JP2005034060A - Instrument for biochemical research - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an instrument with small adsorption of a biologically derived material as a sample, high in organic solvent resistance, and slight in eluate, thus suitable for biochemical experiments. <P>SOLUTION: This instrument for biochemical research consists of a molded form of an ethylene-vinyl alcohol copolymer. Preferably, the ethylene copolymerization ratio of this copolymer is 5-50 mol%. This copolymer is such one as to be subjected to radiation irradiation or plasma exposure. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to instruments such as containers and pipettes used for biochemical research and experiments.

In recent years, in the field of protein research, with the progress of genomics and proteomics, the evaluation and analysis instruments used for research have continued to make remarkable technological innovations. Has been developed to the extent that a single researcher can put out in a few days.
On the other hand, the instruments used for biochemical research and experiments have not made any progress since the transformation from glass to plastic, and classic materials are still being used despite many problems.
At present, there is no particular close-up on the problems of existing instruments, but in the future, a system for large-scale analysis will be provided by informatics such as data processing and use after protein evaluation analysis. As the number of types of substances that are subject to biochemical research increases and a larger amount of biological substances are handled, existing instruments will become overwhelmingly sensitive analytical instruments. It is clear that the performance will be lost.

The biggest problem with existing instruments is the adsorption of the sample onto the container wall. The plastic materials currently used for instruments are polypropylene and polystyrene, but in any of these materials, biologically derived substances such as proteins and peptides are strongly adsorbed on the wall of the container. It has been revealed that about 30% of the protein in the solution is lost by adsorption in the following trace region.
The biological substance to be analyzed is adsorbed to the instruments, so the signal obtained as a result of the analysis becomes low, and even for very small samples, there may be no signal at all, and the performance of the analytical instrument Even if a highly sensitive sample that can detect a very small amount of sample is used, measurement cannot be performed.

For example, when analyzing the structure of a protein using a mass spectrometer in the structural analysis of the protein, the target protein is fragmented into peptides with an enzyme or the like, and then the peptide generated by fragmenting with the mass spectrometer is analyzed for molecular weight. Analytical methods are used to estimate the structure of the protein by separating / detecting the protein, but depending on the type of peptide, there are those that are easily adsorbed to the substrate and those that are relatively difficult to adsorb. It is not always detected, making analysis of the results very difficult.
In order to prevent the above-mentioned adsorption, products whose surfaces have been modified to be hydrophobic with silicon coating etc. are on sale, but the biggest factor when biological substances in solution adsorb to the substrate is the biggest factor Due to the hydrophobic interaction, these products have a very good appearance, but cannot prevent protein adsorption.
However, the surface showing the strongest adsorption actually is not an extremely hydrophobic surface like Teflon (R) or fluororesin, but protein adsorption on the surface where some polar groups are introduced to the surface of polypropylene or polystyrene by irradiation. It is known that the quantity shows a maximum value.
That is, the adsorption is maximum in the intermediate region between superhydrophobic and superhydrophilic, and polypropylene and polystyrene are slightly more hydrophobic surfaces than the region showing the maximum, but the instruments molded with these materials are Irradiation for sterilization results in a stronger adsorptive surface.

In addition, there are two required performances other than anti-adsorption: low elution and solvent resistance. The effluent affects the analysis using highly sensitive measuring instruments as a fatal noise.For solvent resistance, if the effluent comes out from the instruments due to the organic solvent used in all stages, Noise problems occur.
A method of modifying the surface of instruments with a coating is not suitable in terms of eluate and solvent resistance.

  An object of the present invention is to provide instruments suitable for biochemical experiments with little adsorption of a biological substance as a sample, excellent resistance to organic solvents, and little eluate.

That is, the present invention
(1) A biochemical research instrument characterized by being molded of an ethylene-vinyl alcohol copolymer,
(2) The biochemical research instrument according to (1), wherein the ethylene-vinyl alcohol copolymer has an ethylene copolymerization ratio of 5 mol% to 50 mol%.
(3) The biochemical research device according to (1) or (2), which is exposed to radiation or plasma.
It is.

  By using the biochemical research instrument of the present invention, there is no loss due to adsorption to the instrument wall, which is a conventional drawback when handling biologically derived substances such as proteins, peptides, and nucleic acids in biochemical research. In addition, since it has resistance to organic solvents, which was a drawback of surface-modified coating processing equipment such as silicon coat, it can perform highly sensitive and accurate analysis.

  The instruments for biochemical research of the present invention are instruments for liquid operation such as pipettes and dispenser chips used for biochemical research, and containers such as centrifuge tubes, cryopreservation tubes, tubes, and multiwell plates.

  The present inventor has devised the idea of injection molding using a hydrophilic resin in order to solve the above-mentioned problems, and as a result of intensive studies, hydrophilicity at a level at which ethylene vinyl alcohol can be injection-molded and protein adsorption can be prevented. It was found that it has a good surface and is also excellent in solvent resistance.

