Development of a Single Quantum Dot-Mediated FRET Nanosensor for Sensitive Detection of Single-Nucleotide Polymorphism in Cancer Cells

Abstract

Single-nucleotide polymorphisms (SNPs) are important hallmarks of human diseases. Herein, we develop a single quantum dot (QD)-mediated fluorescence resonance energy transfer (FRET) nanosensor with the integration of multiple primer generation rolling circle amplification (MPG-RCA) for sensitive detection of SNPs in cancer cells. This assay involves only a linear padlock probe for MPG-RCA. The presence of a mutant target facilitates the circularization of linear padlock probes to initiate RCA, producing three short single-stranded DNAs (ssDNAs) with the assistance of nicking endonuclease. The resulting ssDNAs can function as primers to induce cyclic MPG-RCA, resulting in the exponential amplification and generation of large numbers of linker probes. The linker probes can subsequently hybridize with the Cy5-labeled reporter probes and the biotinylated capture probes to obtain the sandwich hybrids. The assembly of these sandwich hybrids on the 605 nm-emission quantum dot (605QD) generates the 605QD−oligonucleotide−Cy5 nanostructures, resulting in efficient FRET from the 605QD to Cy5. This nanosensor is free from both the complicated probe design and the exogenous primers and has distinct advantages of high amplification efficiency, zero background signal, good specificity, and high sensitivity. It can detect SNPs with a large dynamic range of 8 orders of magnitude and a detection limit of 5.41 × 10 −20 M. Moreover, this nanosensor can accurately distinguish as low as 0.001% mutation level from the mixtures, which cannot be achieved by previously reported methods. Furthermore, it can discriminate cancer cells from normal cells and even quantify SNP at the single-cell level.

High-efficiency and high-fidelity ssDNA circularisation via the pairing of five 3′-terminal bases to assist LR-LAMP for the genotyping of single-nucleotide polymorphisms

“…The circular structure of ssDNA is often exploited in detecting SNPs or microRNAs. [19][20][21] Molecular detection techniques have been developed for c-DNAs. The two most common approaches are rolling circle amplification (RCA) using a single primer and Phi29 DNA polymerase and hyperbranched-RCA (H-RCA) using two primers and Vent® (exo-) DNA polymerase.…”

Recent advances in biological detection with rolling circle amplification: design strategy, biosensing mechanism, and practical applications

“…[40][41][42] Thirdly, the good compatibility of RCA greatly expands the types of targets, which makes it not only to be used to detect nucleic acids but also be used in the detection of proteins, and cell analyses via immune RCA or through in situ proximity ligation assays. [43][44][45] It has been successfully applied in the detection of multiple biosensing targets, including DNA, [46][47][48] RNA, 49-51 SNP, [52][53][54] proteins, [55][56][57] pathogens, [58][59][60] and cells. 34,61,62 In this review, the common construction methods of RCA are summarized, and their applications in the detection of pathogenic bacteria, nucleic acid tumor markers, viruses, and proteins are reviewed, and the future development trend of RCA is forecasted.…”

“…40–42 Thirdly, the good compatibility of RCA greatly expands the types of targets, which makes it not only to be used to detect nucleic acids but also be used in the detection of proteins, and cell analyses via immune RCA or through in situ proximity ligation assays. 43–45 It has been successfully applied in the detection of multiple biosensing targets, including DNA, 46–48 RNA, 49–51 SNP, 52–54 proteins, 55–57 pathogens, 58–60 and cells. 34,61,62…”

CRISPR/Cas9-based coronal nanostructures for targeted mitochondria single molecule imaging

“…Given the high-sensitivity imaging of this strategy, we further explored subcellular level imaging with total internal reflection fluorescence (TIRF)-based techniques. Singlemolecule counting is a highly sensitive quantitative method achieved by counting the fluorescent dots [52][53][54] . This method has the advantage of low sample consumption, based on which our proposed method was carried out to detect mitomiR.…”