Dissertations / Theses on the topic 'Biosensor label-free'

The discovery of monolayer graphene by Manchester group has led to intensive research into a variety of applications across different disciplines. As a monolayer of carbon atoms, graphene presents a high surface to volume ratio and a good electronic conductivity, making it sensitive to its surface bio-chemical environment. This project investigated the fabrication of electronic biosensors using different graphene-based materials. It included the production of graphene, the fabrication of electronic devices, the chemical functionalisation of graphene surface and the specific detection of target bio-molecules. This project first investigated the production of graphene using three different methods, namely mechanical exfoliation, physical vapour deposition and electrochemical reduction of graphene oxide. With respect to the physical vapour deposition method, the production of large area transfer-free graphene from sputtered carbon and metal layers on SiO2 substrate has, for the first time, been achieved. The relationship between growth parameters and the quality of resultant graphene layer has been systematically studied. In addition, a growth model based on the detailed analysis of morphological structures and properties of graphene film was simultaneously proposed. Optical microscopy, Raman spectroscopy and atomic force microscopy were used for the evaluation of the number, the quality and the morphology of resultant graphene layers in each method. To investigate the performance of graphene electronic devices, field effect transistors were fabricated using both exfoliated and chemical vapour deposited graphene. A novel technique for graphene patterning has been developed using deep ultraviolet baking and an improved photolithography method. A new shielding technique for the low damage deposition of Au electrodes on graphene has also been developed in this project. The practical challenges of device fabrication and performance optimisation, such as polymer residue and contact formation, have been studied using Raman spectroscopy and the Keithley 2602A multichannel source meter. For the functionalisation of graphene, a number of chemicals were investigated to provide linking groups that enable binding of bio-probes on the graphene surface. Hydrogen peroxide and potassium permanganate have been demonstrated to have the capability of immobilising oxygen-containing groups onto graphene. The levels of oxidation were estimated by energy dispersive analysis and Fourier transform infrared spectroscopy. In addition, aminopropyltriethoxysilanes and polyallylamine have exhibited good efficiency for immobilising amino groups onto graphene. The resultant graphene was characterised by X-ray photoelectron spectroscopy and cyclic voltammetry measurements. Graphene electrodes modified with electrochemically reduced graphene oxide were developed for the first time which exhibit significantly improved redox currents in electrochemical measurements. Using single stranded DNA immobilised via π-π bonds as probes, these electrodes showed a limit of detection of 1.58 x 10-13 M for the human immunodeficiency virus 1 gene. In parallel, human chorionic gonadotropin sensors were developed by immobilising its antibodies on 1-pyrenebutyric acid N-hydroxysuccinimide ester functionalised graphene field effect transistors. These field effect transistors have been demonstrated to exhibit a quantitative response toward the detection of 0.625 ng/ml antigen. In summary, the fabrications of two types of graphene-based biosensors for the detection of specific DNA sequence and human chorionic gonadotropin have been achieved in this project. Their sensitivity, selectivity, reproducibility and capability of multiple biomarker detection need to be further improved and explored in future work. The outcomes of this project have provided not only ready-made biosensing platforms for the detection beyond these two targets, but also novel techniques applicable to the development of multidisciplinary applications beyond biosensor itself.

Recent advances in nanotechnology offer a new platform for the label free detection of biomolecules at ultra-low concentrations. Nano biosensors are emerging as a powerful method of improving device performance whilst minimizing device size, cost and fabrication times. Nanogap capacitive biosensors are an excellent approach for detecting biomolecular interactions due to the ease of measurement, low cost equipment needed and compatibility with multiplex formats. This thesis describes research into the fabrication of a nanogap capacitive biosensor and its detection results in label-free aptamer-based protein detection for proof of concept. Over the last four decades many research groups have worked on fabrication and applications of these type of biosensors, with different approaches, but there is much scope for the improvement of sensitivity and reliability. Additionally, the potential of these sensors for use in commercial markets and in everyday life has yet to be realized. Initial work in the field was limited to high frequency (>100 kHz) measurements only, since at low frequency there is significant electronic thermal noise (< V2 > = 4kBTR) from the electrical double layer (EDL). This was a significant drawback since this noise masked most of the important information from biomolecular interactions of interest. A novel approach to remove this parasitic noise is to minimize the EDL impedance by reducing the capacitor electrode separation to less than the EDL thickness. In the case of aptamer functionalized electrodes, this is particularly advantageous since device sensitivity is increased as the dielectric volume is better matched to the size of the biomolecules and their binding to the electrode surface. This work has demonstrated experimentally the concepts postulated theoretically. In this work we have fabricated a large area (100 x 5 μm x 5 μm) vertically oriented capacitive nanogap biosensor with a 40 nm electrode separation between two gold electrodes. A silicon dioxide support layer separates the two electrodes and this is partially etched (approximately 800 nm from both sides of each 5 μm x 5 μm capacitor), leaving an area of the gold electrodes available for thiol-aptamer functionalization. AC impedance spectroscopy measurements were performed with the biosensor in the presence of air, D.I. water, various ionic strength buffer solutions and aptamer/protein pairs inside the nanogap. Applied frequencies were from 1Hz to 500 kHz at 20 mV AC voltage with 0 DC. We obtained relative permittivity results as a function of frequency for air (ɛ=1) and DI water (ɛ~80) which compares very favorably with previous works done by different research groups. The sensitivity and response of the sensors to buffer solution (SSC buffer) with various ionic strengths (0.1x SSC, 0.2x SSC, 0.5x SSC and 1x SSC) was studied in detail. It was found that in the low frequency region (< 1 kHz) the relative permittivity (capacitance) was broadly constant, that means it is independent from the applied frequency in this range. With increasing buffer concentration, the relative permittivity starts to increase (from ɛ=170 for 0.1x SSC to ɛ=260 for 1x SSC). The sensor performance was further investigated for aptamer-based protein detection, human alpha thrombin aptamers and human alpha thrombin protein pairs were selected for proof of concept. Aptamers were functionalized into the gold electrode surface with the Self-Assembly-Monolayer (SAM) method and measurements were performed in the presence of 0.5x SSC buffer solution (ɛ=180). Then the hybridization step was carried out with 1 μM of human alpha thrombin protein followed by measurements in the presence of the same buffer (ɛ=130). The response of the sensors with different solutions inside the nanogap was studied at room temperature (5 working devices were tested for each step). The replacement of the buffer solution (ɛ=250) with lower relative permittivity biomolecules (aptamer ɛ=180) and further binding proteins to immobilized aptamer (ɛ=130) was studied. To validate these results, a control experiment was carried out using different aptamers, in this case which are not able to bind to human alpha thrombin protein. It was found that the relative permittivity did not change after the hybridization step compared to the aptamer functionalization step, which indicates that the sensors performance is highly sensitive and reliable. This work serves as a proof of concept for a novel nanogap based biosensor with the potential to be used for many applications in environmental, food industry and medical industry. The fabrication method has been shown to be reliable and consistent with the possibility of being easily commercialized for mass production for use in laboratories for the analysis of a wide range of samples.

