JP2003180330A - Ultrasonic sealing apparatus for microwell array - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an ultrasonic sealing apparatus for sealing a liquid filled in a well without damaging the liquid and preventing the sealing of air and provided a sealing method by using the apparatus. <P>SOLUTION: A plurality of separated wells are arranged on a plane in the form of an array on a tray. The tray is covered with a cover and sealed with an ultrasonic welding means to seal a liquid or specimen in the wells. The cover is laminated on the tray held on a table and ultrasonic vibration is applied by a horn through the cover in pressed state to weld the cover to the tray and seal the wells and obtain a sealed microwell array. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention joins a plurality of sheet-like processed materials made of a thermoplastic material,
The present invention belongs to the field of microwell array formation technology that utilizes ultrasonic welding technology that makes it possible to form a container group in which a very small amount of liquid is sealed by applying ultrasonic vibration and welding.

The microwell array expressed in this specification is defined as "an array of small (micro) wells for containing a small amount of sample arranged in high density".

[0003]

2. Description of the Related Art Conventionally, HTS is one of the techniques for selectively selecting a necessary compound from many compounds.
(High Throughput Screenin
g) There is technology.

[0004] As an example, a DNA microarray for the purpose of gene expression analysis is a target cDNA.
After a hybridization reaction of A and cDNA serving as a probe in a buffer solution, the slide glass is washed and dried, and the fluorescence emitted from each spot is measured by an optical scanner.

Further, depending on the assay, there are cases where the reaction and the detection of the signal have to be carried out in a solution state.
When a large amount of solution is to be reacted in such a case, the reaction reagent is dispensed into a 96-well microtiter plate or a 384-well microtiter plate having a volume of several tens to several hundreds μL per well, The wells need to be sealed and the wells are usually covered with a flexible sheet or film with an adhesive.

For example, in Japanese Patent Publication No. 11-507508 (publicly known example), a flexible pad in which an elastic compressible ridge is formed on the surface of the pad when sealing each well of a microplate, The invention is characterized in that the inside of the well is maintained in a liquid-tight state by being pressed against the opening of the well, and the pad is easily peeled from the well after the heating / stirring step.

On the other hand, an ultrasonic welding technique in which sheet-like processed materials made of a thermoplastic material are superposed on each other, and ultrasonic vibration is induced while applying pressure to perform a welding process is widely known. For example, as shown in FIG. The ultrasonic vibration generated by the ultrasonic oscillator 3 is converted by the converter 4 with respect to the laminated workpieces W1 and W2 placed on the processing table 2 of the ultrasonic welding device 1.
It is possible to weld the horn 6 to the workpieces W1 and W2 with a required pressure in a state where ultrasonic vibration is generated in the horn 6 connected to the booster 5 by transmitting the horn 6 to the workpieces W1 and W2. It was common (prior art).

Also, the booster 5 used in this case is
An annular protrusion 5B is raised at a position near the horn 6 of the large diameter portion 5A, and inner and outer rings 5D and 5E connected to a fixed frame via an O-ring 5C are fitted to the annular protrusion 5B. Then
The pin 5F was inserted through the large-diameter portion 5A together with the inner and outer rings 5D and 5E to be connected, and thereby the ultrasonic vibration could be effectively transmitted to the horn 6.

[0009]

The above-mentioned known example is also considered to be effective as an attempt to make the well liquid-tight.

However, in order to improve the quality of DNA analysis, it is important that the sample in the required well is safely stored without being damaged, and the well is sealed liquid-tight. That is also an essential requirement.

A first problem to be solved by the present invention is to dispense a liquid such as a sample or a reagent containing an ultratrace amount of DNA or protein into a minute well, and use ultrasonic welding means to remove the liquid. The invention is to provide a microwell array that can be hermetically sealed within a well without being damaged by heat.

A second problem to be solved by the present invention is to form a microwell array by sealing a large number of minute well groups uniformly by using ultrasonic welding means without air intrusion. Is to provide things.

A third problem to be solved by the present invention is to provide a microwell array in which a large number of wells are sealed at once by ultrasonic welding means.

A fourth problem to be solved by the present invention is to provide a microwell array in which ultrasonic vibration is applied for a very short time under a high pressurizing state and the welding state is uniform and no welding failure occurs. is there.

