Acid-base chemistry at the single ion limit.

We present the results of acid-base experiments performed at the single ion (H+ or OH-) limit in ∼6 aL volume nanopores incorporating electrochemical zero-mode waveguides (E-ZMWs). At pH 3 each E-ZMW nanopore contains ca. 3600H+ ions, and application of a negative electrochemical potential to the gold working electrode/optical cladding layer reduces H+ to H2, thereby depleting H+ and increasing the local pH within the nanopore. The change in pH was quantified by tracking the intensity of fluorescein, a pH-responsive fluorophore whose intensity increases with pH. This behavior was translated to the single ion limit by changing the initial pH of the electrolyte solution to pH 6, at which the average pore occupancy 〈n〉pore ∼3.6H+/nanopore. Application of an electrochemical potential sufficiently negative to change the local pH to pH 7 reduces the proton nanopore occupancy to 〈n〉pore ∼0.36H+/nanopore, demonstrating that the approach is sensitive to single H+ manipulations, as evidenced by clear potential-dependent changes in fluorescein emission intensity. In addition, at high overpotential, the observed fluorescence intensity exceeded the value predicted from the fluorescence intensity-pH calibration, an observation attributed to the nucleation of H2 nanobubbles as confirmed both by calculations and the behavior of non-pH responsive Alexa 488 fluorophore. Apart from enhancing fundamental understanding, the approach described here opens the door to applications requiring ultrasensitive ion sensing, based on the optical detection of H+ population at the single ion limit.