This research presents a novel biosensor which utilizes the refractive index sensitivity of a fiber optic long-period grating. The long period grating couples light from the forward propagating guided core mode of a single-mode fiber into discrete circularly symmetric cladding modes. Due to imperfections in the cladding surface, loss bands are seen in the transmission spectrum corresponding to the coupled wavelengths. Based on the phase-matching condition between the coupling and coupled modes, the loss bands shift with changes in the refractive index of the surrounding medium. The grating surface is chemically treated to covalently bond antibody to the cladding of the sensor. Treatment with the proper antigen increases the effective index seen by the cladding modes and affects the placement of the loss bands. This sensor demonstrates specific antigen binding capacity with loss band shifts of 10 nm or more. The device offers several advantages over the widely used Enzyme-Linked Immuno-Sorbent Assays. Diagnostic applications can be expanded beyond the tests presented here. / Master of Science

Cette thèse porte sur les mélanges d’onde non linéaires qui peuvent avoir lieu dans le champ évanescent de nanofibres de silice. Nous nous sommes plus particulièrement intéressés à la diffusion Raman stimulée qui est obtenue par l’interaction du champ évanescent très intense et un liquide non linéaire dans lequel baigne la nanofibre. Afin de mettre en évidence la diffusion Raman stimulée« évanescente », nous avons développé un modèle de simulation non linéaire dont le but est de déterminer les caractéristiques des nanofibres à réaliser. Le gain Raman modal est calculé afin de trouver le rayon optimal des nanofibres pour chaque liquide ou mélange de liquides possible. En considérant la puissance critique et le seuil de dommage de nos nanofibres, nous avons déduit la longueur minimale d’interaction. Les conditions d’adiabacité des parties évasées menant à la nanofibre sont également discutées. Ces spécifications nous ont amenés à développer une plateforme de tirage de nanofibres spécifiquement dédiée à ces expériences de non-linéarités évanescentes. Cette palteforme nous permet de tirer des nanofibres de diamètre allant jusqu’à 200 nm sur des longueurs de 10 cm, avec plus de 90% de transmission. Avec ces nanofibres, nous avons mis en évidence le premier ordre Stokes de l’éthanol dans le champ évanescent d’une nanofibre, ainsi que les premier et second ordres Stokes du toluène. Ces premières expériences sont en très bon accord avec nos simulations et ouvrent la voie à de nombreuses expériences en optique non linéaire. / The present PhD thesis explored nonlinear wave mixing with the strong evanescent field of nanofibers. The focus has been on the effect of stimulated Raman scattering which is activated by the interaction between such a strong evanescent field and the nonlinear liquid surrounding the nanofiber. In order to observe the stimulated Raman scattering, we investigated the nonlinear modeling to determine the needed characteristics of the nanofibers. The modal Raman gain was calculated to determine the optimal radius of nanofibers for each possible liquid. Considering the critical power and the damage threshold of our nanofibers, we found the minimum required interaction length. The condition of adiabacity of the tapers was also described. These specifications of nanofibers guide us towards the design of a proper pulling system. Several pulling systems and techniques are investigated for the fabrication of our specific nanofibers. We now are able to fabricate low loss uniform nanofibers of up to 10 cm long, a diameter down to 200 nm, with two identical low loss tapers by using our own designed translation stage pulling platform and implemented with the “variable heat brush” technique. With the achieved nanofibers, the Raman effect induced in the evanescent field was observed in both pure (ethanol) and binary mixture (toluene in ethanol) liquids. These first measurements are in good agreement with our simulation even without any fitting parameters in the modeling.