[0015]

Specific means for solving the above-mentioned problems are as set forth in the claims.

That is, as shown in the embodiment, the sealing device is as follows.

(1) On a tray in which a plurality of wells formed independently are arranged in an array on the same plane, a cover for covering the tray is brought into close contact with the well by ultrasonic welding means, and a sample or a sample is placed in the well. A device for sealing specimens, in which a cover is stacked on a tray supported on a table means, ultrasonic vibration is applied from the cover under pressure with a horn to weld the tray and the cover, and the wells are sealed. An ultrasonic sealing device in a microwell array configured to.

(2) The horn has a quadrilateral shape, and long holes are formed in a cross shape in the height direction so that a uniform ultrasonic vibration can be applied to the cover over its entire area. 1) The ultrasonic sealing device for the microwell array described.

(3) The elongated hole in the horn is a vertically elongated hole having a length of 60% to 70% of the height dimension of the horn and an elongated hole having a length of 30% to 40% of the height dimension of the horn. An ultrasonic sealing device in the constructed microwell array according to the above (2).

(4) The ultrasonic sealing device for a microwell array according to (1), wherein the horn is linked to an ultrasonic oscillator through a booster supported by an annular resonator.

(5) The ultrasonic sealing device for a microwell array according to (4), wherein the annular resonator of the booster is raised on the booster.

(6) The ring resonator of the booster is raised on the booster close to the converter (4)
Alternatively, the ultrasonic sealing device for the microwell array according to (5).

Therefore, the effects described later in the section of the effects of the invention can be achieved.

The sealing method is as follows.

(7) On a tray in which a plurality of wells formed independently are arranged in an array on the same plane, a cover for covering the tray is brought into close contact with the well by ultrasonic welding means so that the liquid is put into the well. An ultrasonic sealing method in a microwell array, wherein the oscillation time of ultrasonic vibration is 250 ms or less.

(8) In the ultrasonic sealing method of (7) above,
An ultrasonic sealing method in a microwell array in which the ultrasonic vibration amplitude is 30 to 40 microns.

(9) In the ultrasonic sealing method according to (7) or (8), the pressure applied to the stacked trays and the cover is 1000 to 1000.
Ultrasonic sealing method for microwell array at 1700 kgf.

Therefore, the same effect as the sealing device can be achieved.

[0029]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Next, the contents of the present invention will be described based on an embodiment of a sealing device 1000 for a microwell array 100 shown in the drawings.

1. Microwell Array 100 In the example of the microwell array 100 shown in FIG. 1, the tray 10 and the cover 20 are integrally connected by ultrasonic welding means.

Here, the tray 10 and the cover 20 are obtained by molding a plastic material such as polycarbonate, polypropylene, polystyrene, and methylpentene copolymer having excellent chemical resistance and heat resistance.

Also, as shown in FIG. 2, 384 independent wells 11 are arranged on the surface of the tray 10 so that the vertical and horizontal intervals are 4.5 mm.

On the other hand, the cover 20 is formed with 384 protrusions 21 so as to be aligned with the well 11.

Also, prior to ultrasonic welding, the tray 1
In order to arrange the 0 and the cover 20 in line, the tray 10 is provided with the guide pin 12 and the receiving hole 13 as shown in FIG. 1, and the cover 20 is provided with the through hole 22 and the hook 23. .

The tray 10 and the cover 20 are ultrasonically welded as will be described later, but in order to liquid-tightly seal the wells 11, it is preferable that the tray 10 and the cover 20 are flat without warpage.

The cover 20 is required to have a minimum thickness for maintaining flatness and a thinness for ensuring thermal conductivity, and a thickness of 0.15 mm to 3.0 mm is preferable. Suitable, more preferably 0.25 mm to 1.5 m
m is preferable.

Next, sectional shapes of the tray 10 and the cover 20 will be described with reference to FIG.

Although the vertical cross-sectional shape of the well 11 is not shown, it has a trapezoidal shape to facilitate insertion of a needle of a spotting device for dispensing a small amount of liquid.
The cross-sectional shape is not necessarily limited to this, and when arranging the wells 11 at a high density, the cross-sectional shape may be rectangular, and depending on the density of the wells 11 and the size and shape of the needles, a polygon such as a triangle or a quadrangle, It is possible to determine the optimum cross-sectional shape such as a semi-circle.

