Plasmonic Interferometry: A New Approach toward Highly-Sensitive Multispectral Biochemical Detection by Jing Feng B. Sc., Nanjing University; Nanjing, China, 2007 M. Sc., Nanjing University; Nanjing, China, 2010 A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in School of Engineering at Brown University PROVIDENCE, RHODE ISLAND May 2017 c Copyright 2017 by Jing Feng This dissertation by Jing Feng is accepted in its present form by School of Engineering as satisfying the dissertation requirement for the degree of Doctor of Philosophy. Date Domenico Pacifici, Ph.D., Advisor Recommended to the Graduate Council Date G. Tayhas R. Palmore, Ph.D., Reader Date Anubhav Tripathi, Ph.D., Reader Approved by the Graduate Council Date Andrew G. Campbell, Dean of the Graduate School iii Vitae Jing Feng was born on March 27th, 1985 in Rudong, Jiangsu, China. She entered Nanjing University in 2003, and received her Bachelor of Science and Master of Science in Materials Science and Engineering in 2007 and 2010, respectively. She continued her graduate studies at Brown University, with a focus on the applications of plasmonic interferometry. In the mean time, she completed a Master of Science in Innovation Management and Entrepreneurship in 2016. She is currently a candidate for the degree of Doctor of Philosophy in Engineering, which will be awarded at the May 2017 commencement. iv Publication List Portions of this thesis have been drawn from the following publications: Jing Feng, Dongfang Li, and Domenico Pacifici. Circular slit-groove plasmonic interfer- ometers: a generalized approach to high-throughput biochemical sensing [invited]. Optical Materials Express, 5(12):2742–2753, 2015. Jing Feng and Domenico Pacifici. A spectroscopic refractometer based on plasmonic interferometry. Journal of Applied Physics, 119(8):083104, 2016. Jing Feng, Vince S Siu, Alec Roelke, Vihang Mehta, Steve Y Rhieu, G Tayhas R Pal- more, and Domenico Pacifici. Nanoscale plasmonic interferometers for multispectral, high-throughput biochemical sensing. Nano letters, 12(2):602–609, 2012. Dongfang Li, Jing Feng, and Domenico Pacifici. Nanoscale optical interferometry with incoherent light. Scientific reports, 6, 2016. Vince S Siu, Jing Feng, Patrick W Flanigan, G Tayhas R Palmore, and Domenico Pacifici. A “plasmonic cuvette”: dye chemistry coupled to plasmonic interferometry for glucose sensing. Nanophotonics, 3(3):125–140, 2014. v Acknowledgements First and foremost, I would like to thank my advisor, Professor Domenico Pacifici, for his continuous support in these six years. His enthusiasm for research has kept me motivated all the time; his confidence in the projects has encouraged me to move forward through trials and errors; his pursuit for perfection has always pushed me to strive for better outcomings. He demonstrates himself as a good mentor, by providing valuable feedback, asking inspiring questions and offering constructive criticism. As a graduate student, I am lucky to work with such an intelligent, optimistic and compassionate advisor. Besides, I would like to thank Professor G. Tayhas R. Palmore, who showed me to see the problems in a different perspective. I also want to thank Professor Anubhav Tripathi for serving on my dissertation committee and providing suggestions on experiment design. I really appreciate the help from Professor Rashid Zia, who offered valuable advices for my researches. I am grateful for the help of Michael Jibitsky, Anthony McCormick and Charles Vickers, who take good care of the clean room, the focused ion beam system and the machine shop respectively. I also appreciate the work of Chantee Weah, Richard Minogue, Sandra Van Wagoner and John Lee, without whom I could not have the needed parts for experiments in time. A special thank you to Dr. Vince S. Siu for being my close friend and col- vi laborator since I came to Brown, working day and night on the same project and manuscripts. Many thanks to the former and current Pacifici Research Group mem- bers: Dr. Dongfang Li, Dr. Pei Liu, Dr. Patrick Flanigan, Dr. Salvo Consentino, Stylianos Siontas, Tianyi Shen, Aminy Ostfeld, Alec Roelke, Vihang Mehta, Kaan Gunay, Abigail Plummer, and Drew Morrill for fruitful discussion and fun time to- gether. Also, thank you to other Palmore Research Group members: Dr. Hyewon Kim, Dr. Steve Rhieu, Dr. Kwang-min Kim, Dr. Sujat Sen, Steven Ahn and Dan Liu, and Zaslavsky Research Group members: Dr. Son Le, Dr. Peng Zhang, and Yang Song, for the help and convenience provided for the experiments. Thank you to all my family and friends. Thanks to my parents’ support, I can come to the United States and pursue this degree oversea. I am also thankful to my husband, who travelled to U.S. with me six years ago. Without his sacrifices and company, I cannot make it today. Especially, I appreciate my sunshine, my little daughter Qiuning, who “makes me happy when skies are grey”. In addition, I am grateful to the friends I made here, Dr. Xinjun Guo, Dr. Mingming Jiang, Xiaoxiao Hou and so on, for the assistance both in research and life. vii Abstract of “Plasmonic Interferometry: A New Approach toward Highly-Sensitive Multispectral Biochemical Detection” by Jing Feng, Ph.D., Brown University, May 2017 Surface plasmon polaritons are hybrid electron-photon modes confined at a metal/dielectric interface. The combination of highly localized electronic waves and propagating opti- cal waves gives the surface plasmon wave unique characteristics, which have attracted growing interest over the past decades and started the research field of “plasmonics”. A sub-field in plasmonics, named “plasmonic interferometry”, which focuses on the interference effects of surface plasmon waves, is hereby presented. Specifically, in this thesis, I discuss the design of plasmonic interferometers for biochemical sensing, utilizing the unique features of surface plasmons. A multispec- tral plasmonic refractometer and a highly-sensitive biochemical detector are built upon plasmonic interferometry, taking advantage of its susceptibility to optical prop- erties of the interface materials. As a proof of concept, the detector is capable of sensing glucose concentrations down to the physiological levels in saliva. Its sensitiv- ity and specificity is further enhanced when coupled to a dye chemistry. I also explore an alternative scheme to achieve detection selectivity by functionalizing the device surface with specific antibodies. In addition, plasmonic interferometry in some other geometries are investigated, e.g., a generalized circular geometry. Finally, with light emitters directly embedded in the subwavelength cavities of plasmonic interferom- eters, active plasmonic interferometry is accomplished, which successfully replaces the previous rigid requirements for a coherent light source. Accordingly, plasmonic interferometry has the great potential for the development of a new generation of mul- tiplexed biochemical sensors that are compact, multi-spectral and ultra-sensitive. Contents Vitae iv Publication List v Acknowledgments vi 1 Introduction 1 1.1 Introduction to surface plasmon polaritons . . . . . . . . . . . . . . . 2 1.2 Overview of this thesis . . . . . . . . . . . . . . . . . . . . . . . . . . 4 2 A spectroscopic refractometer based on plasmonic interferometry 7 2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 2.2 Sample fabrication and experimental setup . . . . . . . . . . . . . . . 9 2.3 Wavelength resolved plasmonic interferogram . . . . . . . . . . . . . . 13 2.4 Plasmonic refractometry . . . . . . . . . . . . . . . . . . . . . . . . . 17 2.5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 3 Plasmonic interferometry for biochemical sensing 24 3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 3.2 Plasmonic interferometry for glucose detection . . . . . . . . . . . . . 26 3.3 Specific glucose sensing achieved by dye chemistry . . . . . . . . . . . 33 3.4 C-reactive protein binding reaction studied by functionalized plas- monic interferometry . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 3.5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 4 Plasmonic interferometers in circular geometry 46 4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 4.2 Circular plasmonic interferometers . . . . . . . . . . . . . . . . . . . 48 4.3 Optical properties of circular plasmonic interferometers . . . . . . . . 50 4.4 Sensing performance comparison . . . . . . . . . . . . . . . . . . . . . 57 4.5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 viii 5 Active plasmonic interferometry with incoherent light 65 5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66 5.2 Fabrication and experimental setup . . . . . . . . . . . . . . . . . . . 68 5.3 Comparison of passive and active plasmonic interferometry . . . . . . 72 5.4 Plasmonic interferometry under different illumination conditions . . . 77 5.5 Biochemical sensing based on active plasmonic interferometry . . . . 81 5.6 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85 6 Conclusion 87 ix List of Tables x List of Figures 1.1 Basics for surface plasmons. . . . . . . . . . . . . . . . . . . . . . . . 2 1.2 Schemes for different SPP excitation methods. . . . . . . . . . . . . . 3 2.1 Plasmonic refractometry. . . . . . . . . . . . . . . . . . . . . . . . . . 10 2.2 Experimental setup. . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 2.3 Normalized transmission spectra (experimental). . . . . . . . . . . . . 14 2.4 Wavelength-resolved plasmonic interferograms. . . . . . . . . . . . . . 16 2.5 Fits of plasmonic interferograms at various wavelengths. . . . . . . . 18 2.6 Plasmonic refractometry: retrieving the optical functions of dielectric materials. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 2.7 SPP excitation amplitude and phase determined by plasmonic inter- ferometry. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 3.1 Representative schematic of a groove-slit-groove plasmonic interfer- ometer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 3.2 Simulated normalized transmission spectra for SG and GSG plasmonic interferomters on a Ag/water interface. . . . . . . . . . . . . . . . . . 29 3.3 Sensing performance of a GSG plasmonic interferometer for the de- tection of glucose in water. . . . . . . . . . . . . . . . . . . . . . . . . 30 3.4 The calibration curve in terms of relative intensity change at λ = 590 nm for a GSG plasmonic interferometer with longer arm lengths. . . . . . 32 3.5 Reactions in an Amplex-red/Glucose-oxidase/Glucose assay. . . . . . 34 3.6 Relative intensity change spectra of a GSG plasmonic interferometer for glucose detection with the dye assay. . . . . . . . . . . . . . . . . 35 3.7 Improved sensitivity for glucose detection with the dye assay. . . . . . 36 3.8 Specificity of glucose detection with the dye assay. . . . . . . . . . . . 37 3.9 Schematic of PrA coated gold surface for site-oriented immobilization of CRP on the surface of a plasmonic interferometer. . . . . . . . . . 39 3.10 The normalized intensity spectra of a plasmonic interferometer after different chemical treatments. . . . . . . . . . . . . . . . . . . . . . . 41 3.11 The relative intensity change at different time steps during CRP binding. 43 4.1 Definition of a circular plasmonic interferometer. . . . . . . . . . . . . 49 4.2 Wavelength resolved plasmonic interferograms for different geometries. 51 4.3 Transmission spectra through single slits. . . . . . . . . . . . . . . . . 56 4.4 Wavelength resolved plasmonic interferograms at different interfaces. 58 4.5 Plasmonic interferogram at λ = 718 nm. . . . . . . . . . . . . . . . . 59 xi 4.6 Figure of merit comparison. . . . . . . . . . . . . . . . . . . . . . . . 61 4.7 The effect of polarization on light transmission. . . . . . . . . . . . . 62 5.1 Comparison of hole-groove plasmonic interferometer in schematics and spectra. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 5.2 Wavelength resolved passive and active transmission interferograms for bullseye structures with different number of grooves. . . . . . . . . 73 5.3 Passive and active transmission interferograms for bullseye structures with different number of grooves at a fixed wavelength 720 nm. . . . . 74 5.4 Fourier transform of the passive and active transmission interferograms. 75 5.5 Comparison of passive and active plasmonic interferometers under bottom illumination. . . . . . . . . . . . . . . . . . . . . . . . . . . . 77 5.6 Fluorescence modulation induced by plasmonic interferometry. . . . . 79 5.7 Biochemical sensing experiment in a microfluidic channel. . . . . . . . 82 5.8 Biochemical sensing experiment on a micro-droplet . . . . . . . . . . 84 xii Chapter One Introduction 2 1.1 Introduction to surface plasmon polaritons Surface plasmon polaritons (SPPs) are electromagnetic waves coupled to electron oscillations occurring at a metal/dielectric interface [1]. As shown in Figure 1.1(a), SPPs at the metal/dielectric interface have a combination of electromagnetic wave and surface charges, forming charge-compression waves propagating along the in- terface. Thus the field component perpendicular to the surface (Ez ) is enhanced locally, with its maximum at the metal/dielectric interface, while decaying exponen- tially away from the surface, as depicted in Figure 1.1(b). δd and δm represent the skin depths at which the field falls to 1/e in the dielectric and metal, respectively. Figure 1.1: Basics for surface plasmons, adapted from W. L. Barnes et al., Reference [2]. (a) The electromagnetic field of SPPs propagates along the metal/dielectric interface. (b) The exponential dependence of the field in perpendicular direction (Ez ), indicating the evanescent nature of SPPs. (c) The dispersion relation of a SPP shows the momentum mismatch problem. Figure 1.1(c) plots the dispersion curve for a SPP wave, represented by a solid line, which lies on the right side of the light line (dashed line). At a given frequency ω, the momentum of the SPP wave (~kSPP ) is larger than that of a free space photon (~k0 ), giving rise to the non-radiative nature of SPPs. Solving Maxwell’s equations yields the dispersion relation for SPP waves: r m d kSPP = k0 , (1.1) m + d where m and d are the frequency-dependent dielectric functions of the metal and 3 the dielectric material. m and d must have opposite signs for the existence of SPPs, in other words, SPPs can only be supported by a metal/dielectric interface. Figure 1.2: Schemes for different SPP excitation methods. (a) The Kretschmann config- uration for SPP excitation. (b) A grating assists couple the incident beam into SPPs. Due to the momentum mismatch, photons can not be directly coupled to SPPs. Traditionally, there are two approaches to bridge the missing momentum. Figure 1.2(a) is a scheme of the Kretschmann configuration [3], where a metal film is de- posited on a prism with high refractive index. Light incident from the prism can excite SPPs on the metal/air interface, satisfying the relation: r m air np k0 sin θc = k0 , (1.2) m + air where np is the refractive index of the prism; θc is the incident angle; and air is the dielectric constant of air. The other approach is to use gratings (Figure 1.2(b)). The grating constant provides the missing momentum, such that: kSPP = nd k0 sin θ ± mG, (1.3) 4 where nd is the refractive index of the incident plane; θ is the incident angle, which is 0 in the figure; m is an integer; and G = 2π/p is the grating constant, with p being the grating period. Recent research shows that subwavelength structures can excite SPPs successfully [4–6], which provides a more convenient way to generate SPPs. SPPs have been attracting extensive interest over tens of years due to its unique features, evidenced by the fact that the number of papers in the field of plasomonics has doubled every five years since 1990 [7]. Plasmonics has been applied in various circumstances, such as metamaterials for perfect lens [8], photovoltaic absorbers [9], nanoscale waveguiding [10], biochemical sensing [11], to name a few. 1.2 Overview of this thesis This thesis describes the design and experimental realization of various devices based on plasmonic interferometry, a sub-field of plasmonics. A plasmonic interferometer is etched in a metal film, with a through slit flanked by several nano-grooves, whose arm length is in the micrometer scale. The grooves are able to generate propagating SPPs efficiently over a broad wavelength range, without requiring prism- or grating- coupling configurations. At the slit location, the incident beam and the SPP waves from the grooves interfere with each other, and the light transmitted through the slit is the result of the interference process, which carries information about the optical properties of the interface materials. Specifically, this thesis is structured as follows: In Chapter 2, a spectroscopic refractometer that employs plasmonic interfer- ometry to measure the material dispersion relation (wavelength dependence of the 5 refractive index) in the visible range is described. The “plasmonic refractometer ” consists of an array of linear slit-groove plasmonic interferometers with arm lengths varying in steps of 25 nm up to ∼8 µm. A model considering the interference of the incident beam and the propagating SPP wave from the adjacent groove is in- troduced. At each wavelength, by fitting the normalized intensity as a function of arm length, which is the normalization of the light intensity transmitted through a slit-groove plasmonic interferometer by that of a single slit, the refractive index of a given material can be determined. Repeating the fits over a wide wavelength range gives the dispersion relation of this material. As proof of concept, the dispersion re- lations of representative materials are reported, such as silver, gold, water, methanol, and ethanol. In Chapter 3, the development of a novel multispectral biochemical sensor based on plasmonic interferometry is reported. An optimized linear groove-slit-groove plas- monic interferometer is capable of detecting glucose concentrations down to physio- logical levels in saliva, benefiting from the unique “three-beam interference” process, the interference between the incident beam and the two propagating SPP waves from the two adjacent grooves. Coupled to an Amplex-Red dye assay, the sensitivity and selectivity of the proposed glucose detector is further enhanced. In addition, surface immobilization, a different method to achieve sensing specificity, enables the kinetics study of the binding interaction between C-reactive protein and Immunoglobulin G using a plasmonic interferometer. The results suggest that