In recent years, optical near-field has received an ever-increasing attention owing to its ability to localise optical signals beyond the diffraction limit. Optical near-field is a non-propagating field existing in the close vicinity of a matter within a range less than the wavelength of the illumination light and it carries the high spatial frequency information showing the fine details of the matter. An optical near-field can be generated by a near-field optical microscope with a nano-aperture or a metal-coated fibre tip. However, common difficulties associated with this approach, such as a fragile probe, a low throughput and signal-to-noise ratio, and a slow response of gap controlling between the probe and the sample, make it less applicable. Alternatively, optical near-field can be produced by total internal reflection (TIR) occurring at the interface of a prism, which is capable of localising the electromagnetic (EM) field in the close vicinity of the interface. However, in this geometry, no confinement of the field can be achieved in the transverse direction, whereas, in most applications such as optical trapping, micro-fabrication and optical data storage, a transverse confinement of the light field is essential. In order to achieve a transverse confinement of the light field, maintaining the high spatial resolution of the optical near-field, and at the same time eliminating the drawbacks associated with the conventional near-field optical microscope, a novel near-field probe based on a high numerical aperture (NA) TIR objective combined with annular illumination has been developed recently. In this arrangement, an obstruction disk is inserted at the back aperture of the objective to block the light with a convergence angle lower than the critical angle determined by the refractive indices of the two media, resulting in a pure focused evanescent field in the second medium. The evanescent field produced by this method provides a useful tool for studying light-matter interaction at the single molecule level not only because of its high resolution but also due to its inherent merits such as no distance regulation, no heating effect and simple experimental setup. But, the most significant advantage that makes this method unique and superior to the other approaches in terms of producing the optical near-field is that it allows the dynamic control of the focal field by simply modulating the phase or amplitude or even the polarisation state of the incident beam before it enters the objective so that complex illumination beams can be generated, whereas in other fibre probe based approaches this goal is extremely difficult to achieve. To make use of such a novel near-field probe, a thorough theoretical and experimental investigation is required. A complete knowledge of the focused evanescent field is a prerequisite for a wide range of applications including single molecule detection, Raman spectroscopy, near-field non-linear imaging and near-field trapping. Therefore, it is not only necessary but also urgent to exploit the focusing properties of a focused evanescent field under complex field illumination both experimentally and theoretically and this is the major aim of this thesis. The complex fields, which are of particular interest in this thesis, are the radially polarised beam and the Laguerre-Gaussian (LG) beam, because the former owns a more compact circularly symmetric field distribution in the focal region when focused by a high NA objective, while the latter is capable of rotating a trapped particle by transferring the orbital angular momentum. Combining them with the focused evanescent field is potentially able to induce novel functions in the near-field region, which cannot be fulfilled by other near-field approaches. In this thesis, in order to generate these two types of beams, a single liquid crystal spatial light modulator (LCSLM) is employed to produce useful phase modulation to the incident beam. Experimental characterisation of an evanescent focal spot is performed with scanning near-field optical microscopy (SNOM), which is capable of providing the direct mapping of the focused evanescent field not only because of its high spatial resolution and its ability to detect the near-field and far-field signals simultaneously, but also due to the motion of the piezzo-stage enables a three-dimensional characterisation of the evanescent focal spot. In this thesis, a SNOM system with an aluminum coated aperture probe is implemented. The field distributions at both the interface and parallel planes with a small distance away from the interface are obtained. To verify the applicability of SNOM as a characterisation methodology, the field