DNA-metal ion interactions are critical for stabilizing conformations of double stranded (ds) DNA and through specific binding sites will influence the interaction of DNA with other molecules. It has been shown that different metal ions bind to different sites within nucleic acids. Work in this thesis exploits the interactions of Zn2+ with nucleic acids that are linked to surfaces. Zn2+ can interact with the phosphodiester backbone and engage in interactions with the purine nucleobases. Electrochemical studies of ds-DNA films have demonstrated that in the presence of Zn2+ films containing a single nucleotide mismatch give rise to a specific electrochemical signature. Electrochemical impedance spectroscopy (EIS) allows the discrimination of mismatched DNA films from those that are fully matched by monitoring differences in the resistance of charge transfer. Scanning electrochemical microscopy (SECM) allows multiplexing of the data acquisition and monitoring of the current response I, which is attenuated as a function of mismatch. In this thesis, various potential factors were explored in detail that may impact the discrimination of nucleotide mismatches in ds-DNA films by EIS and SECM. These factors include the position of the mismatch, its type, the number of mismatches, the length of the DNA duplex, and the length of target sequences. In particular, when the two strands are of unequal length, the resulting nucleotide overhang may mask the mismatch signature. Such overhangs are expected in real biosensor applications, in which the DNA is isolated from cellular targets. Results presented here clearly demonstrate that mismatches are readily distinguished from fully matched strands even in overhang systems, suggesting that this approach has promise for realistic sensor applications.

The thickness shear mode acoustic wave (TSM) sensor, operated in a flow-through format, has been widely used in bioanalytical research. My research is mainly focused on the study of surface-attached biomolecules and cells using the TSM sensor, including lesions in DNA, conformational change of calmodulin, as well as the properties and attachment of rat aortic smooth muscle cells. Aldehydic apurinic or apyrimidinic sites (AP sites) that lack a nucleobase moiety are one of the most common forms of toxic lesions in DNA. In this work, synthesized oligodeoxyribonucleotides containing one, two, or three abasic sites were hybridized to complementary sequences immobilized on the gold electrode of the TSM device by affinity binding. The influence of AP sites on local base stacking energy and geometry caused a dramatic destabilization of the DNA duplex structure, which was detected by the TSM sensor. The signals detected by TSM correlated well with the thermostability of DNA duplexes in solution. The results indicate that both the number of sites and their localization in the double-stranded structure influence the stability of a 19 b.p. duplex. TSM was also used to detect the binding of ions or peptides to surface-attached calmodulin. The interaction between calmodulin and ions induced an increase in resonant frequency and a decrease in motional resistance. In addition, these signal changes were reversible upon washing with buffer. The response was interpreted as a decrease in surface coupling induced by exposure of hydrophobic domains on the protein, and an increase in the length of calmodulin by approximately 3 Å. In addition, the interaction of the protein with peptide together with calcium ions was detected successfully, despite the relatively low molecular mass of the 2-kDa peptide. In addition, the attachment of smooth muscle cells to various surfaces has been monitored by TSM. These surfaces include laminin, fibronectin and bare gold. The results of these experiments in terms of changes of frequency (fs) and resistance (Rm) were analyzed. The responses of the surface-bound cells to the introduction of various ions, depolarisation events and damage subsequent to exposure to hydrogen peroxide were also observed. Morphological changes in the cells, as confirmed by atomic force microscopy and scanning electron microscopy, are correlated with results from the TSM sensor.

