Label-free detection of single biological molecules using microtoroid optical resonators

Being able to detect a single molecule without the use of labels has been a long standing goal of bioengineers and physicists. This would simplify applications ranging from single molecular binding studies to those involving public health and security, improved drug screening, medical diagnostics, and genome sequencing. One promising technique that has the potential to detect single molecules is the microtoroid optical resonator. The main obstacle to detecting single molecules, however, is decreasing the noise level of the measurements such that a single molecule can be distinguished from background. We have used laser frequency locking in combination with balanced detection and data processing techniques to reduce the noise level of these devices and report the detection of a wide range of nanoscale objects ranging from nanoparticles with radii from 100 to 2.5 nm, to exosomes, ribosomes, and single protein molecules (mouse immunoglobulin G and human interleukin-2). We further extend the exosome results towards creating a non-invasive tumor biopsy assay. Our results, covering several orders of magnitude of particle radius (100 nm to 2 nm), agree with the `reactive' model prediction for the frequency shift of the resonator upon particle binding. In addition, we demonstrate that molecular weight may be estimated from the frequency shift through a simple formula, thus providing a basis for an ``optical mass spectrometer'' in solution. We anticipate that our results will enable many applications, including more sensitive medical diagnostics and fundamental studies of single receptor-ligand and protein-protein interactions in real time. The thesis summarizes what we have achieved thus far and shows that the goal of detecting a single molecule without the use of labels can now be realized.

Insights into Neural Crest Evolution

Neural crest cells are unique to vertebrates and essential to the development and evolution of the craniofacial skeleton. Using a combination of DiI cell lineage tracing, transcriptomics, and analysis of key transcription factors of the Sox Family, I examined neural crest development in the sea lamprey, Petromyzon marinus, as the most basal extant vertebrate from which it is possible to get embryos. The results have uncovered distinct cranial and trunk neural crest subpopulations along the anterior-posterior axis of the lamprey embryo, with a clear separation between the two. However, no evidence of the presence of an intermediate vagal neural crest population was uncovered. Comparing cranial neural crest genes between lamprey and chick, either by examining individual candidate genes or whole genome transcriptome analysis, reveals significant changes in the cranial neural crest gene regulatory network of lamprey compared with chick. In particular, the lamprey cranial neural crest is "missing" several gnathostome cranial crest genes. We speculate that these may underlie the evolutionary divergence of craniofacial development between jawed and jawless vertebrates. Despite the absence of vagal neural crest, DiI-labeling shows that trunk neural crest-derived cells, likely homologous to mammalian Schwann cell precursors, contribute to the lamprey enteric nervous system, potentially representing the most primitive form of neural crest cells contribution to the ENS. Finally, I characterized key members of the Sox Family (Sox B-F) due to their importance in neural crest specification in other species. In comparative studies of the SoxC genes (Sox4, Sox11, and Sox12) in both lamprey and Xenopus, I found similar expression patterns and a novel key role in early neural crest specification, suggesting a conserved role of the SoxC genes amongst vertebrates. Taken together, this work represents important progress in characterizing the early evolution of the neural crest in vertebrates and its role in the transition from jawless to jawed vertebrates.