Label-free micro-RNA detection using surface-enhanced Raman scattering and development of a lab-on-a-chip SERS device
Abell, Justin Louis
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Label-free surface-enhanced Raman scattering (SERS) analysis of nucleic acids has been previously impeded by issues relating to the reproducibility of SERS measurements and SERS substrates, interpretation and quantification of the SERS signal of DNA, and development of suitable SERS-based devices. This dissertation will therefore focus on addressing these issues for microRNA (miRNA) biomarker detection. Improved quantitative SERS analysis using silver nanorod (AgNR) substrates fabricated by oblique angle deposition (OAD) is demonstrated using a dynamic measuring technique. To process and analyze multiple DNA and RNA sequences in parallel, a multi-well array SERS chip is fabricated onto a large-area AgNR substrate using a molding method. To detect miRNA with SERS, DNA is immobilized onto the AgNR substrate to act as a probe to specifically capture complementary miRNA. A linear least squares (LS) regression analysis is developed to decompose the resulting SERS spectrum of DNA to the spectra of the pure nucleotides, and determine the relative contribution of each type of nucleotide within the measured DNA signal. This can be used to distinguish if hybridization/capture has occurred or not. Despite the high degree of spectral similarity observed for the immobilized DNA probe before and after capture of the miRNA, the LS method is capable of quantitatively determining if, and how much, hybridization has occurred. In addition, the effect of solvents on the AgNR SERS response is evaluated using thiol test analytes. The AgNRs are discovered to bundle together upon drying of the solvent, which induces the formation of SERS hot spots. With proper surface functionalization of the AgNRs, the nanorod bundling/de-bundling is shown to be a reversible process that depends on the solvent polarity. The feasibility of utilizing the AgNRs as electrodes is also investigated. Applying a negative voltage to the AgNRs yields improved SERS signal of a test analyte, yet does not induce electrochemical degradation of the AgNRs. The OAD fabrication technique is also integrated with traditional lift-off processes to form AgNR microelectrodes, and a novel lab-on-a-chip (LOC) SERS device is constructed.