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Signal amplification in biological systems is achieved by cooperatively recruiting multiple copies of regulatory biomolecules. Nevertheless, the multiplexing capability of artificial fluorescent amplifiers is limited due to the size limit and lack of modularity. Here, we develop Cayley tree-like fractal DNA frameworks to topologically encode the fluorescence states for multiplexed detection of low-abundance targets. Taking advantage of the self-similar topology of Cayley tree, we use only 16 DNA strands to construct n-node (n = 53) structures of up to 5 megadalton. The high level of degeneracy allows encoding 36 colours with 7 nodes by site-specifically anchoring of distinct fluorophores onto a structure. The fractal topology minimises fluorescence crosstalk and allows quantitative decoding of quantized fluorescence states. We demonstrate a spectrum of rigid-yet-flexible super-multiplex structures for encoded fluorescence detection of single-molecule recognition events and multiplexed discrimination of living cells. Thus, the topological engineering approach enriches the toolbox for high-throughput cell imaging.

Simultaneous and non-destructive quantitative detection of intracellular metal ions holds great promise for improving the accuracy of diagnosis and biological research. Herein, novel multicolor DNAzymes-embedded framework nucleic acids (FNAzymes) were presented, which can easily enter cells and achieve simultaneous and quantitative detection of intracellular physiologically related Cu2+ and Zn2+. Two types of DNAzymes, specific to Cu2+ and Zn2+, were encoded in the framework nucleic acids (FNAs) via self-assembly. With the formation of a well-ordered FNAzyme nanostructure, the fluorophore and the quencher were close to each other; therefore, the fluorescence was quenched. In the presence of Cu2+ and Zn2+, the integrated FNAzymes would be specifically cleaved, resulting in the release of fluorophores in cells. Consequently, the fluorescence in living cells could be observed by a confocal microscope and semi-quantitatively analyzed by flow cytometry with low-nanomolar sensitivity for both metal ions. The FNAzymes have high uniformity and structural accuracy, which are beneficial for intracellular detection with excellent reproducibility. This proposed method offers new opportunities for non-destructive, semi-quantitative, multi-target detection in living cells. [ABSTRACT FROM AUTHOR]

Abstract Biosensor is a device that responds to a particular target in a selective way by incorporation of biological recognition as sensing unit. For practical biomedical applications, a digital output (a qualitative YES/NO answer) is highly demanded for certain end‐user or point‐of‐care applications. Boolean logic, which can be applied to any type of information expressed as 0 (NO) and 1 (YES), is widely employed to achieve such qualitative analysis. Over the past decade, owing to DNA's advantages of stability, accessibility, and manipulability, significant research efforts have been focused on the design and application of DNA logic gates (DLG)‐based biosensors capable of implementing logic‐gated biomedical functions. This review summarizes the existed representative examples and the advanced developments of DLG biosensors, and discusses the limitations and the future directions on the development of novel nanosensors based on DNA logic operations for realizing highly efficient diagnosis. [ABSTRACT FROM AUTHOR]

DNA nanostructures have attracted considerable attention as drug delivery carriers. However, the transmembrane kinetics of DNA nanostructures remains less explored. Herein, the dynamic process of transporting single tetrahedral DNA nanostructures (TDNs) is monitored in real time using a force‐tracing technique based on atomic force microscopy. The results show that transporting single TDNs into living HeLa cells need ≈53 pN force and ≈25 ms duration with the average speed of ≈0.6 µm s−1. Interestingly, the dynamic parameters are irrelevant to the size of TDNs, while the larger TDNs rotated slightly during the transporting process. Meanwhile, both the results from single‐molecule force tracing and ensemble fluorescence imaging demonstrate that the different size TDNs transmembrane transporting depends on caveolin‐mediated endocytosis. [ABSTRACT FROM AUTHOR]