Real-Time Spatiotemporal Measurement of Extracellular Signaling Molecules Using an Aptamer Switch-Conjugated Hydrogel Matrix

Abstract

AbstractCells rely on secreted signaling molecules to coordinate essential biological functions including development, metabolism, and immunity. Unfortunately, such signaling processes remain difficult to measure with sufficient chemical specificity and temporal resolution. To address this need, we have developed an aptamer-conjugated hydrogel matrix that enables continuous fluorescent measurement of specific secreted analytes – in two dimensions, in real-time. As a proof of concept, we performed real-time imaging ofDictyostelium discoideumcells, a well-studied amoeba model wherein inter-cellular communication is performed though cAMP signaling. We engineered a set of aptamer switches that generate a rapid and reversible change in fluorescence in response to cAMP signals. By combining multiple switches with different dynamic ranges, we can measure cAMP concentrations spanning three orders of magnitude in a single experiment. These sensors are embedded within a biocompatible hydrogel on which cells are cultured and their cAMP secretions can be imaged using fluorescent microscopy. Using this aptamer-hydrogel material system, we achieved the first direct measurements of oscillatory cAMP signaling that correlate closely with previous indirect measurements. Using different aptamer switches, this approach could be generalized for measuring other secreted molecules to directly visualize diverse extracellular signaling processes and the biological effects that they trigger in recipient cells.