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Electrochemical Control of pH in Attoliter Volumes to Study pH-dependent Single Enzyme Activity
Friday, March 12, 2021: 8:30 AM - 8:50 AM
Speaker(s)
Description
Electrochemistry at the single entity level (such as individual nanoparticles, molecules) are continuously gaining interest because of its ability to reveal heterogeneous behavior as opposed to the averaging result obtained in the ensemble or bulk experiment. In this work, we study the effects of confinement on chemical reactivity using the most fundamental of reactions – protonation – deprotonation through electrochemical reduction of H+. Our approach relies on the construction of an electrochemically active version of a nanophotonic structure – the zero-mode waveguide (E-ZMW) with a characteristic active volume ~1 aL. The extraordinarily small volumes makes it possible to address one or a few H+ or OH- species through their reaction with a few molecules (~10 molecules/E-ZMW) of the pH sensitive dye, fluorescein, Fl2-/Fl-. Comparing Monte Carlo simulation to experiment reveals how averaging over stochastic fluctuations in (a) H+ ion population, (b) the presence of the Fl2-/Fl- within the optical excitation volume, and (c) the Fl2-/Fl- equilibrium produces the familiar acid-base behavior we observe macroscopically. In addition, surprisingly when the initial solution pH is 3, we observed anomalously large fluorescence intensities at the most negative (reduction) potentials, behavior which is attributed to the formation of H2 nanobubbles as small as 12 nm (radius) within the E-ZMW nanopores. Apart from enhancing fundamental understanding, the approach described here opens the door to applications requiring ultrasensitive ion sensing such as DNA sequencing, based on the optical detection of H+ population at the single ion limit. We extended this approach to study pH-dependent single enzyme activity in situ by electrochemically controlling the pH within the E-ZMW nanopore.
Additional Info
Keywords: Please select up to 4 keywords ONLY:
Microscopy,Single Molecule,Spectroelectrochemistry
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