JP2006247492A - Solid-phase synthesis apparatus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a solid-phase synthesis apparatus without generating troubles such as lowering a yield of a synthesis reaction even if a particle diameter of a carrier particle using for the solid-phase synthesis is small or an amount of carrier particles to be received in a column is plenty. <P>SOLUTION: The solid-phase synthesis apparatus is used for contacting a predetermined liquid to a plurality of carrier particles 2 to thereby synthesize predetermined substances on the surfaces of the carrier particles 2. The solid-phase synthesis apparatus stores the carrier particles 2, and has a column 1 where the liquid passes through and a dispersing mechanism (8, 10, 15, or the like) for making the carrier particles 2 disperse in the column 1. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a solid-phase synthesizer suitable for synthesizing a molecule on a carrier particle by using a solid-phase synthesis method, and in particular, recognizes a large number of functional molecules used for genetic diagnosis and physiological function diagnosis. The present invention relates to a solid-phase synthesizer that is most suitable for producing reaction detection particles, reaction detection chips, and the like.

In a variety of biotechnology application fields, chemically synthesized DNA fragments, ie chemically synthesized oligonucleotides, play an important role. At present, this chemical synthesis is generally performed by a phosphoramidite method using a solid phase method. The phosphoramidite method using the solid phase method is a method of chemically synthesizing oligonucleotides on the surface of carrier particles, which is a solid phase, using a condensation reaction of phosphoramidite. Specifically, the following procedure is used. Consists of.
(1) An active group such as an amino group is introduced on the surface of the carrier particle.
(2) The phosphoramidite corresponding to the first base in the oligonucleotide sequence is bound through this active group.
(3) The trityl group of the phosphoramidite is eliminated with a trichloroacetic acid solution or the like to expose the hydroxyl group (detrityl).
(4) The carrier particles are washed with acetonitrile or the like.
(5) The phosphoramidite corresponding to the base to be bonded next is bonded to the hydroxyl group exposed in step (3) by a condensation reaction using a tetrazole solution or the like as a catalyst.
(6) The carrier particles are washed with acetonitrile or the like.
(7) A hydroxyl group that has not undergone the condensation reaction is acetylated with an acetic anhydride solution or the like to make it inactive (capping).
(8) The carrier particles are washed with acetonitrile or the like.
(9) Oxidation of trivalent phosphoric acid to pentavalent phosphoric acid with iodine solution or the like.
(10) The carrier particles are washed with acetonitrile or the like.
(11) By repeating the condensation reaction cycle steps (3) to (10), the chain is elongated until it becomes a chain having a desired length.
(12) The oligonucleotide chain is completed by subjecting this chain to a protective group elimination treatment using aqueous ammonia or the like.

  As the carrier particles, porous glass particles are usually used. This is because the glass material has mechanical and chemical stability that can withstand the above condensation reaction, the pores of the porous glass become a suitable reaction field for the condensation reaction, excellent surface modification, and activity. This is because it is easy to introduce a group. The phosphoramidite condensation reaction is carried out in a dedicated automatic synthesizer. In this automatic synthesizer, a column is usually used as a reaction chamber, and porous glass particles are accommodated in this column. The column containing the porous glass particles is connected to a tube that supplies a chemical solution such as a phosphoramidite solution, and the porous glass particles are brought into contact with the chemical solution by flowing the chemical solution into the column. At the inlet and outlet of the column, filters that allow the chemical solution to pass but not the porous glass particles are installed.

  The reason why the chemical solution is supplied to the porous glass particles by such a method is that the various types of chemical solutions to be used can be smoothly switched in the column, and a certain amount of pressure is applied to the chemical solution. And that the chemical solution easily penetrates into the porous glass particles, and that it is easy to realize a sealed structure that eliminates atmospheric moisture that adversely affects the reaction. As described above, the conventional automatic synthesizer is designed so that the phosphoramidite method using the solid phase method can be performed satisfactorily, but there is a problem that the particle size of usable porous glass particles is limited. It was. This situation will be described below.

  In the conventional automatic synthesizer, the porous glass particles that can be used have a particle size of 100 μm or more from the viewpoint of easy flow of the chemical in the column. When the porous glass particles are simply used as a synthesis field and the synthesized oligonucleotide chains are used separately from the porous glass particles, such a particle size restriction is not a problem at all.

  On the other hand, when the porous glass particles are used in a state where the synthesized oligonucleotides are supported, and the particle size at this time is required to be less than 100 μm, the restriction of the particle size becomes a problem. For example, this is a case disclosed in Patent Document 1, in which a probe molecule is supported on a surface including pores of porous glass particles, and a reaction detection chip in which the porous particles are arranged and fixed on a substrate is produced. This reaction detection chip has an advantage that it has an overwhelming advantage in the signal intensity from the detection target combined with the probe because the probe is arranged three-dimensionally. For this reason, at present, this type of reaction detection chip has been vigorously developed.

  If the particle size of the porous glass particles used in the production of the reaction detection chip is not several tens μm or less, inconvenience occurs. This is because if the amount of protrusion of the spots formed from the aggregate of porous glass particles from the substrate surface is too large, a small amount of sample solution dropped on the spot array portion during the hybridization is removed from the cover glass. This is because it becomes difficult to spread evenly by covering.

  However, when an oligonucleotide probe is synthesized on a porous glass particle having a particle size of several tens of μm or less with a conventional automatic synthesizer, a problem due to difficulty in flowing a chemical solution in the column occurs. The flow of the chemical solution in the column is defined by the form of the flow path formed from the gap between the accommodated porous glass particles. The cross-sectional area of the flow path is considered to be proportional to the square of the particle diameter of the porous glass particles on average. The number of the flow paths is considered to be proportional to the minus square of the particle diameter of the porous glass particles. The length of the flow path is considered to be constant regardless of the particle size of the porous glass particles. This is because the distance that the flow path detours away from the longitudinal direction of the column is proportional to the particle size, and the frequency of this detour is inversely proportional to the particle size.