The goal of this thesis has been to design, characterise and optimise an electrochemical DNA sensor array. In order to investigate the oligonucleotide probe immobilisation and the hybridisation detection, preliminary experiments with an easy system were performed. This system demonstrated the suitability of oligonucleotide self-assembled monolayers (SAMs) on gold surfaces as immobilisation method. Due to the rapid DNA sensor development towards DNA arrays, a modified strategy was proposed. This strategy was based on the site-directed electrodeposition of biorecognition nanomodules on electrodes of photolithographic resolution. These biorecognition nanomodules, oligonucleotide-modified colloidal gold nanoparticles, were rationally synthesised previously studying the conditions under which colloidal gold suspensions were stable. Fluorescence and colourimetric techniques proved the effectiveness of the conjugation, the functionality of the conjugated probes, and the thermal stability of the modification, which made the biorecognition nanomodules suitable for hybridisation detection. After their characterisation, the biorecognition nanomodules were electrodeposited on different electrode surfaces and the site-directed immobilisation was clearly demonstrated by several techniques, such as light and electron microscopy, and colourimetric, piezoelectric and electrochemical techniques. Additionally, the site-directed deposited biorecognition nanomodules were functional and able to differentiate 4-point mutations in 19-mer oligonucleotides. Despite the promising results, which demonstrated the viability of the directed electrodeposition as arraying technique, the necessity for the electrochemical signal amplification was observed, as system values were very close to blank values. Following two parallel objectives for the electrochemical signal amplification (to intrinsically increase kinetic rates between enzymes and electrodes, and to optimise electrochemical recycling systems), osmium complexes were rationally designed and the kinetics of electron transfer with redox enzymes was evaluated. These kinetic studies showed that more positively charged mediators and with higher redox potentials yielded higher rates, also favoured at high pH and low ionic strength, demonstrating the possibility to amplify electrochemical signals.The thesis is structured in seven chapters. Chapter I is an introduction that establishes the basis of DNA sensors and arrays, explains the behaviour and stability of the colloidal gold suspensions, conjugations and deposition, and presents the theoretical basis for the evaluation of electron transfer rate constants between redox enzymes and mediators. The objective of the thesis, the state-of-the-art, the hypothesis, the methodology, the most important conclusions and the limitations and future work are also presented in this chapter. Chapter II describes the preliminary system for the immobilisation characterisation and hybridisation detection. Chapter III elaborates on the study of colloidal gold suspension stability and the subsequent synthesis of the functional biorecognition nanomodules. Moreover, in this chapter the thermal stability and the functionality of the nanomodules are characterised by various techniques. In Chapter IV, the site-directed electrodeposition of these nanomodules is presented, as well as the characterisation techniques applied for its evaluation. Chapter V covers the rational study of the experimental parameters that affect the electron transfer kinetics between Glucose Oxidase (GOx) and osmium complexes, developed for application in electrochemical signal amplification. Finally, Chapters VI and VII summarise the conclusions,the limitations of the thesis work and proposals for future study. / El objetivo de esta tesis ha sido diseñar, caracterizar y optimizar un array de sensores de ADN electroquímico. Para el estudio de la inmovilización de las sondas de oligonucleótidos y la detección de la hibridación se realizaron experimentos preliminares con un sistema simplificado. Dicho sistema demostró que las monocapas auto-ensambladas (SAMs) en superficies de oro eran apropiadas como método de inmovilización. Debido al rápido desarrollo de los sensores de ADN hacia los arrays de ADN, se modificó la estrategia. La nueva estrategia se basó en la electrodeposición dirigida de nanomódulos de bioreconocimiento en electrodos de resolución fotolitográfica. Dichos nanomódulos, nanopartículas de oro coloidal modificadas con oligonucleótidos, se sintetizaron racionalmente después de haber estudiado las condiciones bajo las cuales las suspensiones de oro coloidal eran estables. Mediante técnicas de fluorescencia y colorimetría se caracterizó la eficacia de las conjugaciones, la funcionalidad de los oligonucleótidos conjugados y la estabilidad térmica de la modificación, experimentos que demostraron