Instrumentation developments and nanomaterial engineering for live cell mapping and bioanalysis

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Intracellular molecular interaction studies in bioanalysis study have attracted great interest among cell biologists. However, the intrinsic resolution of conventional optical microscopy only allows the visualization of subcellular localizations. Therefore, a sensitive technique that can observe molecular interactions down to the molecular level is in great need. By extracting information from molecular diffusion, Fluorescence Correlation Spectroscopy (FCS) gives detailed information on molecular interactions, and has become a noninvasive single-molecule-detection technique that can be applied to the intracellular environment with low detection limits. Therefore, the overall direction of this doctoral research was the development of a self-built FCS setup. Combined with nanomaterial engineering, this project has enabled membrane receptor studies and intracellular measurements in live cells.

The Fluorescence Correlation Spectroscopy instrumentation was to direct measurement of membrane receptor density of the PTK7 protein, an important cancer marker, in its natural physiological environment on the cell surface. A cellular model using a DNA ligand aptamer was designed for specific receptor targeting and labeling, and was used with FCS to determine receptor densities and distributions on the cell surface. With its intrinsic advantages of direct measurement, high sensitivity, rapid analysis and single-cell measurement, this FCS density estimation approach holds great potential for future applications in molecular interaction studies and density estimations for subcellular structures and membrane receptors.

To further understand the structure of PTK7 beyond its spatial distribution and ligand-receptor interactions, a nanoparticle was used as a molecular ruler to measure the distance between two binding sites on the receptor on live cells. Measuring distances at molecular length scales in living systems is a significant challenge. Methods like FRET (fluorescence resonance energy transfer) have significant limitations due to short detection distances and strict orientations. To overcome these limitations and construct a practical nanoruler for measuring distances on live cells, an SET-based nanoruler, using aptamer-gold-nanoparticle conjugates with different diameters, was developed to measure separation distances well beyond the detection limit of FRET.

Since application of fluorescence auto-correlation (FCS) to binding analysis is limited to applications in which the binding event significantly reduces the diffusion of the labeled species, the original FCS setup was upgraded to a novel three-channel Fluorescence Cross-Correlation Spectroscopy (FCCS) setup. This lab-built FCCS not only inherits the single-molecule detection capability from FCS, but also further extends its applications for molecular interaction studies by labeling two species with two spectrally distinct fluorophores. This technique has been establied and is now being adapted for real-time monitoring of intracellular mRNA.

Another aspect of this research was the development of molecular probes based on nanomaterial engineering Two different types of molecular beacon (MB) probes were designed for enzymatic activity studies and protein inhibition studies.

In summary, this research mainly focuses on instrumentation development and nanomaterial engineering for bioanalysis study and biomedical applications, especially for cell membrane receptor studies and intracellular measurements. A successful outcome from these studies will lead to a better understanding of biological events and processe.