Dual-Functional Ag@SiO2 Nanocubes for Biomarker Detection - Nguyen Minh Kha (NTUST)

National Taiwan University of Science and Technology Department of Chemical Engineering Ph. Dissertation Student ID: D10206814 Orientated Ag@SiO2 Core-shell Nanocubes as Dual-functional Plasmonic Substrates for Biomarker Detection Graduate Student : Nguyen Minh Kha Advisor : Prof. Bing-Joe Hwang December, 2016 Abstract The progress in fundamental biological studies of disease has been revealing a variety of new biomarkers that are difficult to be detected by traditional diagnostic tools. Interestingly, plasmonics is an ultrasensitive optical technology applicable in medical diagnostics and biological imaging sensors.

A dual-functional plasmonic substrate with a unique design that allows both the sensitive detection of photoluminescence (PL) via metal-enhanced photoluminescence (MEPL) and the specific Raman fingerprint via surface-enhanced Raman scattering (SERS) is highly desirable to improve accuracy and sensitivity in detection. This dissertation describes the role of coupling agents, spacer effects, and importance characters of various platforms for the optimum bifunctional SERS-MEPL based on Ag@SiO2 core-shell nanocube(s) [NC(s)] for urinary biomarkers detection. In this work, the Ag NCs were synthesized by the polyol method and modified, firstly with different coupling agents, such as 3-mercaptopropyltrimethoxysilane (MPTMS) and 3-aminopropyltrimethoxysilane (APTMS), and secondly with tetraethylorthosilicate (TEOS). The presence of coupling agents greatly modified the Ag NC cores and manipulated the thickness and uniformity of the silica shells.

Ultrathin silica-coated Ag NCs (with a ~1.5 nm silica layer) found to have a SERS intensity 3 fold higher than synthesized Ag NCs. In comparison with MPTMS, it was found that APTMS modified Ag@SiO2 NCs improved significant SERS and MEPL enhancements. Moreover, a ‘dual functionality’ represented by the simultaneous strengthening of SERS and MEPL signals can be achieved by mixing Ag@SiO2 NCs, with a silica shell thickness of ~1. This approach allows both the Ag@SiO2 NCs SERS and MEPL sensitivities to be maintained at ~90% after 12 weeks of storage.

Additionally, it is known that interactions between substrate and plasmonic nanostructures can influence the performance of plasmonic biosensing. Therefore, in the final i approach, a substrate characterized with low refractive index and roughness was first fabricated by creating flower-like alumina on etched Al foil (f-Al2O3/e-Al). The Ag@SiO2 NCs assemble in the edge-edge configuration when they were deposited on this substrate. It is to note that the surface roughness of f-Al2O3/e-Al provides a pathway for the coupling of incident light to surface plasmon.

The Ag@SiO2/f-Al2O3/e-Al substrate exhibits a coupling efficiency of laser light sources into surface plasmon hotspots for both SERS and MEPL. Moreover, the shelf life of this substrate is significantly improved due to reduced oxygen diffusion rate by ultrathin silica spacer and flower-like Al2O3 dielectric layer. Creatinine (CR) and flavin adenine dinucleotide (FAD) are biomolecules present in human blood and urine. With the advanced label-free SERS and MEPL techniques, these biomarkers in urine are detected, and it allows cheap, non-invasive and yet sensitive analysis.

The approaches explored in this dissertation could be developed as a powerful encoding tool for high-throughput bio-analysis. Keywords: SERS; photoluminescence; Ag@SiO2 nanocubes; ultrathin shell; flower-like alumina; plasmonic coupling; edge-edge orientation; stability; dual functionality; Rhodamine 6G; urine; biomarker detection. ii Acknowledgement First and foremost, I would like to express my deep gratitude to my advisor, Prof. Bing- Joe Hwang, for giving me an opportunity to work in his group, and exploring the wonderful plasmonic nanoscale world.

He has taught me not only knowledge in research but also behavior in a new environment. The completion of this dissertation cannot be possible without his intellectual support. I could not imagine having a better advisor and mentor for my Ph. I would like to thank my sincere gratitude to all members at the Nano-electrochemistry Laboratory, Department of Chemical Engineering, National Taiwan University of Science and Technology (NTUST), who assisted me not only my study but also on many occasions.

Special thanks to Prof. Wei-Nien Su who essentially inspired me to do my best in performing my experiment as well as in writing papers. Thank you for your appreciated support, valuable encouragement and considerable recommendations. Additionally, I wish to express my appreciation to Dr.