que los conjugados eran aptos para la detección de la hibridación. Después de su caracterización, los nanomódulos de bioreconocimiento fueron electrodepositados en distintas superficies electródicas y se demostró la inmovilización dirigida mediante varias técnicas, como la microscopía óptica y electrónica, y técnicas colorimétricas, piezoeléctricas y electroquímicas. Además, los nanomódulos de bioreconocimiento depositados eran funcionales y capaces de diferenciar 4 mutaciones en oligonucleótidos de 19 bases. A pesar de los prometedores resultados, los cuales demostraron la viabilidad de utilizar la electrodeposición dirigida como técnica de arraying, se observó la necesidad de amplificar la señal electroquímica, puesto que los valores obtenidos del sistema eran muy parecidos a los valores obtenidos de los blancos. Siguiendo dos objetivos paralelos para la amplificación de la señal electroquímica (aumentar intrínsecamente las constantes de transferencia de electrones entre encimas y electrodos, y optimizar los sistemas de reciclaje electroquímicos), se diseñaron racionalmente complejos de osmio y se evaluó la cinética de transferencia de electrones entre dichos complejos y encimas redox. En estos estudios cinéticos se observó que los mediadores con carga global más positiva y con potenciales redox más altos proporcionaban constantes de transferencia de electrones más altas, también favorecidas a alto pH y baja fuerza iónica, demostrando la posibilidad de amplificar las señales electroquímicas. Esta tesis está dividida en siete capítulos. El primer capítulo es una introducción que explica los principios fundamentales y el estado de la ciencia en el área de los sensores y arrays de ADN, que sienta las bases del comportamiento de las suspensiones de oro coloidal y que presenta la cinética de transferencia de electrones entre mediadores y enzimas. El segundo capítulo describe el desarrollo de un método preliminar para caracterizar la inmovilización y detectar la hibridación en sensores de ADN. En el tercer capítulo se presentan estudios de estabilidad de las suspensiones de oro coloidal, conjugaciones de oligonucleótidos a dichos coloides y la caracterización de la eficacia de dichas conjugaciones, de la estabilidad de las suspensiones de las conjugaciones, y de la funcionalidad de los conjugados. En el cuarto capítulo se demuestra la electrodeposición dirigida y selectiva en varias superficies electródicas mediante varias técnicas de caracterización. En el quinto capítulo se obtienen las constantes de transferencia de electrones entre la Glucosa Oxidasa (GOx) y distintos complejos de osmio, y se analiza el efecto de varios parámetros experimentales en dichas constantes. Finalmente, un sexto capítulo establece las conclusiones de la tesis y un séptimo capítulo propone varias líneas de investigación a desarrollar en un futuro.

In the last years, DNA arrays have attracted increasing attention among medical diagnosis and analytical chemists. The broad range of application that has been found for DNA arrays makes them an important analytical tool. DNA arrays are relevant for the diagnosis of genetic diseases, detection of infectious agents, study of genetic predisposition, development of a personalised medicine, detection of differential genetic expression, forensic science, drug screening, food safety and environmental monitoring.Despite the great promise of DNA arrays in health care and their success in medical and biological research, the technology is still far away from the daily use in the clinic and even more far away from their implementation in home-diagnosis such as glucose biosensors. Their principal problems are the high cost and difficulty of use, because it is required costly laboratory instruments and biology knowledge for the labelling of the DNA prior to the sample injection into the array.On the other hand, the requirements that a biosensor should include are to be easy-to use so that it do not need the previous label of the sample and the addition of reagents. It should give a sensitive response in short time, and it should also include cheap generic multi-analyte detection.The work carried out in this thesis describes new concepts of electrochemical biosensoric platforms based on oligonucleotides for detection of label-free DNA and protein, which include these requirements.Preliminary experiments of direct DNA electrochemical detection of labelled ssDNA were performed to establish a protocol of DNA immobilisation, hybridisation and detection colourimetrically and electrochemically. DNA real samples and multi-analite detection on an array developed by biocopatible photolithography were used.To avoid the analyte labelling to develop an easy to use and low cost device, a label-free electrochemical displacement of DNA sensor was described. The method of detection by displacement requires the pre-hybridisation