JPH08313416A - Ultrasonic treatment apparatus of biological sample - Google Patents

Abstract

PURPOSE: To efficiently perform the ultrasonic treatment operation of samples in many arranged wells by attaching a plurality of horns to a single holder side by side to constitute an osicllator and connecting this oscillator to an ultrasonic vibrator. CONSTITUTION: A large number of horns 31 are moved up and down by the revolving operation of a handle 6 to be simultaneously inserted in a large number of the wells 51 on a plate 5 under the same condition to subject the biological samples in the wells 51 to ultrasonic treatment. In order to subject the samples in the wells 51 to ultrasonic treatment under the same condition by a large number of the horns 31, it is desirable that the horns 31 are made mutually same in shape and the connection states thereof to a holder 32 are made mutually uniform. By making the conditions of the horns 31 same, the positions, lengths and vols. of the horns 31 inserted in the wells 51 become mutually same and ultrasonic energy can be equally transmitted to the samples in the wells 51.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention is used for the operation of crushing and homogenizing biological or chemical samples (hereinafter referred to as biological samples) in operations such as manufacturing, processing, analysis and measurement. The present invention relates to a sound wave processing device.

[0002]

2. Description of the Related Art When manufacturing, processing, analyzing and measuring a biological sample or the like, it is necessary to add various physical operations such as crushing, melting, homogenizing and extracting the sample according to the purpose. However, in recent years, there has been a demand for further improvement in efficiency. Conventionally, ultrasonic waves have been used as one of the energy for such physical operations. The reason for this is that ultrasonic waves make it easy to control the cavity effect that produces crushing force by adjusting the electrical output. This is because it has an advantage that it can be applied to a wide range of objects including samples.

However, since the ultrasonic processing apparatus (ultrasonic homogenizer) which has been commercially available for cell disruption has an oscillator constituted by one horn, it can be used, for example, in wells. For a plate having a total of 96 wells arranged in 12 rows of 8
When the ultrasonic treatment is performed on the sample in each well, the treatment operation must be performed 96 times. Therefore, the work required a long time and was extremely inefficient.
Furthermore, it is very difficult to uniformly apply ultrasonic vibration to 96 wells under the same conditions, and the accuracy is considerably deteriorated.

[0004]

SUMMARY OF THE INVENTION An object of the present invention is to provide an ultrasonic treatment apparatus which can efficiently perform ultrasonic treatment operation on a sample in a well in which a large number of wells are arranged. Another object of the present invention is to provide an ultrasonic treatment apparatus which enables speeding up and improvement in accuracy of various biological tests, biochemical tests and clinical tests.

[0005]

In the ultrasonic processing apparatus of the present invention for achieving the above object, a plurality of horns are attached in parallel to a single holder to constitute an oscillator, and the oscillator is ultrasonically vibrated. It is characterized by being connected to a child. By providing a plurality of horns in this way, it is possible to perform uniform processing operations under the same conditions on a large number of wells at the same time, which can significantly reduce the processing time and improve the accuracy. it can.

Further, when a through hole for heat radiation is provided in the central portion of the holder to which a plurality of horns are attached, the ultrasonic vibrations oscillated from the horns are further equalized and the variations are reduced, so that the accuracy is further improved. be able to. Hereinafter, the present invention will be described in detail with reference to an embodiment shown in the drawings. FIG. 1 shows an ultrasonic processing apparatus according to an embodiment of the present invention.

This ultrasonic processing apparatus is constructed by connecting an ultrasonic oscillator 1, an ultrasonic oscillator 2 and an oscillator 3 to each other. In the illustrated example, the ultrasonic oscillator 1 and the ultrasonic oscillator 2 are configured to be independent of each other, but the both may be integrated. The structure of the ultrasonic oscillator 1 is not particularly limited, and any known oscillator can be used. An output of 100 W to 2 KW and a frequency of 10 to 30 KHz are preferably used. This ultrasonic oscillator 1 is connected to the next ultrasonic transducer 2 via an output connector 11.

