ZERO-MODE WAVEGUIDE FOR SINGLE BIOMOLECULE FLUORESCENCE IMAGING
The disclosed subject matter provides a zero-mode waveguide (ZMW) including a substrate and at least one nano-well thereon and having a bottom surface and a side wall comprising gold. A surface of the side wall is passivated with a first functional molecule comprising polyethylene glycol. The bottom surface of the nano-well can be functionalized with at least one second molecule comprising polyethylene glycol, for example, a silane-PEG molecule. The second molecule can further include a moiety, such as biotin, which is capable of binding a target biomolecule, which in turn can bind to a biomolecule of interest for single molecule fluorescence imaging analysis. Fabrication techniques of the ZMW are also provided.
1 . A zero-mode waveguide, comprising:
a substrate,
at least one nano-well on the substrate, the at least one nano-well having a bottom surface and a side wall comprising gold;
wherein a surface of the side wall is passivated with a first functional molecule comprising polyethylene glycol.
2 . The zero-mode waveguide of claim 1 , wherein the at least one nano-well has a width of from about 25 to about 500 nm.
3 . The zero-mode waveguide of claim 1 , wherein the side wall has a height of from about 50 to about 500 nm.
4 . The zero-mode waveguide of claim 1 , wherein the first functional molecule is attached to the surface of the side wall via a S—Au bond.
5 . The zero-mode waveguide of claim 1 , wherein the first functional molecule form a monolayer on the side wall.
6 . The zero-mode waveguide of claim 1 , wherein the first functional molecule further comprises polyalkylene.
7 . The zero-mode waveguide of claim 6 , wherein the polyethylene glycol of the first functional molecule comprises from about 1 to about 200 ethylene oxide units.
8 . The zero-mode waveguide of claim 1 , wherein the bottom surface of the at least one nano-well is functionalized with at least one second molecule comprising polyethylene glycol.
9 . The zero-mode waveguide of claim 8 , wherein the polyethylene glycol of the second functional molecule comprises from about 1 to about 200 ethylene oxide units.
10 . The zero-mode waveguide of claim 8 , wherein the at least one second functional molecule comprises a molecule having a moiety capable of binding with a target biomolecule.
11 . The zero-mode waveguide of claim 10 , wherein the moiety comprises a biotin moiety.
12 . The zero-mode waveguide of claim 11 , wherein the target biomolecule is streptavidin.
13 . The zero-mode waveguide of claim 8 , wherein the bottom surface comprises silica, and the at least one second functional molecule is attached to the bottom surface via a Si—O—Si linkage.
14 . The zero-mode waveguide of claim 8 , wherein the at least one second functional molecule comprises a mixture of (1) a molecule comprising polyethylene glycol and having a moiety capable of binding with a target biomolecule, and (2) a molecule comprising polyethylene glycol and having no moiety capable of binding with the target biomolecule.
15 . The zero-mode waveguide of claim 13 , further comprising the target biomolecule bound to the moiety.
16 . The zero-mode waveguide of claim 1 , further comprising a layer of titanium or chromium disposed between the substrate and the side wall of the at least one nano-well.
17 . A method for fabricating a zero-mode waveguide, comprising:
forming at least one nano-well on a substrate, the nano-well having a bottom surface, and a side wall comprising gold; and
passivating a surface of the side wall with a first functional molecule comprising polyethylene glycol.
18 . The method of claim 17 , wherein the first functional molecule comprises a thiol end group, and wherein the passivating comprises reacting the thiol end group with the surface of the side wall to form a S—Au bond coupling the first functional molecule with the surface.
19 . The method of claim 17 , further comprising functionalizing the bottom surface of the at least one nano-well with at least one second molecule comprising polyethylene glycol.
20 . The method of claim 19 , wherein the at least one second functional molecule comprises a silane end group, wherein the bottom surface of the at least one nano-well comprise silica, and wherein the functionalizing comprises reacting the silane end group with the bottom surface to form a Si—O—Si bond coupling the second functional molecule with the bottom surface.
21 . The method of claim 19 , wherein the functionalizing comprises functionalizing the bottom surface with a mixture of (1) a molecule comprising polyethylene glycol and having a moiety capable of binding with a target biomolecule, and (2) a molecule comprising polyethylene glycol and having no moiety capable of binding with the target biomolecule.
22 . The method of claim 21 , wherein the moiety comprises a biotin moiety, and the target biomolecule is streptavidin.
23 . The method of claim 17 , wherein the substrate is a silica substrate, wherein the forming further comprises:
applying a photoresist on the surface of the silica substrate;
forming at least one nano-column in the photoresist by etching;
depositing a thin layer of titanium on the substrate;
depositing a layer of gold onto the layer of titanium; and
removing the at least one nano-column in the photoresist, thereby creating the at least one nano-well having a bottom surface and a side wall comprising gold.