of the capture probe immobilised on the electrode surface with a sub-optimum mutated oligonucleotide labelled with a redox molecule. Due to the higher affinity of the target that is fully complementary to the capture probe, the sub-optimum label can be displaced when the complementary target is introduced in the system. The decrease of the signal would verify the presence of the target and should be proportional to its concentration. Sub-optimum hybridisation displacement detection was demonstrated colourimetrically and electrochemically with a sub-optimum mutated oligonucleotide labelled with horseradish peroxidase (HRP), and a ferrocene sub-optimum mutated oligonucleotide was also detected electrochemically, which do not required the addition of reagents for its detection.Furthermore different strategies to develop an electrochemical oligonucleotide (aptamer) based sensor for reagentless and label-free protein detection was carried out. The most sensitive aptasensor achieved 30 fM of detection limit in just 5 minutes. / En els últims anys, els xips d'ADN han atret una atenció creixen en els camps de la diagnosis mèdica i la química analítica, degut a la seva portabilitat, sensibilitat, especificitat, ràpida resposta i l'ampli ventall d'aplicacions. Els xips d'ADN són rellevants per la diagnosis de malalties genètiques, detecció d'agents infecciosos, estudis de predisposició genètica, desenvolupament de medicina personalitzada, detecció d'expressió genètica diferencial, medicina forense, exploració de medicaments, seguretat alimentaria, defensa militar i monitorització mediambiental. Encara que els xips basats en oligonucleòtids per la detecció d'ADN i proteïnes siguin una gran promesa en medicina i recerca biològica, aquesta tecnologia es encara molt lluny del seu ús diari en el camp clínic i encara més lluny de poder ser comercialitzada per ús domèstic com ho han estat el biosensors de glucosa. Els seus principals problemes són el seu alt cost i la seva dificultat d'ús. Ja que per la seva utilització és necessari, previ a la injecció de l'analit en el biosensor, costosos instruments de laboratori i tècnics especialitzats en bioquímica pel marcatge i amplificació de les mostres d'ADN. En canvi els requeriments que un biosensor ha d'incloure són, ser fàcil d'utilitzar, per tant que l'analit no necessiti un marcatge previ i l'addició de reactius per la seva detecció. Aquest ha de donar una resposta ràpida i sensible a baix cost i ha de permetre la detecció en el mateix equip de diferent tipus d'analits.El treball fet en aquesta tesis descriu el desenvolupament de nous concepte de plataformes biosensòriques electroquímiques basades en oligonucleòtids per la detecció d'ADN i proteïnes no marcades prèviament, els quals inclouen aquest requeriments. Experiments preliminars per la detecció de l'hibridació d'ADN marcat es van portar a fi per tal d'establir un protocol per la immobilització, hibridació i detecció d'ADN colorimètricament i electroquímicament. És van utilitzar mostres reals d'ADN i sistemes de detecció de multi-analits en un xip desenvolupat per fotolitografia biocompatible.Per tal de no necessitar un marcatge previ de la mostres d'ADN i així simplificar i reduir el cost del futur biosensor es va desenvolupar un sistema electroquímic de desplaçament. El mètode lliure de marcatge es basa en el desplaçament de molècules d'oligonucleòtid mutat i marcat, els quals encara que continguin certes mutacions són capaços d'hibridar amb la sonda d'oligonucleòtid immobilitzat, però quan aquestes es troben en presència de l'analit desplaça la molècula mutada i marcada, disminuint així la senyal de manera proporcional en la concentració del analit. El sistema de desplaçament ha estat demostrat colorimètricament i electroquímicament utilitzant un marcatge d'HRP sobre el mutat i utilitzant un marcatge de ferrocè en l'oligonucleòtid mutat per tal de no necessitar afegir cap reactiu per la detecció de l'analit, També es van portar a fi diferents estratègies per desenvolupar un biosensor electroquímic basat en oligonucleòtids (aptamers) per la detecció de la proteïna trombina sense el previ marcatge d'aquest analit i sense necessitat d'afegir reactius per la detecció del analit. En el sistema mes sensible es va obtenir un límit de detecció de 30 fM en un temps de resposta de sols 5 minuts. / En los últimos años, los chips de ADN han atraído una atención creciente diferentes campos, debido a su portabilidad, sensibilidad, especificidad y rápida respuesta. Los chips de ADN son aplicados en diagnosis de enfermedades genéticas, detección de agentes infecciosos, estudios de predisposición genética, desarrollo de medicina personalizada, detección de expresión genética diferencial, medicina forense, exploración de medicamentos, columnas de separación, seguridad alimentaría, defensa militar y monitorización medioambiental. Aunque