The continue request in medical field of methods for the diagnosing and the monitoring of diffuse pathologies like cancer, Alzheimer and muscular dystrophy, has pushed the scientific research to focus its interest in he design of biosensors for fast and in-situ assays. Although several typology of biosensors has been proposed, label-free immunosensors are good candidates in the biomarkers detection thanks to a high bio-selective recognition and a simple read-out. This thesis presents the research activity about the design, fabrication and testing of an immunosensor based on a Si3N4 2-D photonic crystal (PhC) in membrane configuration and further optimizations of the fabrication process of PhC membranes for biosensing applications. The structure has been optimized by means of the finite difference time domain method (FDTD) in order to achieve peaks of reflectivity in the visible-near infrared spectrum. Subsequently, a nano-fabrication protocol exploiting e-beam lithography and dry/wet etching has been optimized, obtaining high resolution structures. Finally, the immunosensor has been functionalized with a layer of antibodies for the detection of the IL-6 protein and experimental tests has been performed, achieving a sensitivity of 1.5 pg/ml. A further step has been the optimization of the fabrication processes of PhC membranes for biosensing applications and their transferring from rigid substrate to flexible polymeric layer in order to achieve high integrable and biocompatible devices.

This dissertation investigates a label-free sensing platform which can be used to detect DNA, enzyme or protein, based upon electrochemical detection which is suitable for implementation in microarray form. Two implementations are proposed based on mixed Ferrocene self-assembled monolayer (SAM) and the Azurin (metalloprotein) SAM. We have shown for the first time that electro-active SAM, functionalized with suitable receptors, can be employed for the detection of biomolecular interactions. Detection of streptavidin by biotin-functionalized Ferrocene SAM was successfully demonstrated. These results were made possible by the development of the fabrication protocols that optimize the SAM stability and reproducibility. Reliable samples, combined with theoretical modelling and modification of existing published model for electro-active SAM, has enabled us to experiment and analyse in depth various electrochemical detection techniques, based on changes in capacitance, voltammetric formal potential and current, Open Circuit Potential (OCP). It was found that AC voltammetry and OCP are the best measurement techniques. The use of OCP with an electro-active SAM had not been previously demonstrated and the theoretical basis for this technique was presented. Essential for this technique was the development of micro-electrodes to reduce parasitic capacitances that would reduce the available signal, enabling real-time detection of bio-molecular interaction. We also made possible to characterize the binding of a protein (streptavidin) to a biotin-functionalized Azurin SAM. Also a numerical analysis has been developed to analyse the effect of design parameters of the platform, such as the probe density and buffer concentration, which can greatly affect the assay sensitivity. This is achieved using 3D simulation with finite element method in COMSOL.

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

DNA detection technology has developed rapidly due to its extensive application in clinical diagnostics, bioengineering, environmental monitoring, and food science areas. Currently developed methods such as surface Plasmon resonance (SPR) methods, fluorescent dye labeled methods and electrochemical methods, usually have the problems of bulky size, high equipment cost and time-consuming algorithms, so limiting their application for in vivo detection. In this work, an intrinsic Fabry-Perot interferometric (IFPI) based DNA sensor is presented with the intrinsic advantages of small size, low cost and corrosion-tolerance. This sensor has experimentally demonstrated its high sensitivity and selectivity. In theory, DNA detection is realized by interrogating the sensorâ s optical cavity length variation resulting from hybridization event. First, a microgap structure based IFPI sensor is fabricated with simple etching and splicing technology. Subsequently, considering the sugar phosphate backbone of DNA, layer-by-layer electrostatic self-assembly technique is adopted to attach the single strand capture DNA to the sensor endface. When the target DNA strand binds to the single-stranded DNA successfully, the optical cavity length of sensor will be increased. Finally, by demodulating the sensor spectrum, DNA hybridization event can be judged qualitatively. This sensor can realize DNA detection without attached label, which save the experiment expense and time. Also the hybridization detection is finished within a few minutes. This quick response feature makes it more attractive in diagnose application. Since the sensitivity and specificity are the most widely used statistics to describe a diagnostic test, so these characteristics are used to evaluate this biosensor. Experimental results demonstrate that this sensor has a sensitivity of 6nmol/ml and can identify a 2 bp mismatch. Since this sensor is optical fiber based, it has robust structure and small size ( 125μm ). If extra etching process is applied to the sensor, the size can be further reduced. This promises the sensor potential application of in-cell detection. Further investigation can be focused on the nanofabrication of this DNA sensor, and this is very meaningful topic not only for diagnostic test but also in many other applications such as food industry, environment monitoring.
Master of Science

Medical diagnostics is in constant search of new tools and devices able to provide in short time, accurate and versatile tests performed on patients. Nanotechnology has contributed largely in developing biosensors of smaller size at a lower cost by using a minimal amount of sample. Biosensors aim to monitor and diagnosticate “in situ” the patient status and the diseases caused by alteration of the body metabolism by, for example, the detection of gene mutations, alteration of gene expression or alteration of proteins. The aim of this work is the development of biosensors that satisfy the requirements which are critical for applications. A biosensor must be i) easy to use, ii) economically convenient, and therefore preferentially label free, iii) highly sensitive, iv) reversible, v) and suitable for Point of Care Testing, that is to be used ”in situ” on the patient. We have focused on biosensors based on the optical properties of nanostructured metals as Au or Ag, in particular by using on Localized Surface Plasmon Resonance (LSPR) spectroscopy. Nanostructured metals under irradiation of electromagnetic wave (as light) exhibit intense absorption bands as results of the localized electronic charges of the metal surface coming into resonance with the incident energy. According to the Mie’s theory, the LSPR absorption band feature changes when the refractive index of the media surrounding the metal nanostructures is varied. Of particular interest for our purpose are the possible changes of the LSPR band features taking place under molecular interactions occurring at the nanostructures surfaces: the shift of LSPR bands is the “transducer” of molecular interactions. These changes can be easily detected by conventional UV-Vis spectroscopy, in transmittance mode. While a large number of studies have been carried out on monodisperse nanoparticles suspended in solution, gold nanoparticles (NPs) deposited on a transparent surface open the possibility to fabricate biosensor based on multiplex array platforms. Nonetheless, one of the major problems in using these plasmonic materials for biosensing purpose is related to the stability of the metal NPs in different solvents and in particular in aqueous solutions. In this study we demonstrate i) the possibility to achieve highly stable NPs by simple thermal evaporation of Au on a substrate commercially available, the Fluorine Tin Oxide (FTO) (Chapter 2); ii) a reproducible variation of the LSPR bands under formation of organic selfassembled monolayers (SAMs), iii) reversible changes in the features of the LSPR bands, (Chapter 3), iv) a specific and reproducible LSPR band changes under molecular interactions occurring at NPs surfaces, as DNA hybridization (Chapter 4). This work demonstrates that the plasmonic material based on Au NPs deposited on FTO surfaces represents a convenient platform for biosensors because of i) inexpensive fabrication, ii) stability of this material in various solvent, including water, of, iii) the easy way to detect the molecular interaction, and iv) the good sensitivity to molecular interactions.

