structure 31 into the chip housing 19a described above. The experimental method was performed according to the operation procedure of the pretreatment device 1 described above. Thereafter, PCR (polymerase chain reaction) is performed for confirmation using the sample liquid 13 obtained in the purified product storage container 21 as a sample,
Electrophoresis and fluorescent staining were performed for evaluation.

Table 1 shows the conditions for forming the seed structures 31 manufactured in the first, third, fifth, and sixth embodiments and the results of nucleic acid recovery. The results of the second and fourth embodiments are substantially the same, and thus are omitted here.

As shown in Table 1, each of the structures was 50
% Was obtained. In some cases, a recovery rate of 70% or more was obtained. As described above, by using the chip 19 of the present invention, the DN can be easily obtained at a high recovery rate.
It was found that A could be separated.

Further, in order to confirm the effectiveness of the formed structure, the following two types of chips were prepared using loose composite particles, and confirmation experiments were performed.

As shown in FIG. 8 (a), a chip in which the composite particles are sandwiched between two filters with no gap from above and below and fixed so that the composite particles do not move.

As shown in FIG. 8 (b), a chip in which the composite particles are sandwiched from above and below so that the two filters keep a fixed distance so that loose composite particles can freely move between the filters. .

Using these two chips, a nucleic acid capture experiment was performed by the method described above. As a result, there was no large difference between (a) and (b) in the recovery of nucleic acid from a sample solution having a high nucleic acid concentration, but (a) was better in recovering nucleic acid from a sample solution having a low nucleic acid concentration ( The nucleic acid recovery was better than in b).

[0114] This is because, at a low concentration where the absolute amount of the nucleic acid is small, the chip (a) through which the sample solution passes through the narrow and narrow gap formed by the particles is better than the chip (b) where the particles move freely. Since the frequency of contact between the nucleic acid and silica is high, the recovery rate of the nucleic acid is improved. From these results, it is understood that the structure in which the particles are immobilized as in the present invention and the liquid passes through the gap is effective for nucleic acid recovery. In the case of loose particles, it is necessary to use magnetic force to collect the particles or to use centrifugal separation, which is a difficult problem in configuring the apparatus. There is no problem of particle recovery.

As described above, it was confirmed that the nucleic acid can be easily separated at a high recovery rate by using the chip 19 containing the structure of the present invention. Further, it was found that the smaller the particle size of the resin particles 32a serving as the base particles, that is, the larger the surface area, the better the recovery rate.

[0116]

According to the structure of the present invention, a liquid sample can be easily passed without the need for evacuation or pressurization. In addition, since silicon oxide for adsorbing nucleic acids has irregularities in a portion serving as a flow path of the liquid sample, nucleic acids can be separated from the liquid sample at a high recovery rate without defects such as cutting, Since the recovery rate is high, only a small amount of the sample is required.

[Brief description of the drawings]

FIG. 1 is a plan view of a pre-processing apparatus for gene diagnosis of the present invention.

FIG. 2 is a schematic diagram showing a flow path of a nucleic acid separation unit of the present invention.

FIG. 3 is a view showing a step of forming a structure of the present invention.

FIG. 4 is a view showing another step of forming the structure of the present invention.

FIG. 5 is a view showing a chip including the structure of the present invention.

FIG. 6 is a view showing another step of forming the structure of the present invention.

FIG. 7 is a view showing another step of forming the structure of the present invention.

FIG. 8 shows a chip in which two filters are provided above and below and composite particles are provided therebetween.

FIG. 9 is a view showing another step of forming the structure of the present invention.

[Explanation of symbols]

1 ... Genetic test pretreatment device, 2, 3 ... syringe, 4, 5
... Piping, 6, 7 ... Nozzle holder, 8, 9 ... Nozzle, 1
0, 11: arm, 12: sample rack, 13: sample liquid,
14: dispensing rack, 15: dispensing tip, 16: reaction vessel rack, 17: reaction vessel, 18: tip rack, 19 ...
Chips, 20: purified product rack, 21: purified product storage container,
22: Washing liquid bottle, 23: Eluent liquid bottle, 24: Diluent liquid bottle, 25: Binding promoter bottle, 26: Washing part, 2
7: chip removal, 28: waste liquid port, 29, 30: liquid receiver,
31 ... structure, 32a ... resin particles, 32b ... fiber, 33
... mixture of silica particles and ethyl silicate solution, 34 ... type, 3
5: Organic solvent, 36: resin particles, 37: SiO2 fine particles, 38: non-permeable adhesive, 39: holding material, 40: composite particles, 41: fused resin particles, 42: glass tube, 43
... glass particles.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Shinichi Fukuzono 882 Ma, Hitachinaka-shi, Ibaraki Pref.Measurement Division, Hitachi, Ltd. (72) Inventor Yoshishige Endo 502, Kandamachi, Tsuchiura-shi, Ibaraki Japan, Ltd. (72) Inventor: Uki Aono 502, Kandate-cho, Tsuchiura-shi, Ibaraki Pref.Mechanical Laboratory (72) Inventor: Yasuhiro Yoshimura 502, Kandachi-cho, Tsuchiura-shi, Ibaraki Machinery, Inc. In the laboratory (72) Inventor Takao Terayama 882 Ma, Hitachinaka-shi, Ibaraki Pref.Hitachi, Ltd.Measurement Instruments Division (72) Inventor Tetsuo Yokoyama 882-Hita, Hitachinaka-shi, Ibaraki Pref.Hitachi, Ltd. (72) Inventor Kenji Yasuda 882 Ma, Hitachinaka City, Ibaraki Prefecture Hitachi Measuring Instruments Co., Ltd. Work part in the F-term (reference) 4B024 AA19 BA80 HA19 4B029 AA27 4D017 AA11 BA07 CA05 CB10 DA02 EA10 EB01 EB05 4G019 FA11 FA13

