Luận án: Ghép cặp cộng hưởng quang học nano cho xét nghiệm sinh hóa định lượng

Thesis for the Degree of Doctor of Philosophy Nano-dimensional Resonant Optical Coupling for Quantitative Optical Biochemical Assays By Nhu Hoa Thi Tran Department of Nanoscience and Technology Major in Nano-Physics Graduated school Gachon University Thesis for the Degree of Doctor of Philosophy Nano-dimensional Resonant Optical Coupling for Quantitative Optical Biochemical Assays A dissertation submitted to Gachon University in partial fulfillment of requirements for the Degree of Doctor of Philosophy By Nhu Hoa Thi Tran December, 2017 Department of Nanoscience and Technology Major in Nano-Physics Graduated school Gachon University This dissertation is dedicated to my big family TABLE OF CONTENTS List of figures Abbreviations ABSTRACT CHAPTER 1. INTRODUCTION TO OPTICAL TECHNOLOGY FOR BIOCHEMICAL DIAGNOSIS. Surface plasmon resonance (SPR). Fluorescence for biomedical diagnosis.

Waveguide devices at a sensing platform. Advantage of optical technology. Label-free sensors. Fluorescence-based sensors.

General properties of SPR. Fundamentals of surface plasmons. Excitation of surface plasmons. Surface plasmon polariton (SPP) dispersion relation.

SPR sensor performances. Fluorescence detection and its enhancement. Waveguide mode and the power transmission. SURFACE PLASMON RESONANCE ENHANCED FLUORESCENCE DETECTION.

Motivation and aim of enhanced fluorescence detection. Experimental apparatus, method and techniques. Materials and reagents. The optical configuration for biosensing.

Results and discussion. Theoretical model to determine the bimetal and their thicknesses. Highly sensitive detection of human genomic DNA via SPR metal coated prism on a reflection geometry. Fluorescence system and enhancement factors.

Dielectric metal based multilayers for SPR with biological diagnosis application. LABEL-FREE IMMUNO-BIOSENSOR BASED ON LIQUID CLADDING MODULATION IN OPTICAL FIBER. Motivation and aim of label-free optical biosensors via fiber cladding modulation. Experimental apparatus and techniques.

Materials and reagents. Surface modification for BSA bonding. Label-free fiber optical sensor system. Capture probe and analyte liquid preparation.

Results and discussion. Absorbance water-glycerin solutions of the core polymeric SiO2 material used as sensitive cladding. Analysis of sensitivity of interaction between under the plastic fiber core on the multimode fiber and test medium BSA protein. CONCLUSION AND OUTLOOK.

PEER REVIEWED JOURNAL PUBLISHCATIONS. 138 iii List of figures Figure 1.1 Schematic presentation of the surface plasmon oscillations and electromagnetic fields of SPs propagating on a surface in the x direction.2 Magnetic field exponential decay with the distance from interface in both medium.3 Schematic of excitation of SPR: (a) Otto.4 The steps in ELISA approache: (1) recognition element (antibody) is coated to the substrate; (2) the present antigen links to the capture antibody; (3) labelled antibody binds to antigen; (4) the enzyme changes the optical properties of a substance (absorption).5 BIACoreTM system based on SPR configuration.6 (a) What was expected to happen to the light. (b) What actually happened to the light [Crisp et al.7 History of Attenuation [Briley, 1988].8 Waveguide coupler in end fire configuration.9 (a) An asymmetric slab waveguide with guided mode: cs    900. (b) Cover radiation mode cs    cl.

(c) Substrate-cover radiation mode    cs .1 Schematic of surface-plasmon wave propagating along the interface between metal and dielectric layers.2 Surface plasmon excitation conditions. (a) Surface plasmon waves are excited by incident light using a prism in Kretschmann’s configuration. (b) Illustration of phase matching between the incident light and surface plasmon waves.3 SPP dispersion is the solid black line. The asymptotic surface plasmon frequency is shown as the dashed line.

