loading . . . Morphable âStitchedâ Sensors for Simultaneous Spatiotemporal Tracking of Correlated Bioanalytes in Living Cells Correlated changes in molecular levels and distributions are associated with all life processes and importantly regulate key decision-making events in biology. Spatiotemporal dynamics of biomolecules are essential for functions like cell signaling, transport, immunity, and recycling, and are either affected by or cause diseases such as cancers, inflammation, and neurodegeneration. In this backdrop, the ability to catch molecules of life in action using optical imaging is extremely powerful. Simultaneous tracking of biologically correlated analytes in living cells necessitates cell-permeable, multianalyte sensors. Key criteria for achieving simultaneous, correlated tracking of multiple analytes are that sensors for these analytes should enter cells at the same time and concomitantly reach the same location where we want to detect the analytes. Separate sensors, either small-molecule- or macromolecule-based, cannot fulfill these requirements directly. We introduce morphable âstitchedâ sensors, where fluorescent sensors for single analytes can be strategically joined via native chemical ligation (NCL) on a made-to-order basis. Morphable âstitchedâ sensors are built from a library of single-analyte sensing units conjugated to short peptide scaffolds. The use of peptide-based scaffolds in combination with NCL allows generation of modular, biocompatible, water-soluble, and importantly cell-permeable multianalyte sensors tailored to address specific biological questions. We report five proof-of-concept multianalyte sensors created from a common single-analyte sensor library using this âstitchingâ strategy. âStitchedâ sensors enable simultaneous imaging and temporal tracking of bioanalytes via time-lapse imaging. In our pilot studies we image different combinations of analytes including protons, hydrogen peroxide, and enzyme activity, in live cells, affording insights into pH- and hydrogen peroxide-dependent enzyme activity. Cellular uptake studies show that the analyte-sensing modules in âstitchedâ sensors enter cells simultaneously and as rapidly as 5 min, exhibit synchronized uptake dynamics, and are internalized in equal proportions, resulting in a uniform distribution across the cell population. Our novel morphable âstitchingâ platform therefore offers a universal approach toward live-cell multianalyte imaging. https://pubs.acs.org/doi/10.1021/jacsau.5c01271
