Dissertations / Theses on the topic 'Biosensors'

Thesis (M.S.)--West Virginia University, 2008.<br>Title from document title page. Document formatted into pages; contains vi, 120 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 91-100).

The objective of this research was to develop the initial constituents of a highly scalable and label-free electronic biosensing platform. Current immunoassays are becoming increasingly incapable of taking advantage of the latest advances in disease biomarker identification, hindering their utility in the potential early-stage diagnosis and treatment of many diseases. This is due primarily to their inability to simultaneously detect large numbers of biomarkers. The platform presented here - termed the electronic microplate - embodies a number of qualities necessary for clinical and laboratory relevance as a next-generation biosensing tool. Silicon nanowire (SiNW) sensors were fabricated using a purely top-down process based on those used for non-planar integrated circuits on silicon-on-insulator wafers and characterized in both dry and in biologically relevant ambients. Canonical pH measurements validated the sensing capabilities of the initial SiNW test devices. A low density SiNW array with fluidic wells constituting isolated sensing sites was fabricated using this process and used to differentiate between both cancerous and healthy cells and to capture superparamagnetic particles from solution. Through-silicon vias were then incorporated to create a high density sensor array, which was also characterized in both dry and phosphate buffered saline ambients. The result is the foundation for a platform incorporating versatile label-free detection, high sensor densities, and a separation of the sensing and electronics layers. The electronic microplate described in this work is envisioned as the heart of a next-generation biosensing platform compatible with conventional clinical and laboratory workflows and one capable of fostering the realization of personalized medicine.

Aptamers are single stranded nucleic acids, typically composed of between twenty to eighty nucleotides in length, capable of binding selectively to non-nucleic acid ligands. Aptamers are selected through a combinatorial chemistry process called Systematic Evolution of Ligands by Exponential enrichment (SELEX), which is composed of successive cycles of selection based on target affinity, followed by amplification. This results in the Darwinian evolution of the nucleic acid library resulting in increasing library homogeneity and target affinity over time. Aptamers have been extensively investigated for potential application as sensing molecules, with roles similar to those traditionally occupied by antibodies. Aptamers and monoclonal antibodies have similar sensitivity in the pico to micro molar range. However aptamers have a number of advantages over protein antibodies, such as greater thermal stability, ease of chemical amplification, and amenability to modification, especially at the 5' and 3' prime ends. The work performed in this Thesis is divided into three categories. The first section describes the development of voltammetric Kanamycin and Tetracycline biosensors based on electrode immobilized, redox label bioconjugated nucleotide molecular beacons. These sensors relies on the target-aptamer binding induced spatial displacement of the redox label towards or away from the electrode surface as a means of signal generation. Further study was conducted to test the feasibility of this sensor design under likely field operation environments such as in soil sample analysis for microbial product discovery and in agricultural effluence for regulatory purposes. The biosensor was also enhanced by gel encapsulation for defense against nuclease degradation. Negative control was performed against structurally similar antibiotics of the same family in order to prove the specificity of the biosensor. Lastly, the sensor was moved onto an automated platform in a multichannel format in order to improve the utility of the sensor. The second section describes the development of a voltammetric biosensor based on Enzyme-Linked Oligonucleotide Assay (ELONA) technology. Two sub-types of ELONA-like biosensors were originally envisioned, based respectively on direct and indirect ELONA. Both sub-types depend on the mass of redox label rich Gold Nanoparticles (GNP) at the electrode surface as a means of signal generation. Negative controls was performed against globular proteins Bovine Serum Albumin and Lysozyme, the former since it is the most ubiquitous protein component of serum (the most likely biosensor operational environment), the latter as a worst case scenario for non-specific false positive results due to its positive charge. The last section describes an attempt to develop an automated SELEX device based on mesofluidic flow channels. It was hoped that by using flow channels of a millimeter scale it would be possible to retain both the advantages of the conventional auto sampler based SELEX protocols (large library and sequence variation), while also gaining the primary advantages of microfluidic SELEX (reduced contamination risk, low initial cost and maintenance). Essential components of the SELEX device, such as thermal cycler, liquid handling, electronics infrastructure, and software control were designed, tested and integrated. Lastly an attempt was made to perform automated SELEX against Lysozyme targets using the device, though no nucleic acid with high affinity to target had yet been successfully isolated by the end of this study.

This PhD thesis focuses on improving the limit of detection (LOD) of Raman biosensors by using surface enhanced Raman scattering (SERS) and/or hollow core photonic crystal fibers (HC-PCF), in conjunction with statistical methods. Raman spectroscopy is a multivariate phenomenon that requires statistical analysis to identify the relationship between recorded spectra and the property of interest. The objective of this research is to improve the performance of Raman biosensors using SERS techniques and/or HC-PCF, by applying partial least squares (PLS) regression and principal component analysis (PCA). I began my research using Raman spectroscopy, PLS analysis and two different validation methods to monitor heparin, an important blood anti-coagulant, in serum at clinical levels. I achieved lower LOD of heparin in serum using the Test Set Validation (TSV) method. The PLS analysis allowed me to distinguish between weak Raman signals of heparin in serum and background noise. I then focused on using SERS to further improve the LOD of analytes, and accomplished simultaneous detection of GLU-GABA in serum at clinical levels using the SERS and PLS models. This work demonstrated the applicability of using SERS in conjunction with PLS to measure properties of samples in blood serum. I also used SERS with HC-PCF configuration to detect leukemia cells, one of the most recurrent types of pediatric cancers. This was achieved by applying PLS regression and PCA techniques. Improving LOD was the next objective, and I was able to achieve this by improving the PLS model to decrease errors and remove outliers or unnecessary variables. The results of the final optimized models were evaluated by comparing them with the results of previous models of Heparin and Leukemia cell detection in previous sections. Finally, as a clinical application of Raman biosensors, I applied the enhanced Raman technique to detect polycystic ovary syndrome (PCOS) disease, and to determine the role of chemerin in this disease. I used SERS in conjunction with PCA to differentiate between PCOS and non-PCOS patients. I also confirmed the role of chemerin in PCOS disease, measured the level of chemerin, a chemoattractant protein, in PCOS and non-PCOS patients using PLS, and further improved LOD with the PLS regression model, as proposed in previous section.

The cross-sensitivity of microcantilever sensors presents a major obstacle in the development of a commercially viable microcantilever biosensor for point of care testing. This thesis concerns electrothermally actuated bi-material microcantilevers with piezoresistive read out, developed for use as a blood coagulometer. Thermal properties of the sensor environment including the heat capacity and thermal conductivity affect the ‘thermal profile’ onto which the higher frequency mechanical signal is superimposed. In addition, polymer microcantilevers are known to have cross-sensitivity to relative humidity due to moisture absorption in the beam. However it is not known whether any of these cross sensitivities have a significant impact on performance of the sensor during pulsed mode operation or following immersion into liquid. When analysing patient blood samples, any change in signal that is not caused by the change in blood viscosity during clotting could lead to a false result and consequently an incorrect dose of anticoagulants may be taken by the patient. In order to address these issues three aspects of the operation of polymer bi-material strip cantilevers has been researched and investigated: relative humidity; viscosity/density, and thermal conductivity of a liquid environment. The relative humidity was not found to affect the resonant frequency of a microcantilever operated in air, or to affect the ability of the cantilever to measure clot times. However, a decrease in deflection with increasing relative humidity of the SmartStrip microcantilever beams is observed at 1.1 ± 0.4 μm per 1% RH, and is constant with temperature over the range 10 – 37 °C, which is an issue that should be considered in quality control. In this study, the SmartStrip was shown to have viscosity sensitivity of 2 cP within the range 0.7 – 15.2 cP, and it was also shown that the influence of inertial effects is negligible in comparison to the viscosity. To investigate cross-sensitivity to the thermal properties of the environment, the first demonstration of a cantilever designed specifically to observe the thermal background is presented. Characterisation experiments showed that the piezoresistive component of the signal was minimised to -0.8% ± 0.2% of the total signal by repositioning the read out tracks onto the neutral axis of the beam. Characterisations of the signal in a range of silicone oils with different thermal conductivities gave a resolution to thermal conductivity of 0.3 Wm-1K-1 and resulted in a suggestion for design improvements in the sensor: the time taken for the thermal background signal to reach a maximum can be increased by increasing the distance between the heater and sensor, thus lessening the impact of the thermal crosstalk within the cantilever beam. A preliminary investigation into thermal properties of clotting blood plasma showed that the sensor can distinguish the change between fresh and clotted plasma.

