Morphable ‘Stitched’ Sensors for Simultaneous Tracking of Bio-analytes in Living Cells

Changes in molecular levels and distribution are associated with all life processes and importantly regulate key decision-making events in biology. Thus, correlated spatiotemporal dynamics of bio-molecules are central to biological function ranging from cell-signaling, transport, immunity, and recycling, to most pathophysiological conditions including cancers, inflammation, and neurodegeneration. In this backdrop, the ability to catch molecules of life in action using optical imaging is extremely powerful. Simultaneous imaging and tracking of bio-analytes require multiplexed chemical tools. We introduce the concept of morphable ‘stitched’ sensors where fluorescent sensors for single-analytes can be selected and joined on a made-to-order basis. The approach involves developing a library of single-analyte sensing units conjugated to short peptide-scaffolds. The single-analyte sensing peptide-scaffolds are ‘stitched’ via native chemical ligation (NCL). Thus, single-analyte sensors can be selected from the same library, based on the biological question to be addressed, in a context dependent manner. The advantage of using short-peptide scaffolds in combination with NCL lies in the ability to generate cell-permeable, modular, bio-compatible multiplexed probes in one go. Here, we report five proof-of-concept multiplexed sensors generated from the same library of single-analyte sensors using our novel morphable ‘stitched’ sensing strategy. Representative bio-analytes, protons and hydrogen peroxide, in combination with the activity of an enzyme Cathepsin B, have been imaged in live cells simultaneously, using cell-permeable multiplexed probes generated using our ‘stitching’ strategy via logic-gate/activity-based sensing schemes. The morphable ‘stitched’ sensing idea is general and can be used to develop multiplexed probes, theranostic agents, and combination therapeutics in the future.