Ching-Hsiang Chen, Dr. Chun-Jern Pan, Dr. John Rick, Dr. Meng-Che Tsai, and Dr.

Alok Mani Tripathi for giving their time and effort in evaluating my work. I have benefited from their constructive comments on my report. My sincere thanks also goes to Prof. Hongjie Dai at Stanford University for supporting and suggesting the work to improve my knowledge in the field of biochemistry.

I am thankful to the Department of Chemical Engineering and Department of Materials Science and Engineering at NTUST for their help and research facilities. Also, many thanks to the Faculty of Chemical Engineering of Ho Chi Minh University of Technology (HCMUT), Vietnam, and Office of International Affairs at NTUST for all the best things that they have offered me during my study and research. I would like to acknowledge National Taiwan University of Science and Technology for giving me the chance to improve my knowledge in the graduate school and the scholarship iii during my Ph. Also, gratefully acknowledge financial support from the Ministry of Science and Technology (MOST), and the Top University Projects of Ministry of Education (MOE).

It is my pleasure to thanks my friends at Vietnamese Student Association who made my study and stay at NTUST more colorful. Last but not least, I would like to thank my family with love and gratitude for supporting me spiritually throughout studying Ph. and my life in general. iv Table of Contents Abstract.

iii Table of Contents. v List of Figures. ix List of Tables .xvii List of Acronyms .1 Overview about plasmonic technologies.2 Statement of the problem .3 Objective of the study .4 Significance of the study .5 Structure of the dissertation.1 A brief overview of SERS and MEPL .1 Light scattering and optical properties of metals .2 Normal Raman and surface-enhanced Raman scattering .3 Photoluminescence and metal-enhanced photoluminescence .2 The major concerns related to SERS and MEPL enhancements .1 Type of metal .4 Interacting objects, gaps, and coupled plasmon resonances .3 The research on SERS-MEPL bifunctional mode.1 Solutions for the integrated SERS and MEPL .2 Applications of SERS and MEPL .4 Controlling the fabrication of nanostructures for plasmonic applications .1 General nanofabrication techniques .2 Preparation of Ag nanostructures.3 Surface coating for protection of nanostructures. Materials and Methods.2 Fabrication of the plasmonic substrates .1 Synthesis of Ag NCs .2 Synthesis of Ag@SiO2 core-shell NCs .3 Fabrication of Ag@SiO2/f-Al2O3/e-Al and Ag@SiO2/Al2O3/Al substrates .4 Preparation of artificial urine .5 Preparation of SERS and MEPL measurements .3 Characterization and measurements .2 SERS and MEPL measurements .3 Calculation enhancement factor of the substrate.

SERS and MEPL of Ag@SiO2 Nanocubes with Ultrathin Silica Shells .2 Results and discussion.1 Synthesis of Ag NCs .2 Ultra-coating silica shell for Ag NCs cores .3 The SERS and MEPL of the Ag@SiO2 NCs vs. Sensitivity and Stability of Bifunctional SERS-MEPL from the Mixture of Ag@SiO2 Nanocubes .2 Results and Discussion .1 Characterization of the Ag NCs and Ag@SiO2 NCs .2 Bifunctional SERS-MEPL activity of Ag@SiO2 NCs .3 The stability for dual SERS-MEPL activity .4 Direct label-free SERS-MEPL detection of the mixture of creatinine and flavin adenine dinucleotide. A Plasmonic Coupling Substrate Based on Ag@SiO2/f-Al2O3/e-Al for Sensitive Detection of Biomarkers in Urine .2 Results and discussion.1 Fabrication of the substrates and their characterization .2 SERS and MEPL activities of the substrates .3 Urinary biomarkers detection. Conclusion and Future Perspectives.

123 vii List of Figures Figure 1.1 Models of healthcare delivery in chronic disease management.2 The outcomes from the diagnostic process.3 Plasmonic-based technologies for versatile biosensor applications.4 A schematic illustration of the averaged EFs obtained in the visible region from different types of SERS substrates: Au, Ag and Cu, transition metals, and core-shell (transition metals @ noble metals) NPs.5 Poynting vector or energy flow (lines) around a subwavelength metallic colloid illuminated at the plasmon wavelength (left) and at a wavelength longer than the plasmon wavelength (right). The vertical lines on the left indicate the diameter of the cross sections for absorption.1 Schematic diagram representing the SPP at a metal dielectric interface.2 Schematic diagram representing the LSP.[20] The equilibrium distribution of electron cloud in a metal NP is modified in the presence of an external E-field.3 Coupled surface plasmon modes (a) propagating along a thin metallic film and (b) formed by the Bragg scattering on periodically modulated surface.4 The optical path schematic of a Raman system.5 Energy-level diagram for Rayleigh, Raman and resonance-Raman scattering. S0 and S1 are the singlet electronic ground and first excited states.6 Raman peak shift ranges for organic bonds.7 Jablonski energy-level diagram for absorption and PL. Quenching represents a non- radiative decay process.8 Free-space emission.9 Simplified Jablonski diagram for a fluorophore in absence of (top) and in presence of (bottom) a metal.10 Plasmon controlled emission.11 Schematic representation of the relative intensities of fluorescence and SERS for molecules at different distances from a metal surface.12 A simplified periodic table of the elements.

The part marked in black are ‘free- electron metals’ and those marked in grey are transition metals.13 Plot of the (A) real, εr , and (B) imaginary, εi, components of the dielectric function of Ag, Au, and Si as a function of wavelength.14 Dielectric constants of Ag and Pt.15 Quality factor of the LSPR for a metal/air interface. The shaded area represents the region of interest for many plasmonic applications.16 Extinction (black), absorption (red), and scattering (blue) spectra calculated for Ag nanoparticles of different shapes: (A) a sphere, (B) a cube, (C) a tetrahedron, and (D) an octahedron.17 (a) Gap width dependence of the SERS intensity normalized by extinction values at the incident laser wavelength and Raman scattering wavelength; and (b)-(d) The gap width dependence of the averaged enhancement factor by multiplying FDTD intensity maps monitored at the incident laser wavelength by the map monitored at the Raman scattering wavelength.18 The synthesis procedures of AuNRs functionalized for both SERS and MEPL. DSPE = 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, PEG = poly(ethylene glycol), and OTMS = octadecyltrimethoxysilane.19 MEPL, SERS and bright field images of the precipitates acquired from: (A)-(C) the goat anti-human IgG immobilized nanoprobes and magnetic nanobeads (MBs) x mixed with human, mouse or bovine IgG molecules; and (B)-(D) the goat anti- mouse IgG immobilized nanoprobes and MBs mixed with mouse, human or bovine IgG molecules.20 Schematic illustration of real-time multiplexed imaging using the FRES.[142] (A) The mode of dual modal detection with fluorescence and Raman scattering. (B) Illustration of the in vivo multiplexed molecular imaging procedure.22 Polyol method for synthesizing Ag nanostructures.

In (A) the reduction of Ag+ ions by ethylene glycol leads to the formation of nuclei that are highly volatile. These seeds are then grown into different nanostructures like (B) spheres, (C) cubes, (D) truncated cubes, (E) right bipryamids, (F) bars, (G) spheroids, (H) triangular plates, and (I) wires.23 Strategies for surface-coating SERS tags include coating with (A) denatured BSA, (B) SH-PEG, (C) amphiphilic diblock copolymer, (D) liposome, and (E-H) silica shells via different encapsulation routes.1 Schematic illustration of Ag NCs synthesis.2 Schematic illustration of Ag@SiO2 core-shell nanocube preparation.3 Schematic diagram for the preparation and chemical structures of the Ag@SiO2/f- Al2O3/e-Al substrate.1 Characterizations of Ag NCs with different reaction times. (A)-(D) SEM images; (E) Uv-vis spectra; and (F) Linear relationship of maximum wavelength absorbance and edge length of Ag NCs.2 Raman spectra with the excitation laser 532 nm of: (A) silicon wafer and 0.1 M R6G on a silicon substrate (10 times); (B) SERS intensity and (C) SERS EF of Ag NCs from various reaction times (excitation laser 532 nm). (B) XRD pattern of Ag NCs – CB 72R mixture.4 Calibration curve of standard Ag+ solution.5 UV-Vis spectra of: (a) Ag NCs and Ag@SiO2 NCs prepared by adding different chemicals ((b) 1mM MPTMS and 1mM TEOS, (c) 1mM TEOS, (d) 1mM MPTMS, and (e) 1mM APTMS and 1mM TEOS, respectively).

The inset is the UV-Vis spectra, expanded in the range 454 nm to 466 nm.6 TEM images of Ag@SiO2 NCs prepared by adding different chemicals: (A) 1mM MPTMS and 1mM TEOS, (B) 1mM TEOS, (C) 1mM MPTMS, and (D) 1mM APTMS and 1mM TEOS, respectively.