  According to Hagen-Poiseuille's law regarding the flow rate in a circular pipe, the flow resistance is proportional to the minus square of the cross-sectional area of the flow path and proportional to the length of the flow path. Therefore, when the particle size of the porous glass particles is reduced from 100 μm to 10 μm, the flow resistance per channel is 1/100 times the channel cross-sectional area and the channel length is 1 time. 10,000 times. Then, considering that the number of flow paths is 100 times, it is estimated that the flow resistance of the porous glass particle population is 100 times.

  In the actual phosphoramidite method, it has been confirmed that when the particle size of the porous glass particles is reduced from 100 μm to 10 μm, the liquid flow resistance of acetonitrile or the like becomes about 100 times. Thus, when the particle size of the porous glass particles is reduced to 1/10, the liquid flow resistance (resistance to the liquid flow) in the column increases by a factor of 100. The problems associated with this increase will be described separately for cases where the chemical solution is fed with a constant pressure pump and with a constant flow pump.

(1) When a constant pressure pump is used When the liquid flow resistance in the column increases 100 times, the chemical flow rate decreases to 1/100. Therefore, unless the liquid feeding time corresponding to the reaction time is changed, the synthesis yield is greatly reduced. On the other hand, if the liquid feeding time is increased by 100 times, the synthesis yield is improved to some extent, but the processing time is naturally greatly increased by 100 times. And when reaction time becomes 100 times longer, side reactions other than the objective will occur and the synthetic | combination yield will be reduced.

(2) When a constant flow pump is used When the resistance to the liquid flow in the column is increased 100 times, the chemical pressure is increased 100 times in the column and the upstream liquid supply passage. By receiving this high chemical pressure, the porous glass particles are more densely packed. Therefore, a gap is generated between the inner wall of the column and the porous glass particle lump. Since this gap becomes a flow path with a small flow resistance, the chemical solution is unevenly distributed. Therefore, the chemical solution is excessively supplied outside the porous glass particle mass, and the chemical solution is excessively supplied inside the porous glass particle mass. Therefore, the synthesis yield decreases when viewed from the whole column. Further, the increase in the chemical pressure causes an excessive load on the liquid supply flow path such as the pump, the liquid supply tube, and the connector, and also accelerates the deterioration of the apparatus.

  As described above, when an oligonucleotide is synthesized on porous glass particles having a particle size of several tens of μm or less, the conventional automatic synthesizer has a problem. In addition, even when the porous glass particles have a particle size of 100 μm or more, when a large amount of porous glass particles is synthesized in one column, the resistance to liquid flow in the column increases, so that the small particle size The same problem as in the case of.

JP 2001-281251 A

  The present invention has been made in view of the above-described problems. That is, even if the particle size of the carrier particles used for the solid phase synthesis is small or the amount of carrier particles accommodated in the column is large, there is no problem such as a decrease in the yield of the synthesis reaction. It is an object of the present invention to provide

  In order to achieve the above-described object, one aspect of the present invention is a solid-phase synthesis apparatus that synthesizes a predetermined substance on the surface of a carrier particle by bringing a predetermined liquid into contact with the plurality of carrier particles. A solid phase synthesizer comprising a column that contains carrier particles and through which the liquid passes, and a dispersion mechanism that disperses the carrier particles in the column.

In a preferred aspect of the present invention, the dispersion mechanism causes the carrier particles to exert a force that counteracts the resistance force that the carrier particles receive from the liquid flow.
In a preferred aspect of the present invention, the canceling force is a centrifugal force.
In a preferred aspect of the present invention, the dispersion mechanism includes a column installation table on which the column is installed, and a rotation mechanism that rotates the column installation table, and the column has a radial direction of the column installation table. The rotation mechanism generates a centrifugal force by rotating the column installation table around a rotation axis perpendicular to the radial direction.

In a preferred aspect of the present invention, the rotational speed of the column mounting table is varied around a value corresponding to the canceling force.
In a preferred aspect of the present invention, the apparatus further includes a vibration mechanism that vibrates the column in the radial direction.
In a preferred aspect of the present invention, the rotation mechanism repeatedly rotates the column mounting table alternately clockwise and counterclockwise.

In a preferred aspect of the present invention, the canceling force is a force that the carrier particles receive from an external field.
In a preferred aspect of the present invention, the strength of the external field is varied around a value corresponding to the canceling force.
In a preferred aspect of the present invention, the column is vibrated in the flow direction of the liquid.

In a preferred aspect of the present invention, the carrier particles are magnetic particles, and the external field is a magnetic field.
In a preferred aspect of the present invention, the carrier particles have a particle size of 50 μm or less.
In a preferred aspect of the present invention, the carrier particles are porous glass particles.
In a preferred aspect of the present invention, the predetermined substance is synthesized on the surface of the carrier particle using a phosphoramidite method.

In the present invention, the carrier particles are dispersed in the column by acting on the carrier particles a force that counteracts the resistance force that the carrier particles receive from the liquid flow. Furthermore, the dispersion of the carrier particles is promoted by vibrating the carrier particles in a balanced state of force. This dispersion action induces the following two effects.
(A) Since the gap between the carrier particles accommodated in the column widens, the resistance to the liquid flow in the column decreases.
(B) Since the carrier particles do not agglomerate due to aggregation, uneven distribution of the liquid flow around and inside the mass, which occurs when the carrier particles are agglomerated, is prevented, and the liquid flow becomes uniform in the column.
Therefore, even when small carrier particles are used or when a large amount of carrier particles are accommodated in the column, an increase in resistance to the liquid flow in the column is prevented, and problems during synthesis are eliminated.

