Surface immobilized polyelectrolyte with multiple functional groups capable of covalently bonding to biomolecules
View Patent ↗A polyelectrolyte having multiple exposed functional groups, each such group being capable of covalently bonding to a molecule, is immobilized on a surface for the purpose of bonding to a biomolecule. The biomolecule can be, for example, a nucleic acid, e.g., an amine functionalized oligonucleotide. The polyelectrolyte can include, e.g., BSA (Bovine Serum Albumin) which is bound to a functionalized surface using a covalent immobilization strategy, e.g., reaction with the surface of a tosyl-activated microparticle. Following such reaction, exposed reactive functional groups on the protein, such as amine, carboxyl, thiol, hydroxyl groups can further be utilized to covalently couple the oligonucleotide of interest using suitable chemistry.
1. A method of producing a solid microparticle having nucleic acid molecules attached, said microparticle being made of a polymer, a polymer resin, glass or latex, wherein a surface of the microparticle is coated with Bovine Serum Albumin under conditions whereby the Bovine Serum Albumin has an increased number sites for covalent attachment of nucleic acid molecules, comprising:
covalently attaching Bovine Serum Albumin to said surface at a temperature of about 65° C.; and
covalently attaching nucleic acid molecules to said Bovine Serine Albumin.
2. The method of claim 1 wherein the nucleic acid is attached to the Bovine Serum Albumin through the Bovine Serum Albumin carboxyl or amine groups.
3. The method of claim 1 wherein the nucleic acid is an oligonucleotide.
4. The method of claim 1 wherein the nucleic acid molecules are bound to the Bovine Serum Albumin through an amide linkage, formed by an 1-ethyl-3-(3-imethylaminopropyl) carbodiimide reaction.
5. The method of claim 1 wherein the surface of the microparticle is activated with tosyl.
6. The method of claim 3 wherein the oligonucleotide is DNA, RNA, a peptide nucleic acid, a locked nucleic acid, or mixtures thereof.
7. The method of claim 3 wherein the oligonucleotide is modified at its 3′ or 5′ end with a functional group.
8. The method of claim 7 wherein the functional group is a primary amino group.
9. A method of making a support coated with macromolecules in a first configuration capable of altering their conformation and exposing an increased number of covalent binding sites in response to an increase in temperature and capable of covalently binding oligonucleotides, comprising:
adsorbing to the support, at a first temperature, said macromolecules in said first configuration; and
covalently attaching the adsorbed macromolecules to the support at a second temperature at which said adsorbed macromolecules change to a second conformation in which additional sites for covalent binding of oligonucleotides are exposed.
10. The method of claim 9 wherein a single layer of the macromolecules are adsorbed to the support.
11. The method of claim 9 wherein the macromolecules are selected from the group consisting of protein, polypeptide and glycoprotein.
12. The method of claim 11 wherein the protein is bovine serum albumin.
13. The method of claim 9 wherein the support is a microparticle.