Probe-Encapsulated and Cell-Membrane-Anchored DNA Nanotoolboxes Enable <i>In Situ</i> Imaging of Extracellular Biochemical Components in Complex Biological Media

Cell-membrane-anchored DNA probes are powerful tools for membrane-localized biosensing, bioregulation, and biotherapy studies. However, the poor biostability, membrane-anchoring stability, and effectiveness of membrane-anchored DNA probes in complex physiological environments have seriously hindered their widespread applications. In this work, probe-encapsulated DNA nanotoolboxes were developed by immobilizing DNA probes into DNA cuboids to improve the performance of cell-membrane-anchored DNA probes in complex biological media. Encapsulating DNA probes into small-sized DNA cuboids enhanced the resistance of DNA probes to nuclease degradation and nonspecific protein binding, as well as avoiding nonspecific adsorption of DNA probes by the cell membrane. In addition, cross-linking the membrane-anchored DNA nanotoolboxes with DNA linkers significantly improved their membrane-anchoring stability in biological media. Two distinct types of DNA nanotoolboxes were constructed by fixing ATP aptamer probes and G-quadruplex-based K⁺ probes into DNA cuboids, respectively, and were successfully applied to <i>in situ</i> imaging of extracellular ATP and K⁺ in physiological buffer systems. Furthermore, monitoring of cell apoptosis was also achieved through <i>in situ</i> imaging of endogenously released ATP using DNA nanotoolboxes. Benefiting from the high designability and diversity of DNA probes, DNA nanotoolboxes are expected to provide a powerful platform for various membrane-localized biochemical studies in complex physiological systems.