  A specific method for dispersing the carrier particles in the column is described below. FIG. 1 is a cross-sectional view showing a column incorporated in the solid phase synthesis apparatus of the present invention. As shown in FIG. 1, the column 1 has a cylindrical shape, and an upstream filter 4 and a downstream filter 5 are disposed at both ends of the column 1. The carrier particles 2 are accommodated in a space defined by the inner peripheral surface of the column 1, the upstream filter 4, and the downstream filter 5. The upstream filter 4 and the downstream filter 5 are formed with minute openings, respectively, so that the upstream filter 4 and the downstream filter 5 allow only the liquid to pass without passing through the carrier particles 2. . The upstream filter 4 and the downstream filter 5 are respectively attached to the distal ends of the connectors 6, and the upstream filter 4 and the downstream filter 5 are attached to the column 1 by fitting these connectors 6 into the column 1. It is like that. A liquid feed tube (not shown in FIG. 1) is connected to the connector 6, and a liquid (chemical solution or the like) sent from the liquid feed tube passes through the column 1.

  In the present invention, the carrier particles 2 are accommodated between the upstream filter 4 and the downstream filter 5 of the column. In this case, the carrier particles 2 are accommodated by providing a certain gap so that the carrier particles 2 are dispersed in the column 1. Then, an acting force (canceling force) for conveying the carrier particles 2 to the upstream side against the liquid flow is given to the carrier particles 2. Without this acting force, the carrier particles 2 are unevenly distributed on the downstream filter side due to the liquid flow.

  The gap for dispersion is preferably increased from the viewpoint of increasing the effect (a). However, from the viewpoint of reducing the amount of liquid used, it should be avoided to make it extremely large. This is because, when replacing the liquid with another liquid, it is necessary to supply the liquid by an amount corresponding to the gap. Therefore, the volume of the void is preferably set to about 1 to 10 times the volume of the filled part of the carrier particles.

Next, let us consider the acting force for conveying the carrier particles upstream against the liquid flow. The carrier particles dispersed in the column, that is, the carrier particles floating without being biased downstream or upstream, receive the following force f from the liquid flow.
f = v / β = 6πηav (β = 1 / (6πηa)) (i)
Here, v is the speed of the liquid flow, η is the viscosity coefficient of the chemical solution, and a is the radius of the carrier particles.
The ratio of this force f to gravity mg (m: mass of carrier particles, g: gravitational acceleration) is as follows.
f / mg = (9ηv) / (2a ² ρg) (ii)
It becomes. As typical values, when η = 10 ⁻² g / (cm · s), v = 1 cm / s, a = 5 μm = 5 × 10 ⁻⁴ cm, ρ = 2 g / cm ³ is substituted into the above formula (ii), f / mg is determined to be about 100.

  That is, when the particle size of the carrier particles is 10 μm, which is 1/10 of the normal size, the resistance force received from the flow is about 100 times that of gravity, while the particle size of the carrier particles is 100 μm, which is normal. In the case of a size, the resistance force received from the flow is about the same as gravity. Therefore, when the particle size of the carrier particles is a normal size, for example, the resistance force received from the flow can be canceled by gravity only by standing the column vertically and allowing the liquid to flow from the bottom to the top. On the other hand, when the particle size of the carrier particles is as small as 10 μm, a force as large as 100 times the gravity is required to cancel out the resistance force received from the liquid flow, and special measures must be taken. This idea is described below.

[Centrifugal force]
One method for canceling the resistance force is to fix the column to the disk so that the flow direction of the liquid in the column coincides with the radial direction of the disk, and to connect the disk to the axis of rotation perpendicular to the disk. By rotating around, a centrifugal force is exerted on the carrier particles in the column. In this case, if the liquid is fed into the column from the outside of the disk toward the center, the resistance force received by the carrier particles from the liquid flow is opposite to the centrifugal force. Therefore, it is possible to balance the resistance force and the centrifugal force by adjusting the magnitude of the centrifugal force. In order to make this centrifugal force 100 times the gravity, for example, a column may be fixed at a position 10 cm from the center of the disk, and this disk may be rotated at 1000 min ⁻¹ .

[Action of external field]
Another way to counteract the drag force is to force the carrier particles from an external field. For example, a magnetic field is applied to the magnetic carrier particles to cancel the resistance force received from the liquid flow by the magnetic force. When the carrier particles are porous glass particles, examples of a method for imparting magnetism to the carrier particles include a method of dispersing iron nanoparticles in glass during the production of porous glass. In general, since electromagnetic force can be made much larger than gravity, means using a magnetic field or an electric field is also effective from the viewpoint of canceling a large resistance force received from a liquid flow. Further, the apparatus configuration can be simplified as compared with the apparatus using the disk described above. However, the carrier particles that can be used are greatly limited.

[vibration]
As yet another method for canceling the resistance force, a method of vibrating the column in parallel with the flow direction of the chemical solution can be considered. The vibration energy and vibration mode required to counteract the resistance force that the carrier particles receive from the liquid flow, which is as large as 100 times the gravity, will be described below.
In this method, it is the collision action between the downstream filter and the carrier particles that counteracts the resistance force received from the liquid flow. Downstream filter oscillating at Asinωt is, momentum given to the carrier particles by the collision, when the speed immediately before the collision of the downstream carrier particles as viewed from the filter and v _c, a 2 mv _c Assuming perfectly elastic collision. Therefore, the momentum exerted by the downstream filter on the entire carrier particles in the column per unit time, that is, the collision force Fc is calculated by the following equation considering that the collision occurs when the downstream filter is displaced toward the upstream side. It is represented by
Fc = 2m <v _c > · 2AS · n · ν (iii)
Here, S: column cross-sectional area, n: carrier particle concentration, ν = ω / (2π). In the formula (iii), <v _c > represents an average value of v _c of the carrier particles that collide.