The well 11 has a raised portion 1 having a convex top.
4 is provided.

The liquid escape groove 15 of the tray 10 is formed around the well 11 so as to surround the well 11.
As long as the liquid extruded from the liquid pools, its shape and position are not limited.

A protrusion 21 for pushing out the liquid of the cover 20
Is for pushing the liquid injected into the well 11 into the escape groove 15 so that air does not remain in the well 11, and as described later with reference to FIG. It is possible to seal the liquid so that the air does not remain even if the protrusion 21 is not formed (see FIG. 10).

The protrusion 21 also has a function of preventing the raised portion 14 melted during welding from blocking the upper part of the well 11.

At the time of ultrasonic welding, the resin forming the raised portion 14 melts out and spreads out between the tray 10 and the cover 20, but by forming the protrusion 21, the melted resin covers the well 11 above. This is effective in preventing the above and maintaining the light transmission.

2. Ultrasonic sealing device 1000 (1) Overall configuration and function As shown in FIGS.
0 is the ultrasonic vibration imparting means 1100 which is disposed stationary.
Table means 1200, which can be engaged with the ultrasonic vibration imparting means 1100 so as to be accessible to the ultrasonic vibration applying means 1100, are arranged in an array, and in the ultrasonic welding process work, as shown in FIG. The tray 10 having the wells 11 containing the liquid containing the genomic DNA is covered with a cover 20.
Are placed on the table means 1200, the table means 1200 is lifted based on an instruction from an operator, and the ultrasonic vibration applying means 1100 is superposed on the ultrasonic vibration applying means 1100 while being pressed against the horn 1110 of the ultrasonic vibration applying means 1100. Ultrasonic welding is performed by generating ultrasonic waves to heat and melt the tray 10, and details thereof will be described later in the section "Welding procedure".

(2) In order to facilitate understanding of the configuration contents of each part, the configuration of each part will be described in the following order. Support frame means 1300 Table means 1200 Ultrasonic vibration applying means 1100 Control means 1400

Support Frame Means 1300 As shown in FIGS. 4 and 5, a box-shaped main frame 131.
On the base frame 1311 of 0, table means 1200 to be described later
A guide portion 1313 that guides the guide portion 1225 of the table 1220 that is moved up and down by the lifting drive portion 1210 in the height direction is provided on the vertical frame 1312 of the main frame 1310.

Further, an ultrasonic vibration applying means 1100, which will be described later, is provided on the top frame 1314 which is continuously provided on the top of the main frame 1310.
Are installed in series.

Table Means 1200 As shown in FIG. 6, a table 1220 is connected to an elevating / lowering drive unit 1210 of a table means 1200 composed of an air cylinder and other fluid cylinders, and a fixed frame 1221 is mounted on the table 1220. , Jig plate 1
222 is fixedly installed.

The jig plate 1222 is horizontally positionable so that it can be aligned and opposed to the horn 1110 of the ultrasonic vibration applying means 1100, which will be described later.
Second adjusting screw portion 122 for correcting the inclination of the adjusting screw portion 1223 of the jig and the jig plate 1222 in the height direction.
4 is installed.

A guide portion 1225 is provided in the height direction on the side portion of the table 1220, and the guide portion 1225 is formed so as to be guided by the guide frame 1315.

Ultrasonic Vibration Applying Unit 1100 As shown in FIG. 6, the converter 1130 and the booster 1140 of the ultrasonic vibration oscillating unit 1120 are connected to the horn 1110 described above, and the horn 1110 is mounted on the jig plate 1222. The booster 1140 is to be pressed against the cover 20 placed thereon and ultrasonically welded thereto.
The horn 1110 will be described in more detail below.

(A) Booster 1140 The point that the booster 1140 shown in FIG. 7 is common with the booster 5 of the prior art shown in FIG. 11 is that it is made of a titanium alloy or an aluminum alloy, and its central large diameter portion (a) is formed. The first reduced diameter portion (b) on one side is connected to the converter 1130, and the second reduced diameter portion (c) on the other side is connected to the horn 1110, which is a feature of the booster 1140 shown in FIG. 7. The following points can be cited as typical configurations.