The structure of the ultrasonic oscillator 2 is not particularly limited, and a known nickel magnetostrictive oscillator, ferrite magnetostrictive oscillator, bolted Langevin type PZT oscillator or the like can be used. Further, although the energy is very weak, a vibrator made of a polymer piezoelectric film may be used when giving uniform energy in the MHz band. Biochemically, this polymer piezoelectric film oscillator has an acoustic impedance equal to that of a living organism and has good compatibility.

A plurality of oscillators 3 (eight in the illustrated example)
The horns 31 are arranged side by side in parallel with a single holder 32, and the holder 32 is connected to the ultrasonic transducer 2 via a booster 33 (see FIG. 2). As for the number of horns 31 to be attached, the number of wells 51 provided in one plate 5 is usually 8 × 12 rows = 96, so one row or 8 It is good to provide in multiple rows. That is, the number of horns 31 to be attached may be a multiple of 8 or 12.

A plurality of horns 31 provided in this way
Are vertically moved by the turning operation of the handle 6, so that they are simultaneously inserted into the plurality of wells 51 on the plate 5 placed on the plate base 4 under the same condition.
The biological sample in 1 is sonicated. In order for these plural horns 31 to sonicate the sample in each well 51 under the same conditions, the shapes of the horns 31 should be the same, and the connection state of the horns 31 with the holder 32 should be uniform. It is desirable to do. By making the conditions identical in this way, the positions, lengths, and volumes of the horns 31 inserted into the wells 51 become the same,
Ultrasonic energy can be evenly transmitted to the sample in each well 31.

Further, the holder 32 for connecting the plurality of horns 31 is provided with a through hole 35 for heat radiation corresponding to a substantially central position of the entire width where the plurality of horns 31 are arranged in parallel. By providing such a through hole 35, it is possible to positively radiate the heat that is going to be accumulated in the central portion of the holder 32, and to evenly transmit the ultrasonic energy to each of the plurality of horns 31.

According to the above-described ultrasonic treatment apparatus, for example, when ultrasonic treatment is performed on each biological sample dispensed into 96 (= 8 × 12 rows) wells 51 provided on the plate 5, the conventional ultrasonic treatment is performed. In a 1-horn type ultrasonic treatment device, it is necessary to perform 96 repeated treatment operations, but only 1
It only needs to be repeated twice. In addition, if the ultrasonic processing device is equipped with 12 horns 31, only 8
Ultrasonic treatment of all wells can be performed by repeating the operation once.

The ultrasonic processing apparatus of the present invention has the following applications:
In solution systems, it promotes chemical reactions of biological and chemical samples,
Examples of such operations include dispersion, homogenization, extraction, crushing, DNA or RNA cleavage, welding, embedding, and caulking. Particularly, it is effective when ultrasonically treating a biological sample. In addition, specifically, the biological samples such as cells, organs and tissues placed in 96 wells 51 are subjected to ultrasonic treatment in a solution to homogenize the sample, extract cell components and enzymes, lipid / protein / sugar. Extraction and solubilization of quality, extraction and cleavage of nucleic acid (for solubilization, digestion, color development, biosynthesis, etc.)
The work of accelerating the reaction with various reagents such as Through this work, quantification of biological activities of enzymes and cytokines, cell growth promoters and cell growth inhibitors,
It is possible to evaluate anticancer agents and antiviral agents, quantify cell metabolizers and cell function modifiers, speed up various clinical tests, and improve accuracy and reproducibility.

The quantification and evaluation applicable to this work widely include biochemical quantification and clinical examination. For example, various hormones, various antibodies, tumor-associated antigens, myocardial proteins, collagen, cell surface antigens, intracellular enzymes, intracellular lipids, intracellular carbohydrates, serum proteins, CAMP, CGMP, intracellular nucleic acid components, viral antigens, viruses It includes measuring nucleic acid components, bacterial cell components, bacterial nucleic acid components and the like.