The aim of the thesis was to identify and hence investigate the physical properties of liquid crystals that influence their potential as components of biosensor devices. Silicon surfaces presenting photolithographically fabricated arrays of 50nm thick gold spots were used as the model for a biosensor that detects the surface binding of a biological analyte. The spots ranged in diameter from 2μm to 16μm and their spatial separation varied between 5μm to 40μm. A Self Assembled Monolayer (SAM) of the thiol 3-mercaoptopropionic acid was used to control the surface chemistry of the gold. The responses of the nematic liquid crystals 5CB, E7, ZLI 1695, ZLI 1132 and MDA 01-2012 to were measured by optical microscopy. The spots were seen to induce a tilted planar alignment in the liquid crystals in their nematic phase for spot diameters down to 4μm and for all separations. Anchoring transitions between different tilt angles were observed between spots for some arrays. This was linked to a change in anchoring energy at the gold, possibly stemming from the angle of gold deposition. When heated through the nematic to isotropic phase transition cross defects were observed to nucleate on the gold spots for all spot sizes above 4μm. On cooling through the transition grid patterns of defects were observed to nucleate pinned between the spots for arrays of spots with length scales between 10μm and 20μm. The birefringence and elastic constants K11 and K33 of the liquid crystals were measured for temperatures up to their nematic to isotropic transition points. The birefringences of the liquid crystals at the transition were found to range between 0.003 and 0.007. The device thickness was varied between 7μm and 40μm. Values for the elastic constants were found to range between 1pN and 4pN. The intensity of monochromatic light (670nm) reflected from the arrays as the liquid crystals were cooled through the phase transition was found to increase for smaller values of the elastic constants and found to be highest where the grid of defects on the array was observed most clearly. The effect on the intensity of the birefringence and cell thickness was shown to be small compared to the effect of elasticity. Two possible biosensor designs are proposed. The first would identify the presence of a biological analyte at a surface by the change in alignment of a liquid crystal. This type of sensor would be optimised by carefully controlling the anchoring energy of the liquid crystal at the surface to minimise the quantity of surface binding required to induce an anchoring transition. The second would detect the presence at a patterned surface of an analyte by the defects that form over the pattern as the liquid crystal changes between the nematic to isotropic phases. This type of sensor would be optimised by choosing a liquid crystal with small elastic constants at the phase transition and by designing a patterned surface with length scales between 10microns and 20microns.

Aptamers are single-stranded DNA or RNA molecules isolated in vitro by a selection and amplification method. Aptamers bind with high specificity and affinity to a wide range of target molecules, with dissociation constant comparable to antibodies. In this work aptamers were employed as a new kind of bio-recognition element in affinity biosensors for the detection of clinically relevant proteins in heterogeneous assay, using Piezoelectric Quartz Crystal Microbalance and Surface Plasmon Resonance as transducers. The work was focused on two case studies, i.e. the Thrombin-binding aptamer and the aptamer against C-Reactive Protein. From an analytical point of view, the work was devoted to the optimisation of the analytical performance of a piezoelectric and an optical aptasensor for Thrombin and C-Reactive Protein detection, respectively. Efforts towards the application of these aptasensors in complex matrices, such as human plasma and serum, were also undertaken, in order to demonstrate the wide applicability of aptamers, as an alternative to antibodies. In this work, the possibility of introducing a computationally-assisted method to study aptamer-protein interaction and aptamer selection was also evaluated. For this purpose, the Thrombin-binding aptamer was chosen as a model and a retrospective docking study was performed by comparing the affinity of mutated sequences for thrombin with that of the Thrombin-binding aptamer, on the basis of a computationally-derived binding score. Finally, the reliability of computational results was tested by experimental measurements. For this purpose, the Thrombin-binding aptamer and other mutated sequences, selected on the basis of their binding score, were employed for the development of optical biosensors and the resulting analytical performances were compared. Even if further studies should be carried out in order to validate the proposed computational approach to aptamer selection, this work can have a significant impact on future aptamers selection for sensors and diagnostics.

Recognition of chiral molecules in biological assemblies has been a subject of extensive research. The aim of this work was to fabricate and characterise biocompatible composite materials suitable for chiral recognition. Collagen, the most abundant chiral, extracellular protein, was chosen as a possible matrix. The chiral recognition properties were evaluated by a comparative study in collagen, collagen incorporated in tetramethyl orthosilicate (TMOS) and TMOS. In electrochemical studies, ferrocene was incorporated to facilitate electron transfer. The recognition characteristics of two chiral enzymes, L-lactate oxidase and D-glucose oxidase were tested using circular dichroism (CD), Fourier Transform Infra-Red (FTIR) spectroscopy and electrochemical methods. A surprising result revealed an inversion of chiral selectivity. The effect of various parameters such as immobilisation, temperature, chemical modification, solvent systems, on enantioselectivity is well known. Stereoinversions caused by the ‘sergeants and soldiers’ effect in gel-forming p-conjugated molecules caused by co-assembly has been reported by several groups. The inversion of stereoselectivity observed in this study is probably due to a combination of the microenvironment and electrostatic interactions of the enzyme, mediator and substrate with the chiral collagen matrix. The results may have important implications for biosensing, asymmetric syntheses and understanding the nature of chiral interactions in biological systems.

L’objectif de cette thèse était d’explorer le potentiel des biocapteurs à base de transistors à 1 électron. Depuis l’invention de l’électrode de verre il y a plus de 100 ans, la réponse monotone du potentiel de surface avec le pH est devenue universelle. Aussi, il est bien connu que les mesures de la concentration en ions dans des solutions complexes, de grande importance pour le domaine biomédical, requière des membranes sélectives aux ions. En utilisant ces transistors nanométriques, nous montrons une rupture dans ces concepts avec l’observation d’une réponse en U au pH et la mesure sélective des cations Na+, K+, Ca2+ et Mg2+ dans le sérum sanguin, sans avoir recours aux membranes sélectives. Par ailleurs, les ions divalents ont été mesurés avec une sensibilité deux fois supérieure à la limite de Nernst. Les équations proposées, à l’origine d’une nouvelle méthode pour les mesures sélectives d’ions, peuvent-être étendues à la mobilité électrophorétique. Nous suggérons que ces nanotransistors 0D devraient également permettre des études biomimétiques de la compensation de charge des protéines. Nous montrons enfin que ces composants peuvent être intégrés sur un laboratoire sur puce en PDMS de 1.5 mm x 1.5 mm, qui promet un système de diagnostic sanguin peu couteux et très intégré<br>The aim of this thesis was to explore the potential of 0D nanotransistor biosensors. Since the invention of glass electrode a century ago, the monotonic decrease of oxides surface potential with pH has become universal. Also, it is well known that the measurement of ions concentration in complex solutions, of great importance for biomedical field, requires ion-selective membranes. Using these nanometric transistor biosensors, we report a rupture in these concepts with the observation of a U-shape pH response and the selective measurement of Na+, K+, Ca2+ and Mg2+ cations in blood serum, without falling back on selective membranes. In addition, divalent ions were measured with a sensitivity twice of that of Nernst limit. Proposed equations, at the origin of the new method for ion selective measurements, can be extended to the electrophoretic mobility. We also suggest that 0D nanotransistor biosensors are a relevant test bed for biomimetic studies of proteins charge compensation. We finally show that these devices can be ultimately integrated on a mm² PDMS-based lab-on chip, which promises for a cheap and small blood diagnosis system

This work describes the development of mediated amperometric biosensors that are able to monitor the metabolic activity of both single and mixed microbial populations, with applications in toxicity assessment and wastewater treatment plant protection. Biosensor systems have been constructed incorporating either the single-species eubacteria Escherichia coli or Pseudomonas putida, Bioseed®, or a mixture of activated sludge organisms from wastewater treatment plants, as the sensing components immobilised on disposable screen printed electrodes in stirred reaction vials. The biosensor approach is generic allowing for a wide range of microbial cell types to be employed. Appropriate bacterial species can be selected for specific sensor applications in order to confer validity and relevance to the test, hence the biosensor can be tailor-made to assess the toxicity in a particular environment and provide diagnostically valid and relevant results. The biosensors have been used to assess the toxicity of a standard toxicant and toxicant formulations and in blind testing of a range of industrial effluents, in parallel with a number of bioassays including Microtox® and activated sludge respiration inhibition. The biosensor results generally show significant correlation to the appropriate conventional toxicity tests. In this study, an activated sludge based biosensor assay was developed and used to assess the toxicity of industrial process and site effluents with the specific purpose of wastewater treatment plant protection. Data generated compared significantly with those from an activated sludge respiration inhibition test, with added advantages of rapidity, safety and ease of use.

Optical biosensors are increasingly being considered for lab-on-a-chip applications due to their benefits such as small size, biocompatibility, passive behaviour and lack of the need for fluorescent labels. The light guiding mechanisms used by many of them result in poor overlap of the optical field with the target molecules, reducing the maximum sensitivity achievable. This thesis presents a new platform for optical biosensors, namely slotted photonic crystals, which engender higher sensitivities due to their ability to confine, spatially and temporally, the peak of optical mode within the analyte itself. Loss measurements showed values comparable to standard photonic crystals, confirming their ability to be used in real devices. A novel resonant coupler was designed, simulated, and experimentally tested, and was found to perform better than other solutions within the literature. Combining with cavities, microfluidics and biological functionalization allowed proof-of-principle demonstrations of protein binding to be carried out. High sensitivities were observed in smaller structures than most competing devices in the literature. Initial tests with cellular material for real applications was also performed, and shown to be of promise. In addition, groundwork to make an integrated device that includes the spectrometer function was also carried out showing that slotted photonic crystals themselves can be used for on-chip wavelength specific filtering and spectroscopy, whilst gas-free microvalves for automation were also developed. This body of work presents slotted photonic crystals as a realistic platform for complete on-chip biosensing; addressing key design, performance and application issues, whilst also opening up exciting new ideas for future study.

