Hyperspectral measurement of surface plasmonic resonance for intracellular single molecule detection
Abstract
The detection of biomolecules at single molecule sensitivity is critical to the study of live cell dynamics to explore a variety of biological processes. In this work, gold nanoparticles were used as intercellular sensors because they do not photobleach, are nontoxic, and rich in electrons which are needed for enhancement to develop a robust method. Another significant advantage is that these nanosensors work in a 3D space, thereby enabling live cell process monitoring. In this research, a hyperspectral imaging system was designed and installed to detect various target biomolecules, and eventually, the intracellular biomolecule detection was demonstrated. It is shown that both dark field and the Raman hyperspectral imaging systems are good for gold nanoparticle hyperspectral imaging. Dark field is a perfect tool to collect surface plasmonic resonance directly by Rayleigh scattering, with many validated applications. On the other hand, Raman spectroscopy is not known as a surface plasmon resonance sensor. In this research, dark field HSI was built for various applications including intracellular mRNA detection. The mechanism of plasmon resonance sensor was also studied using Raman spectroscopy. Sensing modalities using dark field spectroscopy and Raman spectroscopy were also demonstrated: Raman spectra can be used as a powerful tool especially for multiplexing detection and chemical identification, while dark-field spectrum is suitable for very sensitive distance measurement at sub-nanometer scales as well as for single- molecule detection method. When spectroscopy is integrated with mapping (scanning), a very powerful tool for detecting and mapping biological events can be produced. First, the nanostructures including gold nanoparticle dimer were characterized by spectra measurements in this study. Secondly, nanostructures were microinjected into cells and calibration standards for intracellular quantification of mRNA, microRNA and protein were possible. Finally, BRCA1 gene from two different cell lines, nontumorigenic Human Breast Epithelial cell (HBEC) and tumorigenic cell was studied. Towards completion, detection and imaging modalities to monitor both mRNA and microRNA were developed by plasmonic hyperspectral imaging. A parallel Raman nanoruler concept was also developed for DNA and protein detection.
Degree
Ph.D.
Advisors
Irudayaraj, Purdue University.
Subject Area
Biomedical engineering|Biophysics
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