Title: Optimization of surface-enhanced Raman scattering spectroscopy for study of biologically important biomolecules and their interactions Author: Natália Šmídová Department: Institute of Physics of Charles University Supervisor of the doctoral thesis: Doc. RNDr. Marek Procházka, PhD. Abstract: The main goal of this thesis was to optimize surface-enhanced Raman scattering (SERS) spectroscopy for study of biologically important biomolecules. For that purpose we focused on substrates based on gold colloidal nanoparticles immobilized to silanized glass plates. Stable, uniform and highly reproducible SERS-active substrates have been prepared by using aminopropyltrimethoxysilane and citrate- reduced gold nanoparticles thermally stabilized after their immobilization. Model biomolecules 5,10,15,20-tetrakis(1-methyl-4-pyridyl)porphyrin (TMPyP) and 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrin (TSPP) were studied on these substrates by using a classical Raman spectrometer in macro-mode and a confocal Raman microspectrometer. Conditions for SERS spectroscopy of porphyrins were optimized with respect to sensitivity and reproducibility. SERS microspectroscopy showed several advantages over SERS measurements in macromode: possibility of surface spectral mapping, easier manipulation with samples, shorter...

Surface-enhanced Raman scattering (SERS) has long been the interest of researchers in chemistry, physics and engineering, especially since the discovery that SERS can probe into the system down to the single molecule (SM) level. Despite the large number of publications regarding the fabrication of SERS substrates, it has been a challenge in the field to quantify the SERS signal and universally compare substrates. Traditionally, enhancement factor (EF) is used as an indicator of substrate quality, but the EF calculation is hugely dependent on the estimation of the surface coverage and other factors that are determined largely subjectively. Therefore, this thesis aims at discussing other parameters that can also be used to evaluate different substrates. Six different SERS substrates of Ag or Au nanoparticles of different sizes were fabricated by nanosphere lithography (NSL) and characterized by electron microscopy and UV-vis spectroscopy. SERS substrates were mapped for different concentrations of a probe molecule. Through subsequent baseline correction and principle component analysis (PCA), the "intensity" of individual spectrum was obtained and the shapes of intensity histograms of each substrate were acquired. Instead of calculating EF, five criteria (six quantification methods in total) were employed to comprehensively evaluate the six substrates. These were density of hot spots (characterized by the number of zero-intensity events), enhancement (represented by mean intensity), spatial variation (calculated by RSD of intensity), repeatability (realized by cross correlation) and histogram shape (quantified by skewness and kurtosis). These new methods provide insights to the understanding of the properties of SERS substrates in terms of hot spots. Different substrates may exhibit better performance in terms of one criterion but worse in terms of others. Those variations in performance can be explained by their surface morphology. These more elaborated methods are believed to provide a more comprehensive approach to evaluate and compare substrates than the traditional EF values. The thesis also paves the way for future study on SM-SERS and fabricating better SERS substrates. / Graduate

