Strand Displacement Amplification Reaction on Quantum Dot-Encoded Silica Bead for Visual Detection of Multiplex MicroRNAs

Abstract

The combination of microbead array, isothermal amplification, and molecular signaling enables the continuous development of next-generation molecular diagnostic techniques. Herein we reported the implementation of nicking endonuclease-assisted strand displacement amplification reaction on quantum dots-encoded microbead (Qbead), and demonstrated its feasibility for multiplexed miRNA assay in real sample. The Qbead featured with well-defined core-shell superstructure with dual-colored quantum dots loaded in silica core and shell, respectively, exhibiting remarkably high optical encoding stability. Specially designed stem-loop-structured probes were immobilized onto the Qbead for specific target recognition and amplification. In the presence of low abundance of miRNA target, the target triggered exponential amplification, producing a large quantity of stem-G-quadruplexes, which could be selectively signaled by a fluorescent G-quadruplex intercalator. In one-step operation, the Qbead-based isothermal amplification and signaling generated emissive "core-shell-satellite" superstructure, changing the Qbead emission-color. The target abundance-dependent emission-color changes of the Qbead allowed direct, visual detection of specific miRNA target. This visualization method achieved limit of detection at the subfemtomolar level with a linear dynamic range of 4.5 logs, and point-mutation discrimination capability for precise miRNA analyses. The array of three encoded Qbeads could simultaneously quantify three miRNA biomarkers in ∼500 human hepatoma carcinoma cells. With the advancements in ease of operation, multiplexing, and visualization capabilities, the isothermal amplification-on-Qbead assay could potentially enable the development of point-of-care diagnostics.

Bio-Conjugated Quantum Dots for Cancer Research: Detection and Imaging

“…This immunoassay provides a simple, sensitive, specific, and repeatable approach for simultaneously detecting tumor markers in clinical situations. Qu et al used three QDs-encoded microbeads (Qbeads) to simultaneously identify three miRNA biomarkers, miRNA-21, miRNA-221, and miRNA-16, in ~500 human hepatoma cancer cells (55). These tests can be completed in a single step, resulting in low cost and simple operation.…”

Application of Nanomaterials in Isothermal Nucleic Acid Amplification

“…C) Reproduced with permission. [ 75 ] Copyright 2018, American Chemical Society. D) Reproduced with permission.…”

A self-quenching fluorescence probe-mediated exponential isothermal amplification system for highly sensitive and specific detection of microRNAs

“…As shown in Table S2 (ESI †), the detection efficiency, detection range, LOD, and other performance metrics displayed in our approach were better than others. [18][19][20][21][22][23][24][25][26][27] Without a doubt, this ultrasensitive detection threshold can be ascribed to following reasons: (i) the adoption of the self-quenching fluorescence probes makes detective signal-tobackground ratio greatly improved. Thus, the targets at lower concentrations can be easily detected; (ii) only two kinds of probes are employed in the system, the occurrence of non-specific amplification is avoided, and the exponential growth of products and signals is also guaranteed.…”