Heavy metals from natural and man-made sources can be a great threat to human and animal life. As small inorganic ions they are challenging to detect, usually requiring expensive and complicated machinery. Several heavy metals can accumulate in the human body, leading to long term toxic effects on the nervous system. Many bacteria have developed strategies to survive in heavy metal rich environments. One of these strategies is a bacterial operon containing genes for detoxification mechanisms controlled by a promoter and a regulatory protein. In this work some of these promoter-protein pairs, Pars-ArsR, PcopA-CueR, PmerTPAD-MerR and PzntA-ZntR from Escherichia coli have been employed in the design and construction of a set of biosensors aimed at the detection of heavy metals in drinking water. Biosensors usually employ biological recognition elements, transducing the signal from these to produce an output that can be integrated into electronic circuitry. The sensors presented in this work focus on reducing complexity and on providing a controlled sensor reaction. The arsenic biosensor ‘AsGard’ is based on the Pars-ArsR pair and functions by making the dissociation of an ArsR-mCherry fusion protein from its binding site in the Pars promoter visible. In the cell, ArsR dissociates from Pars upon binding of trivalent arsenic ions. Immobilising the relevant part of the Pars sequence on a solid plastic support allows for the mobilisation of previously bound ArsR-mCherry proteins in the presence of arsenic to become the sensor output. The AsGard sensor detects arsenic within minutes in a concentration range overlapping with the arsenic thresholds for drinking water as set by the World Health Organisation. Additional prototype sensors are presented bringing a reporter gene under the control of the aforementioned promoters. These sensors have been tested in vivo and in vitro in a cell free transcription translation system and partially detect metal concentrations close to relevant ranges. The Pars based sensor is tuneable in vitro by modifying the ratio of the supplied regulatory protein ArsR and is able to detect arsenic well within the relevant range. Spinach2, a fluorescent RNA aptamer, may make future designs independent from translation, drastically reducing complexity of cell free biosensors based on cis-trans transcriptional regulation.

Electrochemical methods provide attractive sensing techniques for biology. Electrochemical devices can be easily manufactured, miniaturized and are sometimes the only direct sensing method available. However, stability of these sensors is problematic, as foreign-body type reactions may induce distortions of the signal (biofouling). As a consequence, investigating the interactions with the biological matrix is of paramount importance to achieve reliable sensing. Different types of electrodes (boron doped diamond and different preparations of glassy carbon) and various electrode coatings were tested, in the presence of biological molecules. The results showed that boron doped diamond and fibronectin coated sensors offer good stability, even in the presence of high concentrations of proteins. A generally applicable protocol to assess the quality of electrode materials in biofouling conditions is also presented. Fibronectin has also been found to be a highly biocompatible coating, perfectly suited for cell-based measurements. This fibronectin coating was used on an electrode array to study the pathway leading to angiogenic factor induced nitric oxide release. Vascular endothelial growth factor, a well known angiogenic factor, was initially used and allowed me to setup a reliable and robust protocol for the use of electrode arrays in biology. It was then demonstrated that angiogenin, another angiogenic factor, leads to nitric oxide exocytosis through PI-3 kinase transduction.

Thesis (Ph. D.)--University of Oregon, 2001.<br>Typescript. Includes vita and abstract. Includes bibliographical references (leaves 151-157). Also available for download via the World Wide Web; free to University of Oregon users.

My research broadly covers various important aspects of microfluidic devices and biosensors. Specifically, this dissertation reports: (1) a new and effective room temperature method of bonding polydimethylsiloxane (PDMS) microfluidics to substrates such as silicon and glass, (2) a new microfluidic pump concept and implementation specifically designed to repeatedly drive a small sample volume (<1 µL) very rapidly (~500 µL/min) through a sensor-containing flow channel to significantly decrease sensor response time through advection-driven rather than diffusion-driven mass transport, (3) use of a new microfluidic material based on polyethylene glycol diacrylate (PEGDA) to implement impedance-based dynamic nanochannel sensors for protein sensing, and (4) an investigation of galvanoluminescence and how to avoid it for conditions important to fluorescence-based dielectrophoresis (DEP) microfluidic biosensors. Over the last decade, the Nordin research group has developed a lab-on-a-chip (LOC) biosensor based on silicon photonic microcantilever arrays integrated with polydimethylsiloxane (PDMS) microfluidics for protein biomarker detection. Integration requires reliable bonding at room temperature with adequate bond strength between the PDMS element and microcantilever sensor substrate. The requirement for a room temperature process is particularly critical because microcantilevers must be individually functionalized with antibody-based receptor molecules prior to bonding and cannot withstand significant heating after functionalization. I developed a new room temperature bonding method using PDMS curing agent as an intermediate adhesive layer. Two curing agents (Sylgard 184 and 182) were compared, as well as an alternate UV curable adhesive (NOA 75). The bond strength of Sylgard 184 was found to be stronger than Sylgard 182 under the same curing conditions. Overnight room temperature curing with Sylgard 184 yields an average burst pressure of 433 kPa, which is more than adequate for many PDMS sensor devices. In contrast, UV curable epoxy required a 12 hour bake at 50 °C to achieve maximum bond strength, which resulted in a burst pressure of only 124 kPa. In many biosensing scenarios it is desirable to use a small sample volume (<1 µL) to detect small analyte concentrations in as short a time as possible. I report a new microfluidic pump to address this need, which we call a reflow pump. It is designed to rapidly pump a small sample volume back and forth in a flow channel. Ultimately, the flow channel would contain functionalized sensor surfaces. The rapid flow permits use of advection-driven mass transport to the sensor surfaces to dramatically reduce sensor response times compared to diffusion-based mass transport. Normally such rapid flow would have the effect of decreasing the fraction of analyte molecules in the volume that would see the sensor surfaces. By configuring the pump to reflow fluid back and forth in the flow channel, the analyte molecules in the small sample volume are used efficiently in that they have many opportunities to make it to the sensor surfaces. I describe a 3-layer PDMS reflow pump that pumps 300 nL of fluid at 500 µL/min for 15 psi actuation pressure, and demonstrate a new two-layer configuration that significantly simplifies pump fabrication. Impedance-based nanochannel sensors operate on the basis of capturing target molecules in nanochannels such that impedance through the nanochannels is increased. While simple in concept, the response time can be quite long (8~12 hours) because the achievable flow rate through a nanochannel is very limited. An approach to dramatically increase the flow rate is to form nanochannels only during impedance measurements, and otherwise have an array of nanotrenches on the surface of a conventional microfluidic flow channel where they are exposed to normal microfluidic flow rates. I have implemented such a dynamic nanochannel approach with a recently-developed microfluidic material based polyethylene glycol diacrylate (PEGDA). I present the design, fabrication, and testing of PEGDA dynamic nanochannel array sensors, and demonstrate an 11.2 % increase in nanochannel impedance when exposed to 7.2 µM bovine serum albumin (BSA) in phosphate buffered saline (PBS). Recently, LOC biosensors for cancer cell detection have been demonstrated based on a combination of dielectrophoresis (DEP) and fluorescence detection. For fluorescence detection it is critical to minimize other sources of light in the system. However, reported devices use a non-noble metal electrode, indium tin oxide (ITO), to take advantage of its optical transparency. Unfortunately, use of non-noble metal electrodes can result in galvanoluminescence (GL) in which the AC voltage applied to the electrodes to achieve DEP causes light emission, which can potentially confound the fluorescence measurement. I designed and fabricated two types of devices to examine and identify conditions that lead to GL. Based on my observations, I have developed a method to avoid GL that involves measuring the impedance spectrum of a DEP device and choosing an operating frequency in the resistive portion of the spectrum. I also measure the emission spectrum of twelve salt solutions, all of which exhibited broadband GL. Finally, I show that in addition to Au, Cr and Ni do not exhibit GL, are therefore potentially attractive as low cost DEP electrode materials.

Orientador: Lauro Tatsuo Kubota<br>Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Quimica<br>Made available in DSpace on 2018-08-04T04:12:52Z (GMT). No. of bitstreams: 1 Martins_LucileneDornellesMello_D.pdf: 8841041 bytes, checksum: f4618b120bf384d76c695743eb6ce4cc (MD5) Previous issue date: 2005<br>Doutorado<br>Quimica Analitica<br>Doutor em Quimica

Orientador: Lauro Tatsuo Kubota<br>Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Química<br>Made available in DSpace on 2018-08-08T22:44:52Z (GMT). No. of bitstreams: 1 Kisner_Alexandre_M.pdf: 3973690 bytes, checksum: 2810b47ecfaaac028a1bf271a3fc25a0 (MD5) Previous issue date: 2007<br>Conjuntos de transistores de efeito de campo sensíveis a íons (ISFETs) foram desenvolvidos no presente trabalho. Implementou-se durante a fabricação destes uma etapa adicional de anodização que possibilitou a formação de uma fina camada de alumina porosa sobre suas portas. Esta serviu como dielétrico e também molde para o crescimento de nanocristais de Ag e Au sobre os dispositivos. Os transistores desenvolvidos foram divididos em dois conjuntos, onde as dimensões de porta de cada conjunto foram de 10 x 50 mm e 50 x 50 mm. Utilizando-se um processo simples de anodização, obteve-se sobre a porta dos transistores uma fina camada de alumina de aproximadamente 60 nm de espessura, contendo uma alta densidade de poros (~ 10 poros/cm) com diâmetro médio de 30 + 6 nm e distribuídos de forma regular. A implementação desta possibilitou não só um aumento significativo na área de porta, bem como molde para o crescimento de nanoestruturas de Ag e Au sobre os transistores, atuando assim como nanoeletrodos de porta. Os testes destes como sensores para soluções com diferentes valores de pH, mostraram que os dispositivos apresentam um curto tempo de resposta (t < 30 s) e que as nanoestruturas metálicas são capazes de aumentar a sensibilidade dos dispositivos em relação àqueles formados apenas por alumina. Os primeiros testes para a detecção de moléculas como glutationa, demonstraram que os ISFETs fabricados são capazes de detectar esta, mesmo sendo uma espécie com baixa densidade de carga, em concentrações submicromolares<br>Arrays of ion-sensitive field effect transistors (ISFETs) were developed in this work. An additional step in the fabrication process was employed to implement a thin film of porous anodic alumina on the gate. This porous layer works as dielectric and template to the vertical growth of Ag and Au nanocrystals on the gate. The produced ISFETs were divided in two groups, which the gate dimensions were 10 x 50 mm and 50 x 50 mm. Using a simple anodizing process, a 60 nm thickness porous anodic alumina was developed on the gate. This porous film presented a high density porosity (~ 10 pores/cm) with an average pore diameter of 30 + 6 nm and a regular distribution on the gate of those ISFETs. This porous film lead to a significant increase in the gate area and also worked as a template to the growth of Ag and Au nanocrystals, which were used as gate nanoelectrodes. The results of such sensors to detect different pH of the solutions showed that the produced ISFETs present a short response time (t < 30 s). Moreover, the presence of such Ag and Au nanostructures increased the sensors sensitivity in comparison to those observed without nanoelectrodes. The first results to detect species such as glutathione, indicated that the ISFETs are even sensitive to detect small charged species in a submicromolar concentration range<br>Mestrado<br>Quimica Analitica<br>Mestre em Química