los chips basados en oligonucleótidos para la detección de ADN y proteínas tienen un gran futuro en diagnosis e investigación biológica, esta tecnología está aun muy lejos de su uso diario en el campo clínico y aun mas lejos de poder ser comercializado para uso doméstico como lo han sido los biosensores de glucosa. Sus principales problemas son su alto coste y su dificultad de uso. Para su utilización es necesario, previo a la inyección del analito en el biosensor, costosos instrumentos de laboratorio y técnicos especializados en bioquímica para el marcaje y amplificación de las muestras de ADN. En cambio los requerimientos que un biosensor ha de incluir son, ser fácil de utilizar, por tanto el analito no ha de necesitar un marcaje previo ni la adición de reactivos para su detección. Este ha de dar una respuesta rápida y sensible a bajo coste y ha de permitir la detección en el mismo equipo de diferentes analitos.El trabajo hecho en esta tesis describe el desarrollo de nuevos conceptos de plataformas biosensóricas electroquímicas basadas en oligonucleótidos para la detección de ADN y proteínas no marcadas previamente, los cuales incluyen estos requerimientos.Experimentos preliminares para la detección directa de la hibridación de ADN marcado se llevó a cabo para establecer protocolos para la inmovilización, hibridación y detección de ADN colorimétricamente y electroquímicamente. Se utilizaron muestras reales y sistemas de detección de multi-analitos en un chip desarrollado por fotolitografía biocompatible.Para no necesitar un marcaje previo de la muestra de ADN y así simplificar y reducir el coste del futuro biosensor se desarrolló un sistema electroquímico de desplazamiento. El método libre de marcaje se basa en el desplazamiento de moléculas de oligonucleótido mutado y marcado, el cual aunque contenga ciertas mutaciones es capaz de hibridar con la sonda de oligonucleótido inmovilizado, pero cuando estas se encuentran en presencia del analito desplaza la molécula mutada, disminuyendo así la señal de manera proporcional a la concentración del analito. El sistema de desplazamiento ha sido demostrado colorimétricamente y electroquímicamente utilizando marcaje de HRP sobre el mutado, así como un marcaje de ferroceno que no requiere la adición de reactivos para su detección. También se llevaron a cabo diferentes estrategias para desarrollar un biosensor electroquímico basado en oligonucleótidos (aptámeros) para la detección de trombina sin el previo marcaje de este analito, ni la adición de reactivos para la detección del analito. En el sistema más sensible se obtuvo un límite de detección de 30 fM en un tiempo de respuesta de solo 5 minutos

Las señales de los biosensores se pueden mejorar mediante el diseño de superficies transductoras. En este sentido, se han investigado diversos métodos para la nanoestructuración de superficies. El primero de ellos se basó en la formación inicial de monocapas autoensambladas (SAM) de alcanotioles sobre sustratos bimetálicos, seguida de la desorción reductiva selectiva (SRD) de las SAM de determinados metales. Se consiguió la SRD de 2-mercaptoetanol de dominios de paladio desde una superficie de platino-oro. El segundo método para preparar superficies nanoestructuradas que se investigó fue la nucleación electroquímica secuencial de las nanopartículas metálicas (oro y paladio) sobre electrodos de carbón vidrio para las SAM de alcanotiol y para aumentar la densidad de las nanopartículas sin permitir la formación de agregados. Con este método, las señales redox de las SAM alcanotiol ferrocenil eran seis y cincuenta veces mejores que los electrodos de oro y paladio, respectivamente. Finalmente, se demostró la nanoestructuración de las superficies de los electrodos para mejorar la señal de un biosensor de ADN. / Biosensor signals can be enhanced by specifically designing transducer surfaces. In this thesis, several surface nanostructuring approaches have been investigated. The first approach studied was based on the initial formation of self-assembled monolayers (SAM) of alkanethiols on bi-metallic substrates, followed by the selective reductive desorption (SRD) of the SAM from one of the metals. SRD of 2-mercaptoethanol from palladium domains of a palladium-gold surface was achieved. The second nanostructured surface preparation method investigated was the sequential electrochemical nucleation of metal nanoparticles (gold and palladium) on glassy carbon electrode and SAM formation on the NPs to prevent aggregation and by that increasing the number densities. With this method, a six-fold and a fifty fold enhancement in the ferrocenyl alkanethiol SAM redox signal was achieved in comparison to plain gold and palladium electrodes, respectively. Finally, electrode surface nanostructuring using sequentially nucleated gold nanoparticles for signal enhancement of DNA biosensor was demonstrated.

