The present invention relates to a method for preparing silica-coated magnetic bead.
Bio-functionalized magnetic particles have been applied to biomedicines. Various bio-applications require different sizes of magnetic particles. For example, due to the strong magnetism of each particle, magnetic particles with micrometers in diameter are useful for in-vitro extraction or purification of bio-molecules like antibodies, proteins, and nucleic acids.
Recently small nucleotide plays important roles in different biological pathway, which causes the need to provide silica-coated magnetic beads to capture these important small fragments of nucleotide.
Thus, there is a demand for improved silica-coated particles for binding to small fragments of DNA, especially small nucleotide fragments under 100 base pairs. Further, there is a demand for silica-coated particles having thickness-tunable silica layer capable of being customized for different applications in biomedicine.
The present invention relates to a method of preparing a sedimentation adjustment silica-coated magnetic bead, comprising the following steps: (1) preparing a magnetic core; (2) adding various ratio of silica coating material to coat the magnetic core with silica forming a silica-coated magnetic bead; and (3) washing the silica-coated magnetic beads sequentially by alcoholic solvent and water.
The present invention also relates to a silica-coated magnetic bead prepared by the above method of the present invention, comprising a magnetic core and a tunable silica layer, wherein the thickness of the tunable silica layer is ranging from 0.77-2.31 μm.
The present invention is more fully appreciated by reference to the following description, including the following glossary of terms and the concluding examples. For the sake of brevity, the disclosures of the publications, including patents, cited in this specification are herein incorporated by reference.
As used herein in the specification, “a” or “an” may mean one or more. As used herein in the claim(s), when used in conjunction with the word “comprising”, the words “a” or “an” may mean one or more than one.
The present invention provides a method of preparing a sedimentation adjustment silica-coated magnetic bead, comprising the following steps: (1) preparing a magnetic core; (2) adding various ratio of silica coating material to coat the magnetic core with silica forming a silica-coated magnetic bead; and (3) washing the silica-coated magnetic bead sequentially by an alcoholic solvent and water.
In one embodiment, the step (1) of the above method further comprises the following steps: (a) mixing a ferric salt and a ferrous salt in an alkaline solution and heating from 60° C. to 90° C. to form the magnetic core; (b) cooling the magnetic core to room temperature; and (c) adding the alkaline solution and the alcoholic solvent to the magnetic core.
As used herein, “ferric salt” refers to a ferric iron-containing-compound or a material, also denoted Fe(III) salt. In one preferred embodiment, the ferric salt is FeCl3. As used herein, “ferrous salt” refers to a ferrous iron-containing-compound or a material, also denoted Fe(II) salt. In one preferred embodiment, the ferrous salt is FeCl2.
According to an example embodiment, the ferric salt is 0.02 to 0.42 mole and the ferrous salt is 0.024 to 0.3 mole.
In one embodiment, the alkaline solution is 5 to 25%. In one preferred embodiment, the pH value of the alkaline solution is more than 11.
According to another example embodiment, the added volume of the alkaline solution in the step (c) is 0.05 to 0.1 times based on a mixture volume of the step (a). In another embodiment, the added volume of the alcoholic solvent in step (c) is 1 to 3 times based on a mixture volume of the step (a).
In one embodiment, the alcoholic solvent includes but is not limited to a solvent containing an organic compound in which the hydroxyl functional group (—OH) is bound to a carbon atom. In a preferred embodiment, the alcoholic solvent is ethanol. In another embodiment, the water is ddH2O.
According to a further example embodiment, the various ratio of silica coating material is 0.05% to 2%. In another embodiment, the step (2) of the method of preparing a sedimentation-adjusted silica-coated magnetic bead controls the sedimentation rate of the silica-coated magnetic bead.
The term “silica coating material” used herein means a crosslinking agent in silicone polymers and as a precursor to silicon dioxide, including but not limited to a tetrabutyl orthosilicate, a tetrapropoxysilane, a tetraethyl orthosilicate, a tetraisopropyl orthosilicate or a sodium silicate.
The invention also provides a silica-coated magnetic bead prepared by the method of preparing a sedimentation-adjusted silica-coated magnetic bead of the present invention, comprising a magnetic core and a tunable silica layer, wherein the thickness of the tunable silica layer is ranging from 0.77-2.31 μm.
In one embodiment, the thickness of the tunable silica layer is ranging from 0.65-1.9 μm. In a preferred embodiment, the thickness of the tunable silica layer is ranging from 0.42-1.54 μm.
In one embodiment, the material of the tunable silica layer is a tetrabutyl orthosilicate, a tetrapropoxysilane, a tetraethyl orthosilicate, a tetraisopropyl orthosilicate or a sodium silicate.
According to an example embodiment, the silica-coated magnetic bead further is bound to a small nucleotide. In a preferred embodiment, the length of the small nucleotide is 10˜100 base pair. In a more preferred embodiment, the small nucleotide is RNA or DNA.
The examples below are non-limiting and are merely representative of various aspects and features of the present invention.
Procedure of preparing silica-coated magnetic beads of the present invention:
After obtaining the silica-coated magnetic beads, the particle size of the silica-coated magnetic beads of the present invention were further analyzed. The analysis steps included the following:
Table 1 shows the mean diameters of series of the silica-coated magnetic beads of the present invention.
The different particle sizes of above series of silica-coated magnetic beads came from the various thicknesses of the tunable silica layer. The thickness of the tunable silica layer is calculated by the following formula: (the mean diameter of Type X)−(the mean diameter of Type Y). For example, the difference in thickness of the tunable silica layers between type 5 and type 1 is 1.54 μm (4.47−2.93=1.54). Therefore, the range of the thickness of the tunable silica layer was from 0.42-1.54 μm.
The analyzing steps of the sedimentation rate of the silica-coated magnetic beads:
Table 2 further shows the result of the sedimentation rate of those five silica-coated magnetic beads illustrated on
The procedure of preparing silica-coated magnetic beads binding to DNA fragments:
Those skilled in the art recognize the foregoing outline as a description of the method for communicating hosted application information. The skilled artisan will recognize that these are illustrative only and that many equivalents are possible.