The silicon based sensor is able to detect part per trillion ionic silver in 0.0098% hydrofluoric acid based on the open circuit potential (OCP) measurement. The OCP jump of 100 ppt ionic silver solution is up to 120 mV. The complex agent can effectively suppress the ionic silver concentration and suppress the OCP signal. The ability of complex agent to suppress the OCP signal depends on the formation constant of the complex with silver. The complex adsorbed on the sensor surface induces a second OCP jump, the height of the second jump depends on the formation constant of the complex. The MINEQL chemical equilibrium modeling program is used to calculate the ionic silver concentration when complex agent presents, a discrepancy is found between the MINEQL simulation result and the OCP signal of the silicon based sensor. The toxicity of ionic silver to C. dubia is studied parallel to the OCP signal of silicon based sensor. Less toxicity is found when the complex agent is present similar to the OCP signal. Another discrepancy is found between the MINEQL simulation and the toxicity test when MINEQL simulation is used to predict and control the ionic silver concentration. The data from both biosensor C. dubia and silicon based sensor support each other and both are not in agreement with MINEQL simulation prediction.

Abstract Biopharmaceuticals are growing in medical and economical significance, replacing several chemically synthesized pharmaceuticals and providing new treatments for serious diseases. Research within the biopharmaceutical field is expensive and labor intensive with the majority of work featuring diverse bioprocesses. Miniaturization of bioprocesses into microbioreactors, miniaturized bioprocess vessels for diverse experiments, reduces the amount of necessary reagents as well as time and labor due to the possibility of multiplexing. A crucial step of research is analytics, commonly performed by expensive and time consuming standard laboratory based analytical tools that require larger sample volumes that than microbioreactors can provide. Biosensors are analytical devices that can provide highly sensitive and selective online detection (in situ) and lend themselves to miniaturization. Biosensors detecting glucose, lactate, pyruvate and galactose were developed and investigated herein. 3D printing and laser ablation were used as fabrication techniques, enabling development of unit operations (mixing and pumping) necessary for biosensor integration into microbioreactors. A 3D printed microbioreactor was developed with integrated glucose and optical density sensors for the cultivation of yeast where the biosensor was found to provide critical online data of glucose concentration, thus enabling bioprocess control. Additionally, molecularly imprinted polymers were investigated as novel recognition components due to their increased stability compared to biological molecules within bioprocess environments. A molecular imprinted polymer for folic acid was developed and tested as a sorbent in a microfluidic solid phase extraction system for detection<br>Tiivistelmä Biofarmaseuttisten valmisteiden lääketieteellinen ja taloudellinen merkitys kasvavat. Valmisteet korvaavat kemiallisesti valmistettuja yhdisteitä ja tarjoavat uusia hoitoja vakaviin sairauksiin. Biofarmaseuttinen tutkimus on kallista, ja erityisesti bioprosessien kehittäminen vaatii paljon työtä. Bioprosessien miniatyrisointi mikrobioreaktoreita käyttäen vähentää tutkimuksessa tarvittavaa reagenssien kulutusta, lyhentää aikaa ja pienentää työmäärää, koska mikrobioreaktoreiden avulla voidaan tehdä useita rinnakkaisia kokeita samanaikaisesti. Keskeinen osa bioprosessien tutkimusta on niiden seuraamiseen tarvittava analytiikka. Yleensä seuranta tehdään kalliilla ja aikaa vievillä analyyttisen kemian menetelmillä, jotka vaativat suurempia näytetilavuuksia kuin mitä mikrobioreaktoreista saadaan. Biosensorit ovat erittäin herkkiä ja mittauskohteen tarkasti tunnistavia analytiikkavälineitä, joilla voidaan saada online-mittaustietoa suoraan mittauskohteessa. Ne pystytään myös miniatyrisoimaan. Tässä tutkimuksessa kehitettiin aluksi glukoosin, laktaatin, pyruvaatin ja galaktoosin biosensorimittaukset. Sensorilaitteiden valmistustekniikkoina käytettiin 3D-tulostusta ja laserleikkausta. Näin pystyttiin kehittämään yksiköt myös näytteen sekoittamiseen ja pumppaamiseen. Lopuksi hiivasolujen kasvatusta varten valmistettiin 3D-tulostettu mikrobioreaktori, johon yhdistettiin glukoosisensori ja optisen tiheyden mittaus. Glukoosin mittaus tuotti glukoosin pitoisuudesta onlinetietoa, mikä on erittäin tärkeää prosessin valvonnalle ja ohjaukselle. Lisäksi tutkittiin molekyylipainettujen polymeerien käyttöä mitattavia analyyttejä tunnistavina yhdisteinä. Perinteisiä sensoreissa käytettäviä biomolekyylejä kestävämpinä yhdisteinä ne voisivat soveltua etenkin bioprosessien jatkuvaan seuraamiseen. Tutkimuksessa kehitettiin myös foolihapon tunnistava molekyylipainettu polymeeri, jota käytettiin kiinteän faasin sitoja-aineena mikrofluidisessa detektio- ja uuttojärjestelmässä

En les últimes dècades, la nanociència ha sorgit com una nova tecnologia gràcies a la seva versatilitat per ser emprada en diferents camps. Dins d’aquets grup, un dels nanomaterials més prometedors, els punts quàntics han estat estudiats per la seva extraordinària propietats i la seva versatilitat per utilitzar-los en diferents camps. La present tesis doctoral es centra en la síntesi de diferents punts quàntics, així com en el seu ús en LEDs, cèl·lules solars perovskites i biosensors. S'han sintetitzat tres tipus de punts quàntics: cadmi, perovskites i punts quàntics de carboni. Els dos primers presenten una banda d’emissió estreta i un rendiment quàntic elevat. No obstant, la seva alta toxicitat és un inconvenient que s’ha de tenir en comte. Com alternativa al seu ús, hem sintetitzat punts quàntics fet de carboni. La seva baixa toxicitat i biocompatibilitat és una bona alternativa als nanomaterials que contenen metalls pesants. A més, el material basat en carboni es pot preparar amb productes comuns com ara glucosa o sucrosa i poden ser dissolts en dissolvents no clorats com ara l’etanol o l’aigua. El treball presentat en aquesta tesis es va dur a terme a l'Institut d'Investigació Química de Catalunya (ICIQ) i al centre tecnològic Eurecat de Catalunya, entre març de 2015 i març de 2019.<br>En la última década, la nanociencia se ha convertido en una tecnología novedosa debido a su versatilidad para ser empleada en muchas áreas de investigación. Uno de los nanomateriales más prometedores, los puntos cuánticos coloidales, han sido estudiados en profundidad por su extraordinario optoelectrónico y su versatilidad para usar en diferentes campos. La presente tesis se centra en la síntesis de diferentes puntos cuánticos, así como su uso en LED, células solares de perovskita y biosensores. Se han sintetizado tres puntos cuánticos diferentes: cadmio, perovskita y puntos cuánticos de carbono. Los dos primeros materiales presentan un alto rendimiento cuántico y banda de emisión estrecha. Sin embargo, su alta toxicidad es una inconveniente que se tiene que tener en cuenta. Como alternativa a su uso, sintetizamos puntos cuánticos de carbono. Su baja toxicidad y su biocompatibilidad es una buena alternativa a los nanomateriales que contienen metales pesados. Además, el material a base de carbono se puede preparar utilizando productos de uso diario como azúcar o jugo de naranja y se puede resolver en solventes que no sean de cloro, como etanol o agua. El trabajo presentado en esta tesis se llevó a cabo en el Instituto de Investigación Química de Cataluña (ICIQ) y en Eurecat, el centro tecnológico de Cataluña, entre marzo de 2015 y marzo de 2019.<br>In the last decades, nanoscience has emerged as a novel technology due to its versatility to be employed in many research areas. One of the most promising nanomaterials, colloidal quantum dots have been deeply studied for their extraordinary optoelectronic properties and their versatility in order to use in different fields. The present thesis is focused on the synthesis of different quantum dots as well as their use in LEDs, perovskites solar cells and biosensors. Three different Quantum Dots have been synthetized: cadmium, perovskites and carbon based quantum dots. The first two material present a high quantum yield and narrow emission band. However, their high toxicity is an important drawback. In order to avoid the use of those material we synthetized carbon quantum dots. Their low toxicity and biocompatibility is a good alternative to heavy metal-containing nanomaterials. In addition, carbon based material can be prepared using ordinary products as glucose or sucrose and solved in non-chloro solvents such as ethanol or water. The work discussed in this thesis was carried out at Institute of Chemical Research of Catalonia (ICIQ) and Eurecat the technological center of Catalonia, between March 2015 and March 2019.

