Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals – Carrier is inorganic
Reexamination Certificate
2000-11-01
2003-01-07
Le, Long V. (Department: 1641)
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
Carrier is inorganic
C436S527000, C436S518000, C435S006120, C427S212000, C427S213360, C428S900000, C210S222000, C210S502100, C210S504000, C210S506000, C210S510100, C524S547000, C524S458000, C524S459000, C524S062000
Reexamination Certificate
active
06503762
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a magnetic carrier having polyacrylamide on the surface thereof, having controlled properties of a magnetic material content, a particle shape, a particle diameter, and a pore diameter, and being suitable for adsorption and extraction. The present invention also relates to a process for producing the magnetic carrier. The present invention further relates to a method of extraction of a nucleic acid by use of the magnetic carrier.
2. Description of the Related Art
Particulate silica, and the like particulate matters are well known as an adsorbent, and a solid carrier for supporting an adsorbent. The carrier is recovered after use by centrifugation, filtration, or a like operation, which is not convenient. In the adsorption or extraction operation, the objective substance should be isolated from the adsorbate or extract. However, the conventional separation by centrifugation, column separation, or a like method takes a long time, and requires a large apparatus, which is not convenient.
For separation of an objective substance, for example, JP-A-60-244251 (“JP-A” herein means unexamined published Japanese patent application) discloses a method of recovery of objective particles in which a ferromagnetic material is added to the particles and a magnetic field is applied thereto. However, this method has disadvantages that the ferromagnetic material itself may cause self-aggregation in operation of adsorption, extraction, reaction, and so forth, especially under application of a magnetic field, and that the state of the particles cannot be controlled as desired for uniform dispersion for the operation.
For preventing the self-aggregation of ferromagnetic material, JP-A-61-181967 discloses use of a superparamagnetic material as the magnetic material, and Tokuhyo 4-501957 (“Tokuhyo” herein means published Japanese translations of PCT international publication for patent application) discloses use of magnetic particles containing a superparamagnetic material as a solid phase for fixing a sample in separation and analysis of proteins, cells, and DNA. Japanese Patent No. 2554250 discloses a highly movable reagent carrier constituted of a gel matrix having a superparamagnetic reactive substance fixed thereon. In these methods, the superparamagnetic substance is contained in a state of fine micro-particles smaller than the size of the magnetic domains for keeping a magnetic body like iron oxide permanently magnetic, and the p articles in a solution are made to aggregate by application of an external magnetic field. By these methods, however, the properties of the magnetic particles are not sufficiently controllable. Therefore, a method is demanded which produces magnetic particles most suitable for the respective uses by controlling the properties thereof, particularly the particle diameter, the pore diameter, the pore volume, and the specific surface area of the magnetic particles, the quantity of the magnetic material in the magnetic particles, and silica concentration of the particle surface.
Particulate silica, for use as an adsorbent or an extractant, is modified preliminarily by introduction of a specific functional group or adsorption of nucleic acid or the like on the surface thereof. For example, the gel for high-speed liquid chromatography is modified by introducing specific functional groups therein. In conventional modification, however, the introduced functional group may come off to cause decrease of the amount of the functional group, resulting in deterioration of the performance as the adsorbent, disadvantageously.
In one method of introduction of polyacrylamide into particulate magnetic silica, the gel is mixed with acrylamide, and the acrylamide is polymerized to deposit the polyacrylamide onto the gel. In this method, the polyacrylamide simply covers the gel without direct bonding between the gel and the polyacrylamide, tending to come off from the gel during operation of adsorption or extraction, disadvantageously.
For formation of a bond between a carrier like the gel and the polyacrylamide, a reaction should be caused between the gel and the polyacrylamide. In the reaction, the polyacrylamide can difficultly be introduced in a specified amount by direct reaction with the functional groups on the gel, and the amount of introduced polyacrylamide is not reproducible. Therefore, a method is demanded for introducing polyacrylamide onto the gel with high reproducibility of the amount of the introduction.
JP-A-9-19292 discloses a method which employs particulate silica as a carrier for adsorbing nucleic acid. In this method, the nucleic acid is considered to be adsorbed onto the particles of silica by hydrogen bonding on the silica surface between the hydroxyl group of the silica particles and the basic group of the nucleic acid. However, the silica particles do not adsorb the nucleic acid in a sufficient amount by the surface thereof , or the amount of the adsorption is not reproducible owing to the steric hindrance by the bulkiness of the nucleic acid.
Tokuhyo 4-501959 discloses molecular oligonucleotide on a magnetic particle surface. In this method, an oligonucleotide having a sequence complementary to the nucleic acid to be adsorbed or extracted should be immobilized onto the magnetic particle surface. Therefore, particulate magnetic material should be provided which has a nucleotide complementary to the objective nucleic acid fixed thereon. For employing this method in clinical testing, many kinds of nucleotide-carrying magnetic particles should be prepared, which poses other problems such as the cost.
In the practice of a clinical test, biological samples such as serum, plasma, and humor may contain infective viruses or bacteria. When the nucleic acid is extracted manually from the sample, the operator is exposed to danger of the infection therewith. Thus, for manual extraction of a nucleic acid, the procedure is preferably simple, and is less liable to cause evolution of aerosol or the like from the sample, or more preferably the operation is automated. However, a conventional process of nucleic acid extraction is complicated and cannot readily be automated.
After comprehensive study to solve the above problems, it was found by the inventors of the present invention that silica particles containing a magnetic material (hereinafter referred to as “particulate magnetic silica”) are improved by introducing a prescribed amount of polyacrylamide thereto and adjusting the properties to increase the adsorption capacity and the adsorption speed to be suitable for an adsorbent and extractant for various uses. It was also found that the polyacrylamide-containing particulate magnetic silica can be produced readily by reacting the particulate magnetic silica with a coupling agent and then reacting it with acrylamide and/or polyacrylamide. Further it was found that a nucleic acid in a biological sample can be extracted readily and effectively by use of the particulate magnetic silica thus prepared. Consequently the present invention has been accomplished.
SUMMARY OF THE INVENTION
An object of the present invention is to provide a magnetic carrier which has controlled properties of a magnetic substance content, a shape, a particle diameter, and a pore diameter without the above disadvantages of the prior art.
Another object of the present invention is to provide a simplified process for producing the above magnetic carrier.
A further object of the present invention is to provide a simplified automatable method of extraction of a nucleic-acid with the above magnetic carrier.
The magnetic carrier of the present invention comprises particulate silica containing a magnetic material, and has polyacrylamide on the surface thereof in an amount ranging from 0.3 to 5 mmol/g in terms of monomeric acrylamide.
The process for producing the magnetic carrier of the present invention comprises the steps of (a) providing particulate magnetic silica by addition of a magnetic material to silica or a silica sourc
Kasai Kiyoshi
Yamauchi Syoichi
Do Pensee T.
Le Long V.
Sughrue & Mion, PLLC
Tosoh Corporation
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