Surface-enhanced Raman scattering (SERS) spectroscopy is a powerful analytical technique for ultrasensitive detection of chemicals and biomolecules. As the high sensitivity of SERS requires analytes to be in close contact with a plasmonic substrate, the detectionof analyte molecules with low chemical affinity towards the substrate is thus limited. Cucurbit[n]uril (CB[n]) exhibits strong and selective encapsulation of various guest molecules into its barrel-shaped cavity. In addition, it can function as a precise rigid spacer between metallic nanoparticles (NPs). The larger homologue CB[8] can simultaneously sequester two guest molecules to form ternary complexes, allowing for tailoring of the chemical environment of its cavity to trap specific analytes. CB[n] aggregated metallic NPs provide a powerful platform for the detection of a wide variety of molecules. However, the colloidal instability of this system requires the measurement to be finished within 60 min after the preparation of the substrate. In addition, in situ measurements may involve environments that affect such self-assembly processes. For example, the possible displacement of analytes in the nanogap by non-analyte moieties can give rise to fluctuating backgrounds. Therefore, a SERS substrate that can provide the same levels of detection and functionality but eliminates the need for aggregation is of great demand. This thesis mainly focuses on the preparation and characterisation of CB[n]-engineered nanostructures as SERS substrates with great colloidal stability, high SERS enhancements and sensitivities. Other applications of the prepared nanostructures such as peptide separation and high-performance catalysis are also discussed. In the first chapter, the historical development and the remaining challenges in the field of SERS are discussed. Three types of the most commonly used SERS substrates are introduced, followed by the introduction of rationally designed nanoplatforms for molecules with low chemical affinity towards metallic surfaces. In addition, CB[n] host guest complexation, examples of CB[n]-engineered nanostructures and the application of these nanostructures in SERS sensing are also discussed. The second chapter demonstrates the preparation of surface-bound CB[8] catenanes on silica NPs, where CB[8] is employed as a tethered supramolecular "receptor" to selectively capture target guest molecules. More specifically, CB[8] is threaded onto a methyl viologen (MV2+) axle and immobilised onto silica NPs with a surface density up to 0.56 nm$^{−2}$. Its use as an efficient and recyclable nanoplatform for peptide separation is demonstrated. The peptides captured by the catenanes can be released by reversible single-electron reduction of MV$^{2+}$. The entire process demonstrates high recoverability. Continued in the third chapter, a highly stable free-standing molecular sensor that exploits a catenane-engineered nanostructure is described. CB[8] is tethered onto spiky γ-Fe2O3@Au NPs in a similar approach, to collect target analytes from aqueous media. These target analytes can be detected with high sensitivities, on account of the high SERS enhancement (on the order of 10$^{8}$) of the spiky NPs. This CB[8] catenane-based molecular sensor provides a powerful SERS substrate that shows great promise in the detection of versatile chemicals, biomolecules, controlled substances and auxiliary diagnostics of various diseases. The fourth chapter introduces a facile preparation of monodispersed γ-Fe2O3@Au magnetic nanoraspberry NPs using a one-pot seeded growth method. The obtained nanoraspberry NPs show excellent colloidal stability and high SERS enhancement factors (on the order of 10$^{10}$). By immobilising a dense layer of CB[n]s onto the surface of nanoraspberry NPs, a new type of CB/Au NP SERS substrate is obtained. CB[n]s are located perpendicularly to the NP surface and their cavity maintain the capability to sequester guest molecules from aqueous media. More versatile molecules (both electron rich and electron deficient molecules) can thus be detected with high sensitivities. We envisage that this nanoraspberry-based molecular sensor will provide a powerful platform for SERS detection in various fields, such as chemical and biomolecule analysis, illegal drug detectionand pre-clinical/clinical diagnosis. The fifth chapter focuses on the preparation of CB[7]-based catalytic microreactors, where metallic NPs are immobilised onto microchannels via supramolecular interaction of methyl viologen@CB[7]. This microreactor exhibits remarkable catalytic activity on account of the high surface area to volume ratio of the microchannels and metallic NPs. Superior to most conventional heterogeneous catalytic reactions, separation post reaction and complicated recycling steps of the catalysts are not required. Moreover, CB[7] can complex a variety of metallic NPs onto its portal (e.g. gold, silver, palladium, quantum dot), providing a multifunctional in situ catalysis platform. In the end, a concluding chapter summarises the presented work, also giving a brief outlook of the potential future work.