The necessity of using extremely high sensitivity biosensors in certain research areas has remarkably increased during the last two decades. Optical structures, where light is used to transduce biochemical interactions into optical signals, are a very interesting approach for the development of this type of biosensors. Within optical sensors, photonic integrated architectures are probably the most promising platform to develop novel lab-on-a-chip devices. Such planar structures exhibit an extremely high sensitivity, a significantly reduced footprint and a high multiplexing potential for sensing applications. Furthermore, their compatibility with CMOS processes and materials, such as silicon, opens the route to mass production, thus reducing drastically the cost of the final devices. Optical sensors achieve their specificity and label-free operation by means of a proper chemical functionalization of their surfaces. The selective attachment of the receptors allows the detection of the target analytes within a complex matrix. This PhD Thesis is focused on the development of label-free photonic integrated sensors in which the detection is based on the interaction of the target analytes with the evanescent field that travels along the structures. Herein, we studied several photonic structures for sensing purposes, such as photonic crystals and ring resonators. Photonic crystals, where their periodicity provokes the appearance of multiple back and forth reflections, exhibits the so-called slow-light phenomenon that allows an increase of the interaction between the light and the target matter. On the other hand, the circulating nature of the resonant modes in a ring resonator offers a multiple interaction with the matter near the structure, providing a longer effective length. We have also proposed a novel approach for the interrogation of photonic bandgap sensing structures where simply the output power needs to measured, contrary to current approaches based on the spectral interrogation of the photonic structures. This novel technique consists on measuring the overlap between a broadband source and the band edge from a SOI-based corrugated waveguide, so that we can determine indirectly its spectral position in real-time. Since there is no need to employ tunable equipment, we obtain a lighter, simpler and a cost-effective platform, as well as a real-time observation of the molecular interactions. The experimental demonstration with antibody detection measurements has shown the potential of this technique for sensing purposes
García Castelló, J. (2014). A Novel Approach to Label-Free Biosensors Based on Photonic Bandgap Structures [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/35398
TESIS

Negli ultimi decenni, i biosensori hanno suscitato un crescente interesse in diversi settori grazie alle loro particolari caratteristiche biochimiche e la numerosità dei settori potenzialmente coinvolti, che spaziano dal biomedicale all’industria agroalimentare, dalla difesa, alla sicurezza. In letteratura scientifica sono stati studiati svariati tipi di biosensori basati su diversi principi fisici come elettrochimici, ottici e meccanici (sensibili a cambiamenti di massa). Una delle caratteristiche principali richieste dal mercato è che il sensore sia "label-free", ovvero che l'analisi venga eseguita senza alcuna alterazione degli analiti. Il lavoro descritto in questa tesi è incentrato sulla dimostrazione teorica e lo sviluppo pratico di un nuovo tipo di piattaforma basata su fibra ideata per il biosensing ottico. Le fibre a nucleo cavo ad accoppiamento inibito (HC-ICF) sono caratterizzate da un rivestimento microstrutturato composto da tubi che circondano un nucleo cavo. I fori che le caratterizzano consentono l'infiltrazione di sostanze biologiche e l’adesione alle interfacce aria-dielettrico di strati biologici. Rispetto ad altre fibre cave aumenta ulteriormente la facilità d'infiltrazione e la sensibilità del sensore. Inoltre, negli HC-ICF il particolare meccanismo di guida d'onda, basato sull’accoppiamento inibito, rende le loro proprietà di trasmissione particolarmente sensibili allo spessore di tubi di vetro che compongono il rivestimento microstrutturato. Grazie a ciò, se le interazioni molecolari tra la superficie della struttura di vetro e la molecola da rilevare creano uno strato aggiuntivo che incrementa lo spessore della struttura, è possibile rilevare la molecola misurando lo spettro di trasmissione delle fibre. La tecnica non richiede alcun componente aggiuntivo come reticoli di Bragg, tecniche di amplificazione come nanoparticelle o sorgenti coerenti. Il principio è validato con risultati sperimentali che mostrano la rilevazione di molecole come la proteina streptavidina e il DNA.
During the last decades, biosensors have gained more and more interest in several fields due to their particular features in the biochemical analysis. Involved sectors ranging from biomedical area to agri-food industry passing through defence and security. In the literature several types of biosensors have been studied based on different physical principle such as electrochemical, optical and mechanical (sensitive to mass changes). One of the main feature that the market requires is the “label-free” biosensing: the analysis is performed without any analytes alteration. The work described in this dissertation is focused on the conception and development of a new type of fiber based platform which could be a useful and effective for optical biosensing: Hollow core inhibited coupling fibers (HC-ICFs). They are characterized by a microstructured cladding composed of tubes that surround a hollow core. The holes running along HC-ICFs allow the infiltration of biological substances and for biological layers to attach on the air-dielectric interfaces. Compared to other holey fibers, the presence of a hollow core can further increase the infiltration feasibility and the sensor sensitivity. Moreover, in HC-ICFs the particular waveguiding mechanism, based on inhibition coupling, makes their transmission properties particularly sensitive to the thickness of the glass struts composing the microstructured cladding. Thank to that, if the molecular interactions between the surface of the glass struts and the target to be detected result in a generation of an additional layer which modifies the strut thickness, the target can easily detect by measuring the fiber transmission spectrum. The technique does not require any additional transducer component such as Bragg gratings, amplifying techniques such as nanoparticles, nor coherent sources. The principle is validated with experimental results showing the detection of molecules such as the streptavidin protein and DNA.