Biosensors are chemical sensors that use biological materials to detect the presence of the substance of interest. Optical methods to transduce the interaction between the analyte and the biological detection element are highly sensitive, non-invasive and are not subject to noise from electrical interference. Biosensors based on photonic devices such as lasers, optical fibres and photonic crystals offer the possibility of high sensitivity combined with small size, which is highly advantageous for in-the-field and remote applications. In this thesis, the feasibility of using semiconductor lasers as miniature biosensors has been investigated. Semiconductor laser chips contain a light source and a waveguide. Modification of the laser structure should allow the guided modes inside the laser to interact with material on the surface of the chip, thereby making the mode effective index sensitive to changes in the refractive index of material at the surface. This is the basis of an evanescent field sensor. Simulations have been carried out with the aim of finding an optimum structure for laser waveguides adapted for sensing purposes. Laser waveguides were modelled using RSoft Photonics CAD Suite and the BeamPROP simulation tool. For a ridge waveguide structure, two "windows" were placed either side of the ridge to form sensing areas. The optimum dimensions of these windows, and the optimum ridge width, were found by finding the structure with maximum sensitivity of effective index to a change in the cover index. A laser device was modified using focussed ion beam (FIB) milling to create sensing regions on the surface of the chip, in accordance with the computer model. The free spectral range (FSR) of the laser was measured before and after a polymer film was applied to the sensing regions of the chip, and a reproducible negative shift in the FSR was observed when the polymer was applied, indicating an increase in the effective index of the laser cavity. The sensitivity and detection limit (defined as the smallest measureable change in index of the sample) of the device were estimated as 1.5 x 10-3 and 0.15 respectively, although this could be improved significantly by measuring the heterodyne signal of two DFB lasers. The refractive index at a wavelength of lambda = 1550nm of spin-cast films of four different polymers have been measured by ellipsometry. These polymer films can now be used as standard test layers of known refractive indices for characterising the sensitivity of future laser-based biosensors. Finally an investigation into a new type of biosensor surface has been carried out. Antibody- conjugated latex particles are embedded in a cellulose acetate membrane, forming a biologically active surface to which antigens can selectively bind, without the need for complex surface chemistry to attach the antibodies. The membranes should also be reuseable by selectively dissolving the latex particles, and rebinding fresh particles into the imprints left in the membrane. It was found that the biological activity of the surface was difficult to preserve, but some selective rebinding of the latex particles into the imprinted membrane appeared to take place.

Understanding the temporal and spatial changes in calcium concentration has been a difficult endeavor for many years due to the relatively small changes in calcium concentration during messenging events, the rapid changes upon physiological messenging, and the unavailability of fast, efficient, and sensitive sensors to detect calcium changes. In addition, the key factors in calcium binding have yet to be determined due to the metal-metal interactions, cooperativity, and conformational change involved in calcium binding to natural calcium-binding proteins. To overcome these obstacles and to engineer calcium sensors for in vivo studies of calcium signaling events, calcium binding sites have been engineered into Green Fluorescent Protein. The engineered binding sites demonstrate terbium binding affinity from 2-30 ƒÝM and calcium binding affinity from 50-100 ƒÝM. Site 177 demonstrates green fluorescence when expressed in mammalian cells and produces a response to calcium concentration changes when expressed in the cytosol. Addition of the cycle 3 mutations (M153T, V163A, F99S) to Site 177 allowed for increased brightness in the emission of the chromophore but still exhibited calcium response. The second generation Site 1 demonstrates fluorescence response to calcium concentration changes when expressed both in the cytosol and in the endoplasmic reticulum. Addition of M153T and V163A to Site 1 allowed for expression of fluorescent protein at 37 ¢XC in HeLa cells and at 30 ¢XC in bacteria. Site 1-M153T/V163A exhibits chromophore fluorescence response to calcium with a Kd of 100 ƒÝM and competition with Rhodamine-5N produced a calcium Kd of 107 ƒÝM. This designed sensor, Site 1-M153T/V163A is the first demonstration of a designed calcium binding GFP with calcium response measured both in vivo and in vitro.

Diabetes is one of the leading causes of death and disability in the world. Even though insulin was discovered in 1920, an intense research on diabetes has been conducted during the last five decades and this is because of the market size. The huge demand is creating the need for the development of new approaches. This project involved the research aimed at better understanding and improvements in performance of glucose biosensors. In general, high surface area electrodes are desired as the high surface area provides more active sites for electrochemical reactions, and hence higher kinetic rate capability. Therefore, the determination of the active electrochemical surface area of the electrode is very important. A study has been conducted to determine the real electrochemical surface area of the Pelikan screen printed electrodes (SPEs) and a method has been optimised and established by Pelikan for the evaluation of their SPEs. Another very important issue that most of the current blood glucose monitoring tests are facing is the haematocrit effect, since the haematocrit differences observed in the blood samples can significantly affect glucose measurements. Therefore a study has been conducted in order to observe the absorption of the blood samples into the working electrode paste according to the haematocrit level. The second part of the study included the characterisation of the novel conjugated polymer made of N-(N, N’ diethyldicarbamoyl ethyl amido ethyl) aniline (NDDEAEA), the optimization of the conditions for the electrochemical polymerization, their application in grafting and finally the development of NDDEAEA based glucose biosensor. The new conducting polymer, acted as a matrix for the biosensor fabrication in this study, possesses macroiniferter properties and is capable of initiation free radical initiated addition polymerisation after formation of the polyaniline (PANI) material while preserving or even enhancing some of the PANI’s electrochemical properties. This material can potentially be used in the construction of novel Pelikan electrodes with enhanced integration functionalities, e.g. grafting non adhesive polymer coatings to assure that the poor performance in sensors as a result of impact of blood components can be mitigated. The final study included the development and optimisation of the reaction conditions for grafting a hyperbranched polymer onto the surface of the multi walled carbon nanotubes (MWCNT), using the A3 and B2 approach (described below). The aim of this work was achieving further increase in the sensitivity of Pelikan sensors.

Split enzymes have been used in Protein Complementation Assays (PCA) for several years to study protein-protein interactions. Recently Affimers, non-antibody binding proteins, have been created as new tools for studying molecular interactions. Combining these technologies offers substantial benefits for development of highly sensitive and specific diagnostic devices. For proof-of-principle, two fragments of β-lactamase have been generated with two His tags. The binding of the His tag on each fragment to nickel ions facilitates the association of β-lactamase fragments to generate a functional enzyme capable of substrate turnover. Substrates, such as the cephalosporin nitrocefin giving rise to a colour change (yellow to red) detectable at 492 nm. Following this proof of principle, the project focuses upon a novel split alkaline phosphatase to underpin an amperometric detection system developed with colleagues in the School of Electronic Engineering, University of Leeds. Alkaline phosphatase is a homodimer and the monomeric form of this protein does not turnover substrate. Current work in this study is focused on engineering the dimer interface to generate novel monomers that do not spontaneously associate. This is followed by exploring whether the mutated monomers can become associated and brought together, and catalyse substrate turnover. This study also focuses on preparing Affimers that can bind a target molecule, mGFP, at two separate epitopes. Followed by generating a cross linked protein between the Affimers and alkaline phosphatase. The binding of Affimer to target will be amplified by enzyme activity allowing detection. The system should allow single step detection of a target present at very low concentrations in a complex sample.

Biosensors are used for the detection of a range of analytes for applications in healthcare, food production, environmental monitoring and biodefence. However, many biosensing platforms are large, expensive, require skilled operators or necessitate the analyte to be labelled. Direct electrochemical detection methods present a particularly attractive platform due to the simplified instrumentation when compared to other techniques such as fluorescence-based biosensors. With modern integrated circuit capabilities electrochemical biosensors offer greater suitability for monolithic integration with any necessary signal processing circuitry. This thesis explores micro- and nanogap devices for both redox cycling and dielectric spectroscopy sensing mechanisms. By using two electrodes with interelectrode separation down to distances in the micro- and nanometre scale, several benefits can be realised. Firstly the close proximity of the two electrodes significantly reduces the interdiffusion time. This allows an electroactive species to be rapidly shuttled across the gap and switched between reduced and oxidised states. The result is feedback amplification of the amperometric response, increasing the signal. The second benefit is that the screening effect caused by electric double layers at the electrode–electrolyte interface is reduced due to the electric double layers occupying a larger fraction of the sensing volume. This significantly improves the sensor suitability for dielectric spectroscopy by increasing the potential drop across the biolayer. These two sensing mechanisms are demonstrated using a large area dual-plate microgap device for the detection of two different analytes. Utilising the first mode, detection of cysteine–cystine, an important redox couple involved in the signalling mechanism for the regulation of protein function, interaction and localisation is shown. The microgap device is then used for dielectric spectroscopy sensing of a mannose-specific uropathogenic Escherichia coli strain whilst also demonstrating the effect of ionic concentration on the capacitive response. The response of these devices is highly dependent on the interelectrode separation as well as the surface area of the electrodes. However, fabrication of large-area nanogap devices presents a significant challenge. This meant that careful optimisation and the development of novel techniques was necessary. This work reports the design, fabrication and characterisation of both a vertical and a horizontal coplanar large area nanogap device. The vertical nanogap device is fabricated using an inductively-coupled plasma reactive ion etching process to create a channel in a silicon substrate. A lower electrode is then optically patterned in the channel before anodically bonding a second identical electrode patterned on glass directly above. The horizontal nanogap device uses a different approach, utilising a state-of-the-art electron-beam lithography system to create a long serpentine nanogap with passivation to reduce fringing effects. The design allows the electron-beam lithography step to be substituted with nanoimprint lithography to reduce cost and improve throughput. Both of these devices have integrated microfluidic channels and provide a capacity for relatively high-throughput production.

