Increasing the Sensitivity and Single-Base Mismatch Selectivity of the Molecular Beacon Using Graphene Oxide as the “Nanoquencher”

The detection of single nucleotide polymorphisms (SNPs) is of great significance in the early diagnosis of diseases and the rational use of drugs. Thus, a novel biosensor based on the quenching effect of fluorescence-embedded SYBR Green I (SG) dye and graphene oxide (GO) was introduced in this study. The probe DNA forms a double helix structure with perfectly complementary DNA (pcDNA) and 15 single-base mismatch DNA (smDNA) respectively. SG is highly intercalated with perfectly complementary dsDNA (pc-dsDNA) and exhibits strong fluorescence emission. Single-base mismatch dsDNA (SNPs) has a loose double-stranded structure and exhibits poor SG intercalation and low fluorescence sensing. At this time, the sensor still showed poor SNP discrimination. GO has a strong effect on single-stranded DNA (ssDNA), which can reduce the fluorescence response of probe DNA and eliminate background interference. And competitively combined with ssDNA in SNPs, quenching the fluorescence of SG/SNP, while the fluorescence value of pc-dsDNA was retained, increasing the signal-to-noise ratio. At this time, the sensor has obtained excellent SNP resolution. Different SNPs detect different intensities of fluorescence in the near-infrared region to evaluate the sensor's identification of SNPs. The experimental parameters such as incubation time, incubation temperature and salt concentration were optimized. Under optimal conditions, 1 nM DNA with 0–10 nM linear range and differentiate 5% SNP were achieved. The detection method does not require labeling, is low cost, simple in operation, exhibits high SNP discrimination and can be distinguished by SNP at room temperature.

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Detection of p53 Gene Mutation (Single-Base Mismatch) Using a Fluorescent Silver Nanoclusters

Journal of Fluorescence ◽

10.1007/s10895-017-2083-5 ◽

2017 ◽

Vol 27 (4) ◽

pp. 1443-1448 ◽

Cited By ~ 11

Author(s):

Morteza Hosseini ◽

Shiva Mohammadi ◽

Yasaman-Sadat Borghei ◽

Mohammad Reza Ganjali

Keyword(s):

Gene Mutation ◽

P53 Gene ◽

Silver Nanoclusters ◽

P53 Gene Mutation ◽

Single Base ◽

Single Base Mismatch ◽

Base Mismatch

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Digital-resolution detection of microRNA with single-base selectivity by photonic resonator absorption microscopy

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1904770116 ◽

2019 ◽

Vol 116 (39) ◽

pp. 19362-19367 ◽

Cited By ~ 12

Author(s):

Taylor D. Canady ◽

Nantao Li ◽

Lucas D. Smith ◽

Yi Lu ◽

Manish Kohli ◽

...

Keyword(s):

Limit Of Detection ◽

Signal To Noise Ratio ◽

Resonant Wavelength ◽

High Signal ◽

Single Base ◽

Reflected Light ◽

Single Base Mismatch ◽

Base Mismatch ◽

Digital Resolution ◽

Target Probe

Circulating exosomal microRNA (miR) represents a new class of blood-based biomarkers for cancer liquid biopsy. The detection of miR at a very low concentration and with single-base discrimination without the need for sophisticated equipment, large volumes, or elaborate sample processing is a challenge. To address this, we present an approach that is highly specific for a target miR sequence and has the ability to provide “digital” resolution of individual target molecules with high signal-to-noise ratio. Gold nanoparticle tags are prepared with thermodynamically optimized nucleic acid toehold probes that, when binding to a target miR sequence, displace a probe-protecting oligonucleotide and reveal a capture sequence that is used to selectively pull down the target-probe–nanoparticle complex to a photonic crystal (PC) biosensor surface. By matching the surface plasmon-resonant wavelength of the nanoparticle tag to the resonant wavelength of the PC nanostructure, the reflected light intensity from the PC is dramatically and locally quenched by the presence of each individual nanoparticle, enabling a form of biosensor microscopy that we call Photonic Resonator Absorption Microscopy (PRAM). Dynamic PRAM imaging of nanoparticle tag capture enables direct 100-aM limit of detection and single-base mismatch selectivity in a 2-h kinetic discrimination assay. The PRAM assay demonstrates that ultrasensitivity (<1 pM) and high selectivity can be achieved on a direct readout diagnostic.

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