[ES] En esta tesis se ha abordado el desarrollo de biosensores ópticos label-free, basados en tecnología de disco compacto, permitiendo así abaratar y simplificar su fabricación. El trabajo llevado a cabo ha consistido en estudiar diferentes propiedades físico-químicas desarrolladas por diversos materiales. Ello ha permitido obtener sistemas compactos y accesibles, capaces de sensar con buenas prestaciones analíticas en diferentes escenarios. En el capítulo 1 se presenta un estudio de inhibición enzimática sobre discos Blu-ray como plataforma de ensayo para el cribado de fármacos. Para ello, se inmoviliza orientadamente una glicoenzima, de la familia de las peroxidasas sobre la superficie del disco, cuya actividad se relaciona con los compuestos a cribar. Después de ensayar cada compuesto, se determina el grado de inhibición enzimática mediante la adición de un sustrato. La cantidad de producto obtenido, inversamente proporcional al potencial inhibitorio del compuesto en estudio, es cuantificado con un lector de discos que registra las variaciones en la intensidad del haz laser reflejado debidas al producto enzimático. Ello permite realizar más de 1700 ensayos simultáneos en un único disco Blu-ray lo que muestra su potencial en análisis masivo de alto rendimiento. Además, se plantea una estrategia basada en hipersuperficies para el análisis de la elevada cantidad de datos que se generan en las etapas del proceso de descubrimiento de fármacos. En el capítulo 2 se aborda el estudio de una metodología para reducir el ruido generado en la lectura de resultados obtenidos con biosensores ópticos, mejorando así su sensibilidad. Para ello, se plantea la estructuración del ensayo en forma de franjas en lugar del tradicional microarray, generando una señal periódica sinusoidal al ser escaneadas. Al analizar dicha señal en dominio de frecuencias, ésta se concentra en un pico a la frecuencia del ensayo, mientras que la mayor parte del ruido aparece a frecuencias mucho más altas. Inicialmente se quería reducir al máximo el ruido, para diferenciar las interacciones moleculares en formato label free. Sin embargo, los ensayos realizados con discos DVD no generaron suficiente señal, teniendo que recurrir en esta ocasión al marcaje para la cuantificación de inmunoglobulinas G y de caseína. Pese a ello, la metodología desarrollada también se puede aplicar en biosensores tipo label-free, reduciendo el ruido y mejorando su sensibilidad. El capítulo 3 se centra en el desarrollo de sustratos interferométricos multicapa que varían la intensidad de la luz reflejada al realizar un ensayo analítico en su superficie. Los sustratos fueron fabricados utilizando los materiales que componen los DVD-RW, depositados en capas de espesor controlado con el fin de obtener la máxima respuesta. A su vez, se diseñaron de tal forma que uno de ellos disminuya la intensidad del haz reflejado como respuesta a las interacciones moleculares, mientras que el otro la aumenta. El trabajo incluye la utilización de principios y materiales de la tecnología de disco compacto para el desarrollo del sistema de detección. Para ello, se emplea el cabezal de un lector de DVD, ya que dispone de un láser y de todos los elementos ópticos necesarios para el escaneado vertical. Con este sistema se cuantifican con éxito y sin marcaje inmunoglobulinas G y sulfasalazina, una macromolécula y un fármaco de masa molecular reducida. El capítulo 4 consiste en la fabricación de un cristal fotónico utilizando la estructura de los discos compactos cubiertos con una película de óxido de titanio. Se han estudiado las propiedades físico-químicas de estos sustratos y se han caracterizado sus propiedades fotónicas. Todo ello está en concordancia con los resultados obtenido mediante simulaciones. Para interrogar los cristales fotónicos fueron necesarios una fuente de luz blanca y un espectrofotómetro, además de los elementos ópticos necesarios
[CAT] En aquesta tesi s'ha abordat el desenvolupament de biosensors òptics label-free, basats en tecnologia de disc compacte, permetent així abaratir i simplificar la seua fabricació. El treball dut a terme ha consistit en estudiar diferents propietats fisicoquímiques desenvolupades per diversos materials. Això ha permès obtenir sistemes compactes i accessibles, capaços de sensar, amb bones prestacions analítiques, en diferents escenaris. En el capítol 1 es presenta un estudi d'inhibició enzimàtica sobre discos Blu-ray com a plataforma d'assaig per al cribratge de fàrmacs. Per a això, s'immobilitza de manera orientada una glicoenzima de la família de les peroxidases sobre la superfície del disc, i la seua activitat es relaciona amb els compostos a garbellar. Després d'assajar cada compost, es determina el grau d'inhibició enzimàtica mitjançant l'addició d'un substrat. La quantitat de producte obtingut, inversament proporcional al potencial inhibitori del compost en estudi, és quantificat amb un lector de discos que registra les variacions en la intensitat del làser reflectit degudes al producte enzimàtic. Això permet realitzar més de 1700 assajos simultanis en un únic disc Blu-ray el que mostra el seu potencial en anàlisi massiva d'alt rendiment. A més, es planteja una estratègia basada en hipersuperficies per a l'anàlisi de l'elevada quantitat de dades que es generen en les etapes del procés de descobriment de fàrmacs. En el capítol 2 s'aborda l'estudi d'una metodologia per reduir el soroll generat en la lectura de resultats obtinguts amb biosensors òptics, millorant així la seua sensibilitat. Per a això, es planteja l'estructuració de l'assaig en forma de franges en lloc del tradicional microarray, generant un senyal periòdic sinusoïdal en ser escanejades. En analitzar aquest senyal en domini de freqüències, aquesta es concentra en un pic a la freqüència de l'assaig, mentre que la major part del soroll apareix a freqüències molt més altes. Inicialment es volia reduir al màxim el soroll, per diferenciar les interaccions moleculars en format label-free. No obstant això, els assajos realitzats amb discos DVD no van generar prou senyal, havent de recórrer en aquesta ocasió al marcatge per a la quantificació d'immunoglobulines G i de caseïna. Malgrat això, la metodologia desenvolupada també es pot aplicar en biosensors tipus label-free, reduint el soroll i millorant la seua sensibilitat. El capítol 3 es centra en el desenvolupament de substrats interferometrics multicapa que varien la intensitat de la llum reflectida en realitzar un assaig analític en la seua superfície. Els substrats van ser fabricats utilitzant els materials que componen els DVD-RW, dipositats en capes de gruix controlat per tal d'obtenir la màxima resposta. Al seu torn, es van dissenyar de tal manera que un d'ells disminueixi la intensitat del feix reflectit com a resposta a les interaccions moleculars, mentre que l'altre l'augmenta. El treball inclou la utilització de principis i materials de la tecnologia de disc compacte per al desenvolupament del sistema de detecció. Per a això, es va utilitzar el capçal d'un lector de DVD, ja que disposa d'un làser i de tots els elements òptics necessaris per a l'escanejat vertical. Amb aquest sistema es quantifiquen amb èxit i sense marcatge immunoglobulines G i sulfasalazina, un macromolècula i un fàrmac de massa molecular reduïda. El capítol 4 consisteix en la fabricació d'un cristall fotònic utilitzant l'estructura dels discos compactes coberts amb una pel·lícula d'òxid de titani. S'han estudiat les propietats fisicoquímiques d'aquests substrats i s'han caracteritzat les propietats fotòniques. Tot això està en concordança amb els resultats obtingut mitjançant simulacions. Per interrogar els cristalls fotònics van ser necessaris una font de llum blanca i un espectrofotòmetre, a més dels elements òptics necessaris per guiar la llum.
[EN] In this thesis the development of label-free optical biosensors, based on compact disc technology, has been approached, thus making their manufacture cheaper and simpler. The work carried out has consisted of studying different physical-chemical properties manifested with several materials. This has allowed to obtain compact and accessible systems, capable of sensing with a great analytical performance in different scenarios. Chapter 1 presents an enzymatic inhibition study on Blu-ray discs as a test platform for drug screening. For this purpose, a glycoenzyme of the peroxidase family is immobilized on the surface of the disc whose activity is related to the compounds to be screened. After testing each compound, the degree of enzymatic inhibition is determined by adding the enzymatic substrate. The amount of product obtained is inversely proportional to the inhibitory potential of the compound, and is quantified with a disk reader that records the variations in the intensity of the reflected laser beam due to the enzymatic product. In addition, more than 1700 tests are performed on a single Blu-ray disc as proof of concept for application in high performance analysis and a hypersurface based strategy is proposed for the analysis of the large amount of data generated in the stages of the drug discovery process. Chapter 2 deals with the study of a methodology to reduce noise generated in the reading of results obtained with optical biosensors, hence improving their sensitivity. For this purpose, the structure of the test is proposed in the form of stripes instead of the traditional microarray, generating a sinusoidal periodic signal when they are scanned. When analysing this signal in frequency domain, it is concentrated in a peak at the frequency of the test, while most of the noise appears at much higher frequencies. Initially, the aim was to reduce noise as much as possible in order to differentiate molecular interactions in a label-free format. However, the tests carried out on a DVD did not generate enough signal, having to resort to labelling on this occasion for the quantification of immunoglobulins G and casein. Nevertheless, the methodology developed can be applied to label-free biosensors, reducing noise and improving sensitivity. Chapter 3 focuses on the development of multilayer interferometric substrates that vary the intensity of reflected light when performing an analytical test on their surface. The substrates were manufactured using the materials that make up the DVD-RW, deposited in layers of controlled thickness in order to obtain maximum response. At the same time, they were designed in such a way that one of them decreased the intensity of the reflected beam as a response to molecular interactions, while the other increased it. The work includes the use of principles and materials from compact disc technology for the development of the detection system. For this, the head of a DVD reader is used, as it has a laser and all the optical elements necessary for vertical scanning. With this system, immunoglobulins G and sulfasalazine, a macromolecule and a drug with reduced molecular mass are successfully quantified without labelling. Chapter 4 consists of the fabrication of a photonic crystal using the structure of the compact discs covered with a titanium oxide layer. The physical-chemical properties of these substrates have been studied and their photonic properties have been characterized. All this is in accordance with the results obtained through simulations. To interrogate the photonic crystals, a white light source and a spectrophotometer were needed, as well as the optical elements necessary to guide the light.
Sancho Fornés, G. (2019). Integración de diferentes fenómenos fotónicos en tecnología de disco compacto para el desarrollo de biosensores label-free [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/124819
TESIS