This research developed a biosensor for kinase drug discovery applications. In particular it combined electronic techniques with optical techniques to understand the phosphorylation of proteins. There are two major electronic characteristics of phosphorylation that aid in its detection and subsequently biosensor development: first is the release of a proton upon phosphorylation of a protein (change in pH) and second is the addition of negative charge to the protein upon its phosphorylation. The work in this thesis reports an electrolyte–insulator–semiconductor sensing structures to detect the pH changes associated with phosphorylation and metal–insulator–semiconductor structures to detect the charge change upon phosphorylation of proteins. Major application of the developed devices would be to screen inhibitors of kinase that mediate phosphorylation of proteins. Inhibitors of kinase act as drugs to prevent or cure diseases due to the phosphorylation of proteins. With the advancements in VLSI and microfluidics technology this method can be extended into arrays for high throughput screening for discovering drugs.

The salient issues of integrated biosensors on a complementary metal-oxide semiconductor (CMOS) platform are the limited transducer design and the need for post-processing. To overcome these issues, a heterogeneously integrated system, which employs both CMOS and large-area processing, was proposed and developed. The system presented, could become a rapid, low-cost and disposable sensing platform for point-of-care applications. The heterogeneously integrated system, comprising a CMOS front-end circuit and disposable electrodes, was applied to measure the impedance of suspended DNA at different concentrations. The measurement showed a double sensitivity compared to the one carried out on the CMOS platform only. The noise analysis of CMOS transimpedance amplifiers was performed, and the impact of technology scaling on low-noise transimpedance amplifiers was studied using the Enz-Krummenacher-Vittoz (EKV) model. It was found that the noise performance improves slowly with device scaling down to 90 nm. Further device scaling may increase the gate leakage current noise due to the very thin gate oxide. Disposable electrodes fabricated using large-area processing are low cost and flexible in terms of design. In particular, inkjet-printed silver electrodes on glossy paper and gold electrodes on the glass substrate were characterised. Both electrodes with the same dimension agreed well in determining solution resistance. In addition, the paper-based electrodes presented an improved sensitivity of impedance measurement at low frequencies. The amorphous oxide thin-film transistor (TFT) is promising for implementing active circuits on disposable substrates. In particular, the low-frequency noise of amorphous indium-gallium-zinc-oxide (a-IGZO) TFTs was characterised, and a TFT-based regulated cascade transimpedance amplifier was designed and simulated with the extracted device parameters. The a-IGZO TFT showed a comparable noise performance to the PMOS device in deep submicron processes. The simulated circuit featured a transimpedance gain up to 120 dB, a bandwidth of 29.4 kHz, input-referred noise PSD of 2.91 pA/√Hz, and a power consumption of 18.55 μW, indicating that TFT-based front-end circuits are promising for implementing low-cost, low-noise and low-power biosensors.

Aquesta tesi doctoral té com a objectiu el desenvolupament de diversos biosensors que operen sense necessitat de marcatge addicional basats en dispositius plasmònics òptics per a la detecció directa de medicaments o biomarcadors relacionats amb diferents malalties i que són analitzats directament en mostres humanes com plasma, sèrum, orina o esput. Aquests dispositius biosensors ofereixen un sens fi de beneficis com és la seva alta sensibilitat, facilitat d’operació, l’obtenció de dades quantitatives, detecció sense marcatge en temps real, i comunament només necessiten d’un petit volum de mostra. Tot això converteix els biosensors plasmònics en eines analítiques molt adequades per al diagnòstic de malalties, el control de la medicació o el seguiment de teràpies personalitzades. El nostre grup d’investigació ha demostrat amb èxit la implementació de biosensors òptics basats en plasmònica i en fotònica de silici, inclòs el desenvolupament complet de bioaplicaciones, el que ha aplanat el camí de la seva futura transferència tecnològica per a la seva implementació com a dispositius Point-of-Care ( POC). Els biosensors desenvolupats en aquesta Tesi inclouen la seva optimització i validació completa amb mostres reals, exemplificant alguns desafiaments clínics en els quals aquests biosensors plasmònics poden superar importants limitacions de les tècniques d’anàlisi convencionals actuals, mostrant el seu potencial i versatilitat com a futurs dispositius POC per ser usats en les unitats d’atenció primària en salut o fins i tot en l’entorn domèstic per al propi autocontrol per part dels pacients. La tesi està organitzada en sis capítols. El capítol 1 conté la introducció dels conceptes bàsics i l’estat de l’art sobre els avenços actuals en les tècniques de diagnòstic i control de malalties i / o teràpies i el paper que exerceixen els biosensors per millorar-los. El capítol 2 inclou una descripció detallada de les plataformes biosensoras emprades i una descripció general dels processos metodològics. El Capítol 3 descriu el desenvolupament d’un dispositiu nanoplasmónico per al control terapèutic de l’medicament acenocumarol, un anticoagulant comunament administrat directament en plasma humà. El Capítol 4 es centra en el desenvolupament d’un biosensor plasmónico que serveixi com a control de la dieta lliure de gluten que han de portar els pacients celíacs. El Capítol 5 descriu les estratègies desenvolupades per a la detecció de dos biomarcadors per al diagnòstic primerenc de tuberculosi en mostres d’esput. Finalment, el Capítol 6 explora la detecció de quatre autoanticossos específics associats amb l’aparició de l’tumor directament en el sèrum humà com biomarcadors potencials per al diagnòstic primerenc de el càncer colorectal.<br>Esta Tesis Doctoral tiene como objetivo el desarrollo de diversos biosensores que operan sin necesidad de marcaje adicional basados en dispositivos plasmónicos ópticos para la detección directa de medicamentos o biomarcadores relacionados con diferentes enfermedades y que son analizados directamente en muestras humanas como plasma, suero, orina o esputo. Estos dispositivos biosensores ofrecen un sinnúmero de beneficios como es su alta sensibilidad, facilidad de operación, la obtención de datos cuantitativos, detección sin marcaje en tiempo real, y comúnmente sólo necesitan de un pequeño volumen de muestra. Todo esto convierte a los biosensores plasmónicos en herramientas analíticas muy adecuadas para el diagnóstico de enfermedades, el control de la medicación o el seguimiento de terapias personalizadas. Nuestro grupo de investigación ha demostrado exitosamente la implementación de biosensores ópticos basados en plasmónica y en fotónica de silicio, incluido el desarrollo completo de bioaplicaciones, lo que ha allanado el camino de su futura transferencia tecnológica para su implementación como dispositivos Point-of-Care (POC). Los biosensores desarrollados en esta Tesis incluyen su optimización y validación completa con muestras reales, ejemplificando algunos desafíos clínicos en los que dichos biosensores plasmónicos pueden superar importantes limitaciones de las técnicas de análisis convencionales actuales, mostrando su potencial y versatilidad como futuros dispositivos POC para ser usados en las unidades de atención primaria en salud o incluso en el entorno doméstico para el propio autocontrol por parte de los pacientes. La tesis está organizada en seis capítulos. El Capítulo 1 contiene la introducción de los conceptos básicos y el estado del arte sobre los avances actuales en las técnicas de diagnóstico y control de enfermedades y/o terapias y el papel que desempeñan los biosensores para mejorarlos. El Capítulo 2 incluye una descripción detallada de las plataformas biosensoras empleadas y una descripción general de los procesos metodológicos. El Capítulo 3 describe el desarrollo de un dispositivo nanoplasmónico para el control terapéutico del medicamento acenocumarol, un anticoagulante comúnmente administrado directamente en plasma humano. El Capítulo 4 se centra en el desarrollo de un biosensor plasmónico que sirva como control de la dieta libre de gluten que deben llevar los pacientes celíacos. El Capítulo 5 describe las estrategias desarrolladas para la detección de dos biomarcadores para el diagnóstico temprano de tuberculosis en muestras de esputo. Finalmente, el Capítulo 6 explora la detección de cuatro autoanticuerpos específicos asociados con la aparición del tumor directamente en el suero humano como biomarcadores potenciales para el diagnóstico temprano del cáncer colorrectal.<br>This Doctoral Thesis aims to the development of several label-free biosensing analytical strategies integrated within optical plasmonic devices for the direct detection of drugs or biomarkers related to different diseases in biological samples such as plasma, serum, urine, and sputum. These biosensor devices offer several benefits like their high sensitivity, ease of operation, quantitative data, label-free operation, and real-time detection, and commonly require a small sample volume. All this turn plasmonic biosensors into well-suited analytical tools for diagnosing diseases, monitoring medication, or for personalized therapies follow-up. Our research group has extensively demonstrated the successful conjunction of novel in-house optical biosensor configurations (like plasmonic and photonic-based designs) with the full demonstrations of bioapplications, which has paved the way for their potential technological transfer as Point-of-Care devices (POC) for clinical diagnostics. The biosensor assays here implemented, which include their full optimization and validation with real samples, exemplify clinical challenges where such biosensors can overcome limitations of current conventional analytical techniques. The results show the potential and versatility that plasmonic biosensors can offer as future POC devices placed in primary healthcare units or even in the household environment for patients’ self-monitoring. This thesis is organized into six chapters. Chapter 1 is the introductory one, which explains the basic concepts and the state of the art of the current advances in diagnosis and monitoring techniques of diseases and/or therapies and the role of biosensors to improve them. Chapter 2 includes a detailed description of the biosensor platforms employed and a general description of the methodological processes. Chapter 3 is related to the development of a nanoplasmonic device for the therapeutic monitoring of the drug acenocoumarol, a commonly administered anticoagulant, directly in human plasma. Chapter 4 focuses on the implementation of a plasmonic biosensor that monitors the gluten-free diet in urine in celiac patients. Chapter 5 describes the biosensing strategies developed for the detection of two biomarkers for the early diagnosis of tuberculosis in sputum samples. Finally, Chapter 6 explores the detection of four specific autoantibodies associated with the tumor onset directly in human serum as potential biomarkers for the early detection of colorectal cancer.<br>Universitat Autònoma de Barcelona. Programa de Doctorat en Química