distribution in the focal region of a high NA objective illuminated by a linearly polarised plane wave is measured first. A focus splitting along the direction of incident polarisation is observed threedimensionally near the interface under such a circumstance. It has been demonstrated that the depolarisation effect plays an important role in determining the coupling behaviour of the light into the fibre probe of SNOM. The good match between the experimental results and theoretical predications confirms the validity of SNOM. Theoretical investigation of a tightly focused radially polarised beam is undertaken based on the vectorial-Debye diffraction theory because under the tight focusing of a high NA objective, the vectorial nature of the highly localised field has to be carefully considered in order to represent the field distribution accurately. The calculations on the focusing properties of a radially polarised beam suggest that the longitudinal field component in the focal region plays a dominant role in determining the overall field distribution. Direct measurement of the focused evanescent radially polarised beam in a three-dimensional manner near the interface is performed with SNOM. A highly localised focal spot is achieved in the close vicinity of the coverglass. The measured intensity distributions from SNOM show that correction of the focal spot deformation associated with a linearly polarised beam is achieved by taking advantage of the radially symmetric focal spot of a radially polarised beam. A smaller focal spot is acquired due to the dominant longitudinal polarisation component in the focal region, which possesses a more compact focal intensity distribution than that of the overall field. The experimental results demonstrate a good agreement with the theoretical expectations. The fact that a radially polarised beam is capable of eliminating the focus deformation often presented in the focal region of a high NA objective when a linearly polarised beam is employed can be very useful in many applications, including microfabrication using two-photon photopolymerisation technique. The theoretical study on the two-photon point spread function (PSF) of a radially polarised beam indicates that the focus elongation and splitting associated with a linearly polarised beam are eliminated and the achievable lateral size of the focal spot is approximately a quarter of the illumination wavelength, which is less than half of that under the illumination of a linearly polarised beam. A further reductiont of the lateral size can be expected by using annular radial beam illumination. The investigation on the focusing properties of LG beams has also been one of the major tasks of this thesis. Theoretical investigations of a focused evanescent LG beam suggest that the phase shift induced by the boundary effect when a light beam passes the interface satisfying TIR condition plays a vital role in determining the overall shape of the total field distribution. A severe focal intensity deformation is predicted theoretically in the case of focused evanescent LG beam illumination, which might involve new physical phenomena when applied in the near-field trapping. Such a focal intensity deformation is evidenced experimentally by the direct mapping result obtained from the SNOM probe. A quantitative cross-section comparison with the theoretical predication is conducted, which demonstrates a good agreement. To achieve a controllable optical trap and rotation in the near-field region, complex optical fields such as LG beams carrying orbital angular momentum, have been induced for the manipulation of a polystyrene particle. The influence of the focal intensity deformation on a near-field trapping has been thoroughly investigated. Rotation motion of the particle is examined by mapping the two-dimensional (2D) transverse trapping efficiency of the particle. Theoretical investigation reveals that a significant tangential force component is generated on the particle when it is illuminated by a focused evanescent LG beam. Such findings may prove useful in introducing a rotation mechanism in near-field trapping. The research investigations and methodologies described in this thesis provide a new approach to characterise the near-field focal spot under complex field illumination. It enhances the understanding of the novel near-field probe, thus opening the pathway for numerous near-field applications including optical trapping, two-photon excitation (photopolymerisation) and spectroscopy. The focal field rotation phenomena demonstrated in this thesis may prove particularly beneficial in introducing a rotation mechanism in near-field trapping using a focused evanescent field.