Após setenta anos de sua descoberta, o vírus da zika surgiu no Brasil, espalhou-se rapidamente pelas Américas e trouxe complicações incomuns em doenças causadas por Flavivirus, como a microcefalia. A Organização Mundial da Saúde classifica a zika como a doença viral mais preocupante da atualidade e considera urgente desenvolver novos métodos de diagnóstico para ela e doenças correlatas como a dengue. Embora existam exames para identificar infecções pelos vírus dessas duas doenças, ainda não há um método rápido, específico e de baixo custo para o diagnóstico precoce. Visando preencher essa lacuna, este trabalho teve como objetivo construir dois tipos de biossensores eletroquímicos de DNA para detecção label-free desses dois vírus. Foram fabricados eletrodos descartáveis em substrato de politereftalato de etileno metalizado com filme fino de ouro nas configurações com um e três contatos. As sequências genéticas de iniciadores e sondas de captura foram desenhadas especialmente para este trabalho com base na análise dos genomas dos vírus. O primeiro biossensor utilizou o eletrodo em uma célula eletroquímica e foi capaz de identificar sequências de DNA da zika ou da dengue. As análises por espectroscopia de impedância eletroquímica mostraram que o biossensor é seletivo à sequência alvo com limite de detecção de (9,86 ± 0,89) nmol L-1. O segundo biossensor utilizou um eletrodo de três contatos para identificação de sequências de DNA em uma gota da amostra. No contato central, usado como eletrodo de trabalho, foi imobilizada a sequência de captura e os contatos laterais funcionaram como eletrodos de referência e auxiliar. Nesse sistema as medidas de impedância indicaram limite de detecção de (25,0 ± 1,7) nmol L-1. Os biossensores desenvolvidos mostraram seletividade para identificar o material genético dos vírus da zika e da dengue nos ensaios com DNA sintético e, portanto, são promissores para a análise de amostras reais, principalmente de produtos da polimerase da cadeia reversa.
After seventy years of its discovery, zika virus has emerged in Brazil, spread rapidly throughout the Americas, bringing unusual complications in diseases caused by flaviviruses, such as microcephaly. The World Health Organization classifies zika as the most harmful viral disease today and considers urgent the development of new diagnostic methods for zika and related diseases, such as dengue. Although there are tests to identify both infections, no current diagnostic method is rapid, specific and cost-efective. This thesis describes two types of electrochemical DNA biosensors for label-free detection of these zika and dengue. Disposable electrodes were fabricated on polyethylene terephthalate substrates covered with a nanometric gold layer by thermal evaporation, manufactured in one- and three-contact configurations. Genetic sequences of primers and complementary capture probes were designed based on the analysis of the virus genomes. The first biosensor we developed used the new electrode in an electrochemical cell and was able to identify zika or dengue DNA sequences. Analyses by electrochemical impedance spectroscopy showed that these biosensors are selective for zika or dengue with a detection limit of (9.86 ± 0.89) nmol L-1. A second type of biosensor used a three-contact electrode to identify DNA sequences in a drop of sample. In the central contact, used as a working electrode, the capture sequence was immobilized and the lateral contacts acted as reference and auxiliary electrodes. In this system the impedance measurements indicated a limit of detection of (25.0 ± 1.7) nmol L-1. The developed biosensors showed selectivity for zika and dengue in the synthetic DNA assays, and therefore are promising for the analysis of real samples, especially the polymerase chain reaction amplicon.

