Enzymes catalyze most of the biochemical reactions in our cells. The functionality of enzymes depends on their dynamics starting from small bond vibrations in the fs timescale to large domain motions in the microsecond-millisecond timescale. Understanding the precise and rapid positioning of atoms within a catalytic site by an enzyme’s molecular movements is crucial for understanding biomolecular processes and for realizing synthetic biomolecular machines in the longer term. Hence, sensors capable of studying enzymes over a wide range of amplitudes and timescale and ideally one enzyme at a time are required. Many capable single-molecule techniques have been established in the past three decades, each with its pros and cons. This thesis presents the development of one such single-molecule sensor. The sensor is based on plasmonically enhanced whispering gallery mode resonators and is capable of studying enzyme kinetics and large-scale dynamics over the timescale of ns-seconds. Unlike fluorescence techniques which require labeling of the enzymes with dyes, the technique presented in this work detects single enzymes immobilized on the surface of plasmonic gold nanoparticles. A fast, low-noise, lock-in method is utilized to extract sensor signals in the microsecond timescale. Using a model enzyme, the ability of the sensor to detect conformational fluctuations of single enzymes is shown. Further, the thermodynamics of the enzyme is studied and the relevant thermodynamic parameters are extracted from the single-molecule data. Additionally, we extract the heat capacity changes associated with the enzyme using the single-molecule data. The sensor system presented in this thesis in the future could enable a fast, real-time, rapid throughput, lab-on-chip sensor system for studying single enzymes for both research and clinical use.

We report a comparison of two photonic techniques for single-molecule
sensing: fluorescence nanoscopy and optoplasmonic sensing. As the test system,
oligonucleotides with and without fluorescent labels are transiently hybridized
to complementary docking strands attached to gold nanorods. Comparing the
measured single-molecule kinetics helps to examine the influence of fluorescent
labels as well as factors arising from different sensing geometries. Our
results demonstrate that DNA dissociation is not significantly altered by the
fluorescent label, while DNA association is affected by geometric factors in
the two techniques. These findings open the door to exploiting plasmonic
sensing and fluorescence nanoscopy in a complementary fashion, which will aid
in building more powerful sensors and uncovering the intricate effects that
influence the behavior of single molecules.

AbstractProbing individual chemical reactions is key to mapping reaction pathways. Trace analysis of sub-kDa reactants and products is obfuscated by labels, however, as reaction kinetics are inevitably perturbed. The thiol-disulfide exchange reaction is of specific interest as it has many applications in nanotechnology and in nature. Redox cycling of single thiols and disulfides has been unresolvable due to a number of technological limitations, such as an inability to discriminate the leaving group. Here, we demonstrate detection of single-molecule thiol-disulfide exchange using a label-free optoplasmonic sensor. We quantify repeated reactions between sub-kDa thiolated species in real time and at concentrations down to 100’s of attomolar. A unique sensing modality is featured in our measurements, enabling the observation of single disulfide reaction kinetics and pathways on a plasmonic nanoparticle surface. Our technique paves the way towards characterising molecules in terms of their charge, oxidation state, and chirality via optoplasmonics.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Label-free optical sensor systems have emerged that exhibit extraordinary sensitivity for detecting physical, chemical, and biological entities at the micro/nanoscale. Particularly exciting is the detection and analysis of molecules, on miniature optical devices that have many possible applications in health, environment, and security. These micro- and nanosensors have now reached a sensitivity level that allows for the detection and analysis of even single molecules. Their small size enables an exceedingly high sensitivity, and the application of quantum optical measurement techniques can allow the classical limits of detection to be approached or surpassed. The new class of label-free micro- and nanosensors allows dynamic processes at the single-molecule level to be observed directly with light. By virtue of their small interaction length, these micro- and nanosensors probe light-matter interactions over a dynamic range often inaccessible by other optical techniques. For researchers entering this rapidly advancing field of single-molecule micro- and nanosensors, there is an urgent need for a timely review that covers the most recent developments and that identifies the most exciting opportunities. The focus here is to provide a summary of the recent techniques that have either demonstrated label-free single-molecule detection or claim single-molecule sensitivity.

In this work a refractometric air pressure sensing platform based on spherical whispering-gallery mode microresonators is presented and analyzed. The sensitivity of this sensing approach is characterized by measuring the whispering-gallery mode spectral shifts caused by a change of air refractive index produced by dynamic sinusoidal pressure variations that lie between extremes of ±1.8kPa. A theoretical frame of work is developed to characterize the refractometric air pressure sensing platform by using the Ciddor equation for the refractive index of air, and a comparison is made against experimental results for the purpose of performance evaluation.