Claims (16)

[Claims] 1. A chip provided with a structure for separating nucleic acids from a liquid sample, wherein the flow path contains a structure containing silicon oxides and having holes larger in diameter than the silicon oxides. . 2. A chip provided with a structure for separating nucleic acids from a liquid sample, wherein the structure is such that silicon oxide particles are complexed on the surface of resin particles serving as nuclei, and the composite particles are three-dimensionally formed. A chip which is formed by bonding to the liquid sample and includes the structure in a flow path of the liquid sample. 3. The chip according to claim 1, wherein said resin particles have a diameter of not less than 50 μm and not more than 1000 μm. 4. The chip according to claim 1, wherein said structure is a porous body. 5. A chip provided with a structure for separating nucleic acids from a liquid sample, wherein the structure combines silicon oxide particles on the surface of a particle serving as a nucleus, and the composite particles are bonded in a three-dimensional manner. A chip characterized by the following. 6. A structure for capturing a nucleic acid in the liquid sample by flowing the liquid sample, the structure including silicon oxides and having pores larger in diameter than the silicon oxides. The feature structure. 7. The structure according to claim 6, wherein the structure is a porous body. 8. The structure according to claim 6, wherein said silicon oxide particles have a diameter of 0.001 μm or more.
A structure having a thickness of 0 μm or less. 9. A method for forming a structure for capturing nucleic acids from a liquid sample, comprising the steps of: mixing silicon oxide particles, an organic substance, and a sol-gel solution containing a silicon compound into a mold; A method for forming a structure, comprising: a step of subjecting the sol-gel solution to polycondensation to form a mixed material into a compact, and removing the material from the compact; and a step of heat-treating the compact to burn organic matter. 10. A method for forming a structure for capturing nucleic acid from a liquid sample, comprising: mixing resin particles, silicon oxide particles having a smaller diameter than the resin particles, and an organic solvent; A method for forming a structure, comprising the steps of: placing these in a mold, heat-treating the resin particles at a temperature at which the resin particles fuse to each other, and then removing the mold from the mold. 11. A method for forming a structure for capturing nucleic acids from a liquid sample, comprising: immersing a porous material in a sol-gel solution containing a silicon compound; And subjecting the sol-gel solution to polycondensation. 12. A method for forming a structure for capturing a nucleic acid from a liquid sample, comprising the steps of: combining silicon oxide particles on the surface of resin particles having a diameter larger than that of the silicon oxides; A step of forming the structure by fusing the resin particles to each other by putting the composited particles into a mold and heat-treating the resin particles at a temperature equal to or higher than the heat resistance temperature of the resin particles; and A method for forming a structure, comprising: immersing the structure in a solution; and removing the structure from the sol-gel solution and subjecting the sol-gel solution to polycondensation by heat treatment. 13. A method for forming a structure for capturing nucleic acids from a liquid sample, comprising the steps of: combining silicon oxide particles with resin particles having a diameter larger than that of the silicon oxide particles; A structure having a step of mixing the composited particles with a sol-gel solution containing a silicon compound and placing the mixed particles in a mold; Formation method. 14. The method for forming a structure according to claim 10, wherein said silicon oxide particles have a diameter of 0.001 μm to 10 μm.
A method for forming a structure, wherein the thickness is 0 μm or less. 15. The method for forming a structure according to claim 10, wherein the diameter of the organic substance is not less than 50 μm and not less than 1000 μm. 16. The method for forming a structure according to claim 10, wherein a particle diameter of said silicon oxides is 1/1 of a diameter of said resin particles.
A method for forming a structure, wherein the number is 0 or less.