Abstract Integrated optical (IO) sensor allowing sensitive, label-free, real-time and multi-parameter monitoring of bio-molecular interactions are conventionally fabricated with inorganic dielectrics inherited from CMOS manufacturing technology. Polymers as complement materials to inorganic dielectrics are becoming to have an increasing market share for IO circuits in optical communications networks owing to its good optical properties, versatile processibility and low cost. This work aims at developing disposable low-cost biosensors based mainly on polymeric materials, with a performance comparable to inorganic-dielectric based IO biosensors. This thesis describes the development of polymer IO biosensors based on the Young interferometer (YI) transducer platform for ambient noise compensation and a complete periodic intensity fringe pattern. Three different waveguide configurations were utilized, taking into consideration operational simplicity, fabrication simplicity and enhanced sensitivity. Among the developed polymer biosensors, an unconventional interferometer structure: a vertically placed dual-slab waveguide interferometer and an inverted rib waveguide configuration were employed. To enhance the sensitivity of the waveguides, deposition of Ta2O5 high index coating was performed on the rib waveguide configuration. Along with the development of polymer biosensors based on the inverted-rib waveguide configuration, a fabrication process was also developed featuring UV-imprinting and spin coating. The simple two-step fabrication process demonstrated using a polymer mold is potentially transferable to the roll-to-roll manufacture process. Calibration of the developed sensors was performed by homogeneous refractive index (RI) sensing with glucose de-ionized water solutions. By investigating an antibody – antigen binding interaction involving C-reactive protein and its conjugates, this thesis confirmed the applicability of the developed sensors to specific molecule detection. Moreover, to establish the influence of water molecular absorption on measurement stability, an evaluation was carried out on the polymeric waveguide. Finally, the thesis presented a comparison between the developed sensors, exploring their sensitivities, stabilities, limits of detection (LODs) and other aspects related to operation and fabrication. The results indicated that the Ta2O5-coated polymer waveguide sensor had a high sensing capability. In homogeneous RI sensing, the achieved detection limits were 9×10-7 RIU (refractive index unit), i.e., three times the noise level, and 270 fg/mm2 for surface mass density
Tiivistelmä Integroidulla optiikalla toteutetut anturit mahdollistavat biomolekulaarisen vuorovaikutuksen tutkimisen käyttäen herkkiä moniparametrisia ja merkkiaineettomia menetelmiä. Näiden bioantureiden valmistukseen käytetään tavallisesti CMOS-teknologian piiristä tuttuja epäorgaanisia puolijohteita ja eristemateriaaleja. Viime aikoina on kuitenkin polymeeristen materiaalien käyttöä integroidussa optiikassa tutkittu merkittävästi johtuen polymeerien hyvistä optisista ominaisuuksista, monipuolisesta työstettävyydestä ja edullisista kustannuksista. Tämän työn tarkoituksena on kehittää edullisia, kertakäyttöisiä, pääasiallisesti polymeerisistä materiaaleista valmistettuja bioantureita, jotka vastaavat suorituskyvyltään epäorgaanisista materiaaleista valmistettuja integroidun optiikan antureita. Tässä työssä kehitetyt polymeeriset integroidun optiikan bioanturit perustuvat Youngin interferometriin mahdollistaen ympäristökohinan kompensoinnin ja ne tuottavat pintavuorovaikutusten tutkimiseen jaksoittaisen interferenssikuvion. Työssä hyödynnettiin kolmea erilaista valokanavarakennetta huomioiden niiden käytön helppous, valmistuksen yksinkertaisuus ja mittausherkkyys. Yksi kehitetyistä polymeerisistä bioantureista koostui päällekkäisistä kerrostetuista polymeerikerroksista. Toisen tutkitun rakenteen toiminta puolestaan perustui käänteiseen harjannevalokanavaan. Mittausherkkyyttä parannettiin pinnoittamalla polymeerirakenne Ta2O5-pinnoitteella. Näin muodostui kerrostettu komposiittivalokanava, joka oli tässä työssä tutkittu kolmas sensorirakenne. Itse bioanturien lisäksi kehitettiin myös valmistusprosessi, jossa hyödynnettiin UV-painatusta ja nestefaasipinnoitusta. Tässä työssä havaittiin lisäksi, että kehitetty yksinkertainen valmistusmenetelmä on paitsi toimiva, myös mahdollisesti siirrettävissä rullalta rullalle valmistus- ja tuotantoteknologiaan. Kehitettyjen anturien kalibrointi suoritettiin homogeenisella taitekerroinmittauksella käyttäen liuoksia, jotka valmistettiin glukoosista ja deionisoidusta vedestä. Kehitettyjen anturien soveltuvuus spesifien molekyylien tunnistamista varten todennettiin tutkimalla vasta-aineiden ja antigeenien sitoutumisreaktioita ja vuorovaikutusta C-reaktiivisella proteiinilla ja sen konjugaateilla. Lisäksi työssä tutkittiin veden absorption vaikutusta mittauksen stabiilisuuteen. Tutkimuksessa suoritettiin vertailu kehitettyjen anturien ja niiden ominaisuuksien välillä kiinnittäen huomiota mittausherkkyyteen, stabiilisuuteen, määritys- ja toteamisrajoihin ja muihin anturien valmistukseen sekä käyttöön liittyviin keskeisiin piirteisiin. Tulokset osoittavat, että Ta2O5-pinnoitetun polymeerivalokanavan mittausherkkyys oli suurin vertailluista rakenteista. Homogeenisessä taitekerroinmittauksessa saavutettu määritys- ja toteamisraja oli 9×10-7 taitekerroinyksikköä (RIU). Pintamassatiheysmittauksessa saavuttu tulos oli 270 fg/mm2

It is widely known that early estimates about the binding properties of drug candidates are important in the drug discovery process. Surface plasmon resonance (SPR) biosensors have become a standard tool for characterizing interactions between a great variety of biomolecules and it offers a unique opportunity to study binding activity. The aim of this project was to develop a SPR based assay for pre-screening of low molecular weight (LMW) drug compounds, to enable filtering away disturbing compounds when interacting with drugs. The interaction between 47 LMW compounds and biological ligands were investigated using the instrument BiacoreTM, which is based on SPR-technology.

This thesis presents the design, fabrication and testing on a Silicon-on-Insulator (SOI) “lab on a chip” immunosensors based on interferometer technology capable of label-free, real time, parallel detection and identification of multiple analytes with extremely high sensitivity. The basic principles for the biosensor device are evanescent wave sensing and interferometry. Light is guided through a high index contrast photonic wire by total internal reflection. The high index contrast core-cladding photonic wires supporting a vector TM-like mode can be designed to have extremely high sensing sensitivity for low analyte volumes. The optical field decays rapidly from the surface of the waveguide, penetrating into a test solution by ≈178nm. When a chemical, biochemical or biological reaction takes place in the sensing area, i.e. in the region of the decaying field near the surface, the light propagating through the sensing channel will experience a change in its effective refractive index causing a phase change per unit propagation length. With a reference channel built-in on chip, the resulting phase difference between the sensing and reference channels can be detected with a very high sensitivity of detection. The first sensor studied consists of Mach-Zehnder interferometers (MZIs) fabricated with silicon photonic wires. For a MZI with a sensing length of 1000μm the theoretical sensitivity is 3.1 × 10-7 Refractive Index Units (RIUs). Sensitivity is further increased by incorporating spiral waveguides to increase the length of the interferometer arms within a given wafer footprint. A spiral of four turns gives a sensing arm length of 3145μm, which takes up an area of 0.06mm2 and gives a theoretical MZI sensitivity of 9.9 × 10-8 RIUs. The sensitivity of detection of the biosensors developed is at least 10-100 times more sensitive than that of current commercial products. Parallel detection is achieved by exciting multiple sensors in parallel using a 1xN Multimode interferometer (MMI) where N≤20. The second sensor considered is a label-free self-aligned Plasmonic Interferometer (PI). The interfering plasmonic modes are excited on either side of a thin gold layer embedded into a silicon photonic wire. Only the mode on the upper surface interacts with the analyte. The resonant wavelength is thus sensitive to the analyte index. The PI designed for this project is 13μm in length and gives a predicted system wavelength shift responsivity of 500nm/RIU. To guide the way towards the successful design of these sensors commercial software is used to perform detailed simulation evaluations of straight and curved waveguides, Mach Zehnder Interferometers (MZIs), Plasmonic Interferometers (PIs), Multimode interferometers (MMIs), Directional Couplers (DCs), Y-junction splitters and Spot size converters. So that the proposed biosensors can operate as an immunosensor, two methods of selective surface functionalisation are developed to immobilise the antibodies on to the sensor waveguides. The first method attached a non-uniform layer of amine functional group to surface of silicon via a hydrosilylation process. The second method attached a uniform layer of amine functional group to the surface of silicon dioxide via a hydroxylation and silanisation process. Attaching a fluorescent tag to the amine functional groups allowed the imaging and assessment of the surface modification and selectivity. Four sets of biosensor devices were fabricated and tested showing promising results.