As a result of further investigations, it was found that by limiting the copolymerization ratio of ethylene and vinyl alcohol, it exhibited excellent performance particularly in terms of preventing protein adsorption as a device for biochemical research.
That is, in the ethylene vinyl alcohol copolymer used in the present invention, the copolymerization ratio of ethylene and vinyl alcohol is important, and the copolymerization ratio of ethylene is preferably 5 mol% to 50 mol%.
If the copolymerization ratio of ethylene is less than 5 mol%, the water resistance is remarkably lowered, so it is not suitable for use as a biochemical research instrument, and if it exceeds 50 mol%, protein adsorption can be effectively prevented. A level hydrophilic surface cannot be obtained.

  Another excellent feature of the ethylene vinyl alcohol copolymer according to the present invention is that when a polar group is introduced on the surface of the molded article, the protein adsorptivity is further lowered. When a polar group is introduced on the surface by irradiation of radiation for sterilization, the adsorptivity is not increased like polypropylene and polystyrene, and the protein adsorptivity is further lowered.

The method for forming the instruments of the present invention is not particularly limited, and is used for molding liquid handling instruments such as pipettes and dispenser tips, and containers such as centrifuge tubes, cryopreservation tubes, tubes, and multiwell plates. It is preferable to select a suitable injection molding method.
Further, even when other molding methods such as extrusion molding are used depending on the application, the characteristics of the present invention are not affected at all.

Hereinafter, the present invention will be described more specifically with reference to examples.
(Example 1)
A 2 mL capacity tube was formed by injection molding using an ethylene vinyl alcohol copolymer: ethylene copolymer ratio of 29 mol% (Soarelite (R) M manufactured by Nippon Synthetic Chemical Industry Co., Ltd.).

(Example 2)
A 2 mL capacity tube was molded by injection molding using an ethylene vinyl alcohol copolymer: ethylene copolymerization ratio of 44 mol% (Eval (R) E105, manufactured by Kuraray Eval Company).

(Comparative Example 1)
A polypropylene 2 mL tube (MS-4603, manufactured by Sumitomo Bakelite Co., Ltd.) was used as Comparative Example 1.

(Comparative Example 2)
Comparative Example 2 was obtained by subjecting the tube of Comparative Example 1 to siliconization treatment using a commercially available silicon coating agent.

(Comparison of protein adsorption)
A solution in which peroxidase-labeled avidin (43-4423, manufactured by ZYMED) is diluted to 0.5 μg / mL, and a solution in which peroxidase-labeled anti-bovine albumin antibody (55285, CAPPEL) is diluted at 1.0 μg / mL (whichever is used for dilution) Was added to each of the tubes of Example 1, Example 2, and Comparative Example 1, respectively, and allowed to stand at room temperature for 1 hour, and then 0.05% tween 20 The amount of the protein adsorbed and retained on the wall surface after washing three times with the phosphate buffer solution pH 7.4 was measured using a peroxidase chromogenic substrate solution (ML-1120T manufactured by Sumitomo Bakelite Co., Ltd.).

  The results are as shown in Table 1, and both peroxidase-labeled avidin and peroxidase-labeled anti-bovine albumin antibody showed high adsorptivity for Comparative Example 1, but confirmed that the adsorptivity for Example 1 and Example 2 was low. did.

(Comparison of solvent resistance)
A solvent resistance test was performed using acetonitrile and dimethyl sulfoxide (DMSO) used for protein studies.
The tubes of Example 1 and Comparative Example 2 were each dispensed with 2 mL of acetonitrile and DMSO, allowed to stand at room temperature for 1 hour, then washed with pure water and dried, and the adsorbability of each container before and after the operation ( Evaluation was carried out by the adsorption test used in the comparison of protein adsorptivity.

The results are as shown in Table 2. In Comparative Example 2, the surface adsorption characteristics were greatly changed before and after contact with the organic solvent, and it was considered that the silicon-treated layer was deteriorated.
On the other hand, in Example 1, the adsorption property of the surface was not changed before and after contact with the organic solvent, and it was confirmed to have solvent resistance.

  The present invention relates to instruments used for biochemical research such as genetic engineering, protein engineering, clinical laboratory science, etc., and there is no loss due to sample adsorption, and it is possible to use an organic solvent, with high accuracy and high sensitivity. Therefore, it can be used as a pipette, a dispenser chip, a storage container, a dilution container, and the like, which are instruments for analyzing a biological substance.

Claims (3)

A biochemical research instrument characterized by being molded of an ethylene-vinyl alcohol copolymer. The biochemical research instrument according to claim 1, wherein the ethylene-vinyl alcohol copolymer has an ethylene copolymerization ratio of 5 mol% to 50 mol%. The biochemical research instrument according to claim 1 or 2, which is exposed to radiation or plasma.