That is, in the central large-diameter portion (a) of the booster 1140, an annular resonator (e) is formed along the longitudinal direction of the booster 1140 via the groove (d), and this annular resonator (e) is continued. The outward ribs (f) are connected to the support frame 1150.

(B) Horn 1110 As for the horn 1110 shown in FIGS. 8 and 9, like the booster 1140, it is generally formed in a rectangular parallelepiped shape with a metal material such as titanium alloy or aluminum alloy, and its main body 1111. A welded portion 1112 that is slightly larger than the main body 1111 is formed in the lower part of the above, and the lower surface of the welded portion 1112 is a flat ultrasonic weld surface 1113.

In the main body 1111, a plurality of vertically elongated holes 1114 are formed in parallel in the height direction along the longitudinal direction, and the vertically elongated hole 1 is formed in the height direction along the lateral direction.
Horizontal hole 1115 and round hole 1116 so as to be orthogonal to 114
And are arranged above and below and communicate with each other through a communication hole 1117.

The length of the vertical hole 1114 is set to approximately 60% to 70% of the height of the horn 1110,
The length of the laterally long hole 1115 is equal to that of the horn 11.
It is preferable to form it so as to be approximately 30% to 40% of the height of 10.

Control Unit 1400 As shown in a block diagram in FIG. 6, the control unit 1400 having a high-speed data processing function such as a microcomputer includes a central control unit 1410, an oscillation control unit 1420, and a jig plate 12.
22 is connected to the lift control unit 1430, and the jig plate 1222 is connected based on information from the position sensor 1440.
The position of can be detected.

The operation panel 1450 is constructed so that it can be operated by an operator to instruct the central control means 1410 to execute the welding process.

3. Welding Procedure (1) Preliminary Step As shown in FIG. 10A, an extremely small amount of genomic DNA (specimen) is spotted in advance on the microwell array 100 shown in FIG.
In B, the reagent for reacting with DNA is spotted by a non-contact type dispenser in an amount of about 30 to 90% larger than the volume of the well 11.

The liquid has the effect of surface tension, and the well 1
The liquid in the portion that does not fit in 1 is held so as not to spill out from the well 11 so as to rise.

The tray 10 in which the sample and the reagent have been injected as described above is placed on the jig plate 122 of the table means 1200.
Place on top of 2.

Then, as shown in FIG. 10C, the cover 20 is superposed on it, and is gently pushed down to insert the guide pin 12 into the through hole 22 and engage the hook 23 with the receiving hole 13 to thereby make the tray 10. The cover 20 is aligned on the top and temporarily attached.

In this state, as shown in FIGS. 10C and 10D, the liquid filled in the well 11 can be pushed out of the well 11 by the protrusion 21 of the cover 20 and sealed so that air does not remain in the well 11. I can.

(2) Ultrasonic Welding Step Next, the operation panel 1450 operates the central control means 1410 to activate the elevating control section 1430 to activate the elevating drive section 12.
The table 1220 is lifted by 10 and the horn 111
0 is pressed against the cover 20 on the jig plate 1222.

In this state, the oscillation controller 1420 is excited to generate ultrasonic vibrations in the horn 1110, and the tray 10
Is ultrasonically welded to the cover 20.

During this time, the height position of the jig plate 1222 is monitored by the position sensor 1440, and the data is fed back to the elevation controller 1430 to safely manage the distance between the horn 1110 and the jig plate 1222. This guarantees a high quality welding process.

(3) Step of taking out microwell array Next, after the welding process, the operation panel 1450 is operated to lower the jig plate 1222 to return it to the original position, and the completed microwell array 100 may be taken out. It is a thing.

It should be noted that these series of welding treatment procedures are programmed in advance, and C.I. P. It goes without saying that the U operates sequentially.

By the way, at the time of this welding treatment, at least the following quality requirements are listed.

(1) There is no risk of damaging the DNA sample due to vibration or heat generation during the welding process.

(2) The wells 11 of the tray 10 are evenly adhered to the cover 20 so that the DNA sample and the reagent do not leak out.