Here, the equation of motion for the carrier particles dispersed in the vibrating liquid as in the column is
m · dv _p / dt = − (v _p −ωA cos ωt) / β (iv)
It is. By solving this equation of motion, the velocity v _p of the carrier particles can be obtained as follows.
v _p = (γA / mβ) cos (ωt−θ) (v)
However, γ = mωβ / (1+ (mωβ) ² ) ^1/2 , θ = tan ⁻¹ (mωβ)
Therefore, the _{v c} of the carrier particles,
v _c = | v _p −ωA cos ωt |
= | Mβ · dv _p / dt | (from formula (iv))
= | ΓωAsin (ωt−θ) | (Substitute formula (v)) (vi)
Next, the average value of v _c of carrier particles,
<V _c > = (2 / π) γωA (vii)
Therefore, the following equation is obtained by substituting equation (vii) into equation (iii).
Fc = 16γmnS (νA) ² (viii)

On the other hand, the force Fr with respect to the entire carrier particles in the column, which is necessary for the carrier particles to float and disperse in the column without being pushed downstream, is obtained from the equation (i) as follows.
Fr = (v / β) nSL (ix)
However, L: Column length The conditions for dispersing carrier particles in the column are as follows:
Fc ≧ Fr (x)
Therefore, by substituting the expressions (viii) and (ix) into the expression (x), νA ≧ {vL / (mβγ)} ^1/2 / 4 (xi)
Thus, vibration conditions for dispersing the carrier particles in the column can be obtained.

In the system currently considered, the resistance force v / β that the carrier particle receives from the liquid flow is about 100 times the gravity, that is, 100 × mg, as described above. The column length L is about 1 cm. By substituting these values into equation (xi),
νA ≧ 80γ− ¹ / ² cm / s (xii)
It becomes.

  Γ defined by the equation (v) increases monotonously in the range of 0 to 1 as the frequency ν increases, and can be regarded as 1 when ν is 10 kHz or more. Therefore, the right side of the equation (xii) monotonously decreases from infinity to 80 cm / s as the frequency ν increases. Thus, νA, that is, the lower limit of (frequency) × (amplitude) can be reduced as the frequency ν increases, but it is understood that about 100 cm / s is necessary. This condition means that, for example, when the frequency is 10 kHz, the amplitude must be about 100 μm or more. That is, in order to disperse carrier particles having a particle diameter of 10 μm in a column against a liquid flow, such an unrealistic intense vibration must be applied to the column.

  From the above considerations, centrifugal force and acting force of external field are effective to counteract the large resistance force about 100 times the gravity that the carrier particles receive from the liquid flow, and mechanical vibration to the column is unrealistic. You can see that However, only by canceling the liquid flow resistance force by centrifugal force or external force, the carrier particles are dispersed only by diffusion due to the concentration gradient of the carrier particles in the column, and the state shifts to the dispersed state. Takes a long time. Therefore, it is preferable that the centrifugal force and the external field acting force are not constant but are changed with time. By changing the centrifugal force and the external field acting force, the carrier particles in the column are vibrated, and can be quickly transferred to the dispersed state. Alternatively, centrifugal force or external field acting force may be constant and mechanical vibration may be applied to the column. In this case, in the formula (xi), the value of v / β = | resistance force-centrifugal force (or external field action force) | received from the liquid flow is set to be zero, so that the carrier particles are quickly dispersed. For this purpose, a very small νA, that is, a very gentle vibration is sufficient.

Next, it is specifically shown how the resistance of the liquid flow is reduced by dispersing the carrier particles throughout the column.
A cylindrical column is not filled with carrier particles but is provided with voids. Here, the volume of the void is assumed to be x times the volume of the carrier particle filling portion. The volume of the carrier particle filling portion includes the volume of the gap between the carrier particles. That is, the carrier particles are accommodated by 1 / (x + 1) of the column volume, including the gap volume when stacked. Further, the gap ratio between carrier particles in the carrier particle filling portion (= 1− (carrier particle filling ratio)) is y. Let us calculate how much the liquid flow resistance is reduced by shifting from the state in which the carrier particles are accommodated to the state in which they are dispersed throughout the column.

Again, this is based on a model that considers the connection of gaps between carrier particles as a pseudo channel. In this model, the liquid flow resistance for the entire carrier particles in the column is
Flow resistance ∝ (channel length) x (channel cross-sectional area) ^-2 x (number of channels) ^-1 (xiii)
It can be expressed. The reason why the flow resistance is proportional to the negative square instead of the negative square of the channel cross-sectional area is due to Hagen-Poiseuille's law regarding the flow rate in the circular pipe.

The occupied volume in the column per carrier particle becomes (x + 1) times by shifting to the dispersed state. Therefore, the average distance between the carrier particles is (x + 1) ^1/3 times. Therefore, since the occupied cross-sectional area in the column corresponding to one flow path is (x + 1) ^2/3 times, the number of flow paths can be regarded as (x + 1) ^−2/3 times.

The number of carrier particles per column length is (x + 1) / (x + 1) ^1/3 = (x + 1) ^2/3 times, which means that the flow path length is (x + 1) ^2/3 times. To do. In addition, since the gap volume between carrier particles in the column is (x + y) / y times,
Gap volume = (channel cross-sectional area) × (channel length) × (number of channels) (xiv)
From the relationship, it can be considered that the flow path cross-sectional area becomes (x + y) / y times.

Above by substituting the result into equation (xiii), the liquid flow resistance is ^{y 2 (x + 1) 4/3} / (x + y) is ^doubled. When graphing by substituting y = 0.26 at the time of closest packing, it can be seen that the liquid flow resistance is as shown in FIG. 2, and the liquid flow resistance is reduced to 1/50 at x = 7. That is, when the carrier particles are accommodated in the column by 1/8 of the column volume and dispersed throughout the column, the liquid flow resistance with respect to the entire carrier particles in the column can be reduced to 1/50 when not dispersed. . In other words, even when the particle size is 10 μm, which is 1/10 of the size normally used, and therefore the liquid flow resistance is 100 times the normal, the liquid flow resistance can be converted to a normal level by using this dispersion method. can do.

  According to the present invention, solid phase synthesis with good synthesis yield and synthesis efficiency can be realized even when the particle size of carrier particles for solid phase synthesis is small or when synthesis is performed on a large amount of carrier particles. It becomes possible to provide a solid-phase synthesis apparatus.

Hereinafter, a solid phase synthesizer according to an embodiment of the present invention will be described with reference to the drawings. However, the present invention is not limited only to the following embodiments.
First, the case where centrifugal force is used as the force that counteracts the resistance force that the carrier particles receive from the liquid flow will be described below. FIG. 3A is a front view showing a synthesis unit of the solid phase synthesizer according to the first embodiment of the present invention, and FIG. 3B is a cross-sectional view taken along line III-III in FIG. .

