Fundamental Investigations into Single Molecule Surface Enhanced Raman Spectroscopy

<p>After the first claim of single molecule (SM) detection by surface enhanced Raman spectroscopy (SERS) was published in 1997 and years of debate and maturing, SM-SERS can now be considered as an established subfield of SERS. Besides the obvious promising advances in analytical spectroscopy that SM-SERS enables, some more fundamental studies are now also accessible. The main focus of this thesis is to understand certain aspects and tackle some outstanding issues in SM-SERS, both in methods and applications. In the first part of this thesis, we focus on the application of SM-SERS to the study of the homogeneous broadening of molecular vibrations. We show that the homogeneous linewidth of the dye Nile blue as measured on single molecule SERS spectra is much smaller than the inhomogeneous broadening obtained from the average signal. Individual molecules having the central Raman frequency occurring at slightly different positions is therefore the main cause of the inhomogeneous broadening in this system. Furthermore, we show that the homogeneous broadening of the mode of single molecules exhibits a strong temperature dependence from 80K to 300 K. This is suggestive of the vibrational energy exchange model which explicitly relates the temperature dependence of the linewidth of a vibrational mode to its interaction with other modes of the molecule or its environment. The average signal does not show this temperature dependence, this property is indeed washed out by ensemble averaging and its unravelling is made possible by SM-SERS. This study is the first example of direct measurement and study of the homogeneous broadening of a Raman peak. In the second part of this work, we focus on a particular method to prove single molecule sensitivity and demonstrate the single molecule detection of the iconic C₆₀ by SM-SERS using its peculiar spectral properties regarding isotopic substitution. A change in one unit mass in one of the carbon atoms is readily observed as a detectable frequency shift in the Ag(2) mode on the Raman spectrum of one C₆₀. This remarkable result is a direct consequence of the high symmetry of the molecule and is only accessible experimentally by measuring individual molecules. We perform SM-SERS detection of a isotopically enriched C₆₀ and show how the distribution of frequencies for the Ag(2) mode reflects the isotopic spread of the sample. Density Functional Theory (DFT) calculations support the experimental results. This provides the first ever evidence of single molecule detection of C₆₀ via SERS. Finally, we focus on the photostability of dyes excited resonantly in SERS conditions. Photobleaching of the molecule is an issue when doing SERS (and SM-SERS) at resonance. Nile blue is deposited on a highly ordered gold nanolithographic substrate and the time dependence of the SERS signal is monitored. Using a simple two-level system model to describe the mechanisms of photobleaching and express the photobleaching rate, we analyse the SERS intensity decay at different powers. This study is the first to be dedicated to the photobleaching decay rates of molecules on metallic surfaces and to highlight that the decay dynamics contains rates spanning four orders of magnitude. This work can potentially reveal information on the distribution of SERS enhancement factors on the surface.</p>

Fundamental Investigations into Single Molecule Surface Enhanced Raman Spectroscopy

<p>After the first claim of single molecule (SM) detection by surface enhanced Raman spectroscopy (SERS) was published in 1997 and years of debate and maturing, SM-SERS can now be considered as an established subfield of SERS. Besides the obvious promising advances in analytical spectroscopy that SM-SERS enables, some more fundamental studies are now also accessible. The main focus of this thesis is to understand certain aspects and tackle some outstanding issues in SM-SERS, both in methods and applications. In the first part of this thesis, we focus on the application of SM-SERS to the study of the homogeneous broadening of molecular vibrations. We show that the homogeneous linewidth of the dye Nile blue as measured on single molecule SERS spectra is much smaller than the inhomogeneous broadening obtained from the average signal. Individual molecules having the central Raman frequency occurring at slightly different positions is therefore the main cause of the inhomogeneous broadening in this system. Furthermore, we show that the homogeneous broadening of the mode of single molecules exhibits a strong temperature dependence from 80K to 300 K. This is suggestive of the vibrational energy exchange model which explicitly relates the temperature dependence of the linewidth of a vibrational mode to its interaction with other modes of the molecule or its environment. The average signal does not show this temperature dependence, this property is indeed washed out by ensemble averaging and its unravelling is made possible by SM-SERS. This study is the first example of direct measurement and study of the homogeneous broadening of a Raman peak. In the second part of this work, we focus on a particular method to prove single molecule sensitivity and demonstrate the single molecule detection of the iconic C₆₀ by SM-SERS using its peculiar spectral properties regarding isotopic substitution. A change in one unit mass in one of the carbon atoms is readily observed as a detectable frequency shift in the Ag(2) mode on the Raman spectrum of one C₆₀. This remarkable result is a direct consequence of the high symmetry of the molecule and is only accessible experimentally by measuring individual molecules. We perform SM-SERS detection of a isotopically enriched C₆₀ and show how the distribution of frequencies for the Ag(2) mode reflects the isotopic spread of the sample. Density Functional Theory (DFT) calculations support the experimental results. This provides the first ever evidence of single molecule detection of C₆₀ via SERS. Finally, we focus on the photostability of dyes excited resonantly in SERS conditions. Photobleaching of the molecule is an issue when doing SERS (and SM-SERS) at resonance. Nile blue is deposited on a highly ordered gold nanolithographic substrate and the time dependence of the SERS signal is monitored. Using a simple two-level system model to describe the mechanisms of photobleaching and express the photobleaching rate, we analyse the SERS intensity decay at different powers. This study is the first to be dedicated to the photobleaching decay rates of molecules on metallic surfaces and to highlight that the decay dynamics contains rates spanning four orders of magnitude. This work can potentially reveal information on the distribution of SERS enhancement factors on the surface.</p>

Single-Molecule Detection of SARS-CoV-2 by Plasmonic Sensing of Isothermally Amplified Nucleic Acids

Single-molecule detection of pathogens such as SARS-CoV-2 is key to combat infectious diseases outbreak and pandemic. Currently colorimetric sensing with loop-mediated isothermal amplification (LAMP) provides simple readouts but suffers from intrinsic non-template amplification. Herein, we report that plasmonic sensing of LAMP amplicons via DNA hybridization allows highly specific and single-molecule detection of SARS-CoV-2 RNA. Our work has two important advances. First, we develop gold and silver alloy (Au-Ag) nanoshells as plasmonic sensors that have 4-times stronger extinction in the visible wavelengths and give 20-times lower detection limit for oligonucleotides than Au nanoparticles. Second, we demonstrate that the diagnostic method allows cutting the complex LAMP amplicons into short repeats that are amendable for hybridization with oligonucleotide-functionalized nanoshells. This additional sequence identification eliminates the contamination from non-template amplification. The detection method is a simple and single-molecule diagnostic platform for virus testing at its early representation.Table of Content

Screening for Group A Streptococcal disease via Solid-State Nanopore Detection of PCR Amplicons

Single molecule detection methods are becoming increasingly important for diagnostic applications. Practical Early detection of disease requires sensitivity down to the level of single copies of the targeted biomarkers. Of the candidate technologies that can address this need, solid-state nanopores show great promise as digital sensors for single-molecule detection. Here, we present work detailing the use of solid-state nanopores as downstream sensors for a PCR-based assay targeting group A streptococcus (strep A) which can be readily extended to detect any pathogen that can be identified with a short nucleic acid sequence. We demonstrate that with some simple modifications to the standard PCR reaction mixture, nanopores can be used to reliably identify strep A in clinical samples. We also discuss methodological best practices both for adapting PCR-based assays to solid-state nanopore readout as well as analytical approaches by which to decide on sample status.