Colocalized Particle Counting Platform for Zeptomole Level Multiplexed Quantification

For multiplexed detection, it is important yet challenging to simultaneously meet the requirement of sensitivity, throughput, and implementation convenience for practical applications. Using the detection of DNAs and miRNAs for illustration, we present a colocalized particle counting platform that can realize the separation-free multiplexed detection of 6 nucleic acid targets with a zeptomole sensitivity and a dynamic range of up to 5 orders of magnitude. The presence of target induces the formation of a sandwich nanostructure via hybridization; thus, there is an occurrence of colocalization of two microbeads with two different colors. The sequence specific coding is realized by an arbitrary combination of two fluorescence channels with different emitting colors. The platform presents robustness in detecting multiple nucleic acid targets with a minimal cross talk and matrix effect as well as the ability to distinguish the specific miRNA from members of the same family. The results of simultaneous detection of 3 miRNAs in 3 different cell lines present straight consistency with that of the standard qRT-PCR. This platform can be adapted to other multiplexing designs such as the "turn-off" mode, in which the proportion of colocalized microbeads is decreased due to the strand-displacement reaction initiated by the specific target. This separation-free platform offers the possibility to achieve the on-site multiplexed detection with compatibility to different experimental designs and extensibility to other signal sources for enumeration.