Els dispositius òptics biosensors basats en la detecció d’ona evanescent podrien superar les limitacions dels tests de diagnòstic actuals (que són lents i cars) degut a la possibilitat de realitzar les deteccions a temps reals i fent servir un esquema sense la necessitat de marcatges. Entre els diferents transductors òptics, els interferomètrics són els que posseeixen els millors límits de detecció (LOD) deguts a canvis en el índex de refracció de dissolucions (10-7-10-8 Unitats d’Índex de Refracció, RIU) així com per sensibilitat superficial (en el rang dels pg/ml) i un rang lineal més gran. No obstant, les configuracions interferomètriques (l’interferòmetre Mach-Zehnder o el Young) més usuals fan servir un divisor amb forma de Y, que és essencial per dividir o recombinar la llum, lo qual, degut a les toleràncies de les actuals tècniques de fabricació es una gran desavantatge per la reproduibilitat d’aquests dispositius. Per evitar aquests problemes, hem desenvolupat una configuració interferomètrica més simple basada en un guia de ones recte on dos modes de llum de la mateixa polarització interfereixen entre si. Aquesta configuració elimina la complexitat dels interferòmetres més utilitzats i conseqüentment, el biosensors que s’obtenen són més fiables i reproduïbles. Aquesta tesis esta dirigida al desenvolupament i la caracterització d’un nou transductor fotònic per biosensat d’alta sensibilitat i sense marcatges, el dispositiu de guia d’ona bimodal (BiMW). Amb aquest propòsit, els següents punts han estat plantejats: 1. Disseny, fabricació i caracterització òptica del transductor que opera segons el principi de la interferència de dos modes de llum. 2. Desenvolupament i optimització de les estratègies de funcionalització de la superfície transductora fent servir processos de silanització. 3. Estudi de l’aplicabilitat del biosensor amb la demostració del diagnosis analític de problemes clínics rellevants. El transductor es fabrica a nivell d’oblea a la Sala Blanca, lo qual garanteix la producció en massa del dispositiu així com un preu baix del mateix. El dispositiu és molt sensible a variacions en l’índex de refracció de dissolucions, obtenint un límit de detecció de 2×10-7 RIU. La biofuncionalització de l’àrea sensora es un dels aspectes més importants d’aquest treball. Diferents protocols per immobilitzar els diferents bioreceptor en la superfície del dispositiu (cadenes d’ADN, proteïnes i anticossos) han estat desenvolupats. Aquests protocols s’han fet servir per la demostració de diferents bioaplicacions; la detecció d’hormones, bactèries o seqüències d´ADN complementàries. Els resultats presentats en aquesta tesis han destacat pel superior funcionament d’aquest dispositiu en comparació amb els tests de diagnosis convencionals degut a: i) la possibilitat de monitoritzar les interaccions biomoleculars en temps real i fent servir un esquema sense marcadors reduint el temps i el cost de l’assaig, ii) la fabricació del dispositiu fent servir microtecnologia de silici, possibilitant la producció en massa, iii) l’alta sensibilitat (pg/ml, femtomolar) demostrada per les diferents bioaplicacions avaluades i iv) el dispositiu reuneix els requeriments específics per ser miniaturitzat e integrat en una plataforma de sensat multiplexada. Aquest treball obre la porta a la integració d’aquest transductor en un dispositiu lab-on-a -chip, una feina que inclou l’acoblament/detecció de la llum, un sistema capaç de modular la senyal interferomètrica i la incorporació de canals microfluídics per anàlisis multiplexats. Cadascun d’aquests temes afegeix molta complexitat al dispositiu final, han de ser individualment desenvolupades i optimitzades per ser integrades en un biosensor lab-on-a-chip. Finalment, la possibilitat de detectar simultàniament múltiples analits involucra el desenvolupament de noves tècniques per recollir les múltiples senyals així com desenvolupar noves estratègies de biofuncionalització.
Optical biosensor devices based on evanescent wave detection could overcome the limitations of conventional diagnostic tests (expensive and time-consuming) due to the possibility of carrying out the detection in real-time and using a label-free scheme. Among the different optical transducers, interferometric devices have evidenced the best limit of detection (LOD) for refractive index changes of bulk solutions (10-7-10-8 Refractive Index Units, RIU) and for surface sensing (in the pg/ml range) and a wider linear range. However, usual interferometric transducers (Mach-Zehnder or Young interferometers) employ the Y-junction to split or recombine light, a drawback for the coherence and performance of the device due to standard tolerances of microfabrication techniques. To overcome these problems, we have developed a simple configuration based on a single straight waveguide where two modes of the light of the same polarization are interfering between them. This simple approach avoids the complexity of the usual interferometric transducers and as a consequence, more reliable and reproducible biosensors can be obtained. This thesis is focused on the development and characterization of a new photonic transducer, the Bimodal Waveguide device (BiMW), for label- free and high sensitive biosensing. To achieve this, the following steps have been pursued: 1. Design, fabrication, and optical characterization of an optical transducer operating by two-mode interference principle. 2. Development and optimization of biofunctionalization strategies on the transducer surface using silanization techniques. 3. Study of the applicability of the biosensor with the demonstration of bioanalytical diagnosis of relevant problems. The transducers are fabricated at wafer level in Clean Room facilities, which warrants a cost-effective and mass-production of the sensor chips. The device is highly sensitive to small changes in the refractive index occurring on the sensor area, leading to a detection limit of 2.5×10-7 RIU for bulk changes in refractive index solutions. The biofunctionalization of the sensor area is one of the most crucial aspects of this work. Optimized functionalization procedures have been achieved, which has been employed to immobilize different types of bioreceptors (DNA strands, proteins, and antibodies) on the surface. The optimized protocols have been used for the demonstration of different bioapplications such as the detection of hormones, bacteria, or complementary DNA sequences. The results presented in this work have highlighted the superior performance of this device in comparison with conventional diagnostics tests due to: i) the possibility of monitoring biomolecular interactions in real-time and by using a label-free scheme which reduce the time and cost of the assay , ii) the fabrication of the device using standard silicon microelectronics technology opening the possibility for mass-production, iii) the high sensitivity demonstrated for the different bioapplications assessed achieving detection limits in the pg/ml range (femtomolar), and iv) the device meets the specific requirements to be miniaturized and integrated in a multiplexed platform. This work opens the door for the integration of this transducer in a lab-on-a-chip device, including the in-coupling/out-coupling of light, a system able to modulate the interferometric signal, and the incorporation of microfluidics channels for multiplexing. Each of these subjects adds a great complexity to the final device, and must be independently developed and optimized in order to be successfully integrated at the final lab-on-a-chip biosensor. Finally, the possibility to detect simultaneously multiple analytes will involve further efforts in developing new optical in and outcoupling as well as new biofunctionalization strategies.

"Circulating tumor cells (CTCs) are cells released into the bloodstream from primary tumors and are suspected to be one of the main causes behind metastatic spreading of cancer. The ability to capture and analyze circulating tumor cells in clinical samples is of great interest in prevailing patient prognosis and clinical management of cancer. Carbon nanotubes, individual rolled-up graphene sheets, have emerged as exciting materials for probing the biomolecular interactions. With diameter of about 1 nm, they can attach themselves to cell surface receptors through specific antibodies and hold a great potential for diagnostic cellular profiling. Carbon nanotubes can be either semiconducting or metallic, and the electronic properties of either type rivals the best known materials. Small size of nanotubes and the ability to functionalize their surface using 1-Pyrenebutanoic Acid, Succinimidyl Ester (PASE), enables a versatile probe for developing a platform for capture and analysis of cancer biomarkers and circulating tumor cells. Although nanotubes have previously been used to electrically detect a variety of molecules and proteins, here for the first time we demonstrate the label free capture of spiked breast cancer cells using ultra-thin carbon nanotube film micro-array devices in a drop of buffy coat and blood. A new statistical approach of using Dynamic Time Warping (DTW) was used to classify the electrical signatures with 90% sensitivity and 90% specificity in blood. These results suggest such label free devices could potentially be useful for clinical capture and further analysis of circulating tumor cells. This thesis will go in-depth the properties of carbon nanotubes, device fabrication and characterization methodologies, functionalization protocols, and experiments in buffy coats and in blood. Combination of nano and biological materials, functionalization protocols and advanced statistical classifiers can potentially enable clinical translation of such devices in the future. "