  As shown in FIG. 3A and FIG. 3B, the synthesizer 11 includes the column 1 shown in FIG. 1, the disk-shaped column mounting base 8, and a liquid feeding tube (feeding liquid to the column 1). Liquid flow path) 9. The column 1 is arranged on the column installation table 8 along the radial direction of the column installation table 8. The column mounting table 8 is rotatable in a vertical plane, and both end portions of the liquid feeding tube 9 extend so as to coincide with the rotation axis of the column mounting table 8. Further, the central portion of the liquid feeding tube 9 extends in the radial direction of the column mounting base 8 and further penetrates the column mounting base 8. Both ends of the column 1 are connected to a liquid feeding tube 9 via a connector 6 (see FIG. 1).

  The column 1 of this embodiment has an inner diameter of 5 mm and a length of 10 mm, and 30 mg of porous glass particles (carrier particles) having a particle size of 10 μm are accommodated therein. Between the upstream filter 4 and the downstream filter 5, that is, the effective column length is 10 mm. About 1/4 of this effective column length of 10 mm is a packed portion of the porous glass particles 2, and the remaining about 3/4 is a void portion.

  The main part of the synthesizer 11 is a disk-shaped column mounting table 8 having a diameter of about 30 cm. The column 1 is installed so that the radial direction of the column mounting table 8 coincides with the center line of the column 1. The distance between the center of the column mounting base 8 and the center of the column 1 is about 10 cm. A liquid (for example, a chemical solution) is fed from the outside of the column mounting base 8 toward the center, and simultaneously with the liquid feeding, the column mounting base 8 is rotated about its rotation axis. At this time, the porous glass particles 2 (see FIG. 1) are subjected to a resistance force due to the liquid flow from the outside of the column mounting base 8 toward the center, and from the center of the column mounting base 8 to the outside. Centrifugal force due to rotation of the column mounting base 8 works. The rotational speed of the column mounting base 8 is adjusted so that these forces are balanced. In this way, by applying a centrifugal force to the porous glass particles 2, the porous glass particles 2 float in the column 1 without being pressed against the downstream filter 5.

  The column mounting base 8 is preferably rotated in the vertical plane rather than in the horizontal plane. This is because the rotation in the horizontal plane causes the porous glass particles 2 to settle on the side surface of the column 1 due to gravity, thereby preventing complete dispersion of the porous glass particles 2 in the column 1.

  As shown in FIGS. 4A and 4B, the column mounting table 8 is rotated by four rollers 10 arranged around the column mounting table 8. These rollers 10 are provided on the fixed frame 12 and are arranged so as to be in contact with the outer peripheral surface of the column mounting base 8. Then, one of the four rollers 10 is driven by the driving unit 15. The drive unit 15 is controlled by the drive control unit 14. The fixed frame 12 is supported by a support base 13 on the drive control unit 14. The reason why the driving force is applied to the column mounting base 8 from the outer peripheral portion is to avoid contact between the roller 10 and the liquid feeding tube 9. In the present embodiment, the roller 10 and the drive unit 15 constitute a rotation mechanism, and the column installation base 8, the roller 10, and the drive unit 15 constitute a dispersion mechanism.

  FIG. 5A and FIG. 5B are diagrams showing a solid phase synthesizer showing a state in which a waste liquid recovery unit and a liquid feeding mechanism are connected to a liquid feeding tube. As shown in FIGS. 5A and 5B, the column side end of the liquid feeding tube 9 is connected to the waste liquid recovery unit 17, and the opposite end is connected to the liquid feeding mechanism 18. . Furthermore, the liquid feeding mechanism 18 is connected to a liquid storage unit 20 in which a liquid such as a chemical solution is stored. Since both end portions of the liquid feeding tube 9 rotate around the rotation axis of the column mounting base 8, the liquid feeding tube 9 is connected to the waste liquid collecting unit 17 and the liquid feeding mechanism 18 through the rotary joint 19. Here, it is preferable to make the length of the liquid feeding tube 9 upstream of the column 1 as short as possible. The reason is that the amount of liquid replacement required for liquid switching is small.

  According to the present embodiment, the porous glass particles 2 can be suspended in the column 1 by balancing the liquid flow resistance and the centrifugal force, but in order to quickly shift from the suspended state to the dispersed state, It is preferable to employ the following means.

The first means is to vary the rotation speed of the column mounting base 8 around the rotation speed when the liquid flow resistance and the centrifugal force are balanced. Specifically, the rotation speed of the column mounting base 8 is periodically changed at a change rate of about ± 10% of the rotation speed at which the liquid flow resistance and the centrifugal force are balanced. For example, when the rotational speed to balance the liquid flow resistance and centrifugal force is 1000min ^-1 is a column installing base 8 is rotated at 1000 ± 100 min ^-1, varying the rotational speed in a cycle of about 5 seconds.

  The second means is to provide the column installation base 8 with a vibration mechanism 16 that mechanically vibrates the column 1. FIG. 6 is a front view illustrating a configuration example of a synthesis unit including a vibration mechanism. In this configuration example, the column 1 is vibrated in the radial direction by the vibration mechanism 16 at the same time as the column mounting base 8 is rotated. At this time, since the resistance force from the liquid flow received by the porous glass particles 2 is canceled out by the centrifugal force, the porous glass particles 2 can be quickly dispersed with extremely small vibration energy. As described above, since the magnitude of the vibration energy is extremely loose, the vibration mechanism 16 uses a conversion of revolving motion into reciprocating motion, electromagnetic force, elastic force, piezoelectric / electrostrictive / magnetostrictive, ultrasonic wave, etc. Can be used.

