Label-free plasmonic biosensors for real-time live cell analysis

For decades, the understanding of life has been focused on its most fundamental building blocks: molecules. Deciphering the dynamic activities of protein molecules in human cells is of vital significance for fundamental and clinical studies. The minuscule scale of protein molecules poses a great challenge for precise quantification. Labeling the molecules of interest with fluorescent and enzymatic tags has thus become an effective strategy to indicate their existence. However, the addition of extrinsic fluorescent tags has shown a number of evident side effects interfering with protein functions and cell biology. The extra steps caused by molecular labeling also increase the complexity of the entire assay and decrease the temporal resolution of the measurement from hours to days. These drawbacks of traditional strategies inevitably hinder the real-time monitoring of cellular activities with minimum interval or external interference. This doctoral thesis focuses on the engineering and demonstration of novel label-free biosensing platforms for real-time cell studies: (1) the secretion of protein molecules from live cells, and (2) the characterization of cellular interactions. For the first topic, a biosensor using plasmonic nanohole arrays as the core sensing elements has been designed. These nanohole arrays consist of a thin gold film perforated with periodic nanoholes. They enable extraordinary optical transmission (EOT), an optical phenomenon that has shown promising capabilities for biochemical detection. This thesis demonstrates, for the first time the application of these structures for real-time monitoring of protein secretions from live cells. On the one hand, the secretion of VEGF (a type of growth factor) from live cancer cells has been monitored in real-time at the temporal resolution of seconds without any additional sample treatment. On the other hand, the integration with the advanced microfluidic system has enabled the biosensor to measure secretion events at the single-cell level by solving a number of technical issues (e.g., low analyte abundance, liquid evaporation). In particular, the real-time production of IL-2 (a cytokine) from single lymphoma cells were monitored for hours, which shows the exceptional versatility of this optofluidic nanobiosensor. For the second topic, a multiparametric surface plasmon resonance (SPR) biosensor has been exploited to investigate interactions between T-cell receptors (TCR) and peptide-major histocompatibility complexes (pMHC). This type of interaction plays a central role in T cell-mediated immunity. Notably, intact human T cells - rather than purified recombinant TCR proteins - were directly used as analytes. A biomimicking lipid membrane was created on the sensor surface to present pMHC molecules, enabling the capture of T cells in vitro. Therefore, the affinity (e.g., binding kinetics) between different types of T cells and membrane-bound pMHC molecules can be readily measured in a label-fr