<p>The refractive index sensitivity of plasmonic nanoparticles is utilized in the development of real-time, label-free biodetection. Analyte molecules that bind to receptor-conjugated nanoparticles cause an increase in local refractive index that in turn induces an energy shift in the optical resonance of the particle. Biomolecular binding is quantified by quantitatively measuring these resonance shifts. This work describes the application and optimization of a biomolecular detection system based on gold nanorods as an optical transducer.</p> <p>A microspectroscopy system was developed to collect scattering spectra of single nanoparticles, and measure shifts of the spectra as a function of biomolecular binding. The measurement uncertainty of LSPR peak shifts of the system was demonstrated to be 0.3 nm. An analytical model was also developed that provides the optimal gold nanorod geometry for detection with specified receptor-analyte pair. The model was applied to the model biotin-streptavidin system, which resulted in sensing system with a detection limit of 130 pM - an improvement by four orders of magnitude over any other single-particle biodetection previously presented in the literature.</p> <p>Alternative optical detection schemes were also investigated that could facilitate mulitplexed biosensing. A theoretical model was built to investigate the efficacy of using a multi-channel detector analogous to a conventional RGB camera. The results of the model indicated that even in the best case, the detection capabilities of such a system did not provide advantages over the microspectroscopic approach.</p> <p>We presented a novel hyperspectral detection scheme we term Dual-Order Spectral Imaging (DOSI) which is capable of simultaneously measuring spectra of up to 160 individual regions within a microscope's field of view. This technique was applied to measuring shifts of individual nanoparticles and was found to have a peak measurement uncertainty of 1.29 nm, at a measurement rate of 2-5 Hz.</p> / Dissertation

<p>Noble metal nanoparticles exhibit unique optical properties that are beneficial to a variety of applications, including molecular imaging. The large scattering cross sections of nanoparticles provide high contrast necessary for biomarkers. Unlike alternative contrast agents, nanoparticles provide refractive index sensitivity revealing information regarding the local cellular environment. Altering the shape and composition of the nanoparticle shifts the peak resonant wavelength of scattered light, allowing for implementation of multiple spectrally distinct tags. In this project, nanoparticles that scatter in different spectral windows are functionalized with various antibodies recognizing extra-cellular receptors integral to cancer progression. A hyperspectral imaging system is developed, allowing for visualization and spectral characterization of cells labeled with these conjugates. Various molecular imaging and microspectroscopy applications of plasmonic nanoparticles are then investigated. First, anti-EGFR gold nanospheres are shown to quantitatively measure receptor expression with similar performance to fluorescence assays. Second, anti-EGFR gold nanorods and novel anti-IGF-1R silver nanospheres are implemented to indicate local cellular refractive indices. Third, because biosensing capabilities of nanoparticle tags may be limited by plasmonic coupling, polarization mapping is investigated as a method to discern these effects. Fourth, plasmonic coupling is tested to monitor HER-2 dimerization. Experiments reveal the interparticle conformation of proximal HER-2 bound labels, required for plasmonic coupling-enhanced dielectric sensing. Fifth, all three functionalized plasmonic tags are implemented simultaneously to indicate clinically relevant cell immunophenotype information and changes in the cellular dielectric environment. Finally, flow cytometry experiments are conducted utilizing the anti-EGFR nanorod tag to demonstrate profiling of receptor expression distribution and potential increased multiplexing capability.</p> / Dissertation

An amperometric glucose biosensor was fabricated using electospun polyaniline fibers. Polyaniline was reacted with camphorsulfonic acid to produce a salt, which was then dissolved in chloroform containing polystyrene. Using this solution, fibers were formed and collected by electrospinning. Glucose oxidase was immobilized onto these fibers using an electrostatic layer-by-layer adsorption technique. In this method, poly(diallyldimethylammonium chloride) was used as the counter ion source. The level of adsorption was examined and evidence of layer-by-layer adsorption was obtained using a quartz crystal microbalance technique. A biosensor was fabricated from these fibers as a working electrode, and used to measure the glucose concentration accurately.

