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In recent years, there has been an increasing demand for newer, more accurate, technologies that can detect and identify biomolecules or biological entities related to health, agriculture or the environment. With the discovery of new properties of nucleic acids beyond the storage and transfer of genetic information, a new class of nucleic acid-based biosensors is emerging, using DNA and RNA as target recognition elements with the advantage of being simpler and more cost-effective compared to antibodies-based biosensor. Two sequences, TrG14MC and TrG10SC, with evidence to suggest that they are capable of inhibit the metalloenzyme CIP, were isolated from a selection conducted by Dr. Razvan Nutiu. Here we study the inhibitory properties of these two aptamer candidates and measure the IC50 value, determined as 94 nM for TrG14MC and 83 nM for TrG10SC. Different bivalent constructs, designed to increase the inhibitory effect of the isolated sequences, are studied showing a pronounce influence of the linker length improving the inhibitory effect over CIP. Modulating the interaction of the isolated sequences and the CIP is of key importance in order to develop a successful biosensor. Therefore, we try to recover CIP from the inhibition effect by using antisense sequences complementary to different segments of the construct. The maximum recovery, 75%, was achieved by an antisense sequence fully complemented to the inhibitory bivalent construct. We also study here the use of a linker in the bivalent construct that forms a secondary hairpin structure, and the effect of linearizing that structure with an antisense sequence complementary to the linker. This resulted in as 12% of the inhibitory effect. The purpose of this investigation was to establish the first steps toward the development of a new class of biosensors capable of disinhibiting CIP upon the recognition of a specific target, taking advantage of the suggested CIP-inhibitory properties of the isolated sequences TrG14MC and TrG10SC. / Thesis / Master of Applied Science (MASc)

Lab-on-a-chip devices are changing the way that medical testing is performed by allowing rapid testing with small samples. Optofluidic biosensors are a type of lab-on-a-chip device that use light excitation on a fluid sample. One such application of an optofluidic biosensor is a device that can detect antibiotic resistant bacteria by combining DNA from a sample with fluorescent beads, flowing that sample through a hollow channel, and shining laser light on the channel. If the bacteria tested for is present, the fluorescent beads will give off photons that can be detected as a positive signal. The main method for illumination for these devices has been coupling light through a fiber optic cable to a waveguide on the side of the chip. Though effective, this method is impractical in a real world setting such as a hospital due to the difficulty of aligning to the side of the device. One solution to this problem is the use of illumination from the top of the device. Top-down illumination allows for more alignment flexibility, but also introduces the risk of additional noise or false signal as extra light reflects of the device. This dissertation discusses the viability and development of top-down illumination for optofluidic biosensors. This includes the development of an anti-reflective layer compatible with optofluidic biosensors, comparison of top-down illumination to side illumination, and simulations of various methods of performing top-down illumination. Based on the research and findings discussed in this dissertation, it has been found that top-down illumination is a viable illumination method for optofluidic biosensors. Additionally, the use of a pattern of laser lines combined with a light blocking anti-reflective layer is the recommended method for top-down illumination.

During the COVID-19 outbreak, PCR tests were widely used for large-scale testing and screening. Yet, this technique requires bulky and time-consuming procedures to prepare the samples collected from the patients before their analysis by well-trained experts with expensive and specific equipment. PCR is therefore not competitive as a technique of detection for a widespread and rapid use in point-of-care sites. Thus, the COVID-19 pandemic highlighted the need for cheap and easy-to-implement biosensors. Surface plasmon resonance based sensors were suggested as a promising alternative in recent years. Indeed, they enable real-time and label-free detection of a wide range of analytes. That explains their widespread use in various fields of applications such as pharmacology, toxicology, food safety, and diagnosis. This thesis proposes and demonstrates a new plasmonic configuration of detection, which can address challenges posed by point-of-care settings. The gratings used as transducers in this configuration were fabricated based on laser interference lithography combined with a nanoimprinting process. The responses of these nanostructures interrogated by a p-polarized light beam result in a transfer of energy between two diffracted orders over an angular scan. This optical phenomenon termed as “optical switch”, was theoretically and experimentally investigated and optimized. The principle of detection based on this specific configuration was demonstrated for the detection of small variations in the bulk refractive index with solutions comprised of different ratios of de-ionized water and glycerol. A limit of detection in the range of 10−6 RIU was achieved. In addition, preliminary bio-assays obtained by combining this configuration with a functionalization are presented and demonstrate the selectivity and the potential of this new plasmonic configuration for biosensing applications. This thesis work paves the way for the use of the optical switch configuration as a biosensor aligned with low-cost manufacturing and relevant for diagnosing in point-of-care sites.

Applications of surface plasmon polaritons (SPPs) have thus far emphasized visible and near-infrared wavelengths. Extension into the long-wave infrared (LWIR) has numerous potential advantages for biosensors and waveguides, which are explored in this work. A surface plasmon resonance (SPR) biosensor that operates deep into the infrared (3-11 µm wavelengths) is potentially capable of biomolecule recognition based on both selective binding and characteristic vibrational modes. The goal is to operate such sensors at wavelengths where biological analytes are strongly differentiated by their IR absorption spectra and where the refractive index is increased by dispersion, which will provide enhanced selectivity and sensitivity. Potentially useful IR surface plasmon resonances are investigated on lamellar gratings formed from various materials with plasma frequencies in the IR wavelength range including doped semiconductors, semimetals, and conducting polymers. One outcome of this work has been the demonstration of a simple analytic formula for calculating the SPP absorption resonances in the angular reflectance spectra of gratings. It is demonstrated for Ag lamellar gratings in the 6-11 µm wavelength range. The recipe is semi-empirical, requiring knowledge of a surface-impedance modulation amplitude, which is found here by comparison to experiment as a function of the grating groove depth and the wavelength. The optimum groove depth for photon-to-SPP energy conversion was found by experiment and calculation to be ~10-15% of the wavelength. Hemicylindrical prism couplers formed from Si or Ge were investigated as IR surface plasmon couplers for the biosensor application. Strong Fabry-Perot oscillations in the angular reflectance spectra for these high index materials suggest that grating couplers will be more effective for this application in the LWIR. A variety of materials having IR plasma frequencies were investigated due to the tighter SPP mode confinement anticipated in the IR than for traditional noble metals. First doped-Si and metal silicides (Ni, Pd, Pt and Ti) were investigated due to their inherent CMOS compatibility. Rutherford backscattering spectroscopy, x-ray diffraction, scanning electron microscopy, secondary ion mass spectrometry and four point probe measurements complemented the optical characterization by ellipsometry. Calculation of propagation length and mode confinement from measured permittivities demonstrated the suitability for these materials for LWIR SPP applications. Semimetals were also investigated since their plasma frequencies are intermediate between those of doped silicon and metal silicides. The semimetal antimony, with a plasma frequency ~80 times less than that of gold was characterized. Relevant IR surface plasmon properties, including the propagation length and penetration depths for SPP fields, were determined from optical constants measured in the LWIR. Distinct resonances due to SPP generation were observed in angular reflection spectra of Sb lamellar gratings in the wavelength range of 6 to 11 µm. Though the real part of the permittivity is positive in this range, which violates the usual condition for the existence of bound SPP modes, calculations based on experimental permittivity showed that there is little to distinguish bound from unbound SPP modes for this material. The SPP mode decays exponentially away from the surface on both sides of the permittivity sign change. Water is found to broaden the IR plasmon resonances significantly at 9.25 micron wavelength where aqueous extinction is large. Much sharper resonances for water based IR SPR biosensor can be achieved in the 3.5 to 5.5 µm range. Nano-structured Au films (Au-black) were investigated as IR absorbers and possible solar cell enhancers based on surface plasmon resonance. The characteristic length scales of the structured films vary considerably as a function of deposition parameters, but the absorbance is found to be only weakly correlated with these distributions. Structured Au-black with a broad range of cluster length scales appear to be able to support multiple SPP modes with incident light coupling to the corrugated surface as seen by photoelectron emission microscopy (PEEM) and SPR experiments, supporting the hypothesis that Au-black may be a suitable material for plasmon-resonance enhancement solar-cell efficiency over the broad solar spectrum.

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Over the last 20 years, the incidence of Clostridium difficile infection (CDI) has increased dramatically, making it one of the most common healthcare-associated infections. This has been linked to the emergence of hypervirulent C. difficile strains. Currently, cell cytotoxicity assay (CTA) and toxigenic culture are the gold-standard methods for CDI diagnosis. However, they are time-consuming and labour-intensive. Other methods, like enzyme immunoassays (EIAs) and nucleic acid amplification-based tests (NAATs), allow for rapid testing but have poor sensitivity and/or specificity. Additionally, most of these methods target toxins or their associated genes and are unable to discriminate between epidemic and non-epidemic strains. The work described in this dissertation aims to develop easy-to-use and reliable biosensors for C. difficile, with a particular focus on epidemic strains of C. difficile. The development of the in vitro selection technique has allowed for the discovery of a big array of functional DNA, with excellent ability in both target recognition and enzymatic catalysis. My key interest is to employ functional DNA molecules as target recognition elements to develop colorimetric biosensors for C. difficile detection. The first research project aimed to develop a colorimetric detection platform that can be coupled with functional DNA molecules to achieve hypersensitive detection of different targets. This test should be easy-to-use, have broad target applicability and not require expensive equipment. To do so, a colorimetric biosensing platform was created, which takes advantage of the signal amplification ability of rolling circle amplification (RCA) and the simplicity of the classic litmus test. In the presence of the target of interest, RCA will be triggered, and the biotinylated RCA products can hybridize a number of the urease-labelled single-stranded DNA and immobilize urease onto magnetic beads through streptavidin-biotin interactions. The urease can then be used to hydrolyze urea, resulting in significant pH elevation, that can be detected easily using a litmus test. To prove the concept, we have demonstrated that this platform can be employed to visually detect thrombin and platelet-derived growth factor (PDGF) with high sensitivity, by coupling it with an anti-thrombin aptamer and an anti-PDGF aptamer, respectively. We have also shown that the biosensing platform can be incorporated into simple paper-based devices. The second project focuses on the development of a colorimetric DNA detection method for epidemic strains of C. difficile that utilizes both the polymerase chain reaction (PCR) and the litmus test. The strategy makes use of a modified set of primers for PCR to facilitate ensuing manipulations of resultant DNA amplicons: their tagging with urease and immobilization onto magnetic beads. The amplicon/urease-laden beads are then used to hydrolyze urea, resulting in an increase of pH that can be conveniently reported by a pH-sensitive dye. We have successfully applied this strategy for the detection of two hypervirulent strains of C. difficile, which are responsible for the recent increase in the global incidence and severity of C. difficile infections. Furthermore, the viability of this test for diagnostic applications is demonstrated using clinically validated stool samples from C. difficile infected patients. The goal of the third project was to isolate RNA-cleaving fluorogenic aptazymes (RFAs) targeting an epidemic strain of C. difficile. Four classes of RFA probes were derived using in vitro selection approach where a random-sequence DNA library was reacted with a crude extracellular mixture (CEM) derived from the epidemic C. difficile strain BI/027/NAP1, coupled with a subtractive selection strategy to eliminate cross-reactivities to unintended C. difficile strains and other bacterial species. The isolated RFDs can be used together to generate specific cleavage patterns for strain-specific identification of C. difficile. Lastly, the final project was to characterize a novel RFA probe (RFA13-1) that was isolated unintentionally using in vitro selection. By using CEM prepared from C. difficile glycerol stock contaminated by Klebsiella aerogenes as the positive target for in vitro selection, we isolated a remarkably active RFA probe, RFA13-1, targeting K. aerogenes. Further studies demonstrated that RFA13-1 could be activated by CEM prepared from several bacteria from the Enterobacteriaceae family. Moreover, the molecular target of RFA13-1 has been identified, which is ribonuclease I. RFA13-1 showed high sensitivity and specificity towards RNase I and could be employed as a tool to study RNase I functions and to detect RNase I or RNase I-containing bacteria. In summary, I have investigated novel strategies for building a biosensor that is capable of discriminately detecting epidemic strains of C. difficile. I hope that my work can take us one step closer towards the development of easy-to-use and reliable biosensors that can be used in the clinical diagnosis of CDI. / Thesis / Doctor of Philosophy (PhD)