Modular DNA Tetrahedron Nanomachine-Guided Dual-Responsive Hybridization Chain Reactions for Discernible Bivariate Assay and Cell Imaging

Engineering of multivariate biosensing and imaging platforms involved in disease plays a vital role in effectively discerning cancer cells from normal cells and facilitating reliable targeted therapy. Multiple biomarkers such as mucin 1 (MUC1) and nucleolin are typically overexpressed in breast cancer cells compared to normal human breast epithelium cells. Motivated by this knowledge, a dual-responsive DNA tetrahedron nanomachine ( dr DT-NM) is constructed through immobilizing two recognition modules, MUC1 aptamer (MA) and a hairpin H1* encoding nucleolin-specific G-rich AS1411 aptamer, in two separate vertexes of a functional DT architecture tethering two localized pendants (P M and P N ). When dr DT-NM identifiably binds bivariate MUC1 and nucleolin, two independent hybridization chain reactions (HCR M and HCR N ) as amplification modules are initiated with two sets of four functional hairpin reactants. Among them, one hairpin for HCR M is dually ended by fluorescein and quencher BHQ1 to sense MUC1. The responsiveness of nucleolin is executed by operating HCR N utilizing another two hairpins programmed with two pairs of AS1411 splits. In the shared HCR N duplex products, the parent AS1411 aptamers are cooperatively merged and folded into G-quadruplex concatemers to embed Zn-protoporphyrin IX (ZnPPIX/G4) for fluorescence signaling readout, thereby achieving a highly sensitive intracellular assay and discernible cell imaging. The tandem ZnPPIX/G4 unities also act as imaging agents and therapeutic cargos for efficient photodynamic therapy of cancer cells. Based on dr DT-NM to guide bispecific HCR amplifiers for adaptive bivariate detection, we present a paradigm of exquisitely integrating modular DNA nanostructures with nonenzymatic nucleic acid amplification, thus creating a versatile biosensing platform as a promising candidate for accurate assay, discernible cell imaging, and targeted therapy.