Bioinspired Engineering for Microbial Sensing: The Development of Bacterial Sensing Methodologies Based on Fluorescence Staining and Microfluidic Emission Spectroscopy
- Author(s): Nunez, Vicente
- Advisor(s): Vullev, Valentine I
- et al.
Bacterial infectious diseases remain one of the major health hazards nation- and worldwide. The expedience of detection and identification of bacterial pathogens determines how early the diagnosis is, and hence, what the treatment and the outcome of the illness would be. Conventional culture-based methods that are used to diagnose bacterial colonization and infection depend on the organism's ability to grow in pre- determined conditions. Advances in biotechnology and molecular biology have provided new tools for bacterial identification that provide expedience and specificity. Such techniques as genetics based diagnostics, however, depend on initial prediction for the potential pathogens in a sample. Inherently, such high-specificity assays provide information only about the species that are targeted. Therefore, pathogens present in the sample, which are not targeted by the conducted tests, remain undetected. The first part of my doctoral research sought to address these challenges by: (1) investigating the dynamics of fluorescence enhancement caused by the bacterial uptake; (2) identifying fluorescent dyes, e.g., molecular rotors, that can selectively stain only bacteria while in blood and other biological and environmental fluids; (3) employing biomimetic functionalization of bioinert interfaces for a bacterial immobilization that brings the analysis of fluorescence dynamic staining to a single-cell level; and (4) investigating the excited-state dynamics of the cyanine dye 3, 3'-diethylthiacyanine (THIA) using transient absorption spectroscopy to provide rationale for fluorescence staining kinetics observed during bacterial uptake. In parallel, my graduate research focused on utilizing the flow dynamics in microchannels for developing lab-on-a-chip platforms for time-resolved spectroscopy. Current approaches for investigating the photophysical dynamics of chromophores require cost-prohibitive equipment thus reducing the propagation of such specialized techniques. Using steady-state excitation and imaging, we measured luminescence decay kinetics of lanthanide chelates from the spatial distribution of the emission within a microchannel. From the space-domain data for the chelates recorded at different flow rates we extracted lifetimes that we confirmed using "traditional" time-domain measurements. This validated space-domain microfluidic approach reveals a means for miniaturization of time-resolved emission spectroscopy and bringing it to lab-on-a-chip platforms.