Graphene-enabled DNA biosensors with enhanced sensitivity and specificity
Provided are electronic biosensors based on graphene field effect transistors that utilize engineered hairpin probe DNA that allows for target recycling and hybridization chain reactions.
1 . A detector, comprising:
a first portion of functionalized graphene;
a first hairpin probe (H1) comprising a plurality of nucleotides, the first hairpin probe (H1) being linked through a graphene field effect transistor (GFET) to the first portion of functionalized graphene,
in its hairpin configuration, the first hairpin probe (H1) comprising (a) a bottom portion linked to the portion of graphene, and (b) a paired portion, and
the first hairpin probe (H1) opening upon binding by a target molecule (T) with the paired portion of the hairpin probe (H1), the binding giving rise to an H1-T complex having a paired region formed between the first hairpin probe (H1) and the target (T),
the H1-T complex further having a protruding segment.
2 . The detector of claim 1 , wherein the paired portion of the first hairpin probe comprises an aptamer region, the aptamer region being configured to bind to the target molecule (T).
3 . The detector of claim 1 , further comprising an amount of a first hairpin helper (H2) that comprises a plurality of polynucleotides, the first hairpin helper (H2) further being capable of binding to the protruding segment and the paired region of the H1-T complex so as to displace the target (T) from the H1-T complex and give rise to a H1-H2 complex having a protruding segment.
4 . The detector of claim 3 , further comprising an amount of a second hairpin helper (H3) that comprises a plurality of polynucleotides, the second hairpin helper (H3) further comprising a toehold portion that is capable of binding to the protruding segment of the H1-H2 complex so as open second hairpin helper (H3) and give rise to a H1-H2-H3 complex having a protruding segment.
5 . The detector of claim 4 , further comprising an amount of a third hairpin helper (H4) that comprises a plurality of polynucleotides, the third hairpin helper (H4) further comprising a toehold portion capable of binding to the protruding segment of the H1-H2-H3 complex so as give rise to a H1-H2-H3-H4 complex.
6 . The detector of claim 1 , further comprising a second hairpin probe (H1′) comprising a plurality of nucleotides, the second hairpin probe (H1′) being linked through a graphene field effect transistor (GFET) to the first portion of graphene, in its hairpin configuration, the second hairpin probe (H1′) comprising (a) a bottom portion linked to the first portion of graphene, and (b) a paired portion, the second hairpin probe (H1′) differing from the first hairpin probe (H1) by at least one nucleotide.
7 . The detector of claim 1 , further comprising a monitor in electronic communication with the first portion of graphene, the monitor capable of detecting a signal related to one or more of (a) binding between a target and the first hairpin probe (H1) and (b) formation of a complex that comprises the first hairpin probe (H1) and one or more hairpin helpers.
8 . The detector of claim 7 , wherein the signal is a change in Dirac voltage.
9 . A method of detecting a target, comprising
to a first hairpin probe (H1) comprising a plurality of nucleotides, the first hairpin probe (H1) being linked through a graphene field effect transistor (GFET) to a first portion of graphene, in its hairpin configuration, the first hairpin probe (H1) comprising (a) a bottom portion linked to the portion of graphene, (b) a paired portion,
contacting the first hairpin probe (H1) with a target molecule (T) under such conditions that the target molecule (T) binds with the paired portion of the hairpin probe (H1) and the first hairpin probe (H1) opens, the binding giving rise to an H1-T complex having a paired region formed between the first hairpin probe (H1) and the target (T), the H1-T complex further having a protruding segment.
10 . The method of claim 9 , further comprising contacting the H1-T complex with a first hairpin helper (H2) that comprises a plurality of polynucleotides, the first hairpin helper (H2) being capable of binding to the protruding segment and the paired region of the H1-T complex, the contacting being performed so as to displace the target (T) from the H1-T complex and give rise to a H1-H2 complex having a protruding segment.
11 . The method of claim 10 , further comprising contacting the H1-H2 complex with a second hairpin helper (H3) that comprises a plurality of polynucleotides, the second hairpin helper (H3) further comprising a toehold portion that is capable of binding to the protruding segment of the H1-H2 complex, the contacting being performed so as open second hairpin helper (H3) and give rise to a H1-H2-H3 complex having a protruding segment.
12 . The method of claim 9 , further comprising effecting contact between target (T) displaced from the H1-T complex and a further hairpin probe (H1,2), the second hairpin probe (H1,2) being complementary to the target (T), the contacting giving rise to an H1,2-T complex having a paired region formed between the further hairpin probe (H1,2) and the target (T), the H1,2-T complex further having a protruding segment.
13 . The method of claim 12 , further comprising contacting the H1,2-T complex with a first hairpin helper (H2) that comprises a plurality of polynucleotides, the first hairpin helper (H2) further being capable of binding to the protruding segment and the paired region of the H1,2-T complex, the contacting being performed so as to displace the target (T) from the H1,2-T complex and give rise to H1,2-H2 complex having a protruding segment.
14 . The method of claim 10 , further comprising detecting a signal related to one or more of (a) binding between a target and the first hairpin probe (H1), and (b) formation of a complex that comprises the first hairpin probe (H1) and one or more hairpin helpers.
15 . The method of claim 14 , wherein the signal is a change in Dirac voltage.
16 . The method of claim 10 , wherein the target is present at a concentration of less than about 100 fM.
17 . A method of detecting a target, comprising:
to one or more first hairpin probes (H1 i =1 to n) each comprising a different plurality of nucleotides,
the plurality of first hairpin probes being linked through a graphene field effect transistor (GFET) to and a first portion of graphene,
each of the plurality of first hairpin probes in its hairpin configuration comprising (a) a bottom portion linked to the portion of graphene, (b) a paired portion, and
contacting the plurality of first hairpin probes with one or more target molecules (T j , j=1 to m) under such conditions that one or more of the target molecules binds with the paired portion of one or more first hairpin probes and the first hairpin probe opens,
the binding giving rise to an H1 i -T j complex having a paired region formed between the first hairpin probe (Hi 1 ) and the target (T j ), the H1 i -T complex further having a protruding segment.
18 . The method of claim 17 , further comprising contacting a H1 i -T j complex with one or more first hairpin helpers (H2) that comprises a plurality of polynucleotides, the first hairpin helper (H2) further comprising a toehold portion that is capable of binding to the protruding segment and the paired region of the H1 i -T j complex, the contacting being performed so as to displace the target (T i ) from the H1 i -T i complex and give rise to a H i -H2 complex having a protruding segment, one or more of the first hairpin helpers H2 optionally being unique to a particular H1 i -T j complex.
19 . The method of claim 18 , further comprising contacting the H1 i -H i complex with a second hairpin helper (H3) that comprises a plurality of polynucleotides, the second hairpin helper (H3) further comprising a toehold portion capable of binding to the protruding segment of the H1 i -H2 i complex, the contacting being performed so as open second hairpin helper (H3) and give rise to a H1 i -H2-H3 complex having a protruding segment.
20 . The method of claim 17 , further comprising detecting a signal related to one or more of (a) binding between one or more of the two or more targets (T i ) and one or more first hairpin probes (H1 j ) and (b) a complex that comprises the first hairpin probe (H1 j ) and one or more hairpin helpers.