(3) The well is completely liquid-tight even when the liquid inside the well 11 boils.

In order to meet the above requirements, the sealing device 10
00 requires the following work.

(B) The welding process is performed for a very short time at a high pressure.

(B) The welding process is carried out with high quality, homogeneity and no variation.

Therefore, the countermeasures will be described by dividing this point as follows.

Setting of Welding Treatment Conditions Generally, the welding energy that determines the welding result is said to be the phase product of the welding time, the applied pressure, and the amplitude.

The relational expression is as follows. E = 2 × π × f × A × P × t × H

However, E ... welding energy f ... Vibration frequency H ... Transmission efficiency of vibrations transmitted to the joint surface of adherends A ... Magnitude of horn tip amplitude P ... The magnitude of the load applied to the adherend t ... Oscillation time π ... 3.14

From this relational expression, the oscillation time (t) must be shortened as much as possible in order to prevent the enzyme in the reagent from being damaged by the ultrasonic vibration, but for satisfactory welding, The load condition (P) or the amplitude condition (A) must be increased.

However, the larger the amplitude condition (A), the greater the scattering of the enzyme before the bonding surface of the adherend is softened / melted, and there is a risk that the enzyme hardly remains in the well 11.

Therefore, the oscillation time (t) is set to 250 m, for example.
s or less, generally 6 for welding polycarbonate material
An amplitude of about 0 micron (20 KHz) seems to be ideal, but the amplitude condition (A) is set to 3 to prevent the reagent from scattering.
It is reduced to 5-40 microns.

Further, in order to reduce the oscillation time (t) and the amplitude condition (A) to satisfy the welding, the load condition (P) must be increased, and as a result, the sample in the well 11 is 1.
When about 2 μl to 1.5 μl was filled, a load of 1600 Kgf was required as a load condition.

Therefore, in this sealing device 1000, the following measures are taken in order to cope with a vibration system capable of oscillating under high pressure conditions and a high output power supply.

◎ Booster 1140 (Solid Booster) 1600 kgff load is applied at the nodal point of the solid booster, which is a high-pressure booster.
In order to obtain uniform weldability over the entire surface of a size of 20 × 80, the horn end face amplitude was made uniform.

That is, as shown in FIG. 7, the booster 11
By supporting the annular resonator (e) in 40 by the support frame 1150 near the converter 1130, the axial and radial ultrasonic vibrations are effectively absorbed by the elastic deformation of the annular resonator (e) and a uniform supersonic vibration is obtained. The sound wave vibration can be applied to the cover 20 with high pressure.

Selection of Horn 1110 The configuration of the horn 1110 is as described above, and a large number of wells 11 provided on the tray 10 are covered with the cover 20.
Is provided with a flat rectangular welding surface 1113 so that the welding can be performed without variation, and the vertical and horizontal elongated holes 1114 and 1115 and the round hole 1116 formed by crossing vertically and horizontally allow the ultrasonic waves to be uniformly ultrasonicated over the entire welding surface 1113. Vibration is distributed and induced.

The above welding processing conditions and the horn 111
0 and the booster 1140 allow the reagent to be stably contained in the cell without scattering, and the welding process does not vary, and the ultrasonic welding process can be performed with high quality to achieve the required quality.

At the time of this welding process, the top of the well 11 is made into the tapered ridge 14 and the projection 21 is provided on the cover 20, so that the projection 21 penetrates into the well 11 to melt the plastic. The ultrasonic welding could be completed while the material could be safely prevented from flowing into the well 11.

[0090]

In the above-described embodiment of the present invention, the following points can be given as an example of an optimal embodiment that embodies this.

1. Ultrasonic welding device 1000 ◎ Pressurizing force ... 1600 kgf (max. 2000 kg
f) ◎ Horn end face amplitude: 30-40 microns (frequency: 20 KHz) (optimum value)

[0092] 2. Application (tray 10, cover 20)   (1) Tray 10       Size: 120 mm x 80 mm (rectangle)       Thickness ... 0.8mm-3.0mm       Container (round boss) ◎ Number of pieces 384 (16 × 24)                                   ◎ Height… 0.4                                   ◎ Outer diameter… 2.36φ                                   ◎ Inner diameter: 1.44φ

(2) Cover 20   Size: 120mm x 80mm   Plate thickness: 0.25 mm to 1.5 mm

[0094]

The unique effects brought about by the present invention described above are as follows.

