Zero-Mode Waveguide Arrays for Enzyme and Single-Particle Sensing

Physiological studies are frequently carried out using in vitro measurements designed to model the probed system. These models, however, cannot accurately reflect the in vivo systems they depict in detail, as the model systems are typically based upon average properties of the physiological state. For example, species present at low concentration in vivo or transient conditions, such as fluctuations in pH or species concentration, are difficult to model accurately in vitro. Incorporating nanoscale structures and devices into in vitro measurements is a strategy to greatly enhance the verisimilitude of in vitro to in vivo systems, because the ultrasmall (V = 1 fL) volumes accurately reflect in vivo environments, and they facilitate high signal-to-background ratios in common fluorescence and electrochemical methods. The work in this thesis investigates the characteristics of zero-mode waveguides (ZMW) used as nanophotonic devices for single-entity detection and local concentration manipulation in fluorescence and spectroelectrochemical experiments. First, ZMWs were used for digital detection of single Ag nanoparticles using darkfield microscopy. Scattering from individual ZMW nanopores is visible using darkfield microscopy when Ag nanoparticles enter the nanopore volume allowing digital detection of single nanoparticles, which has not been previously demonstrated in ZMWs. The relatively inexpensive and low technicality of darkfield microscopy suggests this method as a possible alternative to more commonly used fluorescence microscopy techniques. ZMW arrays were then explored as aL-volume chemical reactors for controlling the behavior of the enzyme horseradish peroxidase. The metallic optical cladding layer used in ZMWs was then utilized as a working electrode for the generation and/or elimination of reactive oxygen species (ROS), again using the horseradish peroxidase model system to characterize small-volume effects. Tuning the potential of the working electrode to favor generation or elimination of ROS was also found to tune the enzyme turnover rate, and the ability to simultaneously monitor each nanopore in a nanopore array individually was used to characterize exhibiting the heterogeneity in a population of enzymes. Lastly as an application use case, ZMW arrays were extended to the detection of bacterial extracellular vesicles (bEV) excreted from Pseudomonas aeruginosa cultures as a means of investigating bacterial resistance and cell signaling. The size differential between the ZMW nanopores (10-100 aL, typ.) and the bacterial cells (10-100 fL, typ.), enables discrimination between the bEVs and the cells from which they are obtained, thus allowing crude, liquid culture to be studied directly. These capabilities were extended by modifying the transport dynamics of liposomes within ZMWs by altering the liposome interactions within the nanopore chemically and electrochemically. This work allowed the unprecedented simultaneous monitoring of 441 liposome-bearing nanopores, thus exposing the heterogeneity of the liposome population.