This thesis explores the application of coiled sapphire multimode optical fibers for evanescent wave chemical sensing in both the visible spectrum and the near infrared. As has been suggested in the literature pertaining to silica fibers, bending converts low-order modes to high order ones, which leads to more evanescent absorption and thus a more sensitive chemical detector. By coiling the fiber many times, it was expected that even greater sensitivity would be attained. Experiments were performed to investigate the sensor response to different solutions and to characterize this response. In the first of three experiments, the large absorption peak of water at 3μm was examined in order to compare the sensitivity of a straight fiber versus a coiled one. In the second experiment, the effect of increasing the number of coils was investigated, as was the response of the sensor to varying concentrations of water in heavy water. In the third experiment, methylene blue dye was used to investigate the extent of adsorption of dye molecules on the sapphire fiber and its persistence

Near-membrane and trans-membrane proteins and their interactions with the extracellular matrix (ECM) can yield valuable information about cell dynamics. However, advances in the field of nanoscale cellular processes have been hindered, in part, due to limits imposed by current technology. In this work, a novel evanescent field (EF) imaging technique is designed, modeled, created and tested for near-field imaging in the apical surface of cells. This technique and Förster resonance energy transfer (FRET) were used to investigate interactions between integrins on the cell surface and the ECM protein, fibronectin. The goal was to monitor changes in the integrin density at the cell surface as a function of clustering after binding to fibronectin on the microsphere surface. For the EF technique, quantum dot (QD)-embedded polystyrene microspheres were used to couple light into whispering gallery modes (WGMs) inside the microspheres; the resulting EF at the surface of the microsphere was used as a near-field excitation source with ~50 nm axial resolution for exciting fluorescently-labeled integrins. For FRET measurements (~10 nm axial resolution), QDs (donors) were coated on the surface of microspheres and energy transfer to red fluorescent protein (RFP)-integrin constructs (acceptors) studied. In both techniques, the QD-modified microspheres were mounted on atomic force microscope (AFM) cantilevers, functionalized with fibronectin, and brought into contact with fluorescently-labeled HeLa or vascular smooth muscle (VSM) cells. The results obtained from both methods show the clustering and activity of the integrins and are in good agreement with each other. Amsterdam discrete dipole approximation (ADDA) was used to study the effects of inhomogeneous surrounding refractive index on the quality factor and position of the WGMs due to the attachment of a microsphere to an AFM cantilever. WGMs of various QD-embedded microspheres mounted on AFM cantilevers were experimentally measured and shown to be consistent with the model.

Accurate diagnosis of active tuberculosis is required to improve treatment, reduce transmission of the disease and control the emergence of drug resistance. A rapid and reliable test would make a considerable contribution to the management of the TB epidemic, especially in HIV-burdened and resource-poor countries where access to diagnostic laboratories are limited. Surrogate marker antibody detection to mycobacterial lipid cell wall antigens gave promising results, in particular with cord factor. The specific advantage of using mycolic acids as lipid antigens in comparison to protein antigens is that mycolic acid is a CD1 restricted antigen with the ability to induce proliferation of CD4/CD8 double negative T-cells, which may explain the sustained antibody production in AIDS patients. Traditional end-point assays to detect anti-MA antibodies showed an unacceptable number of false positive and negative test results. Here a much improved biosensor method (the MARTI-assay, i.e. Mycolic acid Antibody Real-Time Inhibition assay) was developed to detect antibodies to mycolic acid in patient sera as surrogate markers of active tuberculosis. The test was assessed on an IAsys optical biosensor and gave an accuracy of 82%. The technology was transferred to an SPR (ESPRIT) biosensor to economise and simplify the assay. Mycolic acid containing liposomes were immobilized on the SPR gold surface pre-coated with octadecanethiol. The following parameters were optimized on the ESPRIT biosensor to enable reliable TB diagnosis: effect of degassed buffer, saponin blocking, first exposure to serum at low concentration and second exposure to antigen inhibited serum at high concentration. The IAsys biosensor system has a weakness in the double channel cuvette system, in which the channels often do not give matching results, while being ten times more expensive than the gold discs provided for the ESPRIT biosensor. The ESPRIT biosensor is provided with an adjustable laser setting to compensate for differences in the channel readings as well as an automated fluidic system that reduces variance from one sample to the next. First indications are that the test can also be used for prognosis of TB during treatment. It is hoped that the ESPRIT biosensor will improve the accuracy of the test to more than 90%. If the MARTI-assay technology could be made amenable for high throughput screening, it may provide the solution to the serodiagnosis of tuberculosis and monitoring of progress during TB treatment both in adult and children, thereby reducing the spread of TB within the communities. / Thesis (PhD)--University of Pretoria, 2009. / Biochemistry / unrestricted

The purpose of this research was to determine the effect of diameter fluctuations on the sensitivity of sapphire multimode optical fibers used as evanescent wave fluorescence sensors. It was predicted that fluctuations in the diameter of the fiber would act as a series of bi-tapers converting lower order modes to higher order modes increasing the evanescent wave penetration depth thereby increasing the excitation of a cladding of fluorescent fluid. Induced fluorescence from the fluid cladding would then couple back into the fiber more efficiently increasing the sensitivity of the sensor. The effect of coiling the fiber on the sensitivity of the sensor was also explored. Coiling the fiber converts lower order modes into higher order modes and increases the sensing length while maintaining a small probe size. However, coiling experiments produced unexpected results and in the course of studying these results a layer of material was discovered coating the surface of the sapphire fibers.

