DNA Hybridization-Accelerated Programmable Immuno-Aggregation of Microbeads Enabling Mix-and-Read Flow Cytometric Protein Analysis

Microbeads (MBs) aggregation-based immunoassay, which is independent of multistep bead washing, signal labeling, and even reporter eluting procedures, has emerged as a promising label-free route for protein biomarker analysis. However, their wide application is not only subjected to challenges from target-actuated low aggregation efficiency derived from the steric hindrance and high weight of micrometer-sized beads but also suffers from the lack of precise method to exactly measure the uncontrollable aggregation process/state. Herein, a new mechanism of metastable DNA hybridization-accelerated programmable immuno-aggregation of MBs is proposed, which enables the facile mix-and-read and flow cytometric detection of protein biomarkers. The introduction of auxiliary metastable DNA hybridization and magnetic facilitation can remarkably boost the immunoreaction efficiency between two kinds of MBs, achieving an ∼200-fold increase of detection sensitivity. What is more, benefiting from the powerful ability of flow cytometry to precisely interrogate the light scattering and fluorescence information on individual events one-by-one, the distinct discrimination and precise quantification of MB aggregates from MB monomers can be easily achieved. Additionally, fluorescent color and intensity coencoded MBs can be easily acquired by simply adjusting the amounts of fluorescent probes to achieve the multiplexed analysis of protein targets. With these advantages, the proposed method demonstrated a successful application for mix-and-read protein detection, showing great potential in diverse biomedical applications.