Ultraspecific Multiplexed Detection of Low-Abundance Single-Nucleotide Variants by Combining a Masking Tactic with Fluorescent Nanoparticle Counting

Abstract

To be able to detect simultaneously multiple single-nucleotide variants (SNVs) with both ultrahigh specificity and low-abundance sensitivity is of pivotal importance for molecular diagnostics and biological research. In this contribution, we for the first time developed a multiplex SNV detection method that combines the masking tactic with fluorescent nanoparticle (FNP) counting based on the sandwich design. The method presents a rivaling performance due to its advantageous features: the masking reagent was designed to hybridize with an extremely large amount of the wild-type sequence to render the assay with high specificity; FNP counting provides a sensitive multiplexed SNV detection; the sandwich design facilitates an easy separation to make the detection free of interferences from the matrix. For single SNV target discrimination, including the 6 most frequently occurring DNA KRAS gene mutations and 2 possible RNA KRAS gene mutations as well as 11 artificial mutations, the discrimination factor ranged from 204 to 1177 with the median being 545. Among the tested 19 SNVs, abundances as low as 0.05% were successfully identified in 14 cases, and an abundance as low as 0.1% was identified for the remaining 5 cases. For multiplexed detection of SNVs in the KRAS gene, abundances as low as 0.05-0.1% were achieved for multiple SNVs occurring at the same and different codons. As low as 0.05% low-abundance detection sensitivity was also achieved for PCR amplicons of human genomic DNA extracted from cell samples. This proposed method presents the potential for ultrahigh specific multiplexed detection of SNVs with low-abundance detection capability, which may be applied to practical applications.

Advances in single‐molecule fluorescent nanosensors

“…Single‐molecule detection is characterized by low sample consumption, good selectivity, and ultrahigh sensitivity, enabling straightforward and visual monitoring of various target molecules (Y. Han, Chen, et al, 2019; B. Li et al, 2020; Ma, Wei, & Zhang, 2019; Pei et al, 2018; Z. Wu et al, 2019). To further improve the performance of the single‐molecule detection, a variety of nanomaterials such as QD, AuNP, FCPNP, UCNP, MnO 2 nanosheet, and MB have been introduced for the construction of novel single‐molecule fluorescent nanosensors.…”

“…In the single‐molecule fluorescent nanosensors, these fluorescent nanomaterials can specifically label the target biomolecule and emit strong fluorescent signal which can be detected via single‐molecule imaging (X. Li et al, 2018; Pei et al, 2018; Zhou et al, 2013).…”

“…(c) FNP‐based and MB‐based single‐molecule fluorescent nanosensor for multiplexed detection of three DNA targets. Reprinted with permission from Pei et al (2018). Copyright 2018 American Chemical Society.…”

“…Pei et al developed a fluorescent polymer nanoparticle (FNP)‐based single‐molecule fluorescent nanosensor to simultaneously detect three single‐nucleotide variants (SNVs) (Pei et al, 2018). They conjugated magnetic bead with capture DNA to obtain the capture DNA‐MB, and conjugated the FNP with signaling DNA to obtain the signaling DNA‐FNP (Figure 1(c)).…”

“…Due to their different compositions, dimensions, sizes, and shapes, the nanoscaled materials exhibit unique chemical, optical, mechanical, and electronic properties, and they can be easily functionalized with different biomolecules through electrostatic binding, physical adsorption, ligand exchange, biorecognition, and covalent bonding for the construction of diverse nanosensors (Bellan et al, 2011; Ma, Zhang, & Zhang, 2020; Rosi & Mirkin, 2005; Sheehan & Whitman, 2005). Recently, a variety of nanomaterials have been employed for the development of novel single‐molecule fluorescent nanosensors (Y. Han, Chen, et al, 2019; Y. Han, Ye, et al, 2019; B. Li et al, 2020; W. Li et al, 2016; X. Li et al, 2018; Ma, Ren, & Zhang, 2017; Ma, Wei, & Zhang, 2019; Pei et al, 2018; Z. Wu et al, 2019; J. Zhou et al, 2013). The use of functional nanomaterials including quantum dots, gold nanoparticles, upconversion nanoparticles, fluorescent conjugated polymer nanoparticles, nanosheets, and magnetic nanoparticles as fluorescent labels, quenchers, separators, and carriers has greatly improve the sensitivity, accuracy, and applications of single‐molecule detection.…”