The measuring methods include RIA, EIA, ELI
SA, color development method, colorimetric method, immunoagglutination method, hemagglutination inhibition method, complement fixation method, neutralization method, fluorescent antibody method, PCR method, PT
-RCR method, various hybridization methods, bacterial agglutination method and the like can be mentioned. Further, it can be used as a pretreatment operation for analysis by liquid chromatography, gas chromatography, or electrophoresis, and further, the plate containing the sample and the ultrasonic treatment device of the present invention are integrated into an automatic analysis device. It can also be produced.

[0016]

Example A plastic microplate (size 127x) having 96 wells (8x12) each having a diameter of 7 mm.
85 × 16 mm), 1 × 10 ⁵ cells / well of mouse 7TD1 cells were seeded in each well, and RPMI1640 medium containing 10% fetal bovine serum was used at 37 ° C. for 3 days.
The culture was performed under 5% CO ₂ . Then, 10 μl of MTT reagent containing 5 mg / ml of phosphate buffered saline was added to each well.
Each well was added and incubated for 5 hours, and then 150 μl of 0.04N hydrochloric acid-isopropanol was added to each well.

The cells cultured as described above were supplied with a nominal output of 1
It has an ultrasonic oscillator of automatic tracking oscillation type with 50 W and oscillation frequency of 28 KHz, and once using the ultrasonic processing device having the structure of FIGS. 1 and 2 in which eight horns are connected in parallel as an oscillator. 8 wells each, 28KHz, 60
It was crushed and homogenized under the treatment condition of W for 1.5 seconds / time.

Next, in order to evaluate the homogeneity of cell disruption, the MTT reagent was reduced in the mitochondria in the cells about 10 seconds after sonication, and the cells appeared blue 59.
The absorbance at 5 mm was measured. On the other hand, for comparison, using a commercially available 1-horn type UR-20P type ultrasonic homogenizer (manufactured by Tommy Seiko), ultrasonic treatment was performed under the treatment conditions of 28 KHz, 8 W and 2 seconds / time, and the same as above. The absorbance at 595 mm was measured.

The results of the above measurements are shown in Table 1. When the ultrasonic treatment apparatus of the present invention is used, almost complete cell disruption is achieved and one plate is used as compared with the conventional ultrasonic treatment apparatus. Processing time per 1/4 to 1/7
Was shortened to. It should be noted that in the processing of this case, 100 cells are
The absorbance was set to 1.0 by% crushing.

[0020]

[Table 1]

[0021]

As described above, according to the ultrasonic processing apparatus of the present invention, since a plurality of horns are arranged in parallel in the holder as oscillators, it is possible to perform uniform processing operations under the same conditions on a plurality of wells at the same time. Therefore, the processing time can be greatly shortened and the accuracy can be improved.

[Brief description of drawings]

FIG. 1 is a perspective view of an ultrasonic processing apparatus according to an embodiment of the present invention.

FIG. 2 is an enlarged view of a main part of an oscillator provided in the ultrasonic processing apparatus of FIG.

[Explanation of symbols]

 1 Ultrasonic oscillator 2 Ultrasonic vibrator 3 Oscillator 31 Horn 32 Holder 35 Through hole

Claims (3)

[Claims] 1. An ultrasonic processing apparatus for a biological sample or the like in which a plurality of horns are attached in parallel to a single holder to form an oscillator, and the oscillators are connected to an ultrasonic transducer. 2. The ultrasonic processing apparatus for a biological sample or the like according to claim 1, wherein the holder main body is provided with a through hole for heat dissipation at a position corresponding to the central portion of the width in which the horns are arranged. 3. The number of attached horns is 8 or 12.
The ultrasonic treatment apparatus for a biological sample or the like according to claim 1 or 2, which is a multiple of.