A single quantum dot-based biosensor for DNA point mutation assay

Abstract

Sensitive and selective detection of point mutation is essential to molecular biology research and early clinical diagnosis. Here, we demonstrate a single quantum dot (QD)-based biosensor for DNA point mutation assay. In this assay, a mutant target (G/C) remains unchanged after the endonuclease treatment, and the polymerase chain reaction (PCR) may be initiated with the assistance of primers and polymerase, generating a large number of mutant targets. The amplified mutant targets can be captured by biotinylated probes during the process of denaturation and annealing, and Cy5-dGTP may be assembled into the biotinylated probe with the catalysis of polymerase, leading to the formation of Cy5-labeled biotinylated probes. The Cy5-labeled biotinylated probes can be further assembled onto the QD surface to obtain a Cy5-DNA-QD complex, resulting in the generation of fluorescence resonance energy transfer (FRET) between the QD donor and the Cy5 receptor. The mutant targets can be quantitatively evaluated by the measurement of Cy5 counts by total internal reflection fluorescence (TIRF) microscopy. While in the presence of wild-type targets (T/A), no Cy5-dGTP can be assembled into the biotinylated probe due to the presence of a mismatch and consequently no FRET is observed. This single QD-based biosensor exhibits high sensitivity with a detection limit of 5.3 aM (or 32 copies) and can even discriminate as low as 0.01% variant frequency from the mixture of mutant targets and wild-type ones. Importantly, this biosensor can be used for genomic analysis in human lung cancer cells, and may be further applied for an early clinical diagnosis and personalized medicine.

Synthesis and Optical Properties of Water-Soluble CdTe:Zn2+ Quantum Dots Prepared by the One-Pot Approach

“…Therefore, surface chemistry and polydispersity are the factors that most influence the heterogeneity of the optical properties of QDs. 18,19 Although QDs can be applied in photovoltaic industries, thermal or light-emitting diodes, its applications in life sciences have revolutionized the state of the art of some biological assays, including fluorescence-linked immunosorbent assays (FLISA), 20 fluorescence resonance energy transfer (FRET) assays, 21 immunosensors, 22 DNA probes 23 or multicolor imaging dyes, 24 commonly being a real alternative to the use of traditional organic dyes. In this context, great effort has been made to synthesize novel nanomaterials, in particular, QDs with high surface quality and biocompatible ability, to enhance and fine tune the optical response, concomitantly becoming less harmful to human health.…”

Biosensors for Point Mutation Detection

“…QDs are not only a high-intensity fluorescent label, but also a nano center, which is used to capture multiple conjugated dye products for signal amplification, which is conducive to highly accurate fluorescence detection. Tang et al ( Tang et al, 2015 ) designed a QD based biosensor for DNA point mutation detection. The biotinylated probe can capture the mutation target of PCR amplification.…”

Nanotechnology-Enhanced No-Wash Biosensors for in Vitro Diagnostics of Cancer

“…Moreover, a single QD-based FRET nanosensor in a separation-free format from Zhang et al 291 was constructed for ultrasensitive detection of target DNA by using a sandwich hybridization of Cy5-labeled capture probes, biotinylated detection probes, target DNA, and streptavidin-coated QD to form a FRET donor−acceptor ensemble. Two similar examples were displayed later for analysis of DNA point mutations related to human epidermal growth factor receptor (EGFR) gene 292 and for quantitative determination of miRNA by coupling with an exponential amplification reaction, 293 respectively. On this premise, Zhang et al 294 explored a multiplexed single QD-based FRET sensor for simultaneous detection of two target DNAs, in which QD were cohybridized with A488-and A647-labeled detection probes through sandwich hybridization.…”

“…was constructed for ultrasensitive detection of target DNA by using a sandwich hybridization of Cy5-labeled capture probes, biotinylated detection probes, target DNA, and streptavidin-coated QD to form a FRET donor–acceptor ensemble. Two similar examples were displayed later for analysis of DNA point mutations related to human epidermal growth factor receptor (EGFR) gene and for quantitative determination of miRNA by coupling with an exponential amplification reaction, respectively. On this premise, Zhang et al .…”