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21 February 2020 Microsecond single-molecule enzymology using plasmonically enhanced optical resonators
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Understanding the kinetics and dynamics of single enzymes is crucial for understanding biomolecular processes at a fundamental level. Sensors that provide sensitivity to single enzyme conformational changes at micro/nanoseconds time resolution are required to study these motions. Here, we present an optical sensor based on plasmonically enhanced whispering gallery modes capable of studying the dynamics of single enzymes over timescales of ns-hours. By combing surface chemistry methods for attachment of single enzymes in a preferred orientation with lock-in measurement techniques, we detect signals from single enzyme-substrate interactions in the microsecond timescale. Specific immobilization of enzymes on gold nanoparticles is achieved via histidine groups added to one of the termini of the enzyme. This enables reversible attachment of recombinantly expressed and purified enzymes. The molecular motions of the enzyme and its interactions with the substrate is measured as a shift in the whispering gallery mode resonance frequency. A Pound-Drever-Hall lock is used to track the resonance shift with a microsecond time resolution. Combined with advanced signal processing and simulations, this will enable studying the functional conformational movements of enzymes at the single-molecule level. Future work includes adding microfluidics for fast and automated sample delivery, addition of multiple channels of excitation by combing different wavelengths and polarizations of light. This work provides a proof-of-concept next generation optical sensor for single-molecule enzymology, fundamental protein research, drug discovery and point-of-care devices.
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Sivaraman Subramanian, Simona Frustaci, and Frank Vollmer "Microsecond single-molecule enzymology using plasmonically enhanced optical resonators", Proc. SPIE 11258, Frontiers in Biological Detection: From Nanosensors to Systems XII, 112580E (21 February 2020);


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