The mid-infrared (MIR) spectral range (2-20 m) is particularly useful for chemical sensing due to the excitation of fundamental rotational and vibrational modes. In the fingerprint region (10-20 m), most organic analytes have unique absorption patterns; absorption measurements in this region provide molecule-specific information with high sensitivity. Quantum cascade lasers (QCLs) present an ideal light source for (MIR) chemical sensing due to their narrow linewidth, high spectral density, compact size, and ease of fabrication of nearly any MIR wavelength. As the emission wavelength is dependent on layer size within the heterostructure rather than material composition, various wavelengths in the MIR can be achieved through bandstructure engineering. High sensitivity measurements have been achieved in both gas and liquid phase by developing integrated sensing systems. The laser emission frequency is selected to match a strong absorption feature for the analyte of interest where no other interfering bands are located. A waveguide is then developed to fit the application and wavelength used. Gas sensing applications incorporate silica hollow waveguides (HWG) and an OmniGuide fiber (or photonic bandgap HWG). Analyte gas is injected into the hollow core allowing the HWG or OmniGuide to serve simultaneously as a waveguide and miniaturized gas cell. Sensitivities of parts per billion are achieved with a response time of 8 s and a sample volume of approximately 1 mL. Liquid sensing is achieved via evanescent wave measurements with planar waveguides of silver halide (AgX) and gallium arsenide (GaAs). GaAs waveguides developed in this work have a thickness on the order of the wavelength of light achieving single-mode waveguides, providing a significant improvement in evanescent field strength over conventional multimode fibers. Liquid samples of L volume at the waveguide surfaces are detected. QCLs have begun to be utilized as a light source in the MIR regime over the last decade. The next step in this field is the development of compact and highly integrated device platforms which take full advantage of this technology. The sensing demonstrations in this work advance the field towards finding key applications in medical, biological, environmental, and atmospheric measurements.

The presented work describes first principles for monitoring gas hydrate formation and dissociation in solution by evaluating state-responsive IR absorption features of water with fiberoptic evanescent field spectroscopy. In addition, a first order linear functional relationship has been derived according to Lambert Beer’s law, which enables quantification of percentage gas hydrate within the volume of water directly probed via the evanescent field. Moreover, spectroscopic studies evaluating seafloor sediments collected from a gas hydrate site in the Gulf of Mexico revealed minimal spectral interferences from sediment matrix components, thereby establishing evanescent field sensing strategies as a promising perspective for monitoring the dynamics of gas hydrates in oceanic environments.

This thesis deals with a measurement and interpretation of NIR spectra of bacterial cells and design of biosensor using this analytical technique. In the first chapter, there is introduction of current state of knowledge in the field of NIR spectroscopy in microbiology and technology of fiber optic biosensors. The summary of this chapter shows that NIR is a suitable technique for direct molecular analysis of bacteria, but it suffers from low sensitivity and insufficient interpretation of bacterial spectra. In the next part of the thesis, there is a theoretical background of spectral analysis techniques and technology of fiber optic sensors. In the practical part of this work, there is suggested the elimination of disadvantages of NIR spectroscopy in microbiology by a series of experiments used for interpretation of NIR spectra of bacteria and design of fiber optic sensor to increase sensitivity of this technique. In this work, spectral regions important for the identification of bacterial strains were determined and partially interpreted and the sensor for bacterial analysis capable of classifying strains based on 105 captured cells was designed. Therefore, the objectives of this work were fulfilled.