SELEX is a selective amplification technique based on the assumption that functional nucleic acids (FNAs) can be found in a pool of chemically synthesized random nucleic acid sequences. These FNAs can either bind to a target of interest (aptamers) or catalyze a chemical reaction (DNAzymes and ribozymes) or both (aptazyme). The aptazymes discussed herein, are RNA-cleaving DNAzymes that become catalytically active upon target recognition at the aptamer domain. In chapter 2, two SELEX experiments were performed in parallel to determine whether it was possible to obtain multiple aptazymes from a single SELEX procedure. Two different crude intercellular mixtures (CIM) each containing 35 unique overexpressed ASKA clone gene products were used. After 10 rounds of SELEX, the sequence pool was analyzed using Illumina Genome Analyzer. The sequences tested appeared to bind to a protein other than the 35 gene products of interest. To redirect these SELEX experiments towards the gene products of interest, 12 gene products from each parallel selection was purified and incubated with sequences from round 6 of the CIM SELEX. A total of 13 rounds of selection were performed using these purified proteins. The sequences were analyzed and found to either not cleave at all or self-cleave. In chapter 3 a similar SELEX approach was explored using 36 purified proteins, from the CIM SELEX to isolate protein-binding DNA aptamers. This SELEX approach served as another means to explore isolating multiple aptamers against multiple targets. However both the RNA-cleaving aptazyme and protein-binding DNA aptamer SELEX experiments both experienced challenges in non-specific binding. Although negative selection steps were taken in order to avoid non-specific binding species, such sequences were still isolated. In order for these approaches to be successful, negative selection steps are required to remove any self-cleaving and non-specific FNAs. Although these studies did not conclusively give rise to the desired FNAs, it has produced some insight to the potential setbacks associated with developing a screen for multiple targets. / Thesis / Master of Science (MSc)

PEC bioanalysis represents a unique and dynamically developing methodology that offers an elegant route for sensitive biomolecular detection. Building on the principle of EC analysis, PEC biosensors harness the unique properties of optically active species to enhance analytical performance. Owing to the current based outputs evolved in both PEC and EC bioanalysis, they can be miniaturized and potentially integrated with handheld and portable analyzers, making them uniquely positioned as tools to build effective POC diagnostics. The commercialization of PEC technology for building POC diagnostics, however, heavily depends on enhancing the stability of the photoelectrodes upon repeated use, lowering the limit-of-detection (LOD) of the PEC biosensor used, enhancing the efficiency of signal transduction and the ability of the device to detect minute amounts of biomolecular target in complex biological matrices. In order to address these constraints, we first developed a new solution-based strategy integrating inorganic semiconductive species (titanium dioxide) in an organic framework to construct photoelectrodes with enhanced signal baselines and adequate stability for the cyclic measurements required in biosensing. These transducers were subsequently used to investigate the interaction mechanisms (wavelength dependency, coverage density dependency and spatial dependency) between plasmonic NPs (Au) and the photoelectrodes —chosen as model materials—with the goal of enabling predictive dual-signal modulation and enhanced limit-of-detection in PEC biosensors. The understanding gained was used to design a dual-signal PEC transduction strategy—operated at a single excitation wavelength and on a single electrode—to detect nucleic acid sequences in urine without direct target labeling, target amplification or target enrichment. Here, Au NP terminated biobarcodes served as dynamic signal amplifiers that enabled a low limit-of-detection (5 fM), a wide linear range (1 fM – 100 pM), and the ability to discern between single and double base-mismatched nucleic acid sequences. In parallel, we also detail the development of an EC biosensor featuring dynamic DNA motifs, capable of reagentless, sensitive and specific detection of N-PEDv (nucleocapsid protein of porcine epidemic diarrhea virus)—a protein target with emerging global significance—in both buffer (LOD ~ 1.08 μg mL-1) and urine (LOD ~ 1.09 μg mL-1) Ultimately, this work presents innovations in material architecture and programmable dual-signal transduction that enhance the performance metrics of biosensors; thus, presenting the potential to design POC molecular diagnostics of the future. / Thesis / Doctor of Engineering (DEng) / To address critical limitations in the field of point-of-care molecular diagnostics, it is vital to develop new tools integrating bio-recognition systems with programmable photoelectrochemical and electrochemical signal transduction that enables the design of more effective biosensors. In photoelectrochemical (PEC) systems, plasmonic materials such as gold nanoparticles are often featured to either amplify or attenuate signal response. While there is a significant amount of literature regarding the interaction of gold nanoparticles (Au NPs) with semiconductive systems, the exact nature of the interaction between the two particles has not yet been fully mapped out. In this thesis, we examine various degrees of freedom—including surface coverage density and separation distance—that influence the design of effective photoelectrochemical systems. The understanding gained is further harnessed to design dual-signal photoelectrochemical systems featuring titanium dioxide (TiO2) photoelectrodes and Au linked dynamic deoxyribonucleic acid (DNA) motifs to enable predictive modulation in response to target identification. An electrochemical (EC) analogue featuring structure switching DNA motifs and redox tagged barcodes was also developed. The resultant PEC and EC biosensing assays were critically examined, and their analytical performance was subsequently evaluated in terms of limit-of-detection, sensitivity, and specificity. Ultimately, new classes of bioassays featuring integrated biorecognition and dual-signalling capability for oligonucleotide (i.e., nucleotide sequences and aptamers) based biomolecular detection in urine and saliva were realized.