Since the ultrasonic wave is applied for an extremely short time to perform the welding process, the enzyme in the sample is safely sealed in the well without being damaged.

By applying uniform ultrasonic vibration with a high pressure, a large number of wells can be hermetically sealed with high quality.

By applying uniform ultrasonic vibration for an extremely short time, there is no risk that the sample or reagent in the well will be scattered.

[Brief description of drawings]

FIG. 1 is an assembled perspective view of a microwell array in an embodiment of a sealing device.

FIG. 2 is a sectional view taken along line AA of FIG.

FIG. 3 is a sectional view of the hook and the receiving hole in FIG.

FIG. 4 is a front view showing an embodiment of a sealing device.

5 is a side view of FIG.

6 is a partially enlarged view of FIG.

FIG. 7 is a partially cutaway enlarged explanatory view of the booster of FIG. 4.

FIG. 8 is an enlarged front view of the horn of FIG.

9 is a side view of FIG.

FIG. 10 is an explanatory diagram of a procedure for welding a microwell array in the embodiment of the sealing device.

FIG. 11 is an explanatory diagram of a conventional technique.

[Explanation of symbols]

1000 Ultrasonic sealing device 1110 horn 1113 Welding surface 1114 Vertical hole 1115 Horizontal hole 1130 converter 1140 booster (E) Annular resonator 1200 table means 10 trays 11 wells 20 cover

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Kiyoshi Hirose             4-3-1 Okada, Atsugi City, Kanagawa Prefecture Japan             Emerson Branson Business Division (72) Inventor Yusuke Nakamura             1-17-33 Azamino, Aoba-ku, Yokohama-shi, Kanagawa (72) Inventor Hideyuki Suzuki             3-16-29-101 Shirokanedai, Minato-ku, Tokyo F term (reference) 2G052 AD06 CA03 CA18 DA06 DA12                       DA13 HA12 HC04 HC32 JA08                       JA16                 4B029 AA08 BB15 BB20 CC01 GA03                       GA08 GB09 GB10                 4E067 AA19 BF00 CA01 DA13 DC07                       EA00                 5D107 BB01 FF03

Claims (9)

[Claims] 1. A sample or specimen is placed in the well by placing a cover for covering the tray on a tray in which a plurality of wells formed independently are arranged in an array on the same plane by ultrasonic welding means. Is a device for sealing a well, and a cover is laminated on a tray carried on a table means, and ultrasonic vibration is applied from the cover under pressure by a horn to weld the tray and the cover to seal the wells. Ultrasonic sealing device in a microwell array configured as described above. 2. The horn has a quadrilateral shape, and elongated holes are formed in a crosswise shape in a height direction thereof so that uniform ultrasonic vibrations can be applied to the entire area of the cover. An ultrasonic sealing device in the described microwell array. 3. The elongated hole in the horn comprises a vertically elongated hole having a length of 60% to 70% of the height dimension of the horn and an elongated hole having a length of 30% to 40% of the height dimension of the horn. The ultrasonic sealing device for a microwell array according to claim 2. 4. The ultrasonic sealing device for a microwell array according to claim 1, wherein the horn is linked to an ultrasonic oscillator via a booster supported by an annular resonator. 5. The ultrasonic sealing device for a microwell array according to claim 4, wherein the annular resonator of the booster is provided on the booster. 6. The ultrasonic sealing device for a microwell array according to claim 4, wherein the annular resonator of the booster is raised on the booster close to the converter. 7. A liquid cover is sealed in a well by ultrasonically welding a cover for covering the tray on a tray in which a plurality of wells formed independently are arranged in an array on the same plane. The ultrasonic sealing method in a microwell array, wherein the oscillation time of ultrasonic vibration is 250 ms or less. 8. The ultrasonic sealing method according to claim 7, wherein the ultrasonic vibration amplitude is 30 to 40 microns. 9. The ultrasonic sealing method according to claim 7 or 8, wherein the pressure applied to the stacked trays and the cover is 1000 to 1000.
Ultrasonic sealing method for microwell array at 1700 kgf.