by appropriately choosing the approach for sensing specificity, a plasmonic interferometer can be tuned into a general detector for any biochemical analyte with high sensitivity and selectivity. In Chapter 4, a class of plasmonic interferometers consisting of a circular slit flanked by a concentric circular groove is demonstrated. The circular plasmonic in- terferometry represents a generalized geometry, embracing the previously discussed 6 linear plasmonic interferometry as an extreme case, i.e., when the radiuses of the slit extends to ∞. These circular slit-groove interferometers show a polarization- insensitive optical response, and overall higher light transmission. The circular plas- monic interferometry can be employed to develop improved biochemical sensors. In Chapter 5, active plasmonic interferometry by directly embedding light emit- ters into slits of linear plasmonic interferometers is comprehensively investigated. A systematic comparison between passive and active plasmonic interferometry is presented. Coherent generation of surface plasmons in the active scheme is demon- strated, even when light with extremely low degrees of spatial and temporal coher- ence is employed. This feature enables novel sensor designs with cheaper and smaller light sources, and consequently increases accessibility to a variety of analytes. Fur- thermore, these nanosensors can now be arranged along open detection surfaces, and in dense arrays, accelerating the rate of parallel target screening used in drug discovery, among other high volume and high sensitivity applications. In Chapter 6, I summarize the work on plasmonic interferometry for sensing applications and suggest some potential directions based on current results. Chapter Two A spectroscopic refractometer based on plasmonic interferometry 8 2.1 Introduction The optical dielectric function (λ) of a material is typically determined by mea- suring changes in the propagation direction (prism-based refractometers), or in the polarization or complex amplitude (ellipsometry and interferometry) of the incident light upon interaction (i.e. refraction or reflection) with the material. For instance, prism-based refractometers (such as Abbe [12–14] or Pulfrich [15– 17]) use Snell’s law to extract the index of refraction by finding the critical angle, or directly measuring the direction of the incident beam propagating through a prism, which is made of, in direct contact with or filled with the material of interest. Higher precision can be achieved using optical interferometers (such as Mach-Zehnder or Michelson) that determine the material refractive index by measuring shifts in the observed interference patterns [18–24]. Spectroscopic ellipsometry has also been widely used to determine the optical constants of thin films over a broad wave- length range by analyzing the polarization changes induced by specular reflection and adopting an appropriate model to describe the optical properties of the material [25–33]. Due to the required bulky optical elements and typically large sample volumes, current methods are not easily scalable to small dimensions without sacrificing pre- cision, wavelength span, or refractive index range. In this chapter, we investigate plasmonic interferometry as a tool to precisely de- termine the optical constants of dielectric materials in a broad wavelength range. By employing an array of nanoscale slit-groove (SG) plasmonic interferometers [4, 34– 38], we determine the relative permittivity of several dielectric materials in contact with the device surface. Plasmonic interferograms are measured by detecting SPP- 9 mediated modulation of light transmission through the slit of several SG plasmonic interferometers, as a function of incident wavelength and slit-groove distance. At each wavelength, the measured oscillation period of the interferogram allows extrac- tion of the SPP refractive index, from which the dielectric constant can be derived. Since the nano-groove in each plasmonic interferometer can excite SPPs at multi- ple frequencies simultaneously, the plasmonic refractometer allows extraction of the material dielectric function at all wavelengths using the same, simple illumination setup, without requiring a priori knowledge of the functional dependence of the re- fractive index vs. incident wavelength. The proposed plasmonic refractometer offers a higher degree of system integration, while retaining the spectroscopic capability, high resolution, and wide applicability range of more conventional methods. 2.2 Sample fabrication and experimental setup The plasmonic refractometer was fabricated by depositing a 4nm-thick Ti film onto a previously cleaned 1mm-thick fused-silica slide, to serve as an adhesion layer, fol- lowed by e-beam deposition of a 300nm-thick film of metal, gold or silver – two well-characterized noble metals that can effectively support the excitation and prop- agation of SPPs in the visible and near infrared range. Plasmonic interferometers were then fabricated by focused ion beam (FIB) milling, using a Ga liquid metal ion source with beam current of ∼100 pA and an accelerating voltage of 30 kV. More than 1 500 plasmonic interferometers were milled consisting of slits and grooves with identical width, length and depth (within 1% fabrication error), over an area of ∼15 mm2 , achieved by running a script to automatically move the FIB stage, while adjusting beam focus and stigmatism at each sample location. The scanning electron microscope (SEM) and FIB systems were calibrated using a silicon test spec- 10 Figure 2.1: Plasmonic refractometry. (a) Schematic cross section of a plasmonic refractometer comprising a microfluidic channel, a metal film milled with thousands of plasmonic interferometers, and a supporting glass substrate. (b) Working principle of a slit-groove plasmonic interferometer. Upon illumination with a spatially coherent optical beam at normal incidence, the groove generates SPPs by diffractive scattering of the incident light. The SPPs propagation toward the slit, interfere with the incident reference beam at the slit location, and then transmit through the slit. (c) Image of plasmonic refractometer under broadband illumination at normal incidence. Scanning electron microscope (SEM) micrograph of (d) a reference individual slit (∼100 nm wide and 15 µm long) and (e) a plasmonic interferometer consisting of a through-slit flanked by a groove with subwavelength width. Light intensity spectrum transmitted through (f) a reference isolated slit as shown in (d), and (g) through the slit of a slit-groove plasmonic interferometer as shown in (e), for the case of a silver/air interface. imen with specific lengths of 1.9 µm and 10 µm from Electron Microscopy Science, and the reported interferometer arm length values were corrected accordingly. This calibration step is critical for accurate measurements of dielectric function values. To minimize the effects caused by uneven sample delivery, we designed, fabri- cated and integrated a microfluidic channel made of polydimethylsiloxane (PDMS). To improve uniformity of sample delivery and detection, we chose a channel depth (i.e. 80 µm) that is several orders of magnitude larger than the SPP skin depth in the dielectric. Figure 2.1(a) shows a schematic cross section of the proposed device, consisting of (i) an 80µm-deep PDMS microfluidic channel to control the flow of 11 dielectric materials in liquid phase, (ii) a metal film with integrated plasmonic inter- ferometers, and (iii) a fused-silica substrate to serve as the transparent supporting layer. Figure 2.1(b) depicts a slit-groove plasmonic interferometer, a nanoslit flanked by a nanogroove, etched in a metal film with complex dielectric function m (λ) in direct contact with a material characterized by a dielectric function d (λ). Upon illumination with a spatially coherent optical beam at normal incidence, the groove generates SPPs by diffractive scattering of the incident light. Following propagation toward the slit, the SPPs accrue a wavelength and material-dependent phase that can be measured by direct interference with a reference beam at the slit location. Light transmission through the slit carries information about the specific optical functions of the metal film and dielectric material supporting the SPP waves. A picture of the real device including a custom-made aluminum sample holder, as well as inlet and outlet tubing and microfluidic channel, is reported in Figure 2.1(c), where the active device area is made visible by white light illumination. In addition to SG plasmonic interferometers, individual slits (Figure 2.1(d)) were also etched for normalization purposes. Figure 2.1(e) shows the SEM image of a representative SG plasmonic interferometer consisting of a 100nm-wide, 15µm-long through slit, flanked by a 200nm-wide, 15µm-long, ∼20nm-deep groove. The center- to-center distance p between the slit and the groove is defined as the arm length of the SG plasmonic interferometer, which is 8.16 µm for the specific interferometer shown in Figure 2.1(e). A total of five columns of 311 structures each were milled; specifically, three identical columns of SG plasmonic interferometers with p varying from 0.26 µm to 8.16 µm in steps of ∼25 nm, and two columns of single slits placed on the left and right sides of the former three columns, respectively, serving as the reference for normalization of the raw transmitted intensity spectra through the slit 12 of SG plasmonic interferometers. In order to improve the statistical significance of the experimental results and validate the proposed method, for any given value of the interferometer arm length p, the spectra of three nominally identical SG plasmonic interferometers were used to calculate a mean value at each wavelength and arm length, and the spectra of two single slits were averaged to obtain the mean reference transmission spectrum. λ = 400-800 nm Mirror Xenon Arc Lamp Aperture Condenser Metal Quartz Plasmonic Interferometer Micro. Obj. Tube Lens Mirror Spectrograph CCD Figure 2.2: Experimental setup. The optical setup to measure the light intensity transmitted through the slit of a plasmonic interferometer. A xenon arc lamp coupled to a microscope condenser lens, as shown in Figure 2.2, was used to illuminate the surface of the plasmonic refractometer with a spatially coherent, normally incident light beam. The transmitted light intensity through the slit of each plasmonic interferometer was collected by a 0.6 NA, 40× objective lens, dispersed using a single-grating monochromator and then detected by a CCD camera. Finally, a customized script was used to automatically translate the op- tical microscope stage and acquire light transmission spectra for all the plasmonic interferometers in the array. 13 2.3 Wavelength resolved plasmonic interferogram The interference between the SPP and the incident beam at the slit location, as shown in Figure 2.1(b), can be modeled by defining a normalized transmitted inten- sity, In (λ, p), that can be obtained experimentally by normalizing the measured light intensity transmitted through the slit of an SG plasmonic interferometer (ISG ) by the transmitted intensity through an individual reference slit (IS ), and analytically expressed as: ISG (λ, p) In (λ, p) = IS (λ) 2π = |1 + βei( λ penSPP +φG ) |2 (2.1)  pn  SPP = 1 + β 2 e−αp + 2βe−αp/2 cos 2π + φG , λ where β and φG are the effective SPP scattering amplitude and phase, and n eSPP is the SPP complex refractive index, given by [39]: s m (λ)d (λ) n eSPP (λ) = nSPP (λ) + iκSPP (λ) = , (2.2) m (λ) + d (λ) where n SPP and κSPP are the real and imaginary parts of n eSPP , and m and d are the complex dielectric functions of the metal and of the material in direct contact with it, respectively. Finally, α = 4πκSPP /λ represents the SPP absorption coefficient, which accounts for the SPP amplitude attenuation induced by propagation losses along the metal/dielectric interface. Aim of this work is to show that the wavelength- dependent dispersion of all of the parameters in Equations (2.1) and (2.2) can be extracted using plasmonic interferometry. Figures 2.1(f)–(g) show the averaged raw transmission spectra through an indi- vidual reference slit (Figure 2.1(d)) and through the slit of an SG plasmonic interfer- 14 Figure 2.3: Normalized transmission spectra (experimental). Examples of the normal- ization method, achieved by dividing the transmission spectrum measured through the slit of a slit-groove plasmonic interferometer by that of a reference isolated slit. Normalized transmission spectra for a plasmonic interferometer with arm length (a) p = 8.16 µm, and (b) 1.62 µm, respec- tively, for gold/air (solid orange line), silver/air (solid black line) and silver/water (dashed black line) interfaces. ometer with p = 8.16 µm (Figure 2.1(e)), for a silver/air interface. The transmission spectrum in Figure 2.1(g) shows significant spectral modulation compared to the sin- gle slit case, as a result of SPP interference. Figure 2.3(a) presents the experimental normalized transmission spectra (In = ISG /IS ) for an SG plasmonic interferometer with p = 8.16 µm for different, representative metal/dielectric interfaces, such as gold/air, silver/air and silver/water. The normalized transmission spectra show en- hancement and suppression as a function of wavelength, due to the constructive or destructive interference at the slit position between the incident beam and the SPP waves. The solid orange curve in Figure 2.3(a) reports data for the gold/air interface case. For wavelengths longer than 650 nm, the normalized spectrum is similar to 15 that obtained for silver/air, both in magnitude and peak positions. However, for wavelengths shorter than 540 nm, the normalized transmission spectrum for the gold/air interface case is constant and equal to 1. This value represents the normal- ized transmission of an isolated reference slit, suggesting that although the groove is able to excite SPPs, their propagation length is strongly reduced due to increased losses induced by interband transitions, occurring below 2.3 eV (i.e. ∼539 nm) for gold [40]. Therefore, the SG plasmonic interferometer behaves as an isolated slit for λ <540 nm, thus explaining the observed value of In =1. Since the interband transition for silver occurs at higher energies, i.e. 3.9 eV (∼318 nm) [40], the intensity oscillations are still visible down to 400 nm when silver instead of gold is used to support SPPs (black solid line, Figure 2.3(a)). The dashed black line in Figure 2.3(a) shows the normalized spectrum for the same interferometer etched in silver with p = 8.16 µm, when water is delivered on top of the device surface using the microfluidic channel. Compared to the silver/air case, a significant red shift is observed in the peak positions as a result of the refractive index increase (black dashed line in Figure 2.3(a)). In addition, the intensity modulation becomes visible at longer wavelengths, since the SPP absorption coefficient is increased as a result of the larger dielectric function of water compared to air, which causes a stronger attenuation of the SPP amplitude for λ <450 nm. Figure 2.3(b) shows the normalized transmission spectra for an SG plasmonic in- terferometer with shorter arm length (p = 1.62 µm) for the three different metal/dielectric interfaces. As expected, fewer oscillations are observed as a result of the shorter in- terferometer arm length. Moreover, SPPs generated at the groove location can now reach the slit with reduced amplitude attenuation due to the shorter propagation distance. 16 Figure 2.4: Wavelength-resolved plasmonic interferograms. Color maps showing experi- mental (a)–(c) and fitted (d)–(f) normalized spectra of light intensity transmitted through the slits of various SG plasmonic interferometers as a function of arm length p (0.26–8.16 µm, x axis, in steps of ∼25 nm) and incident wavelength λ (510–760 nm, y axis) for representative metal/dielectric interfaces: gold/air, silver/air and silver/water. Each color map in (a)–(c) reports 311 (number of devices) × 551 (number of wavelengths) ' 170 000 data points, resulting from an averaging pro- cess over several columns of identical devices. Note that the color maps generated from the fitting procedure were purposely flipped vertically, forming mirror images of the experimental maps for easier visual inspection. Transmission spectra similar to those reported in Figure 2.3 were acquired for all of the interferometers fabricated on the same chip, which allows us to generate wavelength-resolved interferograms, displayed in the form of color maps of normal- ized transmission intensity, In (λ, p), as a function of incident wavelength and arm length, and reported in Figures 2.4(a)–(c) for three different interfaces, i.e. gold/air, silver/air and silver/water, respectively. Each color map originally consisted of 311 (number of devices) × 1 340 (total number of recorded wavelengths) ' 417 000 data points, resulting from an averaging process over five total columns of devices, for a total of more than 2 000 000 data points acquired per color map. The reported wavelength-resolved interferograms show a reduced wavelength range, for a total of 170 000 points, and contain all the information needed to extract the relevant phys- ical parameters, such as the dielectric functions of the materials under investigation. 17 A vertical cut in each color map corresponds to the normalized transmission spec- trum for a plasmonic interferometer with a specific arm length, whereas a horizontal cut reveals the plasmonic interferogram, i.e. the normalized transmitted intensity as a function of arm length for a given incident wavelength. Alternating, oblique red and blue bands in the color maps correspond to constructive and destructive interference, respectively, whereas white color indicates In = 1, i.e. null interference between the SPP wave and the incident beam at the slit position. Figures 2.4(d)–(f) show reconstructed color-mapped interferograms using Equation (2.1) together with the physical parameters as extracted from the fitting procedure described in the fol- lowing. The reconstructed color maps were flipped vertically, thus forming mirror images of the experimental maps for easier comparison and visual inspection. 