The introduction of prostate-specific antigen (PSA) testing about 3 decades ago led to the possibility of early detection of prostate cancer (PCa). Although PSA testing reduced the mortality rate, it is also associated with high risk of over diagnosis in patients with and without PCa. Despite the current drawbacks, it would be a challenge to replace PSA testing entirely. Instead, there is a need to develop parallel testing of other potential biomarkers that can complement the results from PSA tests. To address alternative biomarker sensing, this thesis highlights on the development of oligonucleotide-based biosensors for the detection of different biomarkers of PCa. Using PSA as a gold standard, the first study of this dissertation investigates the use of DNA aptamers to detect PSA using electrochemical impedance spectroscopy (EIS). The study compares 6-mercapto 1-hexanol chemistry with sulfo-betaine chemistry for the development of PSA aptasensor in terms of performance and selectivity. The second study focuses on glycoprofiling in order to complement PSA quantification as an additional information for reliable PCa diagnosis. This strategy was developed in a microfluidic channel with an optical read out using chemiluminescence. This study addresses one of the major problems of cross-reactivity with lectins in glycoprofiling, which can be solved using DNA aptamers. A third study concentrates on the development of an aptasensor for Alpha-Methylacyl-CoA Racemase (AMACR). AMACR has been reported for its high specificity and sensitivity to PCa. For the fabrication of the biosensor, a new strategy using polyethylene glycol was developed by electrochemical grafting it to a polypyrrole film. Since PCa diagnosis can be improved by looking at different biomarkers, an electrochemical platform for miRNA/DNA detection using a gold nanoparticle amplification strategy was also investigated. The sensor was fabricated using peptide nucleic acids (PNA) probes on gold electrodes. The study presents non-Faradaic EIS and amperometric techniques in order to exploit the inherent charges of nucleic acids. In conclusion, this thesis wants to serve as a potential orientation for overcoming the shortcomings of the current PCa testing and contribute towards the development of oligonucleotide-based biosensors for PCa biomarker detection and hopefully enhance the diagnosis and prognosis of PCa.

En els últims anys, a causa de la necessitat de diàgnostics ràpids i de millores en sensat, s’han utilitzat nous elements de reconeixement en biosensors. Un tipus d’aquests nous elements de reconeixement són els aptàmers. Els aptàmers són cadenes sintètiques de ADN o ARN les quals són seleccionades in vitro i tenen la capacitat d’unir-se a proteïnes, ions, cèl.lules, fàrmacs i lligands de baix pes molecular, reconeixent les seves molècules diana amb alta afinitat i especificitat. Diversos biosensors basats en aptàmers, també anomenats aptasensors, han sigut desenvolupats recentment. D’entre totes les tècniques de transducció utilitzades en biosensors, l’Espectrocòpia Electroquímica d’Impedància ha sigut àmpliament emprada como a eina per caracteritzar la superficies de sensors i estudiar esdeveniments en el biosensat en la superficie d’elèctrodes. La característica més important que presenta aquesta tècnica és que no requereix cap espècie marcada per a la transducció, per tant, aquesta tècnica de detecció pot utilitzar-se per dissenyar protocols de detecció directa sense marcatge, evitant assajos més cars i laboriosos. El principal objectiu d’aquesta tesi doctoral va ser el desenvolupament d’aptasensors utilitzant la tècnica electroquímica d’impedància esmentada anteriorment. Per a això, diferents tipus d’elèctrodes van ser utilitzats, tals com elèctrodes de compòsit grafit-epoxi, elèctrodes de biocompòsit grafit-epoxi modificats amb molècules d’avidina i elèctrodes comercials serigrafiats de nanotubs de carboni de paret múltiple. El treball es va dividir principalmente en dues parts d'acord amb la detecció de dues proteïnes diferents. La primera part es va focalitzar en la detecció de trombina. Primer de tot, es van comparar i avaluar diversos aptasensors de detecció directa sense marcatge basat en diferents tècniques d'immobilització dels aptàmers, tals com: adsorció física humida, afinitat avidina-biotina i enllaç covalent mitjançant activació electroquímica de la superfície de l'elèctrode i mitjançant inserció electroquímica. Posteriorment, els elèctrodes de biocompòsit van ser comparats com a plataformes en genosensat i aptasensat. Amb la finalitat d'amplificar el senyal impedimètric obtingut utilitzant elèctrodes de biocompòsit, un protocol sàndwich va ser emprat incloent nanopartícules d'or modificades amb estreptavidina i tractament amplificador de plata. La segona part de l'estudi es va basar en la detecció de citocrom c. Primerament, es va realitzar un simple aptasensor de detecció directa sense marcatge per a la detecció d'aquesta proteïna utilitzant la tècnica d'immobilització d'adsorció física humida. Finalment, i amb l'objectiu d'amplificar el señal impedimètric, es va desenvolupar un assaig tipus sándwich híbrid d’aptàmer i anticòs utilitzant elèctrodes serigrafiats de nanotubs de carboni de paret múltiple. D'aquesta manera, la tesi explora i compara una àmplia gamma de procediments d'immobilització, l'ús de detecció directa sense marcatge o nanomaterial modificat amb biomolècules en diferents protocols directes o d'amplificació, i l'ús de reconeixement directe i sándwich per amplificar la sensibilitat i/o la selectivitat de l'assaig.<br>In the recent years, due to the need for rapid diagnosis and improvements in sensing, new recognition elements are employed in biosensors. One kind of these new recognition elements are aptamers. Aptamers are synthetic strands of DNA or RNA which are selected in vitro and have the ability to bind to proteins, ions, whole cells, drugs and low molecular weight ligands recognizing their target with high affinity and specificity. Several aptamer-based biosensors, also called aptasensors, have been recently developed. Among all the transduction techniques employed in biosensors, Electrochemical Impedance Spectroscopy has widely used as a tool for characterizing sensor platforms and for studying biosensing events at the surface of the electrodes. The important feature presented by this technique is that it does not require any labelled species for the transduction; thus, this detection technique can be used for designing label-free protocols thus avoiding more expensive and time-consuming assays. The main aim of this PhD work was the development of aptasensors using the electrochemical impedance technique previously mentioned for protein detection. For that, different types of electrodes were used, such as Graphite Epoxy Composite electrodes (GECs), Avidin Graphite Epoxy Composite electrodes (AvGECs) and commercial Multi-Walled carbon nanotubes screen printed electrodes (MWCNT-SPE). The work was divided in two main parts according to the detection of the two different proteins. The first part was focused on thrombin detection. First of all, different impedimetric label-free aptasensors based on several aptamer immobilization techniques such as wet physical adsorption, avidin-biotin affinity and covalent bond via electrochemical activation of the electrode surface and via electrochemical grafting were developed and evaluated. Then, AvGECs electrodes were compared as a platform for genosensing and aptasensing. With the aim to amplying the obtained impedimetric signal using AvGECs, an aptamer sandwich protocol for thrombin detection was used including streptavidin gold-nanoparticles (Strep-AuNPs) and silver enhancement treatment. The second part of the study was based on cytochrome c detection. Firstly, a simple label-free aptasensor for the detection of this protein using a wet physical adsorption immobilization technique was performed. Finally, with the goal to amplify the impedimetric signal, a hybrid aptamer-antibody sandwich assay using MWCNT-SPE for the detection of the target protein was carried out. In this way, the thesis explores and compares a wide scope of immobilization procedures, the use of label-free or nanocomponent modified biomolecules in different direct or amplified protocols, and the use of direct recognition and sandwich alternatives to enhance sensitivity and/or selectivity of the assay