Next, a procedure for synthesizing an oligonucleotide on the surface of the porous glass particle 2 by the phosphoramidite method using the solid phase synthesizer according to the present embodiment will be described below.
First, an amino group is introduced into the porous glass particle 2 to bind a phosphoramidite corresponding to guanine. Subsequently, 30 mg of this porous glass particle 2 with guanine is accommodated in the column 1 and set on the column mounting table 8. Using a constant flow pump as the liquid feeding mechanism 18, a chemical solution (see “Background Art” column) used in a normal phosphoramidite method is fed to the column 1. At this time, the flow rate of the chemical solution is adjusted so that the flow rate in the column 1 is 1 cm / s.

Simultaneously with the feeding of the chemical solution, rotation of the column mounting base 8 is started. At this time, the column mounting base 8 is rotated at a rotational speed of 1000 + 100 sin ((2π / 5) t) min ⁻¹ (where the symbol t represents time (seconds)). That is, varying from 900 min ^-1 to rotational speed of the column installation base 8 at a period 5 seconds between 1100min ^-1. In this case, the rotational speed of 1000 min ⁻¹ is a rotational speed at which the centrifugal force acting on the porous glass particles 2 and the resistance force caused by the liquid flow acting on the porous glass particles 2 are just balanced (cancelled).

  In an actual test, it was confirmed that the porous glass particles 2 were completely dispersed in the column 1 after several seconds from the start of liquid feeding. Then, by rotating the column mounting base 8, while maintaining this dispersed state, the phosphoramidite corresponding to guanine is subjected to 19-stage condensation reaction to produce a 20-mer guanine chain as an oligonucleotide.

  Here, the present embodiment in which the porous glass particles 2 are synthesized while being dispersed throughout the column 1, and the conventional method in which the porous glass particles 2 are synthesized while being pressed against the downstream filter 5 of the column 1 by a liquid flow. The test result which compared with is shown. The conventional method corresponds to the case where the synthesis is performed by stopping the rotation of the column mounting base 8 in the synthesis apparatus of the present embodiment.

  First, the pressure of the liquid at the time of liquid feeding upstream from the column 1 was reduced to 1/20 in this embodiment as compared with the conventional method. This is because the gap between the porous glass particles 2 is widened by dispersion, and the resistance of the entire porous glass particle 2 to the liquid flow is reduced to 1/20. Note that this embodiment corresponds to the case of x = 3 in FIG. 2, and the liquid flow resistance is 1/25 in the calculation. This significant pressure reduction reduces the load on the device. Further, since the pressure resistance required for the liquid feeding mechanism (pump) 18, the liquid feeding tube 9, the connector 6 and the like may be low, the degree of freedom in designing the apparatus increases, leading to cost reduction of the apparatus.

  In the present embodiment, a constant flow pump is used as the liquid feeding mechanism 18, but if a constant pressure pump is used instead, the liquid flow rate is 20 times that of the conventional method. Therefore, the synthesis time is reduced to 1/20 compared with the conventional method. That is, according to this embodiment, the synthesis using carrier particles having a small particle size of 10 μm can be performed in the same synthesis time as the normal synthesis using carrier particles having a particle size of 100 μm or more.

  In addition, the condensation reaction yield in each stage obtained from the results of the trityl group elimination monitor averaged 95% in the conventional method and 98% in the present embodiment. From this, it can be seen that the condensation reaction is favorably performed by dispersing the porous glass particles 2 throughout the column 1 during synthesis. The reason for this is that the porous glass particles 2 in the column 1 are not agglomerated due to agglomeration, so that the uneven distribution of liquid flow around and inside the mass, which occurs when agglomerated, is prevented, and the liquid flow in the column 1 is prevented. It is because it became uniform.

  In this embodiment, for the purpose of quickly dispersing the porous glass particles 2 in the column 1, the rotational speed of the column mounting base 8 is varied. It is also achieved by giving to. Since the porous glass particles 2 are in a floating state in which no force is received in the column 1, the restriction of vibration energy is loose. As a vibration mechanism, conversion of rotational motion into reciprocating motion, electromagnetic force, elastic force, piezoelectricity is possible.・ Use of electrostriction, magnetostriction, ultrasonic waves, etc. In order to disperse the porous glass particles 2 quickly, the frequency and amplitude of a certain level or more are required. However, if the frequency is too high, the porous glass particles 2 cannot follow the vibration, which makes no sense. In addition, if the amplitude is too large, the device configuration becomes unreasonable and the supplied energy becomes excessive. From such a viewpoint, it is preferable to determine the frequency of vibration for promoting dispersion in the range of 100 to 10000 Hz and the amplitude in the range of 10 to 1000 μm.

  Next, a second embodiment of the present invention will be described with reference to FIG. FIG. 7 is a front view showing a solid phase synthesis apparatus according to the second embodiment of the present invention. Note that the configuration of the present embodiment that is not particularly described is the same as that of the first embodiment described above, and thus redundant description thereof is omitted. As in the first embodiment, this embodiment uses centrifugal force as a force that counteracts the resistance force that the carrier particles receive from the liquid flow. However, this embodiment exemplifies a method that eliminates the need for using a rotary joint by adopting repetitive rotation that alternately repeats clockwise and counterclockwise rotation, instead of continuously rotating the column mounting base in one direction.

  As shown in FIG. 7, the column mounting base 28 in the present embodiment has a rod shape, and rotates around the rotating shaft 21 alternately clockwise and counterclockwise. That is, the column installation base 28 is configured to be periodically swung around the rotation center 21 by a step motor (rotation mechanism) 34. On the column mounting base 28, the column 1 and the liquid feeding tube 9 are arranged. The liquid feeding tube 9 has a U-shape extending in the radial direction (longitudinal direction of the column mounting table 28). Both ends of the column 1 are connected to the liquid feeding tube 9, and the column 1 is disposed along the radial direction of the column installation base 28. The distance between the center of the column 1 and the rotating shaft 21 is 10 cm.

  Both end portions of the liquid feeding tube 9 are connected to the liquid feeding mechanism 18 and the waste liquid collecting unit 17 via the connection tube 30, respectively. The connection tube 30 is flexible enough to withstand the intense movement caused by repeated rotation of the column mounting base 28. The column mounting base 28 is preferably rotated repeatedly in the vertical plane. This is because when the column mounting base 28 is rotated in a horizontal plane, the carrier particles settle on the side surface of the column 1 due to gravity, thereby preventing complete dispersion of the carrier particles in the column 1.

