Outer membrane vesicles (OMVs) are extracellular structures derived from the outer membrane of bacteria. They carry diverse cargos such as proteins, nucleic acids and enzymes, which are released by bacteria to communicate with each other and with host cells. Understanding the role of OMVs as carriers of signalling enzymes provides insights into intercellular communication, pathogenesis and biofilm formation, among others. Although fluorescence-based techniques have been employed to study vesicles, real-time live monitoring of enzyme-based cellular communication has remained challenging due to undesired effects of photobleaching and interference from labelling agents. Here we report label-free dynamic monitoring of the oscillatory activity of the enzyme azoreductase (AzoR) in individual live bacteria via nanoplasmonic optical antennas. Our nanoplasmonic antennas consist of gold nanorods modified with black hole quencher molecules (BHQ-3), whose scattering cross-section is modulated by the presence of AzoR. The antennas enable long-term (several hours) and distance-dependent (up to 3 μm) detection of AzoR via OMVs released by individual live bacteria. We observe periodic oscillatory enzyme activity in different living environments and at different stages of bacterial growth. We also found that oscillatory enzyme activity exhibits heterogeneous features due to the coupling of oscillation signals between neighbouring bacteria. The dynamic monitoring of signalling enzymes paves the way for a better understanding of mechanisms of bacterial communication, pathogenesis and drug resistance.
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