Pesticides are natural or artificial molecules aimed to kill, or mitigate any harmful organism. Although their use in agriculture provides us with an increased crop yield, remains of chemicals on the products creates health concerns in society. Organophosphates and carbamates are two groups of insecticides. Although they are far more lethal against insects and small animals, they can also cause poisoning in humans through the inhibition of acetylcholinesterase enzyme (AChE) that plays an important role in human nervous system. Therefore, the detection of these compounds is crucial. The conventional methods for the detection of these compounds are expensive, time-consuming and need expertise. In this study, a fast, disposable, cheap and accurate acetylcholinesterase biosensor was developed to detect organophosphate and carbamate-based pesticide residues. By means of adsorption method, AChE, the chromophore 5,5&#039 / -Dithio-bis(2-nitrobenzoic acid) (DTNB) and artificial substrate acetylthiocholine (ATCh) were immobilized on the supporting material. In optimization studies / from 3 to 15U/mL concentrations were experimented for AChE, 1 to 5mM DTNB and 1 to 5mM ATCh concentration gradients were used. v As a result of the optimization studies 12U/mL ACHE concentration, 5mM DTNB concentration and 5mM ATCh concentration were determined for constructing a pesticide biosensor. Detection limit of malathion, an organophosphate-based insecticide was found as 2.5ppm in 5% methanol solution. The biosensor conserved its integrity between pH 4 and 8, and gave false positive results after pH 10. Stability studies showed that, biosensor retained its activity for at least 60 days at 4&deg / C to discrimnate between positive and negative controls.

This research proposes a novel polymer-based optical waveguide made with Polydimethylsiloxane (PDMS) for optical detection applications. Alternative to other fiber-based sensor, the proposed optical sensor uses PDMS waveguide as the main sensing component. PDMS has excellent optical properties which is essential for bio-photonic detection, including highly optical transparency, good flexibility and high bio-compatibility. Uncured PDMS polymer is cast in a Teflon tubing to form the PDMS rod. Since the reflective index of PDMS is as high as 1.43, that the bare PDMS can be an optical waveguide while the reflective index of the surrounding media is smaller than 1.43. The cast PDMS waveguide is then connect with plastic optical fibers to form the proposed optical waveguide system. In order to improve the optical performance of the PDMS waveguide, a surface coating process is used to reduce the surface roughness of the PDMS waveguide. The measured insertion loss with and without performing the surface coating procedure is 1.14 and 1.71dB/cm, respectively. Once the PDMS waveguide is formed, Au nanoparticles (Au-Nps) were coated on the PDMS surface with the assistance of a positive charge polymer of PDDA to form an optical waveguide capable of localized SPR detection. In addition, an atmospheric plasma treating process is used to enhance the coating ratio and speed of Au-Nps. UV-VIS spectrum and the SEM observation of the Au-particle coated PDMS waveguide confirm that the plasma treatment process significantly improves the coating results of Au-Nps. Liquid samples with different refractive index were used to demonstrate the LSPR sensing ability of the fabricated optical waveguide. The label free DNA detection was demonstrated by the system. The thiolated single strand DNA was modify on the PDMS optical waveguide as a DNA probe and bound with target DNA by DNA hybridization. The detection limit is as low as 10 pM. This research provides a simple and fast fabrication method to fabricate waveguide-based LSPR sensors.