A liquid such as a chemical solution flows in the column 1 in a direction toward the rotation axis (rotation center) 21, and at the same time, the column mounting base 28 swings around the rotation axis 21. If the vibration frequency of the column mounting table 28 at this time is ν and the amplitude of the rotation angle of the column mounting table 28 is 30 ° = π / 6 radians, the rotation angle θ of the column mounting table 28 is expressed by the following equation.
θ = (π / 6) sin (2πνt) (xvi)
However, t shows time (second).
Therefore, the time average value <dθ / dt> of the angular velocity in this repeated rotation is
<Dθ / dt> = 2πν / 3 (xvii)
It becomes. Therefore, the time average value of the acceleration of the centrifugal force received by the carrier particles in the column 1 accompanying this repeated rotation is 10 × (2πν / 3) ² cm / s ² . If this value is set equal to the acceleration of the resistance force that the carrier particles receive from the liquid flow, that is, 100 times the gravitational acceleration, the frequency ν of the balance condition is found to be 47 Hz.

  In this embodiment, unlike the first embodiment, there is no need to use a rotary joint. Further, since the angular velocity in repeated rotation varies with time, the carrier particles 2 (see FIG. 1) such as porous glass particles vibrate in the column 1. Therefore, it is possible to quickly disperse the carrier particles in the column 1.

  Next, a solid phase synthesis apparatus according to the third embodiment of the present invention will be described. In the third embodiment, an external field action force is used as a force that counteracts the resistance force that the carrier particles receive from the liquid flow. In addition, this embodiment shows an example in which carrier particles are magnetic particles and a magnetic field is used as an external field.

  In order to apply a magnetic force to magnetic particles by a magnetic field, the magnetic field needs to have a spatial gradient. As a means for generating this magnetic field gradient in the column, there is a method of enclosing the column in a solenoid with a non-uniform number of turns so that the central axes of both coincide. The magnetic field inside the solenoid is approximately parallel to the central axis, and the magnitude can be expressed as (number of turns per unit length) × (current). Therefore, in a solenoid where the number of turns gradually increases along the central axis, the magnitude of the internal magnetic field is not uniform, and a magnetic field gradient is created, which is (the number of turns per unit length (Gradient) x (current). In such a magnetic field, a force acts on the Bohr magneton of the magnetic particles, so that a magnetic force acts on the magnetic particles.

  The direction of the magnetic force is from the smaller magnetic field to the larger magnetic field, that is, from the smaller number of turns to the larger one. Therefore, if the solenoid is arranged with respect to the column such that the side with the larger number of turns is located upstream of the liquid flow, the direction of the magnetic force is opposite to the resistance force that the carrier particles receive from the liquid flow. Therefore, by adjusting the magnitude of the current flowing through the solenoid, this magnetic force can cancel the resistance force that the carrier particles receive from the liquid flow. Hereinafter, a specific configuration of the present embodiment will be described with reference to FIGS. 8 (a) and 8 (b).

  In this embodiment, a cylindrical glass column 1 having an inner diameter of 5 mm and a length of 10 mm is used. Then, the magnetic particles 2 having a particle size of 10 μm are accommodated by ¼ of the length of the column 1, that is, until the thickness becomes 2.5 mm, and the upstream filter 4 and the downstream filter 5 are mounted. This column 1 is connected to a liquid supply path (not shown) such as a liquid supply tube. A solenoid 31 is arranged around the column 1 so that the column 1 exists inside as shown in FIG. That is, the central axis of the solenoid 31 is made to coincide with the central axis of the column 1, and the column 1 is arranged in the center of the solenoid 31. An AC power supply 33 is connected to the solenoid 31.

  As shown in FIG. 8B, the number of turns per unit length of the solenoid 31 is not constant but is increased monotonously. Therefore, the magnetic field generated in the solenoid 31 when energized is parallel to the central axis is not constant and increases as the number of turns increases along the central axis. That is, a magnetic field gradient is formed along the central axis of the column 1. The magnetic force received by the magnetic particles 2 by this magnetic field gradient is used as a force that cancels the resistance force that the magnetic particles 2 receive from the liquid flow.

The solenoid 31 has a diameter of 10 mm, a length of 30 mm, and the number of windings gradually from the end once in the first 1 mm section, twice in the next 1 mm section, three times in the next 1 mm section, and so on. The winding number gradient per unit length is increased to 1 / mm ² = 10 ⁶ / m ² . As shown in FIG. 8A, the solenoid 31 is arranged so that the side with the larger number of windings is located on the upstream side of the liquid flow. In this way, the magnetic force acting on the magnetic particles 2 is directed from the downstream side to the upstream side regardless of the direction of the current flowing through the solenoid 31. The direction of the magnetic force is opposite to the direction of the resistance force that the magnetic particles 2 receive from the liquid flow. Therefore, the resistance force that this magnetic force receives from the liquid flow can be canceled by flowing an appropriate current through the solenoid 31. In the present embodiment, the solenoid 31 constitutes a dispersion mechanism.

In order to quickly disperse the magnetic particles 2 in the column 1, it is preferable to vary the current flowing through the solenoid 31 with time. For example, it can be considered that an alternating current such as I _max sin (2πft) flows through the solenoid 31. In this case, if I _max is selected so that 2I _max / π is equal to the current value that balances the force, the magnetic particles 2 are subjected to vibrational motion in the column 1, and diffusion due to the concentration gradient of the magnetic particles 2. Dispersion proceeds more rapidly than the action alone. The frequency f may be determined within a range of 1 to 1000 Hz.

Here, the magnitude of the current value that balances the force is specifically obtained. Assume that the magnetic particles 2 have magnetism equivalent to that of iron. The mass of the magnetic particles is m, the mass density is ρ, the number of atoms constituting the magnetic particles is N, the Bohr magneton is μ, and the winding gradient per unit length of the solenoid 31 is dn / dx. If the current is I,
Magnetic force = (m / ρ) · 2Nμ · dn / dx · I (xv)
It becomes.