DNA is the substance that encodes the genetic information that cells need to replicate and to produce proteins. The detection of DNA sequences is of great importance in a broad range of areas including genetics, pathology, criminology, pharmacogenetics, public health, food safety, civil defense, and environmental monitoring. However, the established techniques suffer from a number of problems such as the bulky size, high equipment costs, and time-consuming algorithms so that they are limited to research laboratories and cannot be applied for in-vivo situations. In our research, we developed a novel sensing scheme for DNA sequence detection, featuring sequence specificity, cost efficiency, speed, and ease of use. Without the need for labels or indicators, it may be ideal for direct in-cell application. The principle is simple. With capture DNA immobilized onto the probe by layer-by-layer selfassembly, the hybridization of a complementary strand of target DNA increases the optical thickness of the probe. Three kinds of sensors were developed. The optical fiber tip sensor has been demonstrated with good specificity and high sensitivity for target DNA quantities as small as 1.7 ng. To demonstrate the potential of this structure for practical applications, tularemia bacteria were tested. Two other micrometric structures were designed with specific advantages for different applications. The micro-fiber Bragg grating interferometer (Micro-FBGI) has the intrinsic temperature compensation capability. The micro-intrinsic Fabry-Perot interferometer (Micro-IFPI)features simple signal processing due to its simple configuration. Successful DNA immobilization and hybridization have been demonstrated onto the 25μm Micro-IFPI. Both structures have great potential for nanometric protrusion, allowing future in-cell DNA direct detection. In addition, its quick response time leads to the potential for express diagnosis. What's more, the idea of nanoscale probe has a broad impact in scanning near-field optical microscopy (SNOM), intracellular surgery in cell sensing, manipulation, and injection.
Ph. D.

DNA arrays have become a major bioanalytical method as they enable high-throughput screening and they can be manufactured on different surfaces depending on the nature of diagnostic purpose. However, current technologies to produce and detect arrays of DNA probes are expensive due to the requirement of specialized instrumentation. In this study we have established an array platform in sandwich hybridization format for the detection of label-free nucleic acid targets. Unlike direct hybridization, which is the main microarray hybridization principle, sandwich assay enables unlabeled target detection, lowering the cost and assay time. To this end, sequence specific signal development was achieved by a sandwich complex which is composed of a surface immobilized capture DNA probe (Probe1) and a fluorescein-tagged signal DNA probe (Probe 2), which are partially complementary to the sequence to be analyzed (target oligonucleotide). As the solid support of the array platform both 3-aminopropyl-3-methoxysilane (APTMS) activated and commercially purchased poly-L lysine coated glass slides were used and due to the less background noise property the latter one was preferred. Similarly, for the immobilization of the capture Probe (P1) onto the solid support two different methods were tried
heat immobilization and immobilization via a heterobifunctional cross-linker (HBCL). In regard to the experiments, it is observed that using a cross-linker instead of heat immobilization reduces the ratio of false negative control results in a significant manner. Following the solid support and immobilization method choice comparative optimization studies which include cross-linker type, probe concentration, sensitivity of the platform and hybridization conditions (sequence, temperature and duration) were conducted. Optimum hybridization signal was obtained with a 32.5

This thesis describes a detailed study of advanced fibre optic sensors and their applications for label-free biochemical detection. The major contributions presented in this thesis are summarised below. A self-assembly based in-situ layer-by-layer (i-LbL) or multilayer deposition technique has been developed to deposit the 2D material nanosheets on cylindrical fibre devices. This deposition technique is based on the chemical bonding associated with the physical adsorption, securing high-quality 2D materials coating on specific fibre cylindrical surface with strong adhesion as well as a prospective thickness control. Then a " Photonic-nano-bio configuration", which is bioprobes immobilised 2D-(nano)material deposited fibre grating, was built. 2D material overlay provides a remarkable analytical platform for bio-affinity binding interface due to its exceptional optical and biochemical properties. EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) and NHS (NHydroxysuccinimide) were used to immobilise bioprobes. This kind of configuration is considered to have many advantages such as: enhanced RI sensitivity, enrich immobilisation sites, improved binding efficiency, selective detection. Followed by this configuration, several label-free biosensors were developed. For example, graphene oxide coated dual-peak long period grating (GO-dLPG) based immunosensor has been implemented for ultrasensitive detection of antibody/antigen interaction. The GO-LPG based biosensor has been developed for label-free haemoglobin detection. Apart from biosensors, the black phosphorus (BP) integrated tilted fibre grating (TFG) has been proposed, for the first time, as BP-fibre optic chemical sensor for heavy metal (Pb2+ ions) detection, demonstrating ultrahigh sensitivity, lower limit of detection and wider concentration range. Ultrafast laser micromachining technology has been employed to fabricate long period grating (LPG) and microstructures on optical fibre. The ultrafast laser micromachined polymer optical fibre Bragg grating (POFBG) has been developed for humidity sensing, showing the significant improvement with the reduced response time.

The Ph.D. thesis is focused on the development of a label-free optical biosensor based on the excitation of surface electromagnetic modes, Bloch Surface Waves (BSW), sustained by truncated One Dimensional Photonic Crystal (1DPC). BSWs are electromagnetic modes that can be coupled at the interface between a truncated dielectric 1DPC and a homogeneous external medium, for particular combinations of angle and wavelength of an incoming radiation. These modes are confined on the multilayer surface and propagate in the plane, while the electric field amplitude shows an exponential decay in the medium, perpendicularly to the stack. The surface waves can be resonantly excited by a light beam through the coupling with a glass prism in Kretschmann-Raether configuration. In the case of white light impinged on the 1DPC surface, BSW appears as a dip in reflected spectrum corresponding to those wavelengths coupled to BSW at specific incident angles. The BSW resonance is very sensitive to small variation of the refractive index of the medium surrounding the 1DPC, induced by surface and bulk perturbations yielding a shift of the spectral dip position. This can be exploited for sensing purpose, similarly to other standard sensing techniques such as Surface Plasmon Resonance (SPR). Different kinds of measurement modalities can be performed, such as fluorescence-based refractometric label free or scattering based detection. The aim of the project is to develop and optimize a sensitive sensor able to recognize target analytes exploiting an immunoassay scheme.

The contamination of the environment, accidental or intentional, in particular with chemical toxins such as industrial chemicals and chemical warfare agents has increased public fear. There is a critical requirement for the continuous detection of toxins present at very low levels in the environment. Indeed, some ultra-sensitive analytical techniques already exist, for example chromatography and mass spectroscopy, which are approved by the US Environmental Protection Agency for the detection of toxins. However, these techniques are limited to the detection of known toxins. Cellular expression of genomic and proteomic biomarkers in response to toxins allows monitoring of known as well as unknown toxins using Polymerase Chain Reaction and Enzyme Linked Immunosensor Assays. However, these molecular assays allow only the endpoint (extracellular) detection and use labels such as fluorometric, colorimetric and radioactive, which increase chances of uncertainