Indiana University-Purdue University Indianapolis (IUPUI) / Noble metal nanostructures display collective oscillation of the surface conduction electrons upon light irradiation as a form of localized surface plasmon resonance (LSPR) properties. Size, shape, and refractive index of the surrounding environment are the key features that control the LSPR properties. Surface passivating ligands on to the nanostructure can modify the charge density of nanostructures. Further, allow resonant wavelengths to match that of the incident light. This unique phenomenon called the “plasmoelectric effect.” According to the Drude model, red and blue shifts of LSPR peak of nanostructures are observed in the event of reducing and increasing charge density, respectively. However, herein, we report unusual LSPR properties of gold triangular nanoprisms (Au TNPs) upon functionalization with para-substituted thiophenols (X-Ph-SH, X = -NH2, -OCH3, -CH3, -H, -Cl, -CF3, and -NO2). Accordingly, we hypothesized that an appropriate energy level alignment between the Au Fermi energy and the HOMO or LUMO of ligands allows the delocalization of surface plasmon excitation at the hybrid inorganic-organic interface. Thus, provides a thermodynamically driven plasmoelectric effect. We further validated our hypothesis by calculating the HOMO and LUMO levels and work function changes of Au TNPs upon functionalization with para-substituted thiol. This reported unique finding then utilized to design ultrasensitive plasmonic substrate for biosensing of cancer microRNA in bladder cancer and cardiovascular diseases. In the discovery of early bladder cancer diagnosis platform, for the first time, we have been utilized to analyze the tumor suppressor microRNA for a more accurate diagnosis of BC. Additionally, we have been advancing our sensing platform to mitigate the false positive and negative responses of the sensing platform using surface-enhanced fluorescence technique. This noninvasive, highly sensitive, highly specific, also does not have false positives techniques that provide the strong key to detect cancer at a very early stage, hence increase the cancer survival rate. Moreover, the electromagnetic field enhancement of Surface-Enhanced Raman Scattering (SERS) and other related surface-enhanced spectroscopic processes resulted from the LSPR property. This dissertation describes the design and development of entirely new SERS nanosensors using a flexible SERS substrate based on the unique LSPR property of Au TNPs. The developed sensor shows an excellent SERS activity (enhancement factor = ~6.0 x 106) and limit of detection (as low as 56 parts-per-quadrillions) with high selectivity by chemometric analyses among three commonly used explosives (TNT, RDX, and PETN). Further, we achieved the programmable self-assembly of Au TNPs using molecular tailoring to form a 3D supper lattice array based on the substrate effect. Here we achieved the highest reported sensitivity for potent drug analysis, including opioids and synthetic cannabinoids from human plasma obtained from the emergency room. This exquisite sensitivity is mainly due to the two reasons, including molecular resonance of the adsorbate molecules and the plasmonic coupling among the nanoparticles. Altogether we are highly optimistic that our research will not only increase the patient survival rate through early detection of cancer but also help to battle the “war against drugs” that together are expected to enhance the quality of human life.

When exposed to light, metal nanoparticles exhibit a phenomenon known as LSPR, Localized Surface Plasmon Resonance. The wavelengths at which LSPR occurs is very dependent on the refractive index of the surrounding medium. Binding of biomolecules to the surface of gold nanoparticles result in a change in the refractive index that can be detected spectrophotometrically by monitoring the LSPR peak shift. When functionalized with the corresponding ligand(s), gold nanoparticles can be utilized in biosensors to detect the presence and concentration of a predetermined analyte. However, the system must exhibit high specificity and give rise to a detectable shift for analytes in the desired concentration range to be of commercial interest. The aim of the diploma project was to investigate and optimize the biofunctionalization and performance of a fiber optics based LSPR biosensor.  Three ligand systems were investigated for detection of antibodies (IgG), insulin and avidin. Binding of the analyte to the ligand caused a shift of a few nanometers when using spherical gold nanoparticles. The shifts were significantly larger when using gold nanorods. When using the IgG and insulin ligands, only minor unspecific binding was observed. The setup thus shows great potential for use in a wide range of sensing applications.

The terahertz regime has until recently been some what neglected due to the difficulty of generating and measuring terahertz radiation. Terahertz time domain spectroscopy has allowed for affordable and broadband probing of this frequency regime with phase sensitive measurements (chapter 3). This thesis aims to use this tool to add to the knowledge of the interactions between electromagnetic radiation and matter specifically in regard to plasmonics. This thesis covers several distinct phenomena related to plasmonics at terahertz frequencies. The generation of terahertz radiation from metal nanoparticles is first described in chapter 4. It is shown that the field strength of the plasmon appears to relate to the strength of the generated field. It is also shown that the power dependence of the generated terahertz radiation is not consistent with the optical rectification description of this phenomenon. An alternative explanation is developed which appears more consistent with the observations. A simple model for the power dependence is derived and compared to the experimental results. In chapter 5 the parameters that make good plasmonic materials are discussed. These parameters are used to assess the suitability of semiconductors for terahertz surface plasmon experiments. The Drude permittivity of InSb is measured here, leading to a discussion of terahertz particle plasmons in chapter 6. Finite element method modelling is used to show some merits of these over optical particle plasmons. This also includes a discussion of fabrication methods for arrays of these particles. Finally, chapter 7 is a discussion of so called spoof surface plasmons. This includes some experimental work at microwave frequencies and an in depth analysis of open ended square hole arrays supported by model matching method modelling. Perfect endoscope effects are discussed and compared to superlensing. The thesis ends with a brief conclusions chapter where some of the ideas presented are brought together.

This dissertation comprises experimental studies on the synthesis and applications of metallic nanoshells. These are a class of nanoparticles composed of a dielectric core and a thin metallic shell. Metallic nanoshells play an important role in nanotechnology, particularly in nanomedicine, due to their peculiar optical properties. The overall objectives of the dissertation were to improve the fabrication of these nanoparticles, and to demonstrate new applications of these materials in cancer research and spectroscopy. The fabrication of nanoshells is a multi-step process. Previously to our work, the procedures for the synthesis of nanoshells reported in the literature lacked systematic characterization of the various steps. The procedure was extremely time-consuming and the results demonstrated a high degree of size variation. In Chapter 3, we have developed characterization tools that provide checkpoints for each step of the synthesis. We demonstrated that it is possible to control the degree of coverage on the shell for a fixed amount of reagents, and also showed important differences on the shell growth phase for gold and silver. The synthetic optimization presented in Chapter 3 led to an overall faster protocol than those previously reported. Although the improvements presented in Chapter 3 led to a higher degree of control on the synthesis of nanoshells, the variations in the resulting particle population were still too large for applications in single particle spectroscopy and imaging. In Chapter 4, the synthesis was completely reformulated, aiming to narrow the size distribution of the nanoshell colloids. Through the use of a reverse microemulsion, we were able to fabricate ultramonodisperse silica (SiO2) cores, which translate into nanoshell colloids with narrow extinction bands that are comparable to those of a single nanoshell. We then fabricate a library of colloids with different core sizes, shell thicknesses and composition (gold or silver). The localized surface plasmon resonance (LSPR) of these colloids span across the visible range. From this library, two nanoshells (18nm silver on a 50nm SiO2 core, and 18nm gold on a 72nm SiO2 core) were selected for a proof of principle cell imaging experiment. The silver nanoshells were coated with a nuclear localization signal, allowing it to target the nuclear membrane. The gold nanoshells were coated with an antibody that binds to a receptor on the plasma membrane of MCF-7 human breast cancer cells. The nanoshells were easily distinguishable by eye in a dark field microscope and successful targeting was demonstrated by hyperspectral dark field microscopy. A comparison was made between fluorescent phalloidin and nanoshells, showing the superior photostability of the nanoparticles for long-term cell imaging. The results from Chapter 4 suggest that the nanoshells obtained by our new synthetic route present acceptable particle-to-particle variations in their optical properties that enables single particle extinction spectroscopy for cell imaging. In Chapter 5 we explored the use of these nanoshells for single-particle Surface-enhanced Raman spectroscopy (SERS). Notice that particle-to-particle variations in SERS are expected to be more significant than in extinction spectroscopy. This is because particle-to-particle SERS variabilities are driven by subtle changes in geometric parameters (particle size, shape, roughness). Two types of gold nanoshells were prepared and different excitation wavelengths (λex) were evaluated, respective to the LSPR of the nanoshells. Individual scattering spectra were acquired for each particle, for a total of 163 nanoshells, at two laser excitation wavelengths (632.8 nm and 785 nm). The particle-to-particle variations in SERS intensity were evaluated and correlated to the efficiency of the scattering at the LSPR peak. Chapter 6 finally shows the application of gold nanoshells as a platform for the direct visualization of circulating tumor cells (CTCs). 4T1 breast cancer cells were transduced with a non-native target protein (Thy1.1) and an anti-Thy1.1 antibody was conjugated to gold nanoshells. The use of a transduced target creates the ideal scenario for the assessment of nonspecific binding. On the in vitro phase of the study, non-transduced cells were used as a negative control. In this phase, parameters such as incubation times and nanoshell concentration were established. A murine model was then developed with the transduced 4T1 cells for the ex vivo portion of the work. Non-transduced cells were implanted in a control group. Blood was drawn from mice in both groups over the course of 29 days. Antibody-conjugated nanoshells were incubated with the blood samples and detection of single CTCs was achieved in a dark field microscope. Low levels of nonspecific binding were observed in the control group for non-transduced cells and across different cell types normally found in peripheral blood (e.g. lymphocytes). All positive and negative subjects were successfully identified. Chapter 7 provides an outlook of the work presented here and elaborates on possible directions to further develop the use of nanoshells in bioapplications and spectroscopy. / Graduate / 2019-05-03

Thus far, a plethora of analysis on genome-wide gene expression microarray experiments on the cell cycle have been reported. Time series data from these experiments include gene expression profiles that might be periodically expressed. However, the numbers and actual genes that are periodically expressed have not been reported with consistency, analysis on similar experiments reports disparate numbers of genes that are periodically expressed with scant overlap. This work ultimately compares the performance of five spectral estimation schemes in their ability to recover periodically expressed genes profiles. Lomb-Scargle (LS), Capon, Missing-Data Amplitude and Phase Estimation (MAPES), Real Value Iterative Adaptive Approach (RIAA) and Lomb-Scargle Periodogram Regression (LSPR) are rigorously studied and pitted against each other in various simulated testing conditions. Results obtained using synthetic and microarray data reveals that RIAA is an efficient and robust method for the detection of periodically expressed genes in short time series data that might be characterized with noisy and irregularly sampled data points.

[pt] O presente trabalho investiga a utilização de fibras ópticas em biossensoriamento e na indução de uma não-linearidade de segunda ordem para a construção de dispositivos sensores. O biossensor proposto tem por finalidade diagnosticar uma das doenças com maior incidência em países tropicais: a Dengue. Foi construído um sensor a fibra óptica que potencialmente é capaz de diagnosticar, num tempo curto, a presença do vírus da Dengue no sangue de um paciente infectado. Esse sensor usa nanopartículas de ouro, depositadas na extremidade de uma fibra óptica, que foram funcionalizadas com os anticorpos da Dengue (anti-NS1). O sensor é capaz de detectar o antígeno NS1 em diferentes concentrações com um limite de quantificação de 0.074 micrograma por mililitro, podendo ser explorado para uso na fase aguda da infecção.Outra vertente do trabalho é a possibilidade de se realizar modificações estruturais nas fibras ópticas com o intuito de alterar as propriedades ópticas da fibra. Através da técnica de polarização térmica, é possível gravar campos elétricos da ordem de 108 volts por metro no núcleo da fibra óptica, sendo possível utilizar as fibras polarizadas como moduladores de fase e de amplitude, seletores de pulso, chaves ópticas, voltímetros, entre outras. O trabalho de tese aqui descrito apresenta um estudo detalhado da polarização térmica em fibras ópticas através de simulações, utilizando o software COMSOL Multiphysics, considerando-se os diversos parâmetros envolvidos e geometrias diferentes de fibras, visando a obtenção de uma alta não-linearidade de segunda ordem. Além do mais, experimentos foram realizados a fim de se entender o mecanismo presente no processo de polarização térmica face aos resultados obtidos pela simulação. Buscou-se, também, entender o papel dos portadores de cargas presentes no material no processo de geração de não-linearidade de segunda ordem realizando-se experimentos de polarização óptica. / [en] The present work investigates the use of fiber optics in biosensing and the creation of a second order nonlinearity to be use in the development of sensor devices. The goal of the proposed biosensor is to diagnose one of the diseases with highest incidence in tropical countries: Dengue. Dengue is a dangerous disease that every year affects more and more people, despite the efforts made to deal with the transmitter, the mosquito Aedes aegypti. Furthermore, since Dengue symptoms resemble flu symptoms, wrong diagnoses are frequently made. As a consequence, wrong medicines may be prescribed, and that may lead the patient to death. Another problem in diagnosing Dengue is the long time is necessary for the laboratorial exams to give a result. In an attempt to offer a solution that could minimize these problems, an LSPR-based fiber optic sensor was adapted for antigen NS1 detection. This sensor is potentially able to perform a Dengue s virus diagnosis in a short period of time in an infected patient s blood. It uses gold nanoparticles that are functionalized with Dengue s antibodies. The antibody, anti-NS1, was immobilized in gold nanoparticles deposited at the endface of a multimode optical fiber. The LSPR sensor is able to detect different concentrations of the antigen NS1 with a limit of quantification equal to 0.074 microgram per milliliter, and may be used in the acute phase of the infection. Another part of the present work investigates the possibility of performing structural modifications in the optical fiber to change the optical properties of silica. Through the electro-thermal poling technique it is possible to record electric fields as high as 108 volts per meter in the core of the fiber, making possible the use of these modified fibers as phase and amplitude modulators, optical keys, pulse selectors, voltmeters, etc. This work also shows a very detailed study of electro-thermal poling in optical fiber through simulations, using the software Comsol Multiphysics, considering various parameters that are involved in the process in order to obtain high second order nonlinearity. Furthermore, experiments on eletro-optical poling were performed to investigate the mechanism involved in this poling process, in order to understand the role of the carriers present in the material in the generation of the second order nonlinearity.

Dans cette thèse, nous décrivons sommairement les techniques utilisées qui sont l’imagerie parrésonance plasmon de surface (SPRi) et la diffusion Raman exaltée de surface (SERS). Le butprincipal du projet Piranex dans lequel la thèse s’inscrit consiste au développement d’une biopucenanostructurée bimodale permettant le couplage des deux techniques SPRi et SERS. Cettebiopuce est constituée d’un film d’or par-dessus lequel nous avons déposé un réseau carré denanocylindres en or. Un ensemble d’études ont été effectuées pour caractériser ses propriétésplasmoniques du biocapteur afin d’en optimiser le signal SERS. Nous avons ainsi constaté quel’émission du signal était fortement anisotrope, dus à l’excitation du Mode de Bragg et que lechamp proche était principalement exalté sur les bords de la nanostructure. Les propriétés furentégalement comparées avec celles de réseaux identiques déposés directement sur un substrat diélectrique.Par la suite un ensemble d’études plasmoniques et SERS ont été effectuées pourl’aluminium, autre matériaux plasmonique d’intérêt. Enfin, un protocole de détection par SERSde l’ochratoxine basé sur un aptamère fut développé et a permis la détection de l’ochratoxine dès10 pM, bien en dessous de la limite autorisée par les organismes de régulation en agroalimentaire. / In this thesis, we briefly describe the techniques used, which are surface plasmon resonanceimaging (SPRi) and surface enhanced Raman scattering (SERS). The main goal of the Piranexproject in which the thesis is based is the development of a bimodal nanostructured biochipallowing the coupling of the two techniques SPRi and SERS. This bio-chip consists of a goldfilm over which we have deposited a square array of gold nanocylinders. A set of studies hasbeen carried out to characterize plasmonic properties of the biosensor in order to optimize theSERS signal. We have thus found that the emission of the signal was strongly anisotropic, due tothe excitation of the Bragg Mode and that the near field was mainly enhanced on the edges of thenanostructure. The properties were also compared with those of identical gratings depositeddirectly on a dielectric substrate. Subsequently a set of plasmonic and SERS studies were carriedout for aluminum, other plasmonic materials of interest. Finally, a detection protocol by SERS ofochratoxin based on an aptamer was developed and allowed the detection of ochratoxin with adetection threshold of 10 pM, well below the limit allowed by food regulatory agencies

In zahlreichen lebenswichtigen Bereichen haben sich Biosensoren als unverzichtbare Messgeräte erwiesen. Der Nachweis von spezifischen Molekülen im Körper für eine frühzeitige Krankheitserkennung erfordert empfindliche und zugleich zuverlässige Messmethoden. Ein rasantes Fortschreiten im Bereich der Nanotechnologie führt dabei zur Entwicklung von Materialien mit neuen Eigenschaften, und damit verbunden, auch zu innovativen Anwendungsmöglichkeiten im Bereich der Biosensorik. Das Zusammenspiel von Nanotechnologie und Sensortechnik gewährleistet die Konstruktion von Sensoren mit empfindlicheren Nachweisgrenzen und kürzeren Reaktionszeiten. Die Option zur Integration und Miniaturisierung stellen daher einen erfolgreichen Einsatz in direkter Patientennähe in Aussicht, sodass Nanobiosensoren die Brücke zwischen Laborddiagnostik und Standardanwendungen schließen können. Die folgende Arbeit widmet sich der Anwendung von nanostrukturierten Biosensoren für einen empfindlichen und markierungsfreien Nachweis von Zielmolekülen. Ein Hauptaugenmerk liegt dabei auf der kontinuierlichen Messung von Biomarkern mit kompakten Auslesesystemen, die eine direkte Signalmeldung und somit eine Detektion in Echtzeit ermöglichen. Dies erfordert zunächst die sorgfältige Funktionalisierung von Sensoroberflächen mit geeigneten DNA-basierten Rezeptoren. Infolgedessen werden beispielhaft verschiedene Sensorsysteme, Analyten und Charakterisierungsmethoden vorgestellt sowie universelle Strategien für die erfolgreiche Konfiguration von Nanobiosensorplattformen präsentiert. Das erste Anwendungsbeispiel widmet sich einem plasmonischen Biosensor, bei dem vertikal ausgerichtete Gold-Nanoantennen Signale mittels sog. lokalisierter Oberflächenplasmonenresonanz (LSPR) erzeugen. Mit dem Sensor konnte erfolgreich die Immobilisierung, das nachträgliche Blocken sowie die anschließende Hybridisierung von DNA nachgewiesen werden. Mithilfe des LSPR-Sensors wurden gleichzeitig grundlegende Hybridisierungsmechanismen auf nanostrukturierten und planaren Oberflächen verglichen und damit verbunden die einzigartigen optischen Eigenschaften metallischer Nanostrukturen betont. In einem zweiten Anwendungsbeispiel misst ein elektrischer Biosensor kontinuierlich die Konzentration des Stressmarkers Cortisol im menschlichen Speichel. Der direkte, markierungsfreie Nachweis von Cortisol mit Silizium-Nanodraht basierten Feldeffekttransistoren (SiNW FET) wurde anhand zugrunde liegender Ladungsverteilungen innerhalb des entstandenen Rezeptor-Analyte-Komplexes bewertet, sodass ein Nachweis des Analyten innerhalb der sog. Debye-Länge ermöglicht wird. Die erfolgreiche Strategie zur Oberflächenfunktionalisierung im Zusammenspiel mit dem Einsatz von SiNW FETs auf einem tragbaren Messgerät wurde anhand des Cortisolnachweises im Speichel belegt. Ein übereinstimmender Vergleich der gemessenen Corisolkonzentrationen mit Werten, die mit einer kommerziellen Alternative ermittelt wurden, verdeutlichen das Potential der entwickelten Plattform. Zusammenfassend veranschaulichen beide vorgestellten Nanobiosensor-Plattformen die vielseitige und vorteilhafte Leistungsfähigkeit der Systeme für einen kontinuierlichen Nachweis von Biomarkern in Echtzeit und vorzugsweise in Patientennähe.:Kurzfassung I Abstract III Abbreviations and symbols V Content VII 1 Introduction 1 1.1 Scope of the thesis 4 1.2 References 6 2 Fundamentals 9 2.1 Biosensors 9 2.2 Influence of nanotechnology on sensor development 10 2.3 Biorecognition elements 12 2.3.1 Biorecognition element: DNA 13 2.3.2 Aptamers 14 2.3.3 Immobilization of receptors 15 2.4 Transducer systems 17 2.4.1 Optical biosensors - surface plasmon resonance 17 2.4.2 Electric Biosensors – Field-effect transistors (FETs) 21 2.5 Metal oxide semiconductor field-effect transistor - MOSFET 21 2.6 Summary 26 2.7 References 27 3 Materials and methods 33 3.1 Plasmonic biosensors based on vertically aligned gold nanoantennas 33 3.1.1 Materials 33 3.1.2 Manufacturing of nanoantenna arrays 34 3.1.3 Surface modification and characterization 35 3.1.4 Measurement setup for detection of analytes 38 3.2 SiNW FET-based real-time monitoring of cortisol 40 3.2.1 Materials 40 3.2.2 Manufacturing of silicon nanowire field effect transistors (SiNW FETs) 42 3.2.3 Integration of SiNW FETs into a portable platform 42 3.2.4 Biomodification and characterization of electronic biosensors SiNW FETs 42 3.2.5 Electric characterization of FETs 47 3.3 References 50 4 Plasmonic DNA biosensor based on vertical arrays of gold nanoantennas 51 4.1 Introduction - Optical biosensors operating by means of LSPR 53 4.2 Biosensing with vertically aligned gold nanoantennas 56 4.2.1 Sensor fabrication, characterization, and integration 56 4.2.2 Integration of microfluidics 58 4.2.3 Immobilization of probe DNA and backfilling 58 4.2.4 Hybridization of complementary DNA strands 62 4.2.5 Surface coverage and hybridization efficiency of DNA 69 4.2.6 Refractive index sensing 72 4.2.7 Backfilling and blocking 73 4.3 Summary 75 4.4 References 77 5 Label-free detection of salivary cortisol with SiNW FETs 83 5.1 Introduction 85 5.2 Design, integration, and performance of SiNW FETs into a portable platform 89 5.2.1 Structure and electrical characteristics of honeycomb SiNW FETs 89 5.2.2 Integration of SiNW FET into a portable measuring unit 91 5.2.3 Performance of SiNW FET arrays 93 5.3 Detection of biomolecules with SiNW FETs 102 5.3.1 General considerations for biodetection with FETs 102 5.3.2 Sensing aptamers with FETs 103 5.3.3 Biodetection of the analyte cortisol with SiNW FETs 104 5.3.4 Detection of cortisol with SiNW FETs 112 5.4 Summary 119 5.5 References 121 6 Summary and outlook 131 6.1 Summary 131 6.2 Perspectives – toward multiplexed biosensing applications 134 6.3 References 137 Appendix i A.1 Protocols i A.1.1 Functionalization of gold antennas with thiolated DNA i A.1.2 Functionalization of SiO2 with TESPSA and amino-modified receptors i A.1.3 Functionalization with APTES and carboxyl-modified receptors ii A.1.4 Preparation of microfluidic channels via soft lithography ii A.2 Predicted secondary structures iv A.2.1 Secondary structures of 100base pair target without probe-strands iv A.2.2 Secondary structures of 100base pair target with 25 base pair probe-strand x Versicherung xvii Acknowledgments xix List of publications xxi Peer-reviewed publications xxi Publications in preparation xxi Selected international conferences xxii Curriculum Vitae xxiii / Biosensors have proven to be indispensable in numerous vital areas. For example, detecting the presence and concentration of specific biomarkers requires sensitive and reliable measurement methods. Rapid developments in the field of nanotechnology lead to nanomaterials with new properties and associated innovative applications. Thus, nanotechnology has a far-reaching impact on biosensors' development, e.g., delivery of biosensing devices with greater sensitivity, shorter response times, and precise but cost-effective sensor platforms. In addition, nanobiosensors hold high potential for integration and miniaturization and can operate directly at the point of care - serving as a bridge between diagnostics and routine tests. This work focuses on applying nanostructured biosensors for the sensitive and label-free detection of analytes. A distinct aim is the continuous monitoring of biomarkers with compact read-out systems to provide direct, valuable feedback in real-time. The first step in achieving this goal is the adequate functionalization of nanostructured sensor surfaces with suitable receptors to detect analytes of interest. Due to their thermal and chemical stability with the possibility for customizable functionalization, DNA-based receptors are selected. Thereupon, universal strategies for confining nanobiosensor platforms are presented using different sensor systems, analytes, and characterization methods. As a first application, a plasmonic biosensor based on vertically aligned gold nanoantennas tracked the immobilization, blocking, and subsequent hybridization of DNA by means of localized surface plasmon resonance (LSPR). At the same time, the LSPR sensor was used to evaluate fundamental hybridization mechanisms on nanostructured and planar surfaces, emphasizing the unique optical properties of metallic nanostructures. In a second application, an electric sensor based on silicon nanowire field-effect transistors (SiNW FET) monitored the level of the stress marker cortisol in human saliva. Based on evaluating the underlying charge distributions within the resulting receptor-analyte complex of molecules, the detection of cortisol within the Debye length is facilitated. Thus, direct, label-free detection of cortisol in human saliva using SiNW FET was successfully applied to the developed platform and compared to cortisol levels obtained using a commercial alternative. In summary, both presented platforms indicate a highly versatile and beneficial performance of nanobiosensors for continuous detection of biomarkers in real-time and preferably point-of-care (POC).:Kurzfassung I Abstract III Abbreviations and symbols V Content VII 1 Introduction 1 1.1 Scope of the thesis 4 1.2 References 6 2 Fundamentals 9 2.1 Biosensors 9 2.2 Influence of nanotechnology on sensor development 10 2.3 Biorecognition elements 12 2.3.1 Biorecognition element: DNA 13 2.3.2 Aptamers 14 2.3.3 Immobilization of receptors 15 2.4 Transducer systems 17 2.4.1 Optical biosensors - surface plasmon resonance 17 2.4.2 Electric Biosensors – Field-effect transistors (FETs) 21 2.5 Metal oxide semiconductor field-effect transistor - MOSFET 21 2.6 Summary 26 2.7 References 27 3 Materials and methods 33 3.1 Plasmonic biosensors based on vertically aligned gold nanoantennas 33 3.1.1 Materials 33 3.1.2 Manufacturing of nanoantenna arrays 34 3.1.3 Surface modification and characterization 35 3.1.4 Measurement setup for detection of analytes 38 3.2 SiNW FET-based real-time monitoring of cortisol 40 3.2.1 Materials 40 3.2.2 Manufacturing of silicon nanowire field effect transistors (SiNW FETs) 42 3.2.3 Integration of SiNW FETs into a portable platform 42 3.2.4 Biomodification and characterization of electronic biosensors SiNW FETs 42 3.2.5 Electric characterization of FETs 47 3.3 References 50 4 Plasmonic DNA biosensor based on vertical arrays of gold nanoantennas 51 4.1 Introduction - Optical biosensors operating by means of LSPR 53 4.2 Biosensing with vertically aligned gold nanoantennas 56 4.2.1 Sensor fabrication, characterization, and integration 56 4.2.2 Integration of microfluidics 58 4.2.3 Immobilization of probe DNA and backfilling 58 4.2.4 Hybridization of complementary DNA strands 62 4.2.5 Surface coverage and hybridization efficiency of DNA 69 4.2.6 Refractive index sensing 72 4.2.7 Backfilling and blocking 73 4.3 Summary 75 4.4 References 77 5 Label-free detection of salivary cortisol with SiNW FETs 83 5.1 Introduction 85 5.2 Design, integration, and performance of SiNW FETs into a portable platform 89 5.2.1 Structure and electrical characteristics of honeycomb SiNW FETs 89 5.2.2 Integration of SiNW FET into a portable measuring unit 91 5.2.3 Performance of SiNW FET arrays 93 5.3 Detection of biomolecules with SiNW FETs 102 5.3.1 General considerations for biodetection with FETs 102 5.3.2 Sensing aptamers with FETs 103 5.3.3 Biodetection of the analyte cortisol with SiNW FETs 104 5.3.4 Detection of cortisol with SiNW FETs 112 5.4 Summary 119 5.5 References 121 6 Summary and outlook 131 6.1 Summary 131 6.2 Perspectives – toward multiplexed biosensing applications 134 6.3 References 137 Appendix i A.1 Protocols i A.1.1 Functionalization of gold antennas with thiolated DNA i A.1.2 Functionalization of SiO2 with TESPSA and amino-modified receptors i A.1.3 Functionalization with APTES and carboxyl-modified receptors ii A.1.4 Preparation of microfluidic channels via soft lithography ii A.2 Predicted secondary structures iv A.2.1 Secondary structures of 100base pair target without probe-strands iv A.2.2 Secondary structures of 100base pair target with 25 base pair probe-strand x Versicherung xvii Acknowledgments xix List of publications xxi Peer-reviewed publications xxi Publications in preparation xxi Selected international conferences xxii Curriculum Vitae xxiii

Localized surface plasmon resonance (LSPR) describes the collective oscillation of conductive electrons in noble metal nanostructures, such as gold, silver and copper; or in selected doped semiconductor nanocrystals. Nanoplasmonics is emerging as a useful and versatile platform that combines the intense and highly tunable optical responses derived from LSPR with the intriguing materials properties at the nanoscale, including high specific surface areas, surface and chemical reactivity, binding affinity, and rigidity. LSPRs in plasmonic nanoparticles (NPs) can provide large optical cross-sections, and can lead to a wide variety of subsequent photophysical responses, such as localization of electric (E-)fields, production of plasmonic hot charge carriers, photothermal heating, etc. Plasmonic NPs can also be combined with other molecular or nanoscale systems into plasmonic heterostructures to further harvest the resonant E-field enhancement or to prolong the lifetime of plasmonic charge carriers. In this dissertation, we study the photophysical properties of plasmonic Ag and Au NPs, particularly E-field localization and hot charge carrier production performances; and illustrate how they can be optimized towards plasmonic photocatalysis, development of nano-antimicrobials, and surface-enhanced Raman spectroscopy (SERS) sensing. We demonstrate that with a lipid-coated noble metal nanoparticle (L-NP) model, the E-field localization properties could be optimized through positioning molecular photosensitizers or photocatalysts within a plasmonic “sweet spot”. This factor renders the plasmonic metal NPs efficient nanoantenna for resonant enhancement of the intramolecular transitions as well as the photocatalytic properties of the molecular photocatalysts. The enhanced photoreactivity have been applied to facilitate fuel cell half reactions for the enhancement of light energy conversion efficiencies; as well as to facilitate the release of broad-band bactericidal compounds that enables plasmonic nano-antimicrobials. Localized E-fields in L-NPs also enhance the inelastic scattering from molecules through SERS. This is utilized to obtain molecular-level information on the configuration of sterol-based, alkyne-containing Raman tags in hybrid lipid membranes, which enables spectroscopic sensing and targeted imaging of biomarker-overexpressing cancer cells at the single-cell level. In contrast to the localized E-field, plasmonic charge carrier generation mechanism relies on non-radiative decay pathways of the excited plasmons that lead to production of ballistic charge carriers. The plasmonic hot charge carriers directly participate in chemical redox processes with degrees of controllability over the nature of the charge carrier produced and direction of their transfers. The implementation and optimization of these mechanisms are explored, and the significances of some relevant applications are discussed.

Anisotropic plasmonic noble metallic nanoparticles (APMNs) have received enormous attention due to their distinct geometric features and fascinating physicochemical properties. Owing in large part to their tailored localized surface plasmon resonance (LSPR) and the intensive electromagnetic field at the sharp corners and edges, APMNs are exceptionally well suited for biomedical applications such as biosensing, bioimaging, diagnostics and therapeutics. Although a rich variety of surfactant-assisted colloidal routes have been developed to prepare well-defined APMNs, biomedical applications necessitate tedious and rigorous purification processes for the complete removal of toxic surfactants. In this dissertation, we aim to develop generic bioenabled green synthetic methodologies towards APMNs. By applying a series of thermodynamic, kinetic and seed quality control, a series of APMNs with varied morphologies such as branched nanostars and triangular nanoprisms have been successfully prepared. We first presented the preparation of gold nanostars (Au NSTs) through a two-step approach utilizing a common Good's buffer, HEPES, as a weak reducing agent. Single crystalline Au NSTs with tunable branches up to 30 nm in length were produced and the halide ions rather than the ionic strength played a significant roles on the length of the branches of Au NSTs. Then consensus sequence tetratricopetide repeat (CTPR) proteins with increasing number of repeats were used as model proteins to probe the effects of concentration as well as the protein shape on the morphology and resulting physicochemical properties of plasmonic gold nanoparticles. Since the underlying growth mechanism for the biomimetic synthesis of APMNs remains elusive and controversial, the other objective is to elucidate the molecular interactions between inorganic species and biopolymers during the course of NP evolution. Fluorescent quenching and 2D NMR experiments have confirmed the moderate binding affinity of CTPR to the Au(0) and Au(III). We observed that the initial complexation step between gold ions and CTPR3 is ionic strength dependent. Furthermore, we also found that NPs preferentially interact with the negatively charged face of CTPR3 as observed in 2D NMR. Knowledge of binding behavior between biospecies and metal ions/NPs will facilitate rational deign of proteins for biomimetic synthesis of metallic NPs. A modified seed-mediated synthetic strategy was also developed for the growth of silver nanoprisms with low shape polydispersity, narrow size distribution and tailored plasmonic absorbance. During the seed nucleation step, CTPR proteins are utilized as potent stabilizers to facilitate the formation of planar-twinned Ag seeds. Ag nanoprisms were produced in high yield in a growth solution containing ascorbic acid and CTPR-stabilized Ag seeds. From the time-course UV-Vis and transmission electron microscopy (TEM) studies, we postulate that the growth mechanism is the combination of facet selective lateral growth and thermodynamically driven Ostwald ripening. By incorporation of seeded growth and biomimetic synthesis, gold nanotriangles (Au NTs) with tunable edge length were synthesized via a green chemical route in the presence of the designed CTPR protein, halide anions (Br⁻) and CTPR-stabilized Ag seeds. The well-defined morphologies, tailored plasmonic absorbance from visible-light to the near infrared (NIR) region, colloidal stability and biocompatibility are attributed to the synergistic action of CTPR, halide ions, and CTPR-stabilized Ag seeds. We also ascertained that a vast array of biosustainable materials including negatively charged lignin and cellulose derivatives can serve as both a potent stabilizers and an efficient nanocrystal modifiers to regulate the growth of well-defined Ag nanoprisms using a one-pot or seeded growth strategy. The influential effects of reactants and additives including the concentration of sodium lignosulfonate, H2O2 and NaBH4 were studied in great detail. It implies that appropriate physicochemical properties rather than the specific binding sequence of biomaterials are critical for the shaped-controlled growth of Ag NTs and new synthetic paradigms could be proposed based on these findings. Last but not the least, we have demonstrated the resulting APMNs, particularly, Au NSTs and Ag NTs exhibit remarkable colloidal stability, enhanced SERS performance, making them promising materials for biosensing and photothermal therapy. Since the Ag nanoprisms are susceptible to morphological deformation in the presence of strong oxidant, they also hold great potential for the colorimetric sensing of oxidative metal cation species such as Fe3+, Cr3+, etc. / Ph. D.