The shaded region corresponds to the allowed wave vectors photons may have for a given frequency within the dielectric layer.4 Dispersion curve for light propagating in a prism of index n with evanescent coupling to the SPP mode of a metal-air interface. For light of frequency 0 within the prism point corresponds to the critical angle c point b the excitation of SPPs by evanescent coupling and c the grazing of light along the interface with the prism. The cyan shaded region corresponds to the allowed wave vectors within the prism.5 Jablonski diagram showing the de-excitation pathways of a molecule.6 Diagram of Beer-Lambert absorption of a beam of light as it travels through  cuvette of width l.7 Excitation of surface plasmons by waveguide coupling. (b) Photograph of R-mode set up.

(c) Bimetallic layer of gold (Au), and silver (Ag) as the SPR coating material. (d) Bachground signal forming from dark noise. (e) Green color on the surface chamber.2 The transmission geometry based optical system for fluorescence enhancement (T-mode). (b) Photograph of the T-mode set up.3 (a) Schematic of the principle of SPR excitation for enhancement of fluorescence.4 AFM image of the surface of a Ag film (50 nm) deposited on a Si substrate (1 × 1 m2 scanned by a Si3N4 cantilever tip of its tip radius less than 10 nm).

(a) A top view (b) The three-dimensional counterpart.5 AFM images for the surface morphology over a scanned area of 1×1 m2.6 (a) The gel image of amplification results of 283 bp gene fragment in IFN-. Land M shows a 100 bp DNA size maker, and the relative intensity scales of the target amplicons were shown below each gel image. (b) Esimated values of output power signal of dsDNA solutions with versus their concentration.7 Results of DNA conjugated SYBR Green I using different dilution factors for DNA after PCR reaction. Land M shows a 100-bp DNA size maker, and the relative intensity scales of the target amplicons were shown below each gel image.

(b) Visual detection of DNA- conjugated SYBR Green I under UV light.(c) Fluorescence spectra of 10-3 dilution sybr green I with 800 pg/L of DNA after PCR reaction.8 The enhancement factor calculated via R-mode and T- mode configuration using the DNA template concentration of 800 pg/L.9 Three Kretschman prism configurations for excitation of surface plasmon polaritons.10 Reflectance as a function of the incident angle of a pair of doubles layers with 10 nm of Ag and different thicknesses of teflon layer.11 Reflectance as a function of the incident angle (R- curves) of a pair of doubles layers with different thicknesses of teflon and silver layers.12 Dielectric-metal thickness optimization of conventional LRSPR with a pair of doubles layers of (teflon –silver)2.13 Comparison of the depth-to-width ratio (DWR) among three different Teflon 1 layer thickness (550 nm – 600nm – 650 nm).14 R- curves of three different kind of LRSPR configuration.1 Illustration the schematic of the modification of silica surface (a) Yielding an amin modified surface by ATPES and (b), (c) Yielding a carboxyl modified surface.2(a) FTIR result of SiO2 surface (b) FTIR result of the modification of SiO2 surface.3(a) Surface of the cladding mode optical fiber. (b) Schematic of the experiment setup for the label-free optical biosensing.4 Refractive indices of glycerol at various concentrations.5 Schematic diagram of experimental arrangement used to measure the characteristics of the cladding mode coupler (a) Water– glycerol solution (b) BSA-PBS buffer.6 (a) Response of glycerol concentrations (volume to volume ratio) for the cladding mode fiber. (b) Fiber sensor response to the glycerol concentration.7 Waveguide mode mismatch for an optical biosensor.8 Time-dependent optical power transmitted via BSA solutions at various concentrations (a) Concentration range of 10−100 ng/mL. (b) Concentration range of 100−1000ng/mL.

105 viii Abbreviations ATR Attenuated total reflection RI Refractive index RIU Refractive index unit S Sensitivity SPR Surface plasmon resonance NA Numerical aperture DNA Deoxyribonucleic acid LOD Limit of detection PBS Polarizing beam splitter SNR Signal-to-noise ratio PCR Polymerase chain reaction TIR Total internal reflection PDMS Polydimethylsiloxan SPW Surface plasmon wave ix ABSTRACT We report development of optical devices and platforms that rely on nano-dimensional optical coupling, which permit the highly sensitive diagnosis of biochemical and biomedical substances in a simple format. We present two types of optical diagnostic devices with their performance, i., the label-free device and fluorescence detection (label-aided) based ones for quantitative assays of biomolecules including proteins and DNAs. First, we present the label-free optical biosensors based on optical fibers, which sensitively respond to introduction of analyte molecules for point-of-care-testing (POCT) application that demands the real-time quantitative diagnosis of biochemical analyte concentration in a compact device structure. We demonstrate the modulation of fiber optical modes by means of liquid cladding which is formed by introducing analyte liquid into the fiber core.

Unlike other types of label-free optical sensors such as optical fiber sensors based on surface plasmon resonance (SPR), the presented optical sensors do not need any additional coatings on its core, such as thin metal film or high index dielectric films, with the great advantages of x both the realization of the simple structure device and highly reproducible sensor performance. The fiber (waveguide) mode mismatch between interfaces of different claddings can be used for highly sensitive change in optical transmission, allowing the label- free bio-chemical diagnosis to be sensitive enough, making these sensors comparable to the plasmonic fiber sensors. It is also unveiled that the device presented has the great inherent advantages of the device-length independence of sensing performance in contrast to the other types of fiber based sensors. We describe its working principle by building up the mathematical modeling of waveguide mode mismatch and liquid analyte absorption of evanescent fields.

We experimentally achieve the limit of detection (LOD) of glycerol concentration of 0.02 × 10-6 refractive index unit) and LOD of BSA concentration of 3. This dissertation also reports the enhancement of fluorescence by near-field coupling of fluorophores with surface plasmons using two different fluorescence detection platforms, i., an upright and inverted types of a flurorescence microscopes. Surface plasmons that enhance the fluorescence and reduce the photobleaching, can be generated by nanoscale thick films of metal layers deposited on a xi slide glass used in a microscope. We investigate various metal thinckesses of gold, silver and their combination to optimize the decaying depth of the evanescent field SPR wave into the sensing region where fluorophores are dispersed.

It is found that the bimetallic layer of silver (52 nm) and gold (2 nm), which is determined by numerical calculation, provides the maximized depth of the SPR evanescent field with strongest coupling of fluorophores with the plasmons at both spectral channels of excitation (470 nm) and emission (525 nm) wavelengths. We experimentally demonstrate the enhancement of fluorescence emitting from interferon- double stranded DNAs (283 bp, polymerase chain reaction product) conjugated with the fluorophores (SYBR Green I), with the enhancement factor estimated as 45-fold. It is noted that this enhancement is achieved only by plasmonic coupling with fluorophores at the emission channel without excitation enhancement. This enhancement can lead to the LOD of DNAs concentration of ~80 fg/L (1.6 nM) in an upright format of a fluorescence miscroscope.

We also achieve the extended photobleaching-free range of DNA concentrations in the fluororescence based assays, allowing photostable diagnosis of the analytes. xii KEYWORDS: Optical biosensor; label-free sensor; fiber optical sensor; Nano-biophotonics; Surface Plasmon Resonance; fluorescence enhancement; fluorescence microscope, DNA assay. INTRODUCTION TO OPTICAL TECHNOLOGY FOR BIOCHEMICAL DIAGNOSIS 1. Surface plasmon resonance (SPR) Althouhg the phenomenon of SPR was known more than a century ago.

In 1902, Wood observed an uneven distribution of light in a reflected spectrum from a metallic diffraction grating [Wood, 1902]. The 60 years later, Thurbadar recognized the adsorption in the reflectivity of a metal film [Turbadar, 1959]. However, both of them did not identify these observations as surface plasmon resonance (SPR). In 1968, Otto explained with same kind of the results; the drop in reflectivity of the metallic film based on to SPR phenomenon [Otto, 1968].

The original of surface plasmon is based on the Maxwell’s equations, where the free electrons charge of the metal are considered as the plasma. Measurement of the electron-plasma oscillation in metals is highly dependent on the dimensions of aggregative longitudinal 1 excitations of the conductive free-electron gas (see Figure 1.1), and plasmon are the quanta of such charge-density oscillations over a surface. The charge-density fluctuations can be existed in the bound at the interface between metal and dielectric, where they propagate as waves along the interface forming what called surface plasmon.