Articles having localized molecules disposed thereon and methods of producing same
Methods of producing substrates having selected active chemical regions by employing elements of the substrates in assisting the localization of active chemical groups in desired regions of the substrate. The methods may include optical, chemical and/or mechanical processes for the deposition, removal, activation and/or deactivation of chemical groups in selected regions of the substrate to provide selective active regions of the substrate.
1. A method of producing a substrate having an enzyme selectively immobilized in preselected regions thereon, comprising:
providing a substrate having a plurality of nanoscale wells defined thereon and having photoactivatable chemical functional groups immobilized on a surface of the substrate, wherein each of the nanoscale wells limits an ability of light to provide active chemical groups on selected portions of the surface of the substrate, wherein the nanoscale wells substantially prevent portions of the substrate surface other than the selected portions from being exposed to the light, wherein the selected portions are bottom surfaces of zero mode waveguides;
exposing the substrate to the light to selectively provide active chemical groups on the selected portions of the surface of the substrate, wherein the light is selected to activate the photoactivatable chemical functional groups; and
coupling the enzyme to the active chemical groups or to an additional group which is coupled to the active chemical groups, thereby coupling the enzyme to the selected portions of the surface of the substrate; wherein density of the enzyme coupled to the selected portions of the surface of the substrate is 100 times or more greater than density of the enzyme on the portions of the substrate surface other than the selected portions.
2. The method of claim 1 , wherein the photoactivatable chemical functional groups comprise photoactivatable groups selected from the group consisting of: nitroveratryl, 1-pyrenylmethyl, 6-nitroveratryloxycarbonyl, dimethyldimethoxybenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, methyl, methyl-6-nitropiperonyloxycarbonyl, 2-oxymethylene anthraquinone, dimethoxybenzyloxy carbonyl, 5-bromo-7-nitroindolinyl, o-hydroxy-alpha-methyl cinnamoyl, and mixtures thereof.
3. The method of claim 1 , wherein:
the photoactivatable chemical functional groups comprise first photoactivatable chemical functional groups;
the light comprises light at a wavelength selected to activate the first photoactivatable chemical functional groups;
the substrate comprises an opaque layer and a transparent layer;
the nanoscale wells comprise zero mode waveguides disposed through the opaque layer to the transparent layer; and
the exposing step comprises directing the light at the transparent layer of the substrate to activate the first photoactivatable chemical functional groups to provide first active chemical functional groups within the zero mode waveguides that are on or proximal to the transparent layer.
4. The method of claim 3 , further comprising:
capping the first active chemical functional groups provided in the exposing step, to provide second photoactivatable chemical functional groups that are activatable by light of a different wavelength than the light in the exposing step; and
directing light at the transparent layer of the substrate, wherein the light is at a wavelength selected to activate the second photoactivatable chemical functional groups but not the first photoactivatable chemical functional groups;
wherein the directing step is carried out under conditions to activate a portion of the chemical functional groups activated in the first exposing step.
5. The method of claim 1 , wherein coupling the enzyme to the active chemical groups or to an additional group which is coupled to the active chemical groups comprises coupling the enzyme to biotin, avidin, streptavidin, or neutravidin.
6. The method of claim 1 , wherein the enzyme is a polymerase.
7. The method of claim 1 , further comprising providing inert groups on the portions of the substrate surface other than the selected portions.
8. The method of claim 1 , wherein the substrate comprises a planar upper surface having the nanoscale wells defined therethrough; the method comprising: providing the substrate having photoactivatable chemical functional groups on a surface of the substrate by, prior to applying the photoactivatable chemical functional groups on the surface of the substrate, applying inert groups upon the planar upper surface of the substrate without depositing the inert groups into the nanoscale wells.
9. The method of claim 1 , wherein the nanoscale wells are disposed through a planar upper surface of the substrate; wherein each nanoscale well comprises a side wall and a bottom surface; and wherein providing a substrate having photoactivatable chemical functional groups on a surface of the substrate comprises applying the photoactivatable chemical functional groups on the planar upper surface of the substrate and on the side walls and bottom surfaces of the nanoscale wells.
10. The method of claim 3 , wherein the opaque layer comprises a metal oxide outer layer.
11. The method of claim 10 , wherein the transparent layer comprises SiO 2 .