Single-molecule biosensors offer distinct advantages over their ensemble-averaged counterparts by being able to extract information related to rare targets and specific molecular configurations within a sample. In particular, solid-state nanopores embody a promising single-molecule technique that is based on detecting target molecules by the amount of ionic current they block as they pass through a nanoscale aperture across a thin membrane. In this thesis, I present extensions of the basic nanopore system aimed at addressing some of its main limitations at present, namely: 1) the low rates at which nanopores capture molecules from a bulk volume, which restricts their ability to work with dilute (≲ nM) samples, and 2) the difficulty in using nanopores to distinguish small or closely related molecules by their direct current blockage signatures alone. I begin by describing the design and construction of a nanopore-based instrument that integrates an optical detection channel in parallel with ionic current sensing. A particular emphasis was placed on minimizing the electrical noise contributions of the added optical equipment on the original ionic current channel. Measuring the optical signals of translocating molecules together with their current blockages can improve the discrimination of two fluorescently labelled targets (or two configurations of a single target) that normally produce similar ionic current signatures. I next investigate the combination of nanopore sensing with target pre-concentration, specifically, by embedding a nanopore membrane within a fluidic cell that features an insulator-based dielectrophoretic (iDEP) trap. Applying large (≳ 100 V) AC voltages across the iDEP channels of the cells resulted in the accumulation of polarizable targets (dsDNA, polystyrene beads) at the locations of the membranes, thus pointing toward a convenient method for the detection of ultra-dilute target samples in future nanopore devices. Finally, I introduce improved protocols for the synthesis and nanopore signal analysis of dsDNA-based molecular carriers. In a molecular carrier scheme, in order to enhance the target specificity of the system, target molecules are not sensed directly by a nanopore but instead interact specifically with secondary molecules (“carriers”) to recognizably alter the carrier translocation signals. Here, I present proof-of-principle analyses of DNA carrier experiments that highlight the multiplexing capabilities of our carrier design, which are based on separating targets by their interactions with carriers of different lengths. Developments of the nanopore sensing platform such as those presented in this work, which leverage the intrinsic versatility of solid-state nanopores to be integrated within complex devices and to detect a wide range of target molecules, will play an important role in continuing to increase the precision of single-molecule measurements into the future and to expand their breadth of potential applications.

The goal of this project was to design networked arrays of cricket biosensors capable of localizing sources such as footsteps within dangerous environments, with a possible application to earthquake detection. We utilize the cricket's natural ability to localize low frequency (5 Hz - 600 Hz) acoustic sources using hair-covered appendages called cerci. Whereas previous investigations explored crickets' neurological response to near field flows generated by single frequency steady-state sounds, we investigated the effects of transient waveforms, which better represent real world stimuli, and to which the cercal system appears to be most reactive. Extracellular recording electrodes are permanently implanted into a cricket's ventral nerve cord to record the action potentials emanating from the cerci. In order to calibrate this system, we attempt to find the relationships between the frequency and direction of acoustic stimuli and the neurological responses known as spike trains, which they elicit. The degree of habituation to repeated signals that exists in most neurological systems was also experimentally measured. We process the signals to estimate frequency and directionality of near field acoustic sources. The design goal is a bionic cricket-computer system design capable of localizing low frequency near field acoustic signals while going about its natural activities such as locomotion.