On the other hand, the resistance force that the magnetic particles 2 receive from the liquid flow is (9 mηv) / (2a ² ρ) from the equation (ii). If this is equal to the equation (xv), I obtained is the current value to be obtained. N = 8.49 × 10 ²⁸ / m ³ , μ = 4π × 9.27 × 10 ⁻³¹ Wb · m, dn / dx = 10 ⁶ / m ² , η = 10 ⁻³ kg / (m · s), Substituting v = 10 ⁻² m / s and a = 5 × 10 ⁻⁶ m yields I = 0.91A.

Here, since 2I _max / π = I, I _max = 1.4 A, and if power is supplied to the solenoid 31 such that an alternating current of 1.4 sin (2πft) amperes flows through the solenoid 31, magnetic particles Solid phase synthesis can be performed on the magnetic particles 2 in a state where 2 is dispersed in the column 1. For example, if a magnetic particle 2 in which a thiol group is previously introduced and a phosphoramidite corresponding to the first nucleotide is bound is set, the particle size of 10 μm is obtained by the phosphoramidite method using the synthesizer according to the present embodiment. Even if the particle size is 1/10 or less of the particle size normally used in the application, it is possible to perform good solid phase synthesis. As described in the first embodiment, since the synthesis is performed while dispersing the magnetic particles 2 in the column 1, the resistance of the liquid flow to the entire magnetic particles 2 in the column 1 is 1/20. This is due to the fact that the liquid flow is greatly reduced and the uneven distribution of the liquid flow due to the agglomeration of the magnetic particles 2 in the column 1 is prevented.

  The means for promoting dispersion is not limited to the method of modulating the current flowing through the solenoid 31. For example, it is also effective to apply a mechanical vibration to the column 1 in a direction parallel to the central axis of the column 1 as in the first embodiment while supplying a current value that can balance the force.

It is sectional drawing which shows the column integrated in the solid-phase synthesis apparatus of this invention. It is a graph which shows the relationship between the filling degree of the carrier particle in a column, and liquid flow resistance. FIG. 3A is a front view showing a synthesis unit of the solid phase synthesizer according to the first embodiment of the present invention, and FIG. 3B is a cross-sectional view taken along line III-III in FIG. . FIG. 4 (a) is a front view showing the entire solid-phase synthesizer according to the first embodiment of the present invention, and FIG. 4 (b) is a side view of the solid-phase synthesizer shown in FIG. 4 (a). is there. FIG. 5A is a front view showing the solid-phase synthesis apparatus of the present embodiment showing a state in which the waste liquid recovery unit and the liquid feeding mechanism are connected to the liquid feeding tube, and FIG. It is a side view of the solid-phase synthesis apparatus shown in FIG. It is a front view which shows the structural example of the synthetic | combination part provided with the vibration mechanism. It is a front view which shows the solid-phase synthesis apparatus which concerns on the 2nd Embodiment of this invention. FIG. 8A is a schematic view showing the main part of a solid phase synthesis apparatus according to the third embodiment of the present invention, and FIG. 8B is a perspective view of the solenoid shown in FIG.

Explanation of symbols

1 column 2 carrier particles (porous glass particles)
4 upstream filter 5 downstream filter 6 connector 8, 28 column mounting table 9 liquid feeding tube 10 roller 11 synthesis unit 12 fixed frame 13 support base 14 drive control unit 15 driving unit 16 vibration mechanism 17 waste liquid recovery unit 18 liquid feeding mechanism 19 Rotary joint 20 Liquid storage unit 21 Rotating shaft 30 Connection tube 31 Solenoid 33 AC power supply 34 Step motor

Claims (14)

A solid phase synthesizer for synthesizing a predetermined substance on the surface of a carrier particle by bringing a predetermined liquid into contact with a plurality of carrier particles,
A column containing the carrier particles and through which the liquid passes;
A solid phase synthesis apparatus comprising a dispersion mechanism for dispersing the carrier particles in the column.   The solid phase synthesis apparatus according to claim 1, wherein the dispersion mechanism causes the carrier particles to exert a force that counteracts a resistance force that the carrier particles receive from the liquid flow.   The solid-phase synthesis apparatus according to claim 2, wherein the canceling force is a centrifugal force. The dispersion mechanism includes a column installation table on which the column is installed, and a rotation mechanism that rotates the column installation table.
The column is installed along the radial direction of the column installation table,
The solid-phase synthesis apparatus according to claim 3, wherein the rotation mechanism generates a centrifugal force by rotating the column installation base around a rotation axis perpendicular to the radial direction.   The solid-phase synthesis apparatus according to claim 4, wherein the rotation speed of the column mounting base is varied around a value corresponding to the canceling force.   The solid phase synthesis apparatus according to claim 4, further comprising a vibration mechanism that vibrates the column in the radial direction.   The solid-phase synthesis apparatus according to claim 4, wherein the rotation mechanism repeatedly and repeatedly rotates the column installation table clockwise and counterclockwise.   The solid-phase synthesis apparatus according to claim 2, wherein the canceling force is a force that the carrier particles receive from an external field.   The solid-phase synthesis apparatus according to claim 8, wherein the strength of the external field is varied around a value corresponding to the canceling force.   The solid-phase synthesis apparatus according to claim 8, wherein the column is vibrated in the flow direction of the liquid.   The solid phase synthesis apparatus according to any one of claims 8 to 10, wherein the carrier particles are magnetic particles, and the external field is a magnetic field.   The solid phase synthesizer according to any one of claims 1 to 11, wherein a particle size of the carrier particles is 50 µm or less.   The solid phase synthesizer according to claim 12, wherein the carrier particles are porous glass particles.   The solid phase synthesis apparatus according to any one of claims 1 to 13, wherein the predetermined substance is synthesized on the surface of the carrier particle using a phosphoramidite method.

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