Date of Award:
Doctor of Philosophy (PhD)
Biological and Irrigation Engineering
Ronald C. Sims
Timothy A. Gilbertson
Charles D. Miller
Lisa M. Berreau
A fundamental understanding of bio-interfaces will facilitate improvement in the design and application of biomaterials that can beneficially interact with biological objects such as nucleic acids, molecules, bacteria, and mammalian cells. Currently, there exist analytical instruments to investigate material properties and report information on electrical, chemical, physical, and mechanical natures of biomaterials and biological samples. The overall goal of this research was to utilize advanced spectroscopy techniques coupled with data mining to elucidate specific characteristic properties for biological objects and how these properties imply interaction with environmental biomaterials.
My studies of interfacial electron transfer (ET) of DNA-modified gold electrodes aided in understanding that DNA surface density is related to the step-wise order of which a self-assembled monolayer is created on a gold substrate. Further surface modification plays a role in surface conductivity, and I found that electro-oxidation of the DNA involved the oxidation of guanine and adenine nucleotides. Scanning tunneling microscopy (STM) was used to create topography and current images of the SAM surfaces. I also used Raman microspectroscopy (RM) to obtain spectra and spectral maps of DNA-modified gold surfaces.
For studies of bacteria, atomic force microscopy (AFM) and scanning electron microscopy (SEM) images showed similar morphological features of Gram-positive and Gram-negative bacteria. Direct classical least squares (DCLS) analysis aided to distinguish co-cultured strains. Fourier transform infrared (FTIR) spectroscopy proved insightful for characteristic bands for Gram-positive bacteria and a combined AFM/RM image revealed a relationship between culture height/density and peak Raman intensity.
In our mammalian cell studies we focused on human lung adenocarcinoma epithelial cells (A549), metastatic human breast carcinoma cells MDA-MB-435 (435), and non-metastatic MDA-MB-435/BRMS1 (435/BRMS1). RM revealed similarities between metastatic 435 and non-metastatic 435/BRMS1 cells compared to epithelial A549 cells. AFM showed increases in biomechanical properties for 435/BRMS1 in the areas of cell adhesion, cell spring constant, and Young’s modulus. Fluorescent staining illustrates F-actin rearrangement for 435 and 435/BRMS1. Electric cell-substrate impedance sensing (ECIS) revealed that 435 cells adhere tightly to substrata and migrate rapidly compared with 435/BRMS1. For ECIS, ≤10-fold diesel exhaust particles (DEP) concentration exposure caused clastogenic DNA degradation whereas ≥25-fold DEP exposure caused cytotoxic results. Resveratrol (RES) at 10 μM showed minimal to mild protection against DEP before and after exposure and aided in improving injury recovery.
McEwen, Gerald Dustin, "Raman Microspectroscopy, Atomic Force Microscopy, and Electric Cell-Substrate Impedance Sensing For Characterization of Bio-Interfaces: Molecular, Bacteria, and Mammalian Cells" (2012). All Graduate Theses and Dissertations. 1251.
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