Carbohydrates are known to play a large number of significant roles in various biological and pathological processes such as cancer metastasis and cellular communication. This is because of their ability to bind a wide range of hosts within the human body such as proteins and viruses. Due to these important interactions, carbohydrate sensing has long been a main focus of research. These research strategies have included the use of aptamers, non-covalent interactions, and boronic acid-based receptors. Boronic acid-based sensors are of particular interest due to their selectivity for 1,2- or 1,3-diols. Within these boronic acid-based studies, a large variety of techniques were employed for detection including different fluorescent, electrochemical, polymeric, and colorimetric studies, as well as various surface bound sensors. One type of technique that has rarely been applied is Surface Enhanced Raman Spectroscopy or SERS. This strategy would be beneficial as it provides information about functional groups, which would aid in the identification of the bound sugar. In this thesis, we present work based on the development of a boronic acid-based carbohydrate receptor that will be used to study carbohydrate binding through SERS. The receptor design includes an aryl boronic acid for carbohydrate recognition, a nitrogen atom in close proximity to the boron center to enhance binding, and a terminal thiol for attachment to a metal surface for SERS. This sensor will be used to study the binding of different saccharides for sensing applications.

Integrins are transmembrane heterodimer protein receptors that mediate adherence to both the intracellular cytoskeleton and extracellular matrix. They play a major role in cellular adhesion and the breadth of their importance in biology is only recently being understood. The ability to detect concentrations of integrins on the cell surface, spatially resolve them, and study the dynamics of their behavior would be a significant advance in this field. Ultimately, the ability to detect dynamic changes of integrins on the surface of a cell maybe possible by developing a combined device such as an atomic force microscope (AFM) and surface enhanced Raman spectroscopy (SERS) system. However, the focus of this research is to first determine if integrins can be detected using SERS. Surface enhanced Raman spectroscopy (SERS) is technique used to detect the presence of analytes at the nanomolar level or below, through detection of inelastically scattered light. This thesis discusses the detection of integrins employing SERS as the detection modality. Integrins have been detected, in solution, using two silver colloids as the enhancing surface. Two silver colloid preparation methods are compared by ease of formulation and degree of enhancement in this thesis. Citrate and hydroxylamine hydrochloride (HA-HCl) reduced silver colloids were prepared through wet chemistry,compared using UV-Vis light spectroscopy, and tested for surface enhancement using adenine (a strong SERS active molecule), and two different integrins, (alpha)V(beta)3 and (alpha)5(beta)1. Results indicated that both colloids demonstrate SERS activity for varying concentrations of adenine as compared to standard non-enhanced Raman, however, only the citrate reduced colloid showed significant enhancement effect for the integrins.

Integrins are transmembrane heterodimer protein receptors that mediate adherence to both the intracellular cytoskeleton and extracellular matrix. They play a major role in cellular adhesion and the breadth of their importance in biology is only recently being understood. The ability to detect concentrations of integrins on the cell surface, spatially resolve them, and study the dynamics of their behavior would be a significant advance in this field. Ultimately, the ability to detect dynamic changes of integrins on the surface of a cell maybe possible by developing a combined device such as an atomic force microscope (AFM) and surface enhanced Raman spectroscopy (SERS) system. However, the focus of this research is to first determine if integrins can be detected using SERS. Surface enhanced Raman spectroscopy (SERS) is technique used to detect the presence of analytes at the nanomolar level or below, through detection of inelastically scattered light. This thesis discusses the detection of integrins employing SERS as the detection modality. Integrins have been detected, in solution, using two silver colloids as the enhancing surface. Two silver colloid preparation methods are compared by ease of formulation and degree of enhancement in this thesis. Citrate and hydroxylamine hydrochloride (HA-HCl) reduced silver colloids were prepared through wet chemistry,compared using UV-Vis light spectroscopy, and tested for surface enhancement using adenine (a strong SERS active molecule), and two different integrins, (alpha)V(beta)3 and (alpha)5(beta)1. Results indicated that both colloids demonstrate SERS activity for varying concentrations of adenine as compared to standard non-enhanced Raman, however, only the citrate reduced colloid showed significant enhancement effect for the integrins.

