Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals – Carrier is inorganic
Reexamination Certificate
2001-05-02
2003-07-08
Ceperley, Mary E. (Department: 1641)
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
Carrier is inorganic
C435S007920, C436S525000, C436S526000, C436S527000, C436S529000, C436S531000, C530S391100, C530S410000
Reexamination Certificate
active
06589799
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to derivatized solid supports. More particularly, the present invention relates to solid supports derivatized with silanes containing aldehydic functionalities and their use in biological applications.
BACKGROUND OF THE INVENTION
The use of immobilized bio-molecules is an essential technique required for many biological applications. A common method used to immobilize bio-molecules is by reaction of the primary amine groups of the biological molecules with an aldehyde functionality that is bonded to a solid support matrix.
A popular method of introducing aldehydes to the solid support matrix is through the activation of an amine-functionalized surface with a glutaraldehyde solution. Although popular, this method has several disadvantages. First glutaraldehyde is an unstable compound that is difficult to purify. Additionally, two Schiff bases are present in the covalent linkage of the bio-molecule to the support. Additionally, the Schiff base linkage of the glutaraldehyde to the support is susceptible to hydrolysis and thus may lead to ligand leaching. Treatment with a reducing agent such as sodium borohydride or sodium cyanoborohydride to remove the Schiff bases can be performed, but this adds an additional step to the process.
There remains a need for a solid support matrix with bonded aldehydic functionalities for immobilizing bio-molecules that is stable and can be produced in a simple process.
Accordingly, it is desirable to provide a solid support matrix containing bonded aldehydic functionalities that is stable and can be used to immobilize bio-molecules. It is further desirable to provide a simple method for producing such a solid support matrix. It is still further desirable to provide an apparatus and method for using a solid support matrix with aldehydic functionalities to immobilize bio-molecules for biological applications.
SUMMARY OF THE INVENTION
The present invention is directed to a derivatized solid support matrix containing bonded aldehydic functionalities, that is stable and can be used to immobilize bio-molecules, as well as to a method for producing such a derivatized solid support matrix.
The present invention is further directed to an apparatus and method for using a derivatized solid support matrix with aldehydic functionalities to immobilize bio-molecules for biological applications.
In accordance with one embodiment of the present invention, a method of producing a derivatized matrix material with aldehyde groups is provided. A raw support matrix material having a surface area with hydroxyl groups occurring on the surface area is activated with an acid. The activated support material is then exposed to an alkoxy aldehydic silane to produce a derivatized matrix material. The support matrix material can be selected from a number of materials, including but not limited to glasses, agarose, silica, alumina, glass-coated ELISA plates, resin, nickel, aluminum, zinc and paramagnetic iron. The support matrix material may have naturally occurring hydroxyl groups, or the hydroxyl groups may be introduced artificially. A number of mono, di and tri alkoxy aldehydic silanes may be used for producing a derivatized matrix material with aldehyde groups. The alkoxy aldehydic silane is preferably a trialkoxy aldehydic silane.
In another embodiment of the present inventions, an aldehydic derivatized matrix material comprises a support matrix material having a surface area, and a siloxane coating is disposed on at least a portion of the surface area. The siloxane coating may be a mono-layer or a cross linked siloxane polymer coating. The siloxane coating has a plurality of organic substituents containing aldehydic functional groups pendant therefrom. The organic substituents are bound to the siloxane coating via carbon-silicon covalent bonds.
In accordance with another embodiment of the invention, the aldehydic derivatized support matrix material can be incorporated into an apparatus for immobilizing bio-molecules for biological applications. Such biological applications include, but are not limited to, combinatorial chemistry, molecular biology, ELISA (Enzyme-Linked Immunosorbent Assay) plates, and cell sorting and identification.
There are additional features of the invention that will be described below and which will form the subject matter of the claims appended hereto.
It is to be understood that the invention is not limited in its application to the details of construction and to the arrangements of the components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced and carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein, as well as the abstract included below, are for the purpose of description and should not be regarded as limiting.
As such, those skilled in the art will appreciate that the conception upon which this disclosure is based may readily be utilized as a basis for the designing of other structures, methods and systems for carrying out the several purposes of the present invention. It is important, therefore, that the claims be regarded as including such equivalent constructions insofar as they do not depart from the spirit and scope of the present invention.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION
One aspect of the present invention provides a composition comprising a derivatized solid support matrix material having a surface and a siloxane coating on at least a portion of its surface, linked through Si—O bonds. The siloxane coating comprises a plurality of silicon atoms that are either individually mono-linked to the solid support matrix or cross-linked to each other and further bonded to the support matrix through Si—O bonds to form a siloxane polymer. Each Si unit in the coating has a pendant aldehyde containing organic substituent, bound to the siloxane coating via carbon-silicon covalent bonds. The siloxane coating is further bound to the solid support matrix material through Si—O—M bonds, wherein M is the support matrix material.
According to one embodiment of the composition, the siloxane coating comprises a multi layer siloxane polymer 1 to 10 Si units deep, preferably 2 to 5 units deep. When viewed in cross section, this embodiment has the general structure:
where R is an aldehyde containing organic substituent. R
1
is C
1
to C
30
alkyl, C
1
to C
30
alkenyl, phenyl, naphthyl or hydrogen, and M is the support matrix material. It should be recognized that in this embodiment the placement of the individual Si units is random with respect to the support matrix material. The example given is for illustrative purposes only and is not meant to limit the scope of the invention.
In an alternate embodiment, the individual Si units are cross linked to form a siloxane polymer 1 Si unit deep, which when viewed in cross section, has the structure:
where R is an aldehyde containing organic substituent and M is the support matrix material.
In a further embodiment, the individual Si units are mono linked to the solid support matrix material, which when viewed in cross section would have the structure:
where R is an aldehyde containing organic substituent. R
1
is alkyl or alkenyl containing 1 to 30 carbon atoms, phenyl, naphthyl or hydrogen and M is the support matrix material.
The aldehyde containing organic substituent, R, may be a straight chain, branched or cyclic alkane containing from 1 to 30 carbon atoms. Alternatively, R may be aromatic or some other unsaturated aldehyde-containing hydrocarbon. For example, wherein R is formaldehyde, a single layer polymer coating would have the following structure when viewed in cross section:
In an alternative embodiment the aldehydic organic substituents pendant from the Si units may vary between the individual Si units, as shown by:
where R′, R″ and R′″ may all be different aldehyde containing organic substituents.
Suitable materials for the solid supp
Coyne Ann N.
MacMillan John H.
Telepchak Michael J.
Buchanan Ingersoll P.C.
Ceperley Mary E.
Koons, Jr. Robert A.
McWilliams Matthew P.
United Chemical Technologies Inc.
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