DNA-based assays
Described herein DNA-based assays that include nanostructures providing a simple, highly sensitive method for small molecule, biologic and peptide measurements that are optimized for use in a commercial, point-of-care diagnostic settings and systems. These DNA-based assays may include a combination of multiple redox molecules (e.g., methylene blue) such as between 3-8 redox molecules, and a spacer of between about 2 A and 20 A (and in particular between 3 A and 8 A), that results in an assay that has significantly greater signal and greater sensitivity as compared to prior DNA-based assays.
1 . A DNA-based assay comprising:
a continuous DNA molecule comprising a first hairpin structural motif attached to a second hairpin structural motif via a first segment of single stranded DNA;
an anchor recognition moiety coupled to the continuous DNA molecule;
a signal moiety comprising a plurality of redox molecules, wherein the signal moiety is coupled to the continuous DNA molecule,
wherein the signal moiety and the anchor recognition moiety are in effective proximity to each other; and
a thiol-DNA spacer segment attached at a first end to an electrically conductive substrate and at a second end to the first or second hairpin structural motif such that it forms a single stranded tether region at one end of the single continuous DNA molecule extending between 2.72 and 6.8 nanometers (nm).
2 . The assay of claim 1 , wherein the signal moiety comprises between 2 and 15 redox molecules.
3 . The assay of claim 1 , wherein each of the plurality of redox molecule comprises a methylene blue molecule.
4 . The assay of claim 1 , wherein the concentration of the thiol-DNA spacer is between 1 nM and 5 uM.
5 . The assay of claim 1 , wherein the signal moiety comprises between 3 and 8 methylene blue molecules and wherein the single stranded tether region extends between 2.04 and 6.8 nm.
6 . The system of claim 5 , wherein the signal moiety comprises between 4 and 8 redox molecules.
7 . The system of claim 5 , wherein each of the plurality of redox molecule comprises a methylene blue molecule.
8 . The assay of claim 1 , wherein the signal moiety comprises between 3 and 8 methylene blue molecules and wherein the single stranded tether region extends between 2.04 and 6.8 nm.
9 . The method of claim 8 , wherein the signal moiety comprises between 2 and 15 redox molecules.
10 . The method of claim 8 , wherein each of the plurality of redox molecule comprises a methylene blue molecule.
11 . The method of claim 8 , where the single stranded tether region extends between 3.4 and 6.8 nm.
12 . The method of claim 8 , wherein the signal moiety comprises between 3 and 8 methylene blue molecules and wherein the single stranded tether region extends between 3.4 and 6.8 nm.
13 . A system comprising:
a nanostructure comprising:
a continuous DNA molecule comprising a first hairpin structural motif attached to a second hairpin structural motif via a first segment of single stranded DNA;
an anchor recognition moiety coupled to the continuous DNA molecule;
a signal moiety comprising between 3 and 10 redox molecules, wherein the signal moiety is coupled to the continuous DNA molecule, wherein the signal moiety and the anchor recognition moiety are in effective proximity to each other; and
a thiol-DNA spacer segment attached at a first end to the first or second hairpin structural motif such that it forms a single stranded tether region at one end of the single continuous DNA molecule extending between 2.72 and 6.8 nanometers (nm); and
an electrode having a surface, wherein the thiol-DNA spacer of the nanostructure is coupled at a second end to the to the surface in a concentration of between 1 nM and 5 uM.
14 . A DNA nanostructure-based method, the method comprising:
exposing a DNA-based nanostructure to a sample material comprising a target molecule, wherein the DNA-based nanostructure comprises:
a continuous DNA molecule comprising a first hairpin structural motif attached to a second hairpin structural motif via a first segment of single stranded DNA;
an anchor recognition moiety coupled to the continuous DNA molecule, wherein the anchor recognition moiety binds to the target molecule;
a signal moiety comprising a plurality of redox molecules, wherein the signal moiety is coupled to the continuous DNA molecule,
wherein the signal moiety and the anchor recognition moiety are in effective proximity to each other; and
a thiol-DNA spacer segment attached at a first end to an electrically conductive substrate and at a second end to the first or second hairpin structural motif such that it forms a single stranded tether region at one end of the single continuous DNA molecule extending between 2.72 and 6.8 nanometers (nm); and
quantifying an amount of the target molecule by analyzing the electrochemical signal from the DNA-based nanostructure, wherein the electrochemical signal changes in proportion to changes in concentration of the target.