Carbohydrates are known to play a large number of significant roles in various biological and pathological processes such as cancer metastasis and cellular communication. This is because of their ability to bind a wide range of hosts within the human body such as proteins and viruses. Due to these important interactions, carbohydrate sensing has long been a main focus of research. These research strategies have included the use of aptamers, non-covalent interactions, and boronic acid-based receptors. Boronic acid-based sensors are of particular interest due to their selectivity for 1,2- or 1,3-diols. Within these boronic acid-based studies, a large variety of techniques were employed for detection including different fluorescent, electrochemical, polymeric, and colorimetric studies, as well as various surface bound sensors. One type of technique that has rarely been applied is Surface Enhanced Raman Spectroscopy or SERS. This strategy would be beneficial as it provides information about functional groups, which would aid in the identification of the bound sugar. In this thesis, we present work based on the development of a boronic acid-based carbohydrate receptor that will be used to study carbohydrate binding through SERS. The receptor design includes an aryl boronic acid for carbohydrate recognition, a nitrogen atom in close proximity to the boron center to enhance binding, and a terminal thiol for attachment to a metal surface for SERS. This sensor will be used to study the binding of different saccharides for sensing applications.

SERS

Fungal species perform many important roles in biotechnology and recycling and act as agents of disease and decay. Surface-enhanced Raman scattering (SERS) has attracted significant attention as an analytical method for chemical and biological identification. For SERS experiments, it is essential to generate gold nanoparticles (AuNPs) with proper sizes and shapes. Raman and SERS imaging of fungi via in vivo synthesis of AuNPs were used to explore cellular components of Aspergillus nidulans (A. nidulans) cell. Critical parameters including pH, temperature and metal concentration affect the sizes and shapes of the NPs. For better control of NP formation (size, shape and location), pre-formed NP were incubated with A. nidulans colonies. Aspergillus nidulans outer hyphal walls were coated with NPs. Raman and SERS imaging of fungal walls revealed that proteins, carbohydrates and lipids are the main constituents of fungal cell wall. / October 2014

Consensus opinion suggests that apoptosis occurs when the intracellular redox potential reaches its oxidative range, i.e. when the balance between oxidants and reductants is disturbed. An understanding of the links between redox potential and the induction of apoptosis in cells could improve our understanding of the process and help to predict therapeutic responses. This study investigates the changes in redox potential at distinct stages of apoptosis induced in the human cervical cancer cell line, HeLa. Stages of the apoptotic process were defined by loss of mitochondrial membrane polarisation (ΔΨm), membrane phosphatidyl serine exposure, caspase-3 activation, and nuclear fragmentation. To measure real-time redox potential change in apoptotic cells two independent methods were used: (1) expression of redox-responsive green fluorescent protein (roGFP2) measured by flow cytometry and (2) redox-responsive nanosensors detected by surface enhanced Raman spectroscopy (SERS). roGFP2 measurements showed that HeLa cells demonstrate a shift towards an oxidative redox state during the later stages of apoptosis and this was preceded by loss of ΔΨm. The relationship between these two events was investigated by transient inhibition of mitochondrial permeability transition pore opening using the inhibitor bongkrekic acid (BKA) pre-treatment. At the cell population level, transient exclusion of the mitochondrial contribution delayed two key events of apoptosis in the first two hours measured by nuclear fragmentation and loss of ΔΨm. However, BKA treatment did not affect redox potential, reported by roGFP2, when compared with controls. Therefore, this suggests that mitochondria do not contribute towards the overall redox potential change in apoptosis. To gain insight into the significance of redox change at the earliest stages of apoptosis, single cell studies were performed. SERS, employing simultaneous redox potential and intracellular pH measurements using two synthetic nanosensors AQ-NS and MBA-NS, showed that BKA pre-treatment resulted in increased alkalinity and the cells were consequently protected from induction of apoptosis in the first thirty minutes of the kinase inhibitor staurosporine treatment. Measurements with SERS nanosensors allowed for adjustment for pH, which provides a clearer insight into redox potential dynamics, with consideration of the environment, and accurate quantitative assessment of redox at early stages of apoptosis. Together these data suggest that while roGFP2 is a valid method to use at a population level, SERS is a more sensitive method for measuring the redox potential of the cell at the early stages of apoptosis.