Date of Award
Doctor of Philosophy
Jeffrey M. Becker, Robert C. DeNovo, Russ F. Knapp
Advances in modern biosciences and optical biosensor technology have provided exciting new insights and capabilities. The integration of these fields has witnessed revolutionary advances, which include the development of optical nanosensors. Optical nanosensors are devices based on a direct spatial coupling between biologically active molecules and a signal transducer element interfaced to electronic equipment for signal amplification, acquisition and recording. Optical nanosensors consist of biorecognition molecules covalently immobilized onto the nanotips nanoscale optical fiber that serves as the transducing element. By combining the specificity of biorecognition molecules and the excellent sensitivity of laser-based optical detection, optical nanosensors are capable of detecting and differentiating biochemical constituents of complex systems enabling the provision of sensitive and specific identification of specific molecular events inside living cells.
This work explores and focuses on the development and application of novel optical nanosensors for single living cell analysis. In this context, single cell analysis involves the application of optical nanosensor technology to observe and possibly map molecular events inside single living cells. Previous studies have focused on the bulk response of cells and this largely increases the probability of missing critical underlying mechanisms specific to the single cell. The ability to perform single cell analysis can dramatically improve our understanding of basic cellular processes e.g., signal transduction as well as improving our knowledge of the intracellular transport and the fate of therapeutic agents at the single cell level. This is important not only because of the capability to perform minimally invasive analysis, but also to overcome the problem of ensemble averaging. This capability to overcome ensemble averaging has the potential to yield new information that is not available from population averaged cellular measurements.
This work involves the development and application of optical nanosensors for specific and sensitive chemical and protein analysis within single living cells. The ability of these sensors to successfully perform chemical and protein analysis at the single cell level, lay in their design specifications, size, specificity, sensitivity and eliminating interferences. With regard to their specifications, their size was in the nanometer regime, which is relative to the scale of a single mammalian cell (~ 10 µm) to allow non-invasive-to-minimally-invasive measurements in single living cells. In addition, they incorporated biological recognition molecules to achieve specificity and finally, near-field evanescent wave excitation and detection to achieve high sensitivity. High specificity and sensitivity allowed for precise and accurate identification of physicochemically detectable substances in complex matrices to eliminate any potential interference.
The optical nanosensor intracellular measurement process is straightforward and begins with a sparsely distributed cell culture in a petri dish to allow viewing of single cells using an inverted fluorescence microscope. The optical nanosensor is secured onto the manipulating arms of the microscope and gently manipulated toward the single cell, interacting with the cell, penetrating but not disrupting cellular membranes. The optical nanosensor is briefly incubated in a single living cell and the laser is turned on and excitation light is launched into the optical nanosensor and propagated to the near field of the nanotip where the target analyte is excited by evanescent optical waves. The fluorescence signal generated when the target analyte is excited is collected by the optical set-up of the inverted fluorescence microscope, passes through spectral and spatial filters before detection with a sensitive photon counting photomultiplier tube (PMT). The PMT signal is amplified and recorded via a universal counter interfaced to a personal computer (PC). Data acquisition and recording are controlled using an integrated custom-written program, built on LabView platform.
During in vitro and in vivo measurements, the optical nanosensor response is determined in terms of the sensitivity, specificity, linear dynamic range, response time, nanosensor stability, and reproducibility. In the course of experimental measurements, it was evident that optical nanosensors have characteristics including fast response times (msec range), sensitivity (pM range), selectivity, and excellent reproducibility. In addition to the above figures of merit, optical nanosensors demonstrated biocompatibility with no observed detrimental effects on the cell under investigation in control growth conditions. This demonstrated the utility of optical nanosensor technology for minimally invasive measurement of cellular reactions without altering or destroying the chemical make-up of the cell. This work also illustrates the potential of optical nanosensors in playing an important role in elucidating and enhancing our understanding of cell signaling and transduction pathways in real-time.
Kasili, Paul Misiko, "The Development of Optical Nanosensor Technology for Single Cell Analysis. " PhD diss., University of Tennessee, 2004.