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US 8,283,936 B2Granted
Nano-scale biosensors
AssigneeBoard of Regents, The University of Texas System
InventorsSamir M. Iqbal, Swati Goyal, Shawn M. Christensen, Mohammud R. Noor
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Abstract
Devices, systems, and methods for detecting nucleic acid hybridization, including single nucleic base mutations at low concentrations, are disclosed, using surface-tethered hairpin loop oligonucleotide probes and metal-nanoparticles conjugated to a hybridization detection sequence that is capable of binding the stem region of the opened hairpin loop oligonucleotide probe, without the use of labeling or target modification and capable of recycling.
Claims42
A device, comprising:
- a thermally responsive, electrically insulating substrate;
- at least one heating element;
- and
- a first detecting unit, comprising:
- a first electrode and a second electrode separated by a nanogap;
- and
- a plurality of first oligonucleotide probes attached to said substrate in said nanogap;
- wherein said first oligonucleotide probes are in a hairpin loop formation and have the same nucleic acid sequence;
- and
- wherein said first oligonucleotide probes comprise an optional spacer, a loop, and a stem region, said stem region being double-stranded.
7 more elements
A method for detecting nucleic acid hybridization, comprising:
- providing a device, comprising:
- a thermally responsive, electrically insulating substrate;
- at least one heating element;
- and
- a first detecting unit, comprising:
- a first electrode and a second electrode separated by a nanogap;
- and
- a plurality of first oligonucleotide probes attached to said substrate in said nanogap;
- wherein said first oligonucleotide probes are in a hairpin loop formation and have the same nucleic acid sequence;
- and
- wherein said first oligonucleotide probes comprise an optional spacer, a loop, and a stem region, said stem region being double-stranded;
- providing a solution comprising at least one buffer and single-stranded oligonucleotide target under hybridizing conditions;
- wherein said single-stranded oligonucleotide target hybridizes at least some of said first oligonucleotide probes to form elongated oligonucleotide probes;
- providing a plurality of first nanoparticle reporter conjugates under hybridizing conditions;
- wherein said first nanoparticle reporter conjugates comprise at least one nanoparticle and an oligonucleotide complementary to at least a portion of said stem of said first oligonucleotide probes;
- wherein said nanoparticle in said first nanoparticle reporter conjugate is a metal, semiconductor, or magnetic colloidal particle;
- applying a voltage drop across said electrodes;
- and
- measuring a change in conductivity, resistivity, capacitance, or impedance across said electrodes at known locations to determine perfect complementarity of said oligonucleotide target to said first oligonucleotide probes.
16 more elements
A method for detecting nucleic acid hybridization, comprising:
- providing a device, comprising:
- a thermally responsive, electrically insulating substrate;
- at least one heating element;
- and
- a first detecting unit, comprising:
- a first electrode and a second electrode separated by a nanogap;
- and
- a plurality of first oligonucleotide probes attached to said substrate in said nanogap;
- wherein said first oligonucleotide probes are in a hairpin loop formation and have the same nucleic acid sequence;
- and
- wherein said first oligonucleotide probes comprise an optional spacer, a loop, and a stem region, said stem region being double-stranded;
- providing a solution comprising at least one buffer and single-stranded oligonucleotide target under hybridizing conditions;
- wherein said single-stranded oligonucleotide target hybridizes at least some of said first oligonucleotide probes to form elongated oligonucleotide probes;
- providing a plurality of first reporter conjugates under hybridizing conditions;
- wherein said first reporter conjugates comprise an oligonucleotide complementary to at least a portion of said stem of said first oligonucleotide probes;
- reversibly exchanging an imino proton in each base pair of said first reporter conjugate or said stem of said first oligonucleotide probes with a metal ion selected from the group consisting of gold ion, silver ion, platinum ion, and copper ion;
- applying a voltage drop across said electrodes;
- and
- measuring a change in conductivity, resistivity, capacitance, or impedance across said electrodes at known locations to determine perfect complementarity of said oligonucleotide target to said first oligonucleotide probes.
16 more elements
A method for detecting nucleic acid hybridization, comprising:
- providing a device, comprising:
- a thermally responsive, electrically insulating substrate;
- at least one heating element;
- and
- a first detecting unit, comprising:
- a first electrode and a second electrode separated by a nanogap;
- and
- a plurality of first oligonucleotide probes attached to said substrate in said nanogap;
- wherein said first oligonucleotide probes are in a hairpin loop formation and have the same nucleic acid sequence;
- and
- wherein said first oligonucleotide probes comprise an optional spacer, a loop, and a stem region, said stem region being double-stranded;
- providing a solution comprising at least one buffer and single-stranded oligonucleotide target under hybridizing conditions;
- wherein said single-stranded oligonucleotide target hybridizes at least some of said first oligonucleotide probes to form elongated oligonucleotide probes;
- providing a plurality of first nanoparticle reporter conjugates under hybridizing conditions to form a double stranded nucleic acid sequence;
- wherein said first nanoparticle reporter conjugates comprise an oligonucleotide complementary to at least a portion of said stem of said first oligonucleotide probes;
- vectorially depositing silver on said double stranded nucleic acid sequence;
- applying a voltage drop across said electrodes;
- and
- measuring a change in conductivity, resistivity, capacitance, or impedance across said electrodes at known locations to determine perfect complementarity of said oligonucleotide target to said first oligonucleotide probes.
16 more elements
A method for detecting nucleic acid hybridization, comprising:
- providing a device, comprising:
- a thermally responsive, electrically insulating substrate;
- at least one heating element;
- and
- a first detecting unit, comprising:
- a first electrode and a second electrode separated by a nanogap;
- and
- a plurality of first oligonucleotide probes attached to said substrate in said nanogap;
- wherein said first oligonucleotide probes are in a hairpin loop formation and have the same nucleic acid sequence;
- and
- wherein said first oligonucleotide probes comprise an optional spacer, a loop, and a stem region, said stem region being double-stranded;
- providing a solution comprising at least one buffer and single-stranded oligonucleotide target under hybridizing conditions;
- wherein said single-stranded oligonucleotide target hybridizes at least some of said first oligonucleotide probes to form elongated oligonucleotide probes;
- providing a plurality of first reporter molecules under hybridizing conditions to form a double stranded oligonucleotide-stem complex;
- wherein said first reporter molecules comprise an oligonucleotide complementary to at least a portion of said stem of said first oligonucleotide probes;
- providing a solution comprising first nanoparticle polypeptide conjugates;
- wherein said first nanoparticle polypeptide conjugates comprise at least one nanoparticle and a polypeptide that binds to said double stranded oligonucleotide-stem complex;
- wherein said nanoparticle in said first nanoparticle polypeptide conjugates is a metal, semiconductor, or magnetic colloidal particle;
- applying a voltage drop across said electrodes;
- and
- measuring a change in conductivity, resistivity, capacitance, or impedance across said electrodes at known locations to determine perfect complementarity of said oligonucleotide target to said first oligonucleotide probes.
18 more elements
Dependencies are inferred from the claim text.