Oxide layers on silicon substrates for effective confocal laser microscopy
View Patent ↗Methods of performing confocal laser microscopy on a polymer array disposed on a silicon wafer substrate, the method comprising the steps of providing a silicon wafer substrate having a top side and a bottom side, coating the top side of the silicon wafer with an oxide coating to provide an oxide coated wafer, covalently coupling a plurality of probes to the top side of the coated wafer to provide a fixed polymer array, hybridizing the fixed polymer array with a plurality of labeled ligands, and assaying for one or more hybridized ligands using confocal laser fluorescence microscopy to detect hybridization are provided.
1. A method of synthesizing a polymer array, the method comprising:
coating a substrate with an oxide layer, wherein the oxide layer has a thickness of at least 3,500 angstroms, and wherein the oxide layer is substantially transparent;
derivatizing the substrate, wherein derivatization forms a plurality of protected functional groups on the substrate, and wherein a functional group is protected by a labile protecting group;
deprotecting one or more of the protected functional groups, wherein deprotection removes the labile protecting group to produce an unprotected functional group;
coupling each of the one or more unprotected functional groups with a monomer, wherein the monomer comprises a protected functional group;
repeating the steps of deprotecting and coupling to produce the polymer array.
2. The method of claim 1 , wherein the oxide layer has a thickness from approximately 3,500 angstroms to approximately 35,000 angstroms.
3. The method of claim 1 , wherein the oxide layer has a thickness from approximately 35,000 angstroms to approximately 100,000 angstroms.
4. The method of claim 1 , wherein the oxide layer comprises an oxide material selected from the group consisting of silicon oxide, silicon dioxide, borophosphosilicate glass, borosilicate glass, fluorosilicate glass, and tetraethoxysilane.
5. The method of claim 1 , wherein the substrate is a silicon substrate.
6. The method of claim 1 , wherein the substrate is a plurality of beads.
7. The method of claim 1 , wherein derivatizing comprises coating the oxide layer with a functionalized silicon compound.
8. The method of claim 1 , wherein the labile protecting group is a photolabile protecting group.
9. The method of claim 8 , wherein the photolabile protecting group is selected from the group consisting of NNPOC, MBPMOC and NPPOC.
10. The method of claim 1 , wherein the labile protecting group is an acid labile protecting group.
11. The method of claim 10 , wherein deprotecting comprises activating a photoacid generator.
12. The method of claim 1 , wherein the substrate is flat or planar.
13. The method of claim 1 , wherein the polymer array comprises a plurality of different polymer sequences synthesized in different features.
14. The method of claim 13 , wherein the plurality of different polymer sequences comprises a plurality of different oligonucleotides.
15. The method of claim 13 , wherein the different features have a size between 1-10 μm 2 .
16. The method of claim 8 , wherein the photolabile protecting group has an average stepwise coupling efficiency of over 90%.
17. The method of claim 8 , wherein the photolabile protecting group is (2-nitro-naphtalen-1-yl)-phenylmethyloxycarbonyl (NNPOC).
18. The method of claim 7 , wherein coating the oxide layer with a functionalized silicon compound results in a functional group density on the substrate of at least 40 pmoles/cm 2 .
19. An array of polymers synthesized according to the method of claim 1 .
20. An array of polymers synthesized according to the method of claim 16 .