Els biosensors microbians són dispositius analítics que utilitzen microorganismes com elements de reconeixement. Els microorganismes s’immobilitzen a la superfície del transductor de manera que la interacció microorganisme-analit genera una senyal (electroquímica, òptica, entre altres) que pot ser quantificada. Els biosensors microbians es poden fer servir en diferents àmbits d’aplicació com al diagnòstic clínic, la indústria alimentària o la monitorització mediambiental amb l’avantatge de ser portables, simples, barats, i per tant bones alternatives als mètodes de laboratori. Desafortunadament, el desenvolupament de biosensors microbians es veu obstaculitzat per: (i) la baixa reproductibilitat que presenten els protocols d’immobilització de les cèl·lules, (ii) la seva poca sensibilitat, deguda a la dificultat d’immobilitzar elevades concentracions de microorganismes, i (iii) la curta vida útil dels mateixos, deguda a la mort cel·lular durant el procés d’immobilització o emmagatzematge. Aquesta tesi descriu el desenvolupament de dues estratègies d’immobilització que permeten el confinament reproduïble de microorganismes a la superfície d’un elèctrode, amb elevades densitats i de manera reproduïble, i al mateix temps proporciona un entorn fisiològic que permet una adequada difusió dels nutrients, garantint la funcionalitat i la viabilitat dels microorganismes atrapats. En la primera estratègia, les cèl·lules microbianes romanen atrapades en una matriu polimèrica d’alginat-grafit electrodepositada a la superfície de l&#8216;elèctrode mitjançant condicions molt suaus i fisiològiques (temperatura ambient, medi aquós, pH neutre…) de polimerització. Els elèctrodes recoberts d’alginat conductor s’obtenen després de l’electrodeposició potenciostàtica de mostres d’alginat dopades amb grafit (fins al 4% de grafit). La presència de grafit redueix la passivació de l’elèctrode i millora la resposta electroquímica dels sensors recoberts d’alginat. L’atrapament de microorganismes és molt eficient (4.4x107 cèl·lules per elèctrode) i reproduïble (CV <0.5%), sense comprometre la integritat ni l’activitat microbiana. En la segona estratègia, els microorganismes romanen atrapats en la matriu de polietersulfona, un material soluble en solvents orgànics que precipita en contacte amb medis aquosos en un procés anomenat inversió de fase. Hem demostrat que els microorganismes poden incorporar-se durant la formació de la membrana mantenint certa viabilitat. Amb aquest mètode, s’obtenen de manera reproduïble membranes de 300 µm amb 2.6x106 cèl·lules en el seu interior, que mantenen nivells acceptables d’integritat i viabilitat cel·lular. Tots dos sistemes s’han utilitzat per immobilitzar E. coli a la superfície d’elèctrodes serigrafiats per desenvolupar biosensors, en els quals, els microorganismes actuen com a elements de reconeixement. La biodetecció s’ha realitzat electroquímicament mitjançant la respirometria de ferricianur, de manera que els microorganismes atrapats dins de la matriu poden reduir el ferricianur en presència de glucosa i convertir-lo en ferrocianur. S’ha avaluat el rendiment analític dels dos biosensors microbians amperomètrics portant a terme un assaig de toxicitat utilitzant 3,5-diclorofenol (DCP) com a compost tòxic model. En tots dos casos, els biosensors van proporcionar una resposta depenent de la concentració de DCP, amb una dosi efectiva (EC50) de 3.5 ppm (alginat) i 9.2 ppm (polietersulfona), d’acord amb els valors reportats. Aquestes metodologies d’atrapament són susceptibles de producció en massa perquè permeten una producció fàcil i repetitiva de biosensors microbians robusts amb una bona sensibilitat.<br>Los biosensores microbianos son dispositivos analíticos que utilizan microorganismos como elementos de reconocimiento. Los microorganismos están inmovilizados en la superficie del transductor de manera que la interacción microorganismo-analito genera una señal (electroquímica, óptica, entre otras) que puede ser cuantificada. Los biosensores microbianos se pueden aplicar en diferentes campos como el diagnóstico clínico, la industria alimentaria o la monitorización medioambiental, con la ventaja de ser portables, simples, baratos, así como una buena alternativa a los métodos de laboratorio. Desafortunadamente, la implementación de los biosensores microbianos se ha visto obstaculizada por: (i) su pobre reproducibilidad, debido a la poca reproducibilidad de los protocolos de inmovilización de las células, (ii) su poca sensibilidad, por la dificultad de inmovilizar elevadas concentraciones de microorganismos, y (iii) su corta vida útil, debido a la muerte celular durante el proceso de inmovilización o almacenamiento. Esta tesis describe el desarrollo de dos estrategias de inmovilización que permiten el confinamiento reproducible de microorganismos en la superficie del electrodo, con altas densidades y de manera reproducible, al tiempo que proporcionan un entorno fisiológico que permite una adecuada difusión de nutrientes, asegurando la funcionalidad y viabilidad de los microorganismos atrapados. En uno de los sistemas, las células microbianas se atrapan en una matriz polimérica de alginato-grafito electrodepositada en la superficie del electrodo utilizando condiciones muy suaves y fisiológicas (temperatura ambiente, medio acuoso, pH neutro…). Los electrodos recubiertos de alginato conductor se obtienen después de la electrodeposición potenciostática de muestras de alginato dopadas con grafito (hasta el 4% de grafito). La presencia de grafito reduce la pasivación del electrodo y mejora la respuesta electroquímica de los sensores recubiertos de alginato. El atrapamiento de microorganismos es altamente eficiente (4.4x107 células por electrodo) y reproducible (CV <0.5%) sin comprometer la integridad o actividad microbiana. En la segunda estrategia, los microorganismos quedan atrapados en una matriz de polietersulfona, un material soluble en solventes orgánicos y que sólo precipita en contacto con medio acuoso en un proceso llamado de inversión de fase. Hemos demostrado que los microorganismos pueden incorporarse durante la formación de la membrana manteniendo cierta viabilidad. Con este método, se obtuvieron de manera reproducible membranas de 300 µm, con 2.6x106 células en su interior, que mantienen niveles aceptables de integridad y viabilidad celular. Ambos sistemas se han utilizado para inmovilizar E.coli en la superficie de electrodos serigrafiados para desarrollar biosensores en los que los microorganismos actúan como elementos de reconocimiento. La biodetección se ha realizado electroquímicamente mediante respirometría de ferricianuro, de manera que los microorganismos atrapados dentro de la matriz pueden reducir el ferricianuro en presencia de glucosa y convertirlo en ferrocianuro. Se ha evaluado el rendimiento analítico de los dos biosensores microbianos amperométricos llevando a cabo un ensayo de toxicidad utilizando 3,5-diclorofenol (DCP) como compuesto tóxico modelo. En ambos casos, los biosensores proporcionaron una respuesta dependiente de la concentración de DCP con una dosis efectiva (EC50) de 3.5 ppm (alginato) y 9.2 ppm (polietersulfona), de acuerdo con los valores reportados. Esta metodología de atrapamiento es susceptible a la producción en masa porque permite una producción fácil y repetitiva de biosensores microbianos robustos con buena sensibilidad.<br>Microbial biosensors are analytical devices that use microorganisms as recognition elements. Microorganisms are immobilized on the surface of a transducer in such a way that the microorganism-analyte interaction generates a signal (electrochemical, optical, among others) that can be quantified. These microbial biosensors can be applied in the fields of clinical, industrial or environmental diagnosis with the advantage of being portable, simple and inexpensive alternatives to many laboratory-based methods. Unfortunately, development of microbial biosensors has been hindered by important technical limitation related to: (i) poor reproducibility, due to non-reproducible cell immobilization protocols, (ii) low sensitivity, by the difficulty of immobilizing high bacterial concentrations, and (iii) short life-time, due to cell death during immobilization or storage. This thesis describes the development of two immobilization strategies that allow reproducible confinement of microorganisms at the electrode surface, with high densities and in a reproducible manner, while providing a physiological environment that allows adequate diffusion of nutrients, ensuring the functionality and viability of the trapped microorganisms. In one of the strategies, (1) microbial cells have been trapped in an alginate-graphite polymeric matrix electrodeposited at the electrode surface using very soft and biocompatible conditions (i.e. room temperature, aqueous medium, neutral pH, etc.). Conductive alginate-coated electrodes are obtained after potentiostatic electrodeposition of graphite-doped alginate samples (up to 4% graphite). The presence of graphite reduces electrode passivation and improves the electrochemical response of alginate-coated sensors. Bacterial entrapment in the conductive matrix is highly efficient (4.4x107 cells per electrode), reproducible (CV < 0.5%) and does not compromise bacterial integrity or activity. In the second strategy, (2) microorganisms are trapped in polyethersulfone when the polymer, initially dissolved in organic solvents, precipitates in aqueous medium through a process of phase inversion. We have shown that microorganisms can be incorporated during membrane formation and remain viable. With this method, 300 µm PES membranes were reproducibly obtained containing up to 2.3x106 cells per electrode, with an entrapment efficiency of 8.2%, while maintaining acceptable levels of cell integrity or viability. Both systems have been applied to immobilize E. coli at the surface of screen-printed electrodes to develop biosensors in which microorganisms act as recognition elements. Biosensing has been performed electrochemically through ferricyanide respirometry, with metabolically-active entrapped bacteria reducing ferricyanide in the presence of glucose. The analytical performance of the two amperometric microbial biosensors has been assessed carrying out a toxicity assay using 3,5-dichlorophenol (DCP) as a model toxic compound. In both cases, biosensors provided a concentration-dependent response to DCP with half-maximal effective concentration (EC50) of 3.5 ppm (alginate) and 9.2 ppm (polyethersulfone), well in agreement with reported values. This entrapment methodology is susceptible of mass production and allows easy and repetitive production of robust and sensitive microbial biosensors.

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.