2.4 Plasmonic refractometry Figure 2.5 displays three representative plasmonic interferograms for the silver/air interface at λ = 750, 650, and 550 nm, respectively, with the symbols representing averaged intensity data from each SG plasmonic interferometer, corresponding to three different horizontal cuts in Figure 2.4(b). Each intensity profile at a given wavelength can be fitted using Equation (2.1) to determine the spatial period, i.e. λSPP = λ/nSPP , from which nSPP (λ), can be determined. The three solid curves in Figure 2.5 represent the nonlinear least squares fits at these three representative wavelengths. The same fitting procedure can be extended to all wavelengths, thus allowing extraction of the wavelength dependence of nSPP , and all relevant param- eters, for all metal/dielectric interfaces, with a spectral resolution of ∼0.4 nm, as determined by our experimental setup. 18 Figure 2.5: Fits of plasmonic interferograms at various wavelengths. Plots of normal- ized transmission intensity In as a function of arm length p for a silver/air interface, at different wavelengths, i.e. λ = (a) 750 nm, (b) 650 nm, and (c) 550 nm, respectively, corresponding to three different horizontal cuts in the wavelength-resolved plasmonic interferogram color map for a specific interface (i.e. silver/air, Figure 2.4(b)). The symbols represent data and the solid lines show the theoretical fits using Equation (2.1). The best-fit values of nSPP (λ) are presented in Figure 2.6(a) for various metal/dielectric interfaces. The lighter band around each curve indicates the 95% confidence interval as determined by propagation and statistical analysis of the experimental and fit errors. Error bars representing standard deviations are also included, generally well within the symbol size. The open symbols reported on the same graph represent ref- erence data, calculated using the dielectric functions determined with conventional methods [12, 41–44]. The results obtained with plasmonic refractometry agree well with the reference values. By using Equation (2.2), together with the best-fit values of n eSPP (λ) for silver/air and the tabulated air (λ) [44], we can determine the complex dielectric function of 19 Figure 2.6: Plasmonic refractometry: retrieving the optical functions of dielectric materials. (a) Real part of the SPP refractive index, nSPP , vs. incident wavelength λ, in steps of ∼0.4 nm, for different interfaces, as extracted from theoretical fits. The open symbols represent reference values calculated from reported optical dispersion of metal and dielectric materials using conventional refractometry methods [12, 41–43]. (b) Extracted real part of the relative permittivity r for various metal and liquid dielectrics vs. incident wavelength. The open symbols represent reference values for gold (orange triangles) and silver (black squares) [41]. The lightly colored regions shadowing the scatter plots represent the 95% confidence intervals estimated from error analysis and propagation of errors. silver (i.e. silver = silver,r + isilver,i ), which describes the optical properties of the specific silver film we deposited, including any possible contamination originating from film preparation and processing. silver,r (λ) is shown as the black scatter plot in Figure 2.6(b), shadowed by a light-gray band representing the 95% confidence interval. The same procedure can be applied to the experimental results for a gold/air interface to extract the dielectric function of gold gold,r (λ), reported in Figure 2.6(b) as the orange scatter plot. The open triangles represent reference data displayed on the same graph for comparison [41]. From the best-fit values of nSPP (λ) for the various metal/liquid interfaces, re- 20 ported as blue, green, and red scatter plots in Figure 2.6(a), and using the previ- ously determined dielectric functions of the respective metal film m (λ), the dielectric functions d (λ) of the liquids can then be extracted, as shown in Figure 2.6(b) for water (green), methanol (red), and ethanol (blue). Figure 2.6(b) reports a summary of the extracted relative permittivity of various dielectric materials, demonstrating the potential of plasmonic interferometry as an alternative optical refractometer. It is worth noting that fits of the plasmonic inter- ferograms using Equation (2.1) at any given wavelength λ can only determine pnSPP . An error in p would therefore determine errors in the extracted nSPP , affecting m and d as well. Thus accurate p values are vital for the results, as mentioned above. In addition, although water and methanol in Figure 2.6 seem to have the same value at λ ∼700 nm, closer inspection reveals that instead the two functions differ by a measurable ∆ = 3 × 10−3 , corresponding to a refractive index change of ∆n = 1 × 10−3 , a difference that the plasmonic refractometer can easily resolve. The sensitivity of the proposed plasmonic refractometer can be affected by several factors. For example, at longer wavelengths, where the magnitude of m (λ) dominates the expression in Equation (2.2), the SPP refractive index becomes less sensitive to the presence of the dielectric material at the surface. However, the sensitivity of the interferometer can be improved by using longer arms, such that the product pnSPP is proportionally increased, thus leading to measurable shifts in the plasmonic interferogram peak positions. From a theoretical standpoint, the detection limit of the proposed plasmonic refractometer is on par compared with SPR and other SPP-based sensing schemes [45, 46]. Although we have only reported results for the real part of the dielectric function 21 of the material in contact with the metal over a broad range of wavelengths and dielectric constant values, plasmonic interferometry is also capable of determining the imaginary part of d . Indeed, the imaginary part of the refractive index of lossy dielectrics can be extracted by fitting the exponential decay of the plasmonic in- terferograms envelope amplitude using Equation (2.1) to estimate α, i.e. the SPP absorption coefficient (provided sufficiently long interferometers are employed in or- der to observe a significant decrease in the interferogram envelope amplitude). From α (related to the interferogram amplitude decay) and nSPP (governing the oscillation period), both the real and the imaginary parts of the material dielectric function can be calculated. Figure 2.7: SPP excitation amplitude and phase determined by plasmonic interferom- etry. (a) SPP excitation efficiency, β, and (b) phase shift upon excitation, φG , as a function of incident wavelength λ in steps of ∼0.4 nm, as extracted from fits of the interferometric data, for various metal/dielectric interfaces. The shadowed, colored regions surrounding the scattered data represent the 95% confidence bands. From the fitting procedure, other relevant parameters can also be determined, namely the SPP excitation efficiency β(λ) and scattering phase φG (λ), as reported 22 in Figure 2.7 for different metal/dielectric interfaces. β decreases as the wavelength increases and is also affected by the materials at the interface. φG is relatively insensitive to the presence of different materials at the metal interface, and it has a value close to π/2 over the entire spectral range. β and φG are fundamental parameters that describe the light scattering process at the groove responsible for the SPP excitation. β and φG can be used together with n eSPP and Equation (2.1) to reconstruct the plasmonic interferograms at each wavelength, as well as the two- dimensional color maps, as already reported in Figures 2.4(d)–(f), showing excellent agreement with the experimental color maps. The geometry of the proposed refractometer is intrinsically planar and is amenable to integration into a relatively compact device, as shown in Figures 2.1(a)–(c). The proposed system does not require prisms or other bulky optical means to couple incident light into SPPs, since the subwavelength groove acts as a broadband and lo- calized source of SPPs in a wide wavelength range. Detection of the refractive index dispersion can be achieved by performing a simple measurement of light transmission through the slit of each interferometer, which could also be accomplished by inte- grating the sensor chip onto a complementary metal-oxide-semiconductor (CMOS) photodiode array. Since the slits are identical, the transmission does not need to be corrected for the collection efficiency of the optical set-up or detector, thus greatly simplifying the data acquisition process. Another advantage of the proposed method in comparison with existing technologies is that much smaller sample volumes can be used to determine the optical properties of the material. Considering the skin depth of the excited SPPs —which for a silver/water interface varies between ∼0.1–0.4 µm in the 400–800 nm range— the estimated sampled volume is ∼6 nL. The sampled volume can be further reduced by employing a denser, spatially optimized array of circular (rather than linear) plasmonic interferometers as seen in Chapter 4. 23 2.5 Conclusion In conclusion, we have designed, fabricated, and tested an optical refractometer con- sisting of a planar array of slit-groove plasmonic interferometers etched in a metal film (silver or gold) with device densities > 104 /cm2 . This alternative approach en- ables detection of the refractive index of dielectric materials over a broad wavelength range, without a-priori knowledge of the dispersion model for the dielectric func- tion of the material. Compared to ellipsometry and other refractometric techniques, plasmonic interferometry can prove useful when ultra-small sample volumes and de- tection areas are required. This platform could help generate tabulated functions for chemical analytes, biological markers, and other materials that are difficult to char- acterize with more conventional methods. Moreover, thanks to the intrinsic planarity of the fabrication process, and lack of bulky in-coupling optical elements to excite SPPs at multiple wavelengths simultaneously, the proposed plasmonic refractometer can be further developed into a portable spectroscopic tool. Chapter Three Plasmonic interferometry for biochemical sensing 25 3.1 Introduction SPPs are electromagnetic fields coupled to plasma oscillations supported by free electrons in metal films, and with an evanescently decaying field amplitude away from the metal surface [39, 47]. Accordingly, the propagation constants of SPPs depend on the dielectric material above the metal surface. Taking advantage of this property, we accomplished the multispectral refractometer, which was discussed in Chapter 2. On the other hand, extensive efforts have been devoted to the development of various surface plasmon resonance (SPR) biosensors [11, 48, 49]. Typical SPR systems utilize a prism [3, 50, 51] or metallic grating [52–55] to couple the light beam incident from a wavelength-specific angle into SPPs. Given the resonant nature of the SPP excitation, these SPR-based schemes are limited in the number and range of wavelengths, thus preventing spectroscopic characterization of the sample. Diffractive scattering by a sub-wavelength groove etched in a metal film can generate SPPs at multiple wavelengths simultaneously, and independently of the angle of incidence [4], offering the potential to add spectroscopic capabilities to SPP- based sensing applications. In this chapter, we design and fabricate multispectral biochemical detectors based on plasmonic interferometry, with glucose and C-reactive protein (CRP) as analytes of interest. Diabetes mellitus is a public health problem affecting ∼415 M people all over the world [56]. The primary symptom of the disease is blood glucose level higher than the normal range. There are numerous complications associated with diabetes, all of which can be greatly reduced through stringent control of blood glucose level through tight monitoring using a glucose meter. Current commercially available glucose meters utilize glucose enzyme electrodes [57], where glucose oxidase (GOx) or 26 similar glucose specific enzymes are immobilized on the electrode surface. Electrons transfer in the electrochemical reaction produces a current signal proportional to glucose concentration. However, the detection limit of this kind of glucose meters is ∼50 mg/dL due to an intrinsic barrier caused by a thick protein layer of the enzyme [58], which prevents their use in other bodily liquids, such as tears or saliva, whose glucose concentrations are typically two orders of magnitude lower than what is found in blood [59, 60]. Here we report a novel sensor based on plasmonic interferometry that is capable of measuring glucose concentration down to its physiological level in saliva, a bodily liquid that can be acquired in an easy and non-invasive way. Furthermore, the specificity for glucose is enhanced by the adoption of a dye assay. Besides, the detection selectivity issue can also be addressed through antibody immobilization. As a proof of concept, we used a functionalized plasmonic inter- ferometer to perform label-free detection of binding interactions between CRP and Immunoglobulin G (IgG, anti-CRP antibody). CRP is a well-known protein synthe- sized by the liver in systematic response to inflammation [61], whose plasma level increases rapidly after acute inflammation. Hence, fast and accurate detection and quantification of CRP is important for the evaluation of inflammatory states, disease progress or treatment effectiveness. 3.2 Plasmonic interferometry for glucose detec- tion A groove-slit-groove (GSG) geometry was chosen for the sensing purpose in this chapter. Figure 3.1(a) displays the SEM image of a representative 10-µm-long GSG plasmonic interferometer, which has one 100-nm-wide through slit flanked by a 200- 27 Figure 3.1: Representative schematic of a groove-slit-groove (GSG) plasmonic inter- ferometer. (a) SEM image of a 10-µm-long GSG plasmonic interferometer, with p1 = 0.57 µm and p2 = 1.85 µm. (b) Working principle of a GSG plasmonic interferometer. nm-wide, ∼20-nm-deep groove on its each side. The center-to-center slit-groove distance is defined as the arm length. For this specific plasmonic interferometer, p1 = 0.57 µm and p2 = 1.85 µm. Figure 3.1(b) depicts the working principle for such a GSG plasmonic interferom- eter. When a spatially coherent optical beam with amplitude E0 is normally incident on a groove, SPPs are generated by diffractive scattering of the incident light. Both the SPP waves from the left groove (ESPP1 ) and the right groove (ESPP2 ) propagate 28 toward the slit. At the slit location, these three waves, i.e., two SPP waves and the incident beam, interfere with each other. Light transmitted through the slit is the result of the “three-beam interference” and carries information about the specific optical property of dielectric material the SPP waves travel through. Any change in the dielectric material would cause observable changes in the transmitted light. The “three-beam interference” at the slit location can also be modeled by nor- malized transmitted intensity In , as described in detail in Chapter 2, normalizing the light intensity transmitted through the slit of a GSG plasmonic interferometer (IGSG ) by the transmitted intensity through an individual reference slit (IS ), analytically given by: IGSG (λ, p1 , p2 ) In (λ, p1 , p2 ) = IS (λ) (3.1) i( 2π p n +φG1 ) i( 2π p n +φG2 ) 2 = |1 + β1 e λ 1 SPP e + β2 e λ 2 SPP e |, where λ is the free-space wavelength; subscript 1 or 2 denotes the resource of the SPP wave, the left or right groove; β1,2 and φG1,2 are the effective SPP scattering amplitude and phase; and n eSPP is the SPP complex refractive index. Comparing with the model for an SG plasmonic interferometer (Equation 2.1), this model accounts for the contribution from one more groove. Figure 3.2 shows the simulated normalized intensity as a function of wavelength for SG and GSG plasmonic interferometers on a Ag/water interface, using the de- scribed analytical models. The GSG plasmonic interferometer (black line) and the SG plasmonic interferometer with a short arm p = 0.57 µm (red line) have a same slit-groove distance, causing an envelope profile (red line) in the normalized trans- mission spectra. The GSG plasmonic interferometer has more periods due to the “three-beam interference”, the contribution from the extra groove. Therefore, the 29 Ag/Water 1.8 p 1 = 0.57 µm, p 2 = 9.75 µm 1.6 p = 0.57 µm p = 9.75 µm 1.4 1.2 In 1 0.8 0.6 0.4 400 500 600 700 800 Wavelength (nm) Figure 3.2: Simulated normalized transmission spectra for SG and GSG plasmonic interferomters on a Ag/water interface. The black line is for a GSG plasmonic interferometer with two grooves, with p1 = 0.57 µm and p2 = 9.75 µm. The red line is for a SG plasmonic interferometer with one groove, p = 0.57 µm. And the blue line is for a SG plasmonic interferometer with one groove, p = 9.75 µm. GSG plasmonic interferometer with smaller full width half maxima (FWHM) will be more sensitive, comparing with the SG plasmonic interferometer. The spectrum for the SG plasmonic interferometer with a long arm p = 9.75 µm (blue line) shows a lower amplitude comparing to that for the GSG plasmonic interferometer, which would result in lower sensitivity in the regard of intensity change. Accordingly, in this chapter, the GSG structure was chosen over SG geometry for the sensing appli- cations. A GSG plasmonic interferometer with p1 = 0.57 µm and p2 = 5.70 µm was used for glucose detection experiment. As described in Chapter 2, a microfluidic channel was used to guide the flow of the glucose water solution onto the device surface, and the light intensity transmitted through the slit of a plasmonic interferometer was measured by an inverted microscope coupled to a spectrometer. Figure 3.3(a) shows the normalized transmission spectra for increasing concentrations of glucose water solution. A systematic shift to longer wavelength is observed since increasing glucose 30 Ag/Glucose Solution 2.5 Glucose Concentration (mg/dL): (a) 0 900 2.0 45 1800 90 3600 180 7200 n I 450 14400 1.5 1.0 Relative Intensity Change (%) (b) 40 20 0 -20 500 600 700 800 W avelength, (nm) Figure 3.3: Sensing performance of a GSG plasmonic interferometer for the detection of glucose in water. Normalized transmission spectra (a) and relative intensity change spectra (b) of a GSG plasmonic interferometer (p1 = 0.57 µm and p2 = 5.70 µm) at different concentrations of glucose in water. 31 concentration results in higher refractive index of the dielectric material that SPP waves propagate through. Figure 3.3(b) displays the relative intensity change for increasing glucose concen- trations, defined as follows: ∆I IGlucose − IWater = × 100%, (3.2) I0 IWater where IGlucose is the light intensity transmitted through the slit of a plasmonic in- terferometer at a fixed glucose concentration; and I0 = IWater is the light intensity transmitted through the same device in water (i.e., zero glucose concentration), which serves as a reference. It is worth noting that at some wavelength, there is no signif- icant intensity change. Even though theoretically every wavelength can be used for sensing, it is important to choose an appropriate wavelength to maximize the relative intensity change. As for this specific plasmonic interferometer, the maximum relative intensity change achieves ∼40 % at the wavelength of 610 nm, while the wavelength shift is just ∼10 nm. In short summary, by monitoring the wavelength shift and rel- ative intensity change, glucose concentration can be detected over broad wavelength and concentration ranges, with relative intensity change as the best figure of merit. Figure 3.4 is a calibration curve of relative intensity change at λ = 590 nm for a GSG plasmonic interferometer with p1 = 5.70 µm and p2 = 9.75 µm. The dashed line is the least-square fit of the data points. The gray boxes denote the physiological ranges of glucose in saliva (light gray) and serum (dark gray), respectively. Typical physiological glucose concentration is 0.36 – 4.3 mg/dL in saliva [59], and 53 – 138 mg/dL in serum [60]. This plasmonic interferometer is able to detect glucose in water solution with a concentration ranging from 0.1 to 400 mg/dL. Clearly this device can measure glucose level in saliva successfully, while the detection limit for 32 Figure 3.4: The calibration curve in terms of relative intensity change at λ = 590 nm for a GSG plasmonic interferometer with longer arm lengths. The relative intensity change as a function of glucose concentration in water solution for a GSG plasmonic interferometer with p1 = 5.70 µm and p2 = 9.75 µm. The gray boxes denote the physiological ranges of glucose in salive (light gray) and serum (dark gray). commercially available glucose meters is ∼50 mg/dL [58]. To better quantify the sensitivity of the plasmonic interferometer, a figure of merit with regard to intensity change can be calculated: ∆I/I0 F OMI = , (3.3) ∆n where ∆n is the refractive index difference between two glucose water solutions with different concentrations. For this plasmonic interferometer, F OMI reaches 884 000 %/RIU and 17 000 %/RIU respectively in the glucose concentration ranges for saliva and serum. The figure of merit for this plasmonic interferometer is at least one order of magnitude greater than published results [62–67], demonstrating the feasibility of using plasmonic interferometry for detection of clinically relevant molecules with low concentration. 33 3.3 Specific glucose sensing achieved by dye chem- istry The GSG plasmonic interferometer is capable of detecting glucose concentrations down to the physiological levels in saliva. However, if directly used on a clinical saliva sample, it cannot give an accurate glucose concentration value, since saliva is a mixture of salts, proteins and urea [68], while SPP wave responds to refrac- tive index change caused by any substance on its interface. In this section, an Amplex-red/Glucose-oxidase/Glucose (AR/GOx/Glucose) enzyme-driven dye assay was coupled to the GSG plasmonic interferometer to add selectivity for glucose de- tection. Horseradish peroxidase (HRP) from Armoracia rusticana, GOx from Aspergillus niger, D-(+)-glucose, urea and dimethylsulfoxide (DMSO) were purchased from Sigma-Aldrich. Stock concentrations of HRP (10 U/mL) and GOx (1 U/mL) were determined spectrophotometrically using their molar extinction coefficients [69, 70]. A stock solution of 0.5 mM D-(+)-glucose was prepared in deionized (DI) water. 10-Acetyl-3,7-dihydroxyphenoxazine (AR) was purchased from Invitrogen. A stock solution of 19.5 mM AR was prepared in anhydrous DMSO, aliquoted and stored at −20◦ C. Figure 3.5 illustrates the reaction mechanism in this assay. Catalyzed by GOx, β-D-gluocse is oxidized into D-gluconolactone, with hydrogen peroxide (H2 O2 ) as a byproduct [71]. Then H2 O2 is utilized by HRP to oxidize the colorless and non- fluorescent AR into the red and fluorescent resorufin [72–74]. In short, 1 mol of resorufin is produced when 1 mol of glucose is consumed. This dye assay has been commonly utilized to determine glucose concentrations [75, 76], using the fluores- 34 Figure 3.5: Reactions in an Amplex-red/Glucose-oxidase/Glucose assay. Glucose is consumed and resorufin (a red-fluoresecent molecule) is produced in a 1:1 stoichiometric ratio. cence property of resorufin for higher signal-to-noise ratio. Here, we employ the absorption property of resorufin, specifically the fact that resorufin exhibits maxi- mum absorption at λ = 571 nm [72]. Thus the incident beam and the propagating SPP waves will be affected by the absorption. Correspondingly, the light transmitted through the slit will show intensity attenuation that can be quantitatively correlated to glucose concentration in solution. In the experiment, an initial mixture of 280±6 µM Amplex red (AR), 5.5±0.1 nM horseradish peroxidase (HRP) and 82.5±0.7 nM glucose oxidase (GOx) in 50 mM sodium phosphate buffer solution (pH 7.4) was reacted with different concentrations of glucose in the dark for 50 min. The reacted solution was then delivered onto the device surface by a PDMS microfluidic channel. Figure 3.6 displays the relative intensity change spectra of a GSG plasmonic interfereometer with p1 = 7.00 µm and p2 = 9.75 µm, for increasing glucose concentrations with the presence of the dye assay. The spectra were flipped vertically for better visual effects. The intensity drops dramatically with increasing glucose concentration, in the region from 510 nm to 590 nm, a wavelength range around the maximum absorption wavelength (571 nm) of resorufin. 35 Figure 3.6: Relative intensity change spectra of a GSG plasmonic interferometer for glucose detection with the dye assay. (Relative intensity change is plotted as a function of wavelength for a GSG plasmonic interferomter (p1 = 7.00 µm and p2 = 9.75 µm) coupled to the dye assay in different concentrations of glucose in solution. Figure 3.7(a) is a comparison of the relative intensity change spectra of the GSG plasmonic interfereometer with p1 = 7.00 µm and p2 = 9.75 µm when the dye assay is present (red solid line) or absent (blue dashed line). The initial glucose concentration is 250±6 µM in both cases. The attenuation of transmitted light intensity due to absorption of resorufin in the range of 510–590 nm is more obvious through compari- son. At the maximum absorption wavelength 571 nm, the absolute value for relative intensity change is 41.7 % with the assay v.s. 2.8 % without the assay. Accordingly, in this region, the absorption of resorufin dominates over plasmonic interference when the dye assay is present, which also illustrates the specificity toward glucose of the dye assay. In the wavelength range below 510 nm, when assay is present, the intensity drops slightly while the interference pattern is also visible, which is a joint effect of absorption and plasmonic interference. At wavelengths above 590 nm, the two spectra overlap, indicating the leading role of plasmonic interference. Figure 3.7(b) plots the relative intensity change as a function of glucose concen- tration with (red squares) and without (blue circles) the dye assay. Note that for the 36 Figure 3.7: Improved sensitivity for glucose detection with the dye assay. (a) Relative intensity change spectra of the GSG plasmonic interferometer (p1 = 7.00 µm and p2 = 9.75 µm) for 250±6 µM glucose detection with (red solid line) and without (blue dashed line) the dye assay. (b) The calibration curves for this plasmonic interferometer with regard to relative intensity change with (red squares) and without (blue circles) the dye assay. The solid lines are the linear fits of the data points. 37 Figure 3.8: Specificity of glucose detection with the dye assay. The resorufin concentration is plotted as a function of reaction time for 13±0.6 µM glucose in modified Fusayama artificial saliva (red circles) and 50 mM phosphate buffer (blue triangles). The control experiment is diluted artificial saliva without glucose (0 µM glucose, black squares). The shadowed, colored regions surrounding the scattered data represent the error for all the data. best sensitivity in each case, the intensity was monitored at 571 nm for the case with dye assay, and at 628 nm without dye assay. Without the assay, the sensitivity from the calibration curve is 0.2 × 105 %/M; with the assay, the fit yields a sensitivity of 1.7 × 105 %/M, which is an 8.5 times improvement. It is interesting to mention that the sensitivity of the GSG plasmonic interferometer used in last section (p1 = 5.70 µm and p2 = 9.75 µm) is calculated to be 0.4 × 105 %/M at 590 nm without the dye assay. Therefore, the presence of the dye assay significantly improves the sensitivity and specificity of the GSG plasmonic interferometers for glucose detection. The specificity of the dye assay in 50 mM sodium phosphate buffer has been demonstrated. However, actual saliva is a more complex mixture, containing en- zymes, urea and salts [68]. To test the specificity of the dye assay in a more realistic sample, a glucose detection experiment was performed using modified Fusayama ar- tificial saliva (MFAS) [77], which differs from actual saliva by the absence of large ( > 12 kDa) proteins. The MFAS solution was diluted 7 times, since there is ∼7 times more urea in the original formula than in actual saliva. Specifically, original formula 38 contains 16.7 mM of urea, while the concentration of urea in actual saliva is typi- cally 2 – 3 mM. Figure 3.8 shows the production of resorufin over time using the dye assay, with the same concentration of glucose in MFAS (red circles) and phosphate buffer (blue triangles). The control experiment (black squares) was ran in MFAS without glucose. In the absence of glucose molecules, there is no resorufin produced. The two curves, red and blue, corresponding to the same glucose concentration in different solutions, are similar, demonstrating the specificity toward glucose of this dye-coupled sensing scheme. There is a ∼7.4 % difference at the end point of the two curves, which might be attributed to reaction kinetics change of the assay in different environments, i.e., resorufin is produced a little faster in MFAS. 3.4 C-reactive protein binding reaction studied by functionalized plasmonic interferometry The dye chemistry scheme presented previously is one possible approach achieving detection specificity. However, the dye must have a large absorption cross-section and be presented in a high concentration to predominate over the intensity change caused by the refractive index of other substances. There are other biospecific in- teractions that can be adopted in plasmonic sensing for the detection of analytes in low concentration, such as antigen-antiboy, protein–protein, or protein–DNA bind- ing interactions, to name a few [48]. In this section, the surface of a GSG plasmonic interferometer was functionalized to study the kinetics of C-reactive protein (CRP)– Immunoglobulin G (IgG) interaction. Protein A (PrA) Agarose from Staphylococcus aureus, ethanolamine hydrochlo- ride, phosphate buffered saline (PBS) and Anti-C-Reactive Protein antibody pro- 39 (a) (f ) p p Au Ti Quartz (b) DSP Au Ti 5 μm Quartz (c) Protein A NH2 (g) O DSP O O N O O Au O N N Ti O O S O O S Quartz Au O O IgG O O O S S N O (d) Au Au (h) O O Ti NH NH Quartz N N O O O O O O CRP O O O O NH2 (e) S S S S Au Au Au Legend: Ti Quartz : Cross-linker NH2 : Protein A : IgG : CRP Figure 3.9: Schematic of PrA coated gold surface for site-oriented immobilization of CRP onto the surface of a plasmonic interferometer. (a)-(e) PrA binds covalently to the DSP-treated gold surface. And then IgG (anti-CRP antibody) binds to the PrA functionalized gold chip in an oriented manner. The antibody bound chip was used for CRP detection. (f) SEM image of a representative GSG plasmonic interferometer with equal arm lengths, p = 8 µm. (g) Reaction mechanism of DSP on a gold surface. (h) Reaction mechanism of PrA on the DSP coated gold surface. 40 duced in rabbit (IgG fraction of antiserum, lyophilized powder) were purchased from Sigma-Aldrich. Dithiobissuccinimide propionate (DSP) was acquired from Pierce. The CRP solution was from Diazyme high-sensitivity CRP control set. A PrA stock solution was prepared by dissolving 250 mg powder in 2.5 mL PBS buffer at pH 7.4; IgG powder was dissolved in 2 mL DI water to make a 15 mg/mL stock solution. Figures 3.9(a)-(e) display all the steps in the protein binding process on a gold surface via thiol-based cross-linker DSP [78]. PrA was used as a faciliator on the DSP-modified device surface to achieve a uniform and stable orientated immobi- lization of IgG for CRP recognition. Before the chemical treatments, the chip was fabricated by pre-cleaning, gold deposition and FIB milling. Figure 3.9(f) is the SEM image of a representative GSG plasmonic interferometer, whose left (p1 ) and right (p2 ) arm lengths are equal, denoted by p, which is 8 µm for this specific inter- ferometer. After milling, the chip was first immersed in a solution of ammonium hydroxide, hydrogen peroixde and DI water mixture in a ratio of 1:1:5 at ∼ 75◦ C for 10 min to remove organic residues [79] and then thoroughly rinsed with DI water and dried with nitrogen. The cleaned chip (Figure 3.9(a)) was immediately soaked in 10 mM DSP freshly prepared in DMSO for 2 hours at room temperature to form cross- linkers on the device surface (Figure 3.9(b)), whose mechanism is shown in Figure 3.9(g). Afterwards, the chip was rinsed with DMSO and PBS buffer at pH 7.4. The PrA covalently attached to the thiol-linked gold surface (Figure 3.9(c)) by 18-hour immersion in 2 mg/mL PrA solution prepared in PBS buffer at 4◦ C. Figure 3.9(h) demonstrates the mechanism for this reaction. The chip was rinsed with PBS buffer and treated by ethanolamine hydrochloride (1 M), pH 8.6, for 1 hour to block the residual protein binding sites. The chip was then thoroughly rinsed with DI water, dried with nitrogen and stored at 4◦ C before use. The functionalized chip stays 41 p = 7.875 μm 1.6 Plain gold Δλ O O O O DSP S 1.5 N N S O O O O PrA 1.4 NH2 IgG 1.3 CRP 1.2 1.1 In 1 0.9 0.8 0.7 0.6 600 620 640 660 680 700 Wavelength (nm) Figure 3.10: The normalized intensity spectra of a plasmonic interferometer (p = 7.875 µm) after different chemical treatments. stable for several days. For the sensing experiments, the chip was first soaked in 0.25 mg/mL IgG solution prepared in PBS buffer for 30 min. IgG is a Y-shaped antibody, made up of three fragments, two antigen binding sites and another Fc unit [80, 81]. PrA specifically recognizes and binds this Fc domain of IgG [82]. Thus a layer of site-directed IgG was immobilized on the chip after the soaking, as shown in Figure 3.9(d). In the end, 10-µL CRP solution was delivered onto the device surface. After a given period of time, some CRP molecules bound with IgG (Figure 3.9(e)); the chip was rinsed with PBS buffer and dried with nitrogen; then the light intensity transmitted through a certain plasmonic interferometer was measured. This procedure was repeated as necessary. Figure 3.10 shows the normalized intensity spectra after each treatment, corre- 42 sponding to the light intensity transmitted through the slit of the GSG plasmonic interferometer with p = 7.875 µm normalized to the light intensity transmitted through an identical single slit after the soaking, rinse and drying process. After each step, the spectral maximum and minimum shift to longer wavelengths, due to the higher refractive index of the dielectric environment caused by adding one more layer of molecules on the chip surface, as shown in Figures 3.9(a)-(e). Comparing the normalized spectra before any reaction (brown solid line) and after CRP binding (green solid line), there is a ∼15 nm shift after the whole process indicated by the gray arrow in the figure, demonstrating that plasmonic interferometry is also a good tool for monitoring chemical reactions in situ, since SPPs are highly sensitive to the dielectric environment, in which they propagate. To avoid possible non-specific adsorption caused by the surface of tubing and PDMS, the standard microfluidic channel setup was not used in this experiment. Instead, a drop of CRP solution (1.89 mg/L) was directly delivered onto the device surface by using a pipette. After a given period of time, the reaction was stopped by rinsing the chip with PBS buffer to wash out free CRP molecules on the device surface and avoid further binding. The light intensity transmitted through the slit of the plasmonic interferometer was measured using the inverted microscope and denoted as the spectrum of this time step. This procedure was repeated at different time to study the CRP–IgG binding interaction over time. To take full advantage of plasmonic interferometer as the tool to study the binding interaction between CRP and its antibody IgG, the concept of relative intensity change is adopted here as well, defined as: ∆I IGSG,t − IGSG,0 = × 100%, (3.4) I0 IGSG,0 43 Figure 3.11: The relative intensity change at different time steps during CRP binding. (a) The relative intensity change spectra at different time steps in the CRP binding process. The gray dashed vertical line is a cut at λ = 683 nm. (b) The relative intensity change at λ = 683 nm as a function of time. The black rings are data points and the red dashed line is an exponential fit of the data. The inset shows the exponential decay of the field in perpendicular direction above the functionalized surface. 44 Here, IGSG,t and IGSG,0 are the transmitted intensity through the slit of the GSG plasmonic interferometer at time t and 0 respectivley. Figure 3.11(a) displays the relative intensity change spectra at different time steps. There are both systematic wavelength shifts and intensity changes in the full wavelength range. However, the intensity change is more prominent, with ∼100 % intensity change at the wavelength of ∼685 nm, while the wavelength shift is just ∼4 nm. Figure 3.11(b) plots the relative intensity change (black circles) as a function of reaction time at λ = 683 nm, corresponding to the vertical cuts indicated by the gray dashed line in Figure 3.11(a). Before 6 min, the intensity increases rapidly over time; after 6 min, the intensity stays stable, which indicates that the binding reaction has reached saturation. The red dashed line is an exponential fit of the experimental data. The inset shows the exponential field decay on a functionalized surface. Considering the skin depth of the SPP wave (∼250 nm at λ = 700 nm) and the length of a bound structure (∼15 nm), the binding interaction happens close to the gold surface with maximized field amplitude, i.e., with highest possible sensi- tivity. Therefore besides as sensitive and specific detectors for biochemical analytes, functionalized plasmonic interferometers can benefit the study of enzyme reaction kinetics as well. Cumbersome SPR setups have been widely used for reaction kinetics studies. Plasmonic interferometry has demonstrated its potential for a compact and multispectral system that is capable of monitoring interactions with high sensitivity, while consuming a sample with ultra-small volume. 45 3.5 Conclusion In conclusion, we report the design and characterization of a biochemical sensor based on GSG plasmonic interferometers in this chapter. Light intensity transmit- ted through the slit of a GSG plasmonic interferometer carries information about the refractive index change of the dielectric environment, which is a result of the “three beam interference” process between the incident beam and the propagating SPP waves originated from the two grooves by diffractive scattering. The reported plasmonic sensor is capable of detecting glucose concentrations down to its phys- iological levels in saliva, achieving a figure of merit of ∼ 106 %/RIU in terms of relative intensity change, with a resolution of ∼ 3 × 10−7 RIU, using a typical sens- ing volume that can be as low as 20 fL. An AR/GOx/Glucose dye assay was further coupled to the established plasmonic sensor for the specficity of glucose detection. Thanks to the strong absorption of resorufin at 571 nm, the sensitivity of the sensor was enhanced to 1.7 × 105 %/M within the physiological range of glucose in saliva. In addition, functionalizing the device surface with specific antibodies, a different methodology toward selective detection was also adopted. As a proof of concept, a GSG plasmonic interferometer with antibody immobilized on its surface was used to study the binding reaction of CRP. A plot of relative intensity change v.s. time illus- trates the kinetics of this enzyme reaction. This chapter demonstrates the first step toward a sensitive and selective biochemical detector based on plasmonic interfer- ometry, opening up the possibility of coupling plasmonic interferometry to different schemes for the specific detection of various analytes in bodily fluids. Chapter Four Plasmonic interferometers in circular geometry 47 4.1 Introduction As mentioned in Chapter 1, generally SPPs can be excited through the use of a prism [3, 50], or metal nanostructures [2, 4, 54, 83]. Conventional SPR implementations rely on the prism-based configuration [84, 85]. Comparably, sensors utilizing plasmonic nanostructures employ simpler optical geometries and thus offer great potential for system miniaturization [49, 86, 87], an essential requirement for the development of fast and portable sensing devices for various biomedical applications. Several researchers have therefore channeled their efforts into the study of different struc- tures [88–90], among which the (linear) SG interferometer and the bullseye structure (consisting of a circular shallow groove flanking a subwavelength hole) have shown great promise for future applications due to high sensitivity and their potential for multiplexing [45, 91, 92]. In this chapter, we propose an alternative geometry: a circular SG structure, consisting of a circular through-slit flanked by a concentric circular shallow groove. The proposed structure represents a generalized geometry, which includes the bulls- eye and the linear SG structures as two extreme cases [4, 35, 45, 91–96]. Accordingly, many benefits of the two well-known structures are retained by this geometry and can be optimized by a proper choice of parameters. The outer groove acts as a broadband source of SPPs by efficiently scattering a normally-incident white light beam into propagating SPPs and redirecting them toward the central slit, where the fields of the directly incident beam and SPP waves interfere with each other, thus modulating the light intensity transmitted through the slit. Furthermore, SPPs excited by any subwavelength segment of the circular slit can also propagate toward other parts of the slit and interfere with the incident light and with the SPPs origi- nating from the groove, to further affect light transmission. Far-field measurements 48 of light intensity transmitted through the slit carry detailed information about the near-field interaction between the SPPs and the dielectric material on the metal sur- face. Hence, circular SG plasmonic interferometers can prove to be useful for the detection of biochemical analytes and the interplay between SPPs generated by both the circular groove and slit can further enhance the device sensitivity. Preliminary results show that, compared to a linear SG plasmonic interferometer, a circular SG plasmonic interferometer can achieve 7 times higher sensitivity. In addition, thanks to the polarization independence property due to its rotational symmetry and the larger transmission area, a circular SG plasmonic interferometer is able to make bet- ter use of the incident light intensity, an advantage over both the bullseye and the linear SG plasmonic interferometer structures. This high light throughput charac- teristic is crucial for the future integration of the proposed general SG interferometer into a real-time, label-free and portable sensing platform. 4.2 Circular plasmonic interferometers Plasmonic interferometers consisting of circular and linear SG pairs were fabricated in a gold film following the same procedure illustrated in Chapter 2. Gold was cho- sen because (1) it can support the excitation of long-range propagating SPPs at λ > 550 nm, and (2) it offers a surface that is stable and resilient upon exposure to various chemical environments, making it a good material choice for sensing appli- cations. The intensity spectrum transmitted through the slit of each SG plasmonic interferometer was measured using the same optical setup demonstrated in Chap- ter 2, except that the light source was changed to a supercontinuum laser (NKT Photonics, SuperK Extreme). 49 Figure 4.1: Definition of a circular plasmonic interferometer. (a) SEM image of a circular SG plasmonic interferometer with RS = 2.5 µm and RG = 13 µm. The arm length of the interfer- ometer, defined as the separation distance between the groove and the slit, is p = 10.5 µm. The slit and groove widths are 100 and 300 nm, respectively. The depth of the groove is ∼20 nm. (b) SEM image of a bullseye structure with a hole and a concentric groove, which is an extreme case of a circular SG plasmonic interferometer with RS = 0. (c) SEM image of a linear SG plasmonic interferometer featured by a linear slit flanked by a groove, which is the other extreme case of a circular SG plasmonic interferometer with RS = RG = ∞. Figure 4.1(a) shows the SEM image of a representative circular SG plasmonic interferometer, consisting of a 100nm-wide circular slit entirely etched through the metal film, with radius RS = 2.5 µm, and a concentric 300nm-wide, 20nm-deep circular groove, with radius RG = 13 µm. The optical path in-between the slit and the groove defines the interferometer arm, with a characteristic length p = RG − RS . The sign of p is defined positive (negative) if the circular slit rests inside (outside) the area defined by the circular groove. For example, the plasmonic interferometer depicted in Figure 4.1(a) has a positive arm length p = 10.5 µm. The bullseye and the linear SG plasmonic interferometers can be thought of as two extreme cases of the more general circular SG structure. For instance, when RS = 0, the circular SG becomes a bullseye interferometer, as shown in Figure 4.1(b); on the other hand, 50 if RS = RG = ∞, the circular SG becomes a linear SG plasmonic interferometer, as shown in Figure 4.1(c). Twelve columns of 246 plasmonic interferometers each were milled with various combinations of the geometric parameters mentioned above. For the circular SG case, each column has a constant RS , while RG varies between 0.75–13 µm in steps of 0.05 µm. For example, p varies from −1.75 to 10.5 µm when RS = 2.5 µm. To improve the statistical significance of the experiment, two identical columns of circular SG plasmonic interferometers were milled for each RS . The spectra of any two nominally identical devices were measured and then used to calculate a mean value at each wavelength. The procedure was repeated for each interferometer. In addition, a column of single through-slits with the same RS was also milled to serve as the reference for normalization of the raw transmitted intensity spectra through SG plasmonic interferometers. The aim of study in this chapter is to determine the functional dependence of the transmitted intensity through a SG plasmonic interferometer ISG (λ, RS , p) as a function of incident wavelength, λ, radius of curvature of the slit, RS , as well as interferometer arm length, p. The scope of work is to experimentally identify the best configuration for sensing applications. 4.3 Optical properties of circular plasmonic inter- ferometers The groove in a circular SG plasmonic interferometer works as an efficient source of propagating SPPs through diffractive scattering of the incident light beam. The circular groove focuses the SPPs in-plane and redirects them toward the inner slit where the incident beam and the SPP waves interfere, resulting in intensity modula- 51 Figure 4.2: Wavelength resolved plasmonic interferograms for different geometries. (a)-(d) Representative SEM images of circular SG plasmonic interferometers with different RS : RS = 0.25 µm (a), 2.5 µm (b), 11 µm (c), RS = RG = ∞ (d). (e)-(h) Respective SEM images of isolated circular slits with different RS : RS = 0.25 µm (e), 2.5 µm (f), 11 µm (g), RS = ∞ (h). (i)-(l) Normalized transmitted intensity spectra for circular SG plasmonic interferometers with p = 5.45 µm, RS = 0.25 µm (i), 2.5 µm (j), 11 µm (k), RS = RG = ∞ (l). (m)-(p) Color maps showing normalized transmission spectra (Au/air interface) for the four kinds of plasmonic interferometers, as shown above, with fixed RS (0.25 µm, 2.5 µm, 11 µm and ∞) and varying p (−1.75 to 8 µm, in steps of 0.05 µm). For the case of RS = 0.25 µm and the linear SG plasmonic interferometer, the experimental p value starts at 0.5 µm. In the absence of experimental data, the region p ¡ 0.5 µm is intentionally left blank in panels (m) and (p). 52 tion of the transmitted spectra through the circular slit, as a function of wavelength and interferometer arm length. The optical properties of circular SG plasmonic interferometeRS , as shown in Figures 4.2(a)-(d), were characterized systematically for four specific types of circular SG plasmonic interferometers, with RS = 0.25, 2.5, 11 µm (circular SG plasmonic interferometers) and RS = ∞ (linear SG interferometers). For each finite value of RS , RG was varied between 0.75–13 µm, in steps of 0.05 µm. For RS = ∞ (corresponding to a linear SG plasmonic interferometer), RG = ∞ as well, since both the linear slit and groove have an infinite radius of curvature. The slit and groove lengths were kept constant at 15 µm, and the slit groove distance p was varied between 0.25 and 8 µm. Figures 4.2(e)-(h) show the corresponding isolated slits. The light intensity transmitted through the slit of each plasmonic interferometer (ISG ) was measured and normalized to the reference light intensity transmitted through an isolated circular slit (IS ) with the same RS , to isolate the interference effects due to SPPs propagating in between the circular slit and groove, as shown in Figures 4.2(i)- (l) for representative SG plasmonic interferometers with p = 5.45 µm. A color map reporting normalized transmitted intensity defined as In = ISG /IS can be constructed by recording and then stacking together the spectra of all the plasmonic interferometers as a function of arm length, p. A vertical cut in such a color map is the normalized transmission spectrum for a plasmonic interferometer with a specific arm length, as shown in Figures 4.2(i)-(l). Normalized intensity color maps for these four different SG structures on a gold/air interface are displayed in Figures 4.2(m)-(p). Since RS is different for the four configurations, even though RG is constant, the range of p will vary accordingly. To be consistent, only spectra for interferometers with p between −1.75 and 8 µm are presented here, in order to provide for a clear comparison between the different devices. For the case of RS = 53 0.25 µm as shown in Figure 4.2(m), p starts at 0.5 µm. For the linear SG geometry, the spectra are symmetric for positive and negative p, assuming normal incidence for the light beam; the region with p < 0.5 µm in Figures 4.2(m) and (p) has been left intentionally blank. In Figure 4.2(o), since p ranges between −10.25 and 2 µm, −p was adopted as the horizontal axis to show a comparable p range for all plasmonic interferometers. Figure 4.2(m) presents the color map of normalized transmitted intensity ob- tained for the circular SG interferometer with RS = 0.25 µm. Since the radius of the circular slit is smaller than the wavelength of the incident light (within the ex- perimental wavelength range), the slit behaves like a subwavelength hole, and the entire circular SG plasmonic interferometer can therefore be thought of as a bullseye structure. In a single-hole/circular-groove system, it is known that the transmission through the hole can be enhanced by constructive interference of SPPs originating at the circular groove and redirected or focused toward the central hole [93–95]. It is interesting to note that the alternating oblique blue and red bands in Figure 4.2(m), corresponding to suppressed (blue) and enhanced (red) light transmission respec- tively, are remarkably similar to the bands observed in Figure 4.2(p) for a linear SG geometry with comparable arm lengths. This suggests that the periodicity observed in the normalized intensity In vs. arm length p at any given wavelength is dictated by SPP interference effects controlled by the changes in SPP propagative phase due to the variation in arm length, p. In simpler terms, as the distance of the groove from the slit is varied, the SPP optical path also changes, thus producing either con- structive or destructive interference with the incident beam at the slit location. Also worth noting in Figure 4.2(m) is the presence of a feeble yet distinct horizontal white band at around λ = 700 nm. This band neither shifts nor is modulated in intensity by a change in arm length, suggesting that its origin cannot be related to SPP waves 54 generated by the circular groove. It is worth noting that a white pixel observed in the color map indicates a normalized transmission equal to 1, corresponding to a condition of neither enhancement nor suppression with respect to a reference single circular slit having the same diameter. The observed white band at ∼700 nm can therefore be the result of the normalization procedure. For instance, at certain wave- lengths, SPPs generated by the circular slit itself are so strong that the interference effect is mainly the result of the addition of the incident beam and these SPPs, and it is not affected significantly by the SPP contribution from the neighboring circular groove, which is a weaker source of electromagnetic fields. Therefore, at these specific wavelengths, the raw intensity measured through the slit of the circular SG inter- ferometer is largely unaffected by the SPP contribution originating from the groove, leading to a value comparable to that of the isolated circular slit. Accordingly, the normalized transmission spectra will have a value of 1 at the resonant wavelength, irrespective of the interferometer arm length, p. Figure 4.2(n) reports the normalized transmission intensity color map for a cir- cular SG plasmonic interferometer with RS = 2.5 µm as a function of incident wave- length and arm length. The radius of the slit was chosen to be 2.5 µm so that the transmission area of the circular slit and the linear slit would be similar. Besides higher normalized transmission intensity, Figure 4.2(n) now shows four instead of one horizontal white bands at around 550 nm, 610 nm, 670 nm and 765 nm for p > 0. These bands do not shift in wavelength and are not modulated in intensity when p is varied, suggesting once again that their origin is unrelated to the presence of the groove and any groove-excited SPP contribution. Observation of these bands fur- ther supports the theory that their origin is related to SPPs excited by the slit itself. Since the diameter of the cavity defined by the slit is 5 µm, more plasmonic resonant modes can be supported, compared to the previous structure with a diameter of 500 55 nm. Interestingly, for p < 0 the white bands experience dramatic wavelength shifts. A negative arm length indicates that the groove is now inside the area defined by the slit, which may introduce an additional phase shift due to the presence of the groove inside the slit-defined cavity area. Figure 4.2(o) shows the color map for a circular SG plasmonic interferometer consisting of a relatively large circular slit (RS = 11 µm ) and varying groove radius. For small arm lengths, i.e. when p = RG - RS  RS , the system behaves locally like a linear SG interferometer, indeed the periodicity features observed in the cor- responding color maps of the two interferometers (Figures 4.2(o) and (p)) are quite similar. Due to the longer radius, the cavity defined by the slit can support more resonant modes, that are responsible for the intensity ripples observed in the color map as faint horizontal bands characterized by short oscillation periods. These bands are more visible in the top-right corner of the color map, corresponding to longer wavelengths and longer interferometer arm lengths, i.e. smaller groove radius. Inci- dent light with longer wavelengths can be diffracted by the slit and generate SPPs that, having longer propagation lengths, can propagate across the entire cavity thus setting up weak yet observable standing wave patterns due to plasmonic resonances. Furthermore, scattering of the slit-generated SPPs by the central groove is reduced for long p, since the relatively small groove does not offer significant scattering area to the propagating SPP; therefore ripples can be observed. As p is decreased, the groove becomes a stronger scatterer for the slit-generated SPPs which reduces the strength and visibility of the horizontal ripples. In order to demonstrate the hypothesis that the horizontal white bands in the color maps actually originate from the interference between the directly incident light and the SPPs excited by the circular slit rather than the groove, the transmission spectra through the isolated slits were directly measured. Figure 4.3(a) shows the 56 Figure 4.3: Transmission spectra through single slits. (a) Raw spectra of light intensity transmitted through an isolated circular slit (RS = 2.5 µm, black line) and a linear slit (RS = ∞, red line). (b) Ratio between the transmitted intensity of the two structures as a function of wavelength. Four peaks are observed at ∼555 nm, 610 nm, 675 nm and 765 nm as denoted by the dashed lines, in good agreement with the horizontal white band positions observed in Figure 4.2(n). Note that the reported ratio in (b) is calculated only for qualitative description and identification of the wavelength position of the resonant peaks; the specific relative intensity ratio values are therefore irrelevant since they are obtained from two totally different structures. transmission spectra through an isolated circular slit with RS = 2.5 µm (black line) and a linear slit (red line), as well as their intensity ratio plotted in Figure 4.3(b). Four resonant peaks at ∼555 nm, 610 nm, 675 nm and 765 nm are clearly observed, as denoted by the gray dashed lines, which exactly match the horizontal white band positions in Figure 4.2(n). This resonant behavior can be explained by the construc- tive interference effect of the directly incident light and SPPs excited by the circular slit itself, which weakens the relative contribution of SPPs induced by the circular groove and results in arm-length independent white bands. However, in the region where destructive interference occurs, the SPPs excited by the groove would play a 57 more important role in the modulation of transmitted light intensity, as confirmed by the dark blue and red regions between the white bands in Figures 4.2(n), attesting stronger interference effects. 4.4 Sensing performance comparison In this section, we compare the performance of two systems, i.e. circular SG plas- monic interferometers with RS = 2.5 µm, and 15µm-long linear SG plasmonic inter- ferometers. Figures 4.4(c)-(f) represent color maps of normalized light transmission for these two geometries (Figures 4.4(a) and (b)) recorded in the presence of air and water, respectively. Figures 4.4(c) and (d) are the same as Figures 4.2(n) and (p), except that a smaller p range was chosen for better comparison. Due to the refractive index change induced by water onto the gold surface, the color maps show a sys- tematic wavelength shift and a change in the normalized intensity, clearly evidenced when comparing panel (c) with (e), and (d) with (f) in Figure 4.4. Specifically, when air is replaced with water as the dielectric material in contact with the gold surface, a higher number of oscillations in normalized intensity is observed at each wavelength, due to the increased refractive index. In addition, the characteristic white bands in Figures 4.4(c) and (e) also shift to longer wavelengths. For a straightforward comparison, representative spectra for the devices with p = 6.85 µm on gold/air and gold/water interface were plotted in Figures 4.4(g) and (h), for the circular and linear geometries, respectively. Clearly, the circular SG plasmonic interferometer reaches higher normalized in- tensity both in air and in water. An evident advantage of the circular SG geometry is that the circular groove can focus the SPPs waves toward the central slit, thus en- 58 Figure 4.4: Wavelength resolved plasmonic interferograms at different interfaces. (a),(b) SEM images of a representative circular (RS = 2.5 µm) and a linear SG plasmonic in- terferometer. (c),(d) Color maps of normalized transmission spectra of the two types of plasmonic interferometers with varying p (0.25–8 µm, in steps of 0.05 µm) on gold/air interface. (e),(f) Color maps of normalized transmission spectra of the two types of plasmonic interferometers with varying p (0.25–8 µm, in steps of 0.05 µm) on gold/water interface. (g) Normalized transmitted spectra for a specific circular SG plasmonic interferometer (RS = 2.5 µm, p = 6.85 µm) on gold/air (black) and gold/water (red) interfaces. (h) Normalized transmitted spectra for a specific linear SG plasmonic interferometer (p = 6.85 µm) on gold/air (black) and gold/water (red) interfaces. hancing light transmission through the slit if the arm length is chosen in such a way as to generate constructive interference. Moreover, the number of subwavelength scatterers making up the groove increases linearly with increasing groove radius, i.e. arm length p, which in turn determines a proportionally higher field intensity at the slit location when p increases. On the other hand, as the distance between the groove and the slit is increased, the amplitude of the SPPs generated by each subwavelength section of the groove reaches the slit with an attenuated amplitude p−1/2 e−αp , where α is the SPP absorption coefficient due to ohmic loss in the metal. The overall net effect is that as the circular SG arm length p is increased, the total SPP amplitude at the slit location slightly increases as evidenced when looking at a horizontal cut at ∼720 nm as a function of p in Figures 4.4(c). This is valid only for interferometer arm lengths p such that αp  1, since, for αp  1 the exponential decay becomes more important, causing the overall intensity to decrease as p is increased. On the contrary, the normalized transmitted intensity of the linear SG interferometer de- 59 creases monotonously as the arm length is increased, as seen from a horizontal cut at the same wavelength in Figure 4.4(d). To clarify this point, Figure 4.5 reports a comparison between the normalized intensities transmitted through circular and linear SG interferometers as a function of arm length, p. From Figure 4.5 it is evi- dent that the envelope function of the periodically varying normalized light intensity increases with p for circular SG interferometers, thus supporting the hypothesis pro- vided above. The magnitude of the increase is reduced since the central slit has a finite diameter (5 µm), while the explanation provided above strictly applies to small nano-apertures placed at the center of the circular groove. In contrast, Figure 4.5 also shows that the linear SG interferometer is characterized by a decreasing inten- sity envelope function as p increases, due to ohmic loss in the metal. Compared to the linear SG interferometer, the circular geometry can therefore mitigate the ohmic loss in the metal. The resonant cavity modes supported within the area defined by the circular slit can be tuned by simply varying the slit diameter and employed to further enhance the sensitivity of the device to small refractive index changes. Figure 4.5: Plasmonic interferogram at λ = 718 nm. Normalized light transmitted intensity measured in air through the slit of a circular SG interferometer with RS = 2.5 µm (solid line) and through the slit of a linear SG interferometer with RS = ∞ (dashed line) as a function of interferometer arm length, p, at λ = 718 nm. To evaluate the sensing performance of each plasmonic interferometer, a figure 60 of merit F OMI can be defined as: In,water − In,air F OMI = | | × 100%/∆n, (4.1) In,air Here, In,water and In,air are the normalized transmitted intensities calculated when the surface of the plasmonic interferometers is in contact with water and air, respec- tively; (In,water − In,air )/In,air is a relative intensity change at a specific wavelength induced by the refractive index change ∆n. Color maps of F OMI for circular and linear SG plasmonic interferometers are shown in Figures 4.6(a) and (b), where 5.6 has been evaluated taking into account the refractive index dispersion of air and water [42, 44]. With specific combination of wavelength and arm length, the circular SG plasmonic interferometers demonstrate several times higher F OMI than that of the linear SG plasmonic interferometers. For example, Figure 4.6(c) plots F OMI as a function of p at the wavelength of 693 nm, which corresponds to the horizontal cuts in Figures 4.6(a) and (b). This plot is useful to determine the best device at a certain wavelength, an advantage of tunability provided by the broadband response of the subwavelength-width slit and groove in the SG plasmonic interferometers. Fig- ure 4.6(d) shows the F OMI as a function of wavelength for a specific interferometer with p = 6.85 µm. At 693 nm, the F OMI of the circular SG plasmonic interferom- eter with p = 6.85 µm reaches 464 %/RIU, compared with 67 %/RIU for the linear plasmonic interferometer, which is a ∼7 times improvement. With optimization of the structure parameters, such as arm length, width and depth of the groove, as well as increased number of circular slits and grooves, the performance can be further enhanced. It is also important to note that the device sensitivity can be higher for smaller changes of the refractive index, as shown for example in Chapter 3. It is well known that linear nano-slits on a metal film can be efficient polarizers [97]: light can transmit through the slits without cutoff under transverse magnetic 61 Figure 4.6: Figure of merit comparison. (a),(b) Color maps showing F OMI for circular (a) and linear (b) SG plasmonic interferometers with varying p (0.25–8 µm, in steps of 0.05 µm). (c) F OMI as a function of arm length for the circular (solid line) and linear (dashed line) SG plasmonic interferometers at 693 nm, corresponding to the horizontal cuts in (a) and (b). (d) F OMI as a function of wavelength for a circular (solid line) and a linear (dashed line) SG plasmonic interferometer with p = 6.85 µm, corresponding to the vertical cuts in (a) and (b). (TM) illumination condition, i.e. when the electric field of the incident electromag- netic wave is perpendicular to the long slit axis; instead, a transverse electric (TE) incident field is highly attenuated [97]. Due to its circular symmetry, randomly po- larized light can be transmitted through a circular slit without a significant loss. This property is beneficial for future integration of the interferometers onto an imaging system such as a CCD camera, which directly captures transmitted light intensity changes caused by biochemical analytes. The optical setup will be simplified with- out the requirement of using the polarized light sources. Even though the bullseye structure is also polarization independent, due to its small aperture, the transmitted intensity is limited comparing with that through a circular SG plasmonic interfer- ometer, since the subwavelength hole cannot support any guided modes, and hence light transmission is cutoff for all polarizations. 62 Figure 4.7: The effect of polarization on light transmission. (a),(b) SEM images of a representative circular (RS = 2.5 µm) and linear SG plasmonic interferometers. The inset shows TM and TE incident polarization. (c) Raw transmitted intensity spectra for TM (red) and TE (black) polarized light through the slit of a circular SG plasmonic interferometer (with RS = 2.5 µm, and p =3.55 µm). (d) Raw transmitted intensity spectra for TM (red) and TE (black) polarized light measured through the slit of a linear SG plasmonic interferometer (with p =3.55 µm). The transmitted intensity of TE-polarized light has been magnified by a factor of 100 for clarity. To better illustrate the advantage of using a circular SG as shown in Figure 4.7(a) to achieve polarization independent light transmission, a linear polarizer (Thorlabs LPVIS) with high extinction ratio (> 106 : 1 over a broad wavelength range) was in- serted into the original light path before the sample surface. The setup was also used for the measurement of light intensity transmitted through a linear SG plasmonic interferometer, as shown in Figure 4.7(b). Figures 4.7(c) and (d) report the raw transmission spectra for TM (red lines) and TE (black lines) illumination conditions through representative circular and linear SG plasmonic interferometers, respectively. As shown in Figure 4.7(c), a circular plasmonic interferometer shows a polarization insensitive light transmission. In contrast, the linear SG plasmonic interferometer shows more than two orders of magnitude higher transmission under TM illumination compared to TE polarization, since the linear slit is characterized by an extinction 63 ratio > 100 : 1 [97]. Devices with high light throughput are important for potential biochemical sen- sors. Compared with the linear SG, the circular SG geometry can make better use of the chip area. Indeed, given a fixed device area, the length of the circular groove can be up to π times that of the linear counterpart, allowing the circular groove to couple more of the free space light intensity into SPPs. The circular slit can also be longer given the same area, and it can transmit more light. In addition, the curvature of the circular groove and slit also reduces the crosstalk between adjacent devices, which enhances the integration capability of the circular geometry for high-density, high-throughput sensing applications. 4.5 Conclusion In conclusion, we reported a detailed experimental study about the design, fabrica- tion and characterization of circular SG plasmonic interferometers in this chapter. These interferometers, consisting of a circular through-slit flanked by a concentric shallow groove, can be thought of as a generalization of two well-established geome- tries, i.e. the bullseye and the linear SG interferometers. Being characterized by an increased light transmission per unit area and a polarization-independence response, the proposed circular SG plasmonic interferometers show a significant advantage over both the bullseye and the linear SG geometries. The transmission properties of various circular SG plasmonic interferometers were studied in detail as a function of slit and groove diameters, as well as slit-groove separation distance, which defines the characteristic interferometer arm length. A circular SG plasmonic interferometer works by employing the circular groove as an efficient source of SPPs. The generated 64 SPPs are then focused toward the central slit location, where the incident beam and the SPP contributions interfere with each other thus generating either constructive or destructive interference. Tuning of the interference conditions can be achieved by simply varying the arm length, the wavelength of the incident light, or even the slit radius. Indeed, the circular slit can also serve as an additional source of SPP resonant modes that can further modulate the transmitted intensity, a capability missing in both linear SG and bullseye plasmonic interferometers. We then assessed the potential of this class of interferometers to perform as sensors and measured their optical response upon variation of the dielectric material on top of the metal surface. Thanks to the synergistic interaction between resonant SPP modes and plasmonic interference effects, the circular SG interferometers show improved sen- sitivity compared to their linear counterpart. For instance, compared to the linear SG plasmonic interferometer with same arm length, the circular SG structure shows a 7-time increase in the sensitivity to refractive index change. Moreover, compared to both the bullseye and the linear slit-groove geometries, circular SG plasmonic in- terferometers show overall increased light transmission per unit area, thus allowing for a higher signal-to-noise ratio when coupled to an integrated photodetector. In addition, a reduced crosstalk between adjacent circular interferometers due to the circular geometry can allow for higher degrees of device integration, with achievable densities up to one million of plasmonic interferometers per square centimeter. In conclusion, the proposed circular SG plasmonic interferometers hold great promise for the realization of compact, sensitive and high-throughput platforms for various point-of-care sensing applications, such as glucose management for diabetic patients [92], or detection of relevant biomarkers. Chapter Five Active plasmonic interferometry with incoherent light 66 5.1 Introduction Plasmonic interferometry, in the form of slit-groove or hole-groove structures on a metallic film, has been extensively studied [98, 99] from enhanced light transmis- sion [100, 101] and beaming light [94] to optical modulator [4] and label-free biosens- ing [34, 45, 91]. In these applications, nanostructures on the metallic surface serve as scattering sources to efficiently couple the incident light into SPPs. This scheme can be defined as passive plasmonic interferometry since its optical response highly depends on the external light source (e.g., polarization, wavelength, coherence, and incident angle) [45, 83, 102]. Generally in most studies [45, 91, 92, 92, 103], only the first order (i.e., SPPs generated by one nanostructure propagate toward another nanostructure to interfere with the incident light) is taken into account. Although in several studies high-order SPPs contributions due to multi-trip propagation between nanostructures have been considered to explain the interference results [4, 104, 105], they have not yet been directly experimentally observed or deconvolved. Recently, several works have employed plasmonic interferometers to engineer the emitters’ radiation pattern [106–110] and/or rate [111] and modulate the emitters’ fluorescence spectra for biochemical sensing [46]. For example, the plasmonic bullseye structure, consisting of a nanohole and circular grooves, was used to beam [106, 107] and sort [109] fluorescence emission. Specifically, a portion of the excited dipole transition in the nanoaperture can couple into SPPs, which propagate towards the groove and then are scattered into free space. By the proper design of the plasmonic structure, the interference of the scattered light results in directional emission. In- deed, despite scattering SPPs into free space, the groove structure can also work as an in-plane mirror to reflect SPPs [112, 113]. By leveraging the relative optical path, the SPPs reflected toward the nanoaperture can constructively or destruc- 67 tively interfere with the emission directly transmitted through the nanoaperture. As a result, the transmitted fluorescence spectra in the far field can be modified [83]. Such a scheme is defined as active plasmonic interferometry. Similarly to the passive scheme, high-order SPPs should also exist in the active scheme. In this chapter, we perform a systematic study of passive and active plasmonic interferometry. In particular, the active scheme is realized by depositing a thin Cr3+ :MgO emitter layer on top of plasmonic interferometers. After measuring the passive transmission and the active fluorescence transmission of an array of inter- ferometers with varied arm length, wavelength resolved interferograms are obtained, from which the distinct SPPs-mediated interference in the two schemes is clearly evidenced by the different oscillation periods. In addition, by applying discrete fast Fourier transform (FFT) to the wavelength resolved interferograms, high-order SPPs (e.g. up to sixth) are evidenced and the corresponding dispersion of SPPs match well the SPP wave vector theoretically calculated by finite difference frequency do- main (FDFD) method. Furthermore, the contribution of different-order SPPs can be leveraged by altering the design of the groove grating. More interestingly, architectures based on passive plasmonic interferometry typ- ically require coherent external light sources that have to be carefully aligned and periodically calibrated, which poses challenges to reliable integrated devices. Here we developed an alternative biochemical sensor that does not require a coherent external light source, precise alignment, or re-calibration, through active plasmonic interferometry, where solid-state light emitters in the nanoaperture serve as the lo- calized light source directly. The optical response of this novel sensor is independent of coherence, spectral bandwidth, and incidence angle of the excitation source. This latter property is particularly advantageous when measuring chemical analytes in micro-droplets, where lensing effects can significantly vary the angle of incidence of 68 the light beam, thus introducing artifacts in the sensor response. 5.2 Fabrication and experimental setup A 300nm-thick silver film was deposited on a quartz substrate previously coated with a 4nm-thick titanium adhesion layer using electron-beam evaporation. Then, bullseye structures, consisting of a hole flanked by one, two or three circular shallow grooves (i.e., H-N G, where N is the number of grooves) were fabricated on the silver film using FIB milling, together with corresponding holes as references. Following the FIB milling, a ∼40nm-thick Cr3+ :MgO (1 wt%) emitter layer was deposited on the chip surface using electron-beam evaporation. Each structure consists of a ∼300nm- diameter hole and several (N ) ∼200nm-wide, ∼40nm-deep concentric grooves. The radius of the inner groove (RG ) was varied from 0.25 µm to 6 µm in steps of 25 nm (231 distinct devices in total for each H-1G, H-2G and H-3G structure), and the pitch for the grooves was designed to be 320 nm (half of the SPPs wavelength in the Cr3+ :MgO fluorescence wavelength range ∼650–800 nm) to keep the reflected SPP in phase for propagation at the silver/air interface. All the isolated holes are identical to the holes in corresponding H-N G structures. Figure 5.1(a) shows a schematic of passive plasmonic interferometry including the possible alternative paths for SPPs generated by the circular groove (odd-order modes) and the central hole (even-order modes) while illuminated from the top side by a normally incident, collimated light beam. The light intensity transmitted through the nanohole is a result of the interference between the directly incident light at the hole position and all the orders of SPPs induced by both the groove and hole. For a representative interferometer with RG = 5 µm, the light spectra of a normally 69 (a) 3 (b) 2 4 2 4 1 5 2M 2M 2M+1 (c) x 10 4 (d) −2 x 10 RG=5μm Hole-Groove RG=5μm Hole-Groove Single hole 2 Single hole IT (a.u.) 10 IF,n 5 1 0 0 650 700 750 800 650 700 750 800 Wavelength, λ (nm) Wavelength, λ (nm) (e) λSPP (f ) λSPP IT,r IF,n,r 800 Wavelength, λ (nm) 750 700 650 1 2 3 4 5 6 IT,r 1 2 3 4 5 6 I F,n,r Arm length, RG (μm) Arm length, RG (μm) IT,r IF,n,r 0 5.0 10 0 1.0 1.8 Figure 5.1: Comparison of hole-groove plasmonic interferometer in schematics and spectra. Top-view schematics of circular hole-groove plasmonic interferometer to illustrate (a) passive (external illumination light source) and (b) active (embedded emitting layer as the inte- grated light source) plasmonic interferometry, evidencing the optical paths of SPPs for multiple trips. Note that for the active interferometry, only even order exists. (c,d) Passive (c) and active (d) transmission spectra for a plasmonic interferometer with arm length RG = 5 µm (solid black and red lines, respectively), together with transmission spectra through a reference individual hole (dashed black and red lines, respectively). (e,f) Normalized intensity interferograms generated by dividing the passive (e) and normalized active (f) intensity transmitted through the hole of a hole- groove interferometer by that transmitted through a reference individual hole, vs: wavelength λ and interferometer arm length RG . The right and top panels in (e) and (f) show the corresponding vertical and horizontal cuts labeled by the dashed lines. 70 incident collimated supercontinuum laser (NKT Photonics, SuperK Extreme) beam transmitted through the nanohole of the H-1G plasmonic interferometer (solid black line IT ) and through an isolated reference hole (dashed black line IT,0 ) are shown in Figure 5.1(c). On the other hand, Figures 5.1(b) is a schematic of an active plasmonic interferometer whose central hole has been filled with light emitters. While pumping the emitters with a quasi-monochromatic laser source ( 590±10 nm selected from the supercontinuum laser), a fraction of the excited emitters decay by generating SPPs [106, 114, 115] that propagate toward the neighboring groove, then are reflected back to either interfere with the directly emitted light at the hole location or be scattered by the hole. For example, Figure 5.1(d) shows the normalized fluorescence spectra transmitted through a representative interferometer (RG = 5 µm, solid red IF,n ) and through an isolated hole (dashed red IF,0,n ). It is worth noting that the normalization process here is different from the treatment in previous chapters. The fluorescence spectra were first normalized to their integrated intensity, in order to eliminate any additional excitation effects due to the direct modulation of the pump intensity caused by the plasmonic interferometer, for example: IF (λ, RG ) IF,n (λ, RG ) = R , (5.1) IF (λ, RG )dλ As illustrated by Figures 5.1(a-d), the transmitted intensity for both passive and active schemes can be modulated by the presence of the shallow groove, which serve as an SPP source and/or in-plane mirror. The fundamental optical properties of plasmonic interferometers can be captured by the ratio (IT/F,r ) between the measured transmission intensity through the nanoaperture of a plasmonic interferometer (IT/F ) and the reference intensity transmitted through an isolated identical nanoaperture 71 (IT/F,0 ): IT/F (λ, RG ) X IT/F,r (λ, RG ) = = |1 + βm ei(k0 mRG nSPP +φm ) |2 , (5.2) IT/F,0 (λ) m where the subscript T/F indicates passive transmission (T) or active fluorescence transmission (F), respectively; λ is the wavelength of free-space incident light or emission; βm is a wavelength-dependent effective SPP scattering coefficient, which also includes the in-plane reflection coefficient of the groove and/or hole for the high- order modes; nSPP is the complex, wavelength-dependent SPP refractive index; k0 is the amplitude of the free-space wave vector; φm is the wavelength-dependent phase change induced by the scattering processes, and m indicates the order number, which is an integer for passive transmission or even integer for the active fluorescence, as shown in Figures 5.1(a,b). Accordingly, the ratio of the normalized fluorescence spectra can then be written as: R IF,n (λ, RG ) IF,0 (λ)dλ IF,n,r (λ, RG ) = = IF,r (λ, RG ) R . (5.3) IF,0,n (λ) IF (λ, RG )dλ It can be seen from Eq. (5.3) that the additional normalization process only shifts the base line of the oscillation for each interferometer while keeping the shape to be the same to that calculated by Eq. (5.2). In both schemes, the lowest order mode (i.e., 1 for passive and 2 for active scheme) dominates the interference effect, while the higher-order modes are generally neglected. 72 5.3 Comparison of passive and active plasmonic interferometry The intensity ratio of a passive plasmonic interferometer with RG =5 µm is ex- tracted using Eq. (5.2) and shown in the right panel of Figure 5.1(e). In contrast, the corresponding result of the active plasmonic interferometer obtained by Eq. (5.3) is plotted in the right panel of Figure 5.1(f). By stacking all the oscillation ratios of 231 H-1G interferometers with distinct arm lengths, passive and active wavelength resolved interferograms are constructed and shown in Figures 5.1(e) and (f), respec- tively. Periodically constructive (red) and destructive (blue) interference is clearly observed. For each individual interferogram (i.e., horizontal cuts in the color maps, for example, top panels of Figures 5.1(e,f) at λ = 720 nm), the oscillation period in active scheme is only half of that in the passive case (i.e., the corresponding SPP wavelength). This is the result of the round-trip effect that dominates the SPP contribution in the active case and effectively doubles the optical path length, as schematically illustrated in Figures. 5.1(a,b). Similarly to the bullseye structure developed for optical beaming and enhanced transmission [54, 91, 94, 101, 106, 110], adding grooves with optimized width, depth, and pitch [112, 113] can lead to larger in-phase SPPs reflection and stronger in- terference at the nanohole location. Figures 5.2(a)-(c) show SEM images of the representative H-N G structures. The wavelength resolved interferograms for passive and active schemes are shown in FiguresFigures 5.2(d)-(f) and 5.2(g)-(i), respectively. Note that Figures 5.2(e,h) are vertically flipped for better comparison. In the passive case, the strong interference in H-1G (see Figure 5.2(d)) is significantly suppressed while adding an extra groove with 320nm separation (i.e., H-2G in Figure 5.2(e)) due to the π phase difference in SPPs generated by the two grooves. While for H-3G 73 passive transmission active fluorescence 800 (a) (d) IT,r (g) IF,n,r Hole-1Groove 10 1.8 750 700 2 μm 8 Wavelength, λ (nm) 650 (b) (e) (h) Hole-2Groove 700 6 750 1.0 4 800 (c) (f ) (i) Hole-3Groove 750 2 700 650 0 0.2 1 2 3 4 5 6 1 2 3 4 5 6 Arm length, RG (μm) Arm length, RG (μm) Figure 5.2: Wavelength resolved passive and active transmission interferograms for bullseye structures with different number of grooves. (a)–(c) SEM images of H-1G, H-2G and H-3G bullseye structures. (d)–(f) Passive transmission (IT,r ) and (g)–(i) active normalized transmission (IF,n,r ) interferograms for H-1G, H-2G and H-3G plasmonic interferometers. Note that the pitch for the N = 2 and 3 grooves is designed to be 320 nm to keep the in-plane SPPs reflected by each individual groove in phase. For the passive transmission, SPPs induced by the two grooves in H-2G interferometers are out of phase and destructive, as shown in (e), while the third groove helps resemble the interference pattern of H-1G structure, as shown in (d) and (f). For active fluorescence, by adding extra grooves the interference effect is gradually increased (g–i). structures as shown in Figure 5.2(f), the oscillation amplitude is almost recovered compared with the result of H-1G. In contrast, while adding grooves with 320-nm pitch in the active scheme, the oscillation amplitude gradually increases as shown by Figures 5.2(g)-(i) . This is due to the dominant second-order SPP-mediated inter- ference in the active scheme and the gradually enhanced SPP reflection coefficient. To better highlight this effect, the interferograms at λ = 720 nm for both passive and active H-N G structures (N = 1, 2, 3) are plotted in Figures 5.3(a) and (b), re- spectively. By comparing the blue (H-1G) and green (H-2G) line in Figure 5.3(a), the SPP contribution of the inner groove of H-2G structures still dominates the in- terference although the outer groove is slightly longer. This can be explained as follows: (1) SPPs generated by the outer groove are partially scattered by the in- 74 passive transmission active fluorescence (a) Hole-1Groove (b) Hole-1Groove λ = 720 nm λ = 720 nm 10 Hole-2Groove 1.5 Hole-2Groove Hole-3Groove Hole-3Groove 8 IF,n,r IT,r 6 1.0 4 2 0 0 1 2 3 4 5 6 1 2 3 4 5 6 Arm length, RG (μm) Arm length, RG (μm) Figure 5.3: Passive and active transmission interferograms for bullseye structures with different number of grooves at a fixed wavelength 720 nm. Passive transmission (a) and active fluorescence transmission (b) interferograms as a function of the arm length at a fixed wavelength 720 nm. In the passive case, since the scatter number is proportional to the circular groove length, the SPP contribution is enhanced while increasing the arm length, thus resulting in stronger oscillation. In active cases, the SPP amplitude generated is not affected by the existence of the groove if removing the pump effect. Therefore, due to the ohmic losses, the SPPs contribution is reduced while increasing the arm length, as evidenced by the the amplitude envelopes in (b). ner groove before reaching the central hole; (2) the groove pitch 320 nm does not accurately match half of SPP wavelength at 720 nm although very close. The en- hanced oscillation amplitude for H-1G and H-3G structures while increasing the arm length is due to the concentration effect of the longer circular groove, which generates stronger SPPs at the nanohole location. However, for the active scheme, the initial amplitude of SPP generated by the decay of the excited emitters at the nanohole location is constant for H-N G with varied arm length due to the identical nanoholes. Because of the ohmic loss, the oscillation amplitude in the active scheme is grad- ually decreased while increasing the arm length. The small oscillation amplitude for short arm length in Figure 5.3(b) is because of the normalization process, where the intensity over most of the fluorescence wavelength range is either enhanced or suppressed. From the above results and discussion, it is clearly shown that (i) for the passive scheme, the first order dominates the SPP contribution; (ii) while for the active scheme, the second-order SPP prevails the interference of the transmitted light. In order to obtain the contribution of the other modes, the interferogram at each 75 passive transmission, FFT active fluorescence, FFT m=1 2 3 4 5 6 m=2 4 6 800 (a) (d) 1.6 Energy (eV) 750 1.7 700 1.8 650 1.9 Wavelength, λ (nm) 1.9 (b) (e) 700 1.8 1.7 6 750 log(amplitude) 1.6 800 4 (c) (f ) 1.6 750 1.7 2 700 1.8 1.9 0 650 0 20 40 60 0 20 40 60 Wave vector, k (μm-1) Wave vector, k (μm-1) Figure 5.4: Fourier transform of the passive and active transmission interferograms. (a,d), (b,e) and (c,f) show the results of H-1G, H-2G and H-3G structures, respectively. For the passive case, up to the fifth order multi-trip is observed. For active case, only even orders (e.g., 2, 4 and 6) are observed. The dashed gray lines denote the theoretically calculated mkSPP for different orders by FDFD. wavelength can be transformed to momentum space through discrete fast Fourier transform (FFT), using the following equations: N −2πi X (j−1)(M −1) fbF/T (M, λ) = IF/T,nr/r (RG,j , λ) × e N , M = 1, 2, ..., N. (5.4) j=1 2π k = (M − 1) , M = 1, 2, ..., N. (5.5) RG,max − RG,min where N is the number of data points in each interferogram (i.e., 231 in this study), RG,j = (j − 1) × 0.025µm + 0.25µm, RG,max and RG,min are the maximum and minimum arm length of all the interferometers. Using Eq. (5.4) for the wavelength resolved interferograms (Figure 5.2), the FFT results for both passive and active schemes are calculated. Then, the results are transformed to the momentum space by Eq. (5.5), as shown in Figure 5.4. Note that for better comparison, Figures 5.4(b,e) 76 are vertically flipped. Different order modes are clearly observed in both cases, as labeled by the black arrows on the top of Figure 5.4. It is seen that only even-order modes exist in the active case, and up to the sixth order is observed in H-3G structure. For the same mode, its amplitude decreases for shorter wavelength because of the larger ohmic losses. The dispersion of SPP wave vector kSPP = 2π/λSPP = nSPP k0 in a three-layer system (air/40nm Cr3+ :MgO/Ag) is numerically calculated using FDFD method [116] and the real part is plotted in dashed gray lines (labeled as m = 1). The calculated results match well with that extracted by FFT (i.e., the first bright color bands in Figure 5.4(a)-(c)). For the higher-order modes, as illustrated in Eq. (5.2), their wave vectors can be expressed as mkSPP and are also plotted in Figure 5.4, which clearly match well all the bright bands and further verify the FFT analysis. The m values are labeled by the corresponding black arrows. Also note that in Eq. (5.2), although the cross terms of two different orders (i.e., m1 and m2 ) slightly affect the lower order (|m1 − m2 |), the higher order (|m1 + m2 |) observed in Figure 5.4 does not change. This further supports that the observed sixth-order mode originates from the six-trip SPPs rather than the combination of two lower orders. In the passive scheme (Figs. 5.4(a-c)), due to the design of the groove grating pitch (∼ λSPP /2), the contributions of the first- and third-order SPPs in H-2G structure are significantly suppressed compared with H-1G and H-3G structures. In other words, the amplitude of the second-order SPPs in H-2G (see Figure 5.4(b)) increased compared to that in H-1G. Thus by the proper design of the groove grating, in specific wavelengths, the second-order SPPs can be tuned to be even stronger than the first order in the passive scheme, as observed in short wavelength range, ∼650 nm in Figure 5.4(b). In addition, although the second-order SPPs originate from a nanohole while the third-order SPPs come from a much longer circular groove, their 77 contributions are comparable to each other in H-1G and H-3G structures. 5.4 Plasmonic interferometry under different illu- mination conditions Figure 5.5: Comparison of passive and active plasmonic interferometers under bottom illumination. (a) and (b) are side-view schematics of bottom illumination of passive and active schemes, respectively. (c) and (d) are the corresponding wavelength resolved interferograms. (d) and (e) are the FFT results, together with the theoretically calculated mkSPP results (dashed gray lines). Only even-order SPPs-mediated interference effects are observed. In addition to top illumination, the optical responses of both passive and active schemes under bottom illumination were also investigated, as shown in the schemat- ics of Figures 5.5(a,b). For the passive case, the chip was flipped; while in the active case, the spectra was acquired through the same objective, used for both excitation 78 and collection of the fluorescence emission. Following the same analysis procedures described above, the measured wavelength resolved interferograms are shown in Fig- ures 5.5(c,d). Note that for simplicity, only the results of H3G structures are shown here. Compared with Figures 5.2(f,i), the results of active scheme are similar, which confirms the theory that the fluorescence modification originates solely from inter- ference effects mediated by SPPs that are generated by excited emitters within the nanohole; the passive results under bottom illumination show similar oscillation pe- riod (i.e., ∼ λSPP /2) to the active scheme, which are totally different from that of passive scheme under top illumination. The corresponding FFT results are shown in Figures 5.5(e,f). For both schemes, only the even-order SPPs (up to the fourth order) are observed and this can be well explained by the mechanism shown in Fig- ures 5.5(a,b). Furthermore, the extracted mkSPP value match well the FDFD results, as indicated by the dashed gray lines. Based on the theory, it is straightforward to infer that the fluorescence modula- tion should be independent of the specific excitation conditions, being only affected by the structural parameters of the interferometer and by changes in the refractive index of the dielectric materials. To demonstrate that coherent excitation is not required to achieve fluorescence modulation, a broadband thermal light source (a Xenon arc lamp combined with a 600 nm short pass filter) was used to excite the emitters in both a H-3G plasmonic in- terferometer and a reference isolated hole, as shown in Figure 5.6(a). The transverse spatial coherence was tuned by employing K¨ohler’s illumination [117]. Specifically, by increasing the diameter of the microscope condenser aperture diaphragm, the transverse wavevector of the incident light can be made to span a broader range, which in turn determines a decrease in the spatial coherence at the hole location [117]. Figure 5.6(b) reports normalized fluorescence transmission intensity ratios for 79 (a) (b) 2.0 Dq = 9° 1.5 Dq = 18° Dq = 36° λexc<600nm IF,n,r 1.0 0.5 0 (c) (d) 2.0 q = 0° q = 5° 580nm<λexc<600nm 1.5 q = 8° q = 12° IF,n,r 1.0 0.5 0 (e) (f ) 2.0 1.5 IF,n,r 1.0 0.5 580nm<λexc<600nm 0 650 675 700 725 750 775 Wavelength, λ (nm) Figure 5.6: Fluorescence modulation induced by plasmonic interferometry: role of coherence, spectral bandwidth, and incidence angle of the external light source. (a) Schematic of a plasmonic interferometer with embedded emitters illuminated by a broadband thermal light source (with excitation wavelengths < 600 nm and variable transverse coherence length, achieved by changing the subtended angle ∆θ, see Methods). (b) Corresponding normalized fluorescence intensity ratios measured at ∆θ = 9◦ (blue), 18◦ (green), and 36◦ (red). (c) Quasi-monochromatic (580 – 600 nm) top illumination with varying angle of incidence θ. (d) Corresponding normalized fluorescence intensity ratios with θ = 0◦ (normal incidence, black), 5◦ (blue), 8◦ (green) and 12◦ (red). (e) Quasi-monochromatic (580 – 600 nm) bottom illumination. (f) Corresponding normalized fluorescence intensity ratio. The fluorescence modulation is independent of the specific excitation conditions (coherence, bandwidth, or angle of incidence), as evidenced by the vertical dashed lines in b, d and f. The arm length of the measured interferometer is 6 µm, the grating pitch is 320 nm. various subtended angles ∆θ = 9◦ (blue), 18◦ (green), and 36◦ (red), showing no ap- preciable changes in the observed modulation. Although the emitters are excited by an external light source with low spatial coherence, they retain temporal coherence since the emission occurs from a subwavelength cavity. Indeed, any given photon de- tected in the far field has to be transmitted through the nanohole and it can originate from two main optical paths, i.e. (1) direct emission from an excited emitter or (2) SPP excitation, round-trip propagation, and diffractive scattering followed by trans- mission through the same nanoaperture. Since the two paths are indistinguishable 80 due to the subwavelength nature of the aperture, their complex amplitude probabil- ities (corresponding to the complex field amplitudes E0 and ESPP ) can be summed up in order to calculate the total far-field fluorescence intensity. This leads to a coherent interference process that is independent of how the emitter is excited (i.e. either coherently or incoherently) provided the temporal coherence of the emitter is sufficiently high. To prove that the fluorescence modulation is also independent of precise align- ment of the external source, a quasi-monochromatic source (580 – 600 nm) was used to illuminate the same active plasmonic interferometer with varying angles of incidence (Fig. 5.6(c)). Figure 5.6(d) shows that the measured normalized fluores- cence intensity ratios are also invariant for different incident angles: θ = 0◦ (normal incidence, black), 5◦ (blue), 8◦ (green) and 12◦ (red). In addition, the measured optical response of the sensor under bottom illumi- nation (Figure 5.6(e)) is shown in Figure 5.6(f). This configuration is especially attractive since for conventional passive plasmonic interferometry the light source and the detector generally need to be placed on opposite sides of the sample[11, 45, 47, 91, 102]. Moreover, to measure biological samples or liquids, a microfluidic channel is typically required to ensure uniform sample delivery and illumination, thus adding complexity to the sensing architecture. Here we show freedom from that constraint in the novel design, whereby pump and detection can be achieved from the same (i.e. the substrate) side of the sensor. This arrangement does not re- quire a microfluidic channel, and solves the common problem arising from potential biological sample damage occurring under direct illumination. Despite the significantly different illumination conditions, the experimental re- sults in Figure 5.6 demonstrate that the optical response of plasmonic interferometers 81 with embedded light emitters is no longer affected by coherence, spectral bandwidth, and incidence angle of the excitation source, thus providing for a more reliable sens- ing architecture compared to passive interferometry. The spectroscopic capabilities can still be retained by employing a broadband light emitter such as Cr3+ :MgO. 5.5 Biochemical sensing based on active plasmonic interferometry Figure 5.7(a) shows a schematic of a sensing experiment performed under top illu- mination. Two representative liquids with slightly different refractive index (∆n ≈ 0.05) were delivered onto the metal surface of the active plasmonic interferometers using a microfluidic channel, resulting in a change of the relative SPP optical path length equal to 2nSPP RG . The pitch of the 3-groove grating in the H-3G plasmonic interferometer was designed to be 230 nm to keep the SPPs reflected by different grooves in phase while flowing the liquid dielectric materials (with n ≈ 1.35). An additional 10nm-thick Al2 O3 film was deposited by atomic layer deposition (ALD) to protect the surface of the emitting layer from potential chemical contamination. The inset to Figure 5.7(b) shows the normalized spectra through an isolated single hole in the presence of water (blue line, n ≈ 1.33) and isopropanol (red line, n ≈ 1.38). No appreciable difference is observed, suggesting that the change of local density of optical states has negligible effects on light emission. In striking contrast, SPP- mediated interference in a H-3G interferometer can determine a dramatic spectral change in the normalized fluorescence spectra and corresponding intensity ratios, as evidenced in Figures 5.7(b) and (c), respectively. A figure of merit for this sensing scheme can be defined as follows: 82 (a) −2 (b) 3 x 10 RG=4.05μm Single water hole IF,n (a.u.) 2 isopropanol 1 0 (c) 2 ~22nm 1.5 IF,n,r 1 0.5 (d) 3,000 2,000 FOMI (%/RIU) 1,000 0 -1,000 650 700 750 800 Wavelength, λ (nm) Figure 5.7: Biochemical sensing experiment in a microfluidic channel: top excitation and bottom detection of fluorescence modulation by plasmonic interferometry. (a) Schematic of a circular H-3G plasmonic interferometer with embedded Cr3+ :MgO emitters under top illumination. Transmitted fluorescence is detected from the bottom side. Molecules of isopropanol are also shown (not to scale). (b) Normalized fluorescence spectra transmitted through the nanohole of a circular H-3G plasmonic interferometer with arm length RG = 4.05 µm when deionized water (blue) or isopropanol (red) are flown in the microfluidic channel (not shown in a). Inset reports the respective reference spectra transmitted through an isolated hole in water (dashed blue) or isopropanol (dashed red). (c) Corresponding normalized fluorescence intensity ratios. A clear wavelength shift is observed as a result of the refractive index change. (d) Representative figure of merit F OMI for this specific plasmonic interferometer, calculated according to data in c and using equation (5.6). 83 IF,n,r,isop. − IF,n,r,water 100% F OMI = × , (5.6) IF,n,r,water ∆n describing the relative intensity change per refractive index unit (RIU). Figure 5.7(d) shows the estimated F OMI , with values up to 2,600% RIU−1 . A ∼22 nm red shift is also observed when going from water to isopropanol, with a ∼4% refractive index change sufficient to turn constructive interference into destructive, and vice versa (see Figure 5.7(c)). The spectral figure of merit of this sensor can therefore be calculated as F OMλem = ∆λem /∆n = 440 nm RIU−1 . To illustrate the advantage of the same-side illumination and fluorescence detec- tion scheme as previously discussed in Figures 5.6(e) and (f), we performed sensing experiments by delivering a single micro-droplet of liquid with different concentra- tions of isopropanol dissolved in water directly on top of an individual H-3G interfer- ometer (with RG = 5 µm and 230 nm grating pitch), as schematically shown in Fig- ures 5.8(a) and (b). The embedded emitters were illuminated from the bottom side, and fluorescence emission was detected from the same side. Normalized intensity ratios are displayed in Figure 5.8(c) for individual water/isopropanol micro-droplets with varying isopropanol concentrations, from 0 to 50%, in steps of 10% correspond- ing to a refractive index change of ∼0.4%. Clear wavelength shifts are observed, with a red shift of sim11 nm determined by a refractive index change of ∼0.025, in agreement with the results reported in Figure 5.7. The estimated sensitivity to refractive index change of the proposed sensing scheme (limited by the resolution of our spectrometer) is < 5×10−4 , using <1 pL sample volumes. The performance of the proposed sensing scheme may be limited by the low flu- orescence intensity compared with the external pump light and the requirement for spectral analysis as described above. However, such shortcomings are not intrinsic to the proposed platform. For example, by using more efficient light emitters, the fluo- 84 (a) (b) (c) 736 nm 796 nm 765 nm 1.5 50% 40% 30% IF,n,r 20% 10% 1.0 0% 725 nm 753 nm 783 nm 710 720 730 740 750 760 770 780 790 800 Wavelength, λ (nm) Figure 5.8: Biochemical sensing experiment on a micro-droplet: same-side (i.e. bottom) excitation and detection of fluorescence modulation by plasmonic interferometry. (a) Schematic of a micro-droplet on an array of H-3G plasmonic interferometers with embedded light emitters. Selective detection of the droplet is accomplished by bottom illumination of a specific plasmonic interferometer and detection of the modulated fluorescence (red) transmitted through the nanohole. (b) Schematic showing bottom illumination and fluorescence detection for an active H-3G plasmonic interferometer. (c) Normalized fluorescence intensity ratios vs. λem measured through the nanohole of a H-3G plasmonic interferometer with RG = 5 µm, sequentially covered by single micro-droplets with different concentrations of isopropanol: 0% (deionized water), 10%, 20%, 30%, 40%, and 50%. Red shifts of the constructive interference peaks are clearly observed (the dashed lines are guides to the eye). For clarity, the curves corresponding to 10–50% isopropanol have been vertically shifted by a constant offset value, in multiples of 0.1. 85 rescence signal can be greatly enhanced. The light intensity throughput can also be enhanced by integrating a large number of identical interferometers as well as using different configurations (e.g. circular slit-groove plasmonic interferometers that are characterized by a polarization-independent response and higher throughput [102] compared with the bullseye structure employed in this work). Moreover, by em- ploying shorter wavelength excitation (e.g. ultraviolet light sources), together with bandpass filters or emitters with narrow fluorescence wavelength range, the spectral analysis required here due to the small pump effect can be entirely eliminated. 5.6 Conclusion In conclusion, we have systematically studied and compared the optical responses of the passive and active hole-groove plasmonic interferometers. Different from the passive scheme where SPPs are generated by both the nanohole and the circular groove, in the active scheme, where solid-state light emitters are embedded in the subwavelength cavities, SPPs are generated by the excited emitters in the nanohole location, which results in the existence of only even-order modes. By proper design of the groove grating pitch and the number of grooves, even- and odd-order modes can be selectively altered. The wavelength resolved interferogram, together with FFT, allows the observation of up to the sixth-order (i.e., optical path ∼ 6RG ) mode. In addition, we have realized optical interference at the nanoscale with incoher- ent external light sources through the active scheme. The spatial confinement of the emitters results in the generation of coherent SPPs regardless of the coherence state of the external excitation source. Such a platform enables a novel sensor design that allows measurements in a broader set of environments than previously thought 86 possible. In contrast to conventional plasmonic interferometry, this sensing architec- ture eliminates the need for a coherent, broadband, and precisely aligned external light source, without sacrificing the figures of merit (2,600% RIU−1 and 440 nm RIU−1 ). And such a sensing scheme enables same-side excitation and detection of fluorescence modulation which allows for direct exposure of the top surface to the analyte of interest. This eliminates the need for a microfluidic channel to control sample flow, and addresses the problem of misalignment induced by lensing effects arising from the droplet surface curvature. Finally, the proposed sensing technology can be further extended to include various light emitters other than Cr3+ :MgO, such as semiconductor quantum dots [118–120], as well as electrically excited emitters [121–123] to realize fully integrated, electrically-driven biochemical sensors. Chapter Six Conclusion 88 In this thesis, the features and applications of plasmonic interferometry, a new sub- field of plasmonics, have been thoroughly investigated. A plasmonic interferometer is composed of a through slit milled on a metal film flanked by several grooves on its side/sides. When coherent light is normally incident upon the plasmonic interferom- eter, diffractive scattering by the groove will excite propagating surface plasmons. At the slit location, the incident beam and the propagating surface plasmon waves originating from the adjacent groove/grooves would interfere with each other. The light transmitted through the slit carries information about the optical properties of the interface materials. A plasmonic refractometer was built upon this feature. Confined at the metal/dielectric interface, surface plasmon waves are very sensitive to the environment they travel through and would reflect any dielectric change in the form of intensity change and wavelength shift of the transmitted light. A plasmonic interferometer was designed and fabricated, which is capable of detecting glucose concentrations down to the physiological levels in saliva. Coupled to dye chemistry, the detection sensitivity and selectivity was further enhanced. In addition, function- alizing the surface of a plasmonic interferometer with given antibodies, an alternative approach to improve detection specificity is demonstrated. A detailed study on the kinetics of an antibody-antigen interaction, the binding of C-reactive protein to Im- munoglobulin G, is presented. To better understand plasmonic interferometry for developing biochemical sensors with improved performance, the plasmonic interfer- ometers in a generalized circular geometry was explored, which embraces the linear plasmonic interferometers as a subsection. In then end, with light emitters directly embedded into cavities of plasmonic interferometers, active plasmonic interferome- try was accomplished, which generates surface plasmons even by a light source with extremely low coherence. Plasmonic interferometry demonstrates great potential for the development of 89 next-generation ultra-sensitive biochemical sensors. From this thesis, some potential efforts can be exerted toward this goal in the following aspects: (1) Miniaturization. Comparing with traditional surface plasmon resonance systems based on the Kretschmann configuration, which works in a single wave- length and consumes the sample in a relative large volume, the proposed plasmonic interferometry is capable of providing results over a broad wavelength range with a sample volume as low as femtoliter. For active plasmonic interferometry, the light source and the imaging system can be coupled on the backside of the interferometers, which can be achieved using a dichroic filter or optical fiber. So in a system with active plasmonic interferometry coupled to surface functionalization, the prototype can come in the form of a small box equipped with a light source and a camera, and a functionalized chip above it, whose surface is exposed to biochemical analytes of interest. (2) Multiplexing. Since a plasmonic interferometer takes merely an area of 15 × 25 µm2 , thousands of interferometers can be accomandated in a chip with a surface of 1 cm2 . 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