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Numerous reports have been published detailing a wide variety of strategies for the production of many different prototype screen-printed biosensors, hmvever, few of these devices have been developed to the commercialisation stage. There is an unquestionable need for disposable biosensors suitable for decentralised analysis that can be mass-produced at low cost by a simple process; screen-printed carbon electrodes (SPCEs) fulfil both of these criteria. Conventional methods for producing biosensors based on this technology usually involve the deposition of a biological recognition element (typically an enzyme) onto a SPCE which has been printed using an organic solvent-based ink. The removal of organic solvents from the manufacturing process is a highly desirable goal as it should result in improved health and safety and also the possibility of incorporation of enzymes directly into the ink. The latter is difficult to achieve with conventional screen-printing inks as enzymes are inactivated by both the organic solvents themselves and the elevated temperatures required in the curing step. The studies described in this thesis utilise a screen-printing ink which incorporates a water-based binder and the electro catalytic mediator cobalt phthalocyanine (CoPe.) It is demonstrated that the addition of different oxidase enzymes directly into this ink allows for the one-step manufacture of biosensors with desirable performance characteristics, notably high precision and outstanding stability. A water-based carbon ink incorporating CoPC was used to produce robust and precise SPCEs which were found to act as effective sensors for H20 2• The sensors were operated in stirred solutions at an applied potential of +0.5 V, which was shown to be a significant reduction in the potential required for H20 2 detection at an un-modified electrode. The ink was modified further by adding glucose oxidase (GOD) to its bulk prior to printing. This allowed for the one-step printing of glucose biosensors which dried at room temperature. These biosensors were investigated using amperometry in stirred solution which revealed long-term operational stability and a shelf-life of at least 18 months. The analytical signal was shown to arise from the electro catalytic oxidation of the H20 2 produced by the enzyme in the presence of glucose and O2. Using these biosensors with a background correction technique, it was possible to determine the concentration of glucose present in a bovine serum sample with a good degree of both accuracy and precision. This demonstrated that the newly-developed glucose biosensors were capable of operating reliably in a biological sample. In order to extend the linear range, the ink composition was re-formulated and chronoamperometry was used as the measurement technique. The sensitivity of these new glucose biosensors was found to be comparable to that of the earlier system, and the upper limit of the linear range was successfully extended. A simple method for interference removal was developed, which involved the use of 'dummy' sensors that did not contain any enzyme. Using this system, it was possible to quantify glucose in dilute human plasma samples which had been spiked with glucose in order to represent diabetic samples. The generic nature of the CoPC modified water-based ink was illustrated by adding a different enzyme, lactate oxidase (LOD,) which is known to be much more delicate than GOD, into the ink. Even under un-optimised conditions, the LOD-containing biosensors gave a measurable response to lactate over a clinically useful range. Owing to their unusual properties, the study of materials with dimensions smaller than 100 nm is playing an increasingly important role in the development of biosensors. In an attempt to extend the linear range of the GOD-containing biosensors to higher glucose concentrations, the possibility of using nanoscaled cobalt phthalocyanine (n-CoPC) as a mediator was investigated. It was shown that the response of the n-CoPC containing sensors towards H20 2 was superior to those incorporating the bulk mediator. GOD was added to the n-CoPC modified ink, and the resulting glucose biosensors displayed a superior sensitivity and linear range to the bulk-CoPC containing biosensors produced earlier. The increased sensitivity was attributed to the increased sensitivity of the base transducer, and further experiments were conducted to determine the reason for the extended linear range. Remarkably, it was discovered that the n-CoPC modified biosensors were capable of operating in the absence of O2, which implied that the n-CoPC must be interacting in some way with the redox centre of GOD. Such direct electron transfer has been reported for biosensors incorporating other types of nanomaterials but, as far as is known, never for CoPC. In order to investigate the possibility of determining another clinically important analyte, cholesterol oxidase was introduced into the n-CoPC modified water-based ink. Although the resulting cholesterol biosensors did not display the same Orindependent operation, a good sensitivity and a linear response up to at least 2 mM cholesterol was achieved. Free cholesterol was determined in 40 dilute human plasma samples which had been spiked with cholesterol to represent a clinically useful range of total cholesterol concentrations; the results agreed well with a standard reference method (R2 = 0.95.)

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