A Framework Nucleic Acid-Mediated Multimodal Tandem Multivariate Signal Amplification Strategy for Simultaneous Absolute Quantification of Three Different MicroRNAs in a Single Cell.

Abstract

The simultaneous quantification of multiple microRNAs (miRNA) in a single cell can help scientists understand the relationship between different miRNA groups and different types of cancers from an miRNA omics perspective at the single-cell level. However, there currently remains a challenge in developing techniques for the simultaneous absolute quantification of multiple miRNAs in single cells. Herein, we propose a framework nucleic acid (FNA)-mediated multimodal tandem multivariate signal amplification strategy for simultaneous absolute quantification of three different miRNAs in a single cell. In this study, DNA hexahedron FNAs (DHFs) and DNA tetrahedron FNAs (DTFs) were first prepared, multiple DNA hairpins and substrates were then connected to the hexahedron frame nucleic acid as the target recognition units, and three substrates with labeled FAM fluorophores on the tetrahedral frame nucleic acid served as signal output units. After the two types of FNAs entered the cell, they reacted with three different miRNAs (miRNA-155, miRNA-373, and miRNA-21) and multimodal tandem multivariate signal amplification was initiated simultaneously, reducing the detection limit of the three miRNAs to 8 × 10-15, 2 × 10-15, and 1 × 10-15 M, respectively. The detection sensitivity of the three miRNAs was simultaneously increased by six orders of magnitude, reaching the quantitative requirement of trace miRNAs in single cells. Combined with single-cell injection, membrane melting, and intracellular component separation technology on a microchip electrophoresis platform, we achieved the simultaneous absolute quantification of three different miRNAs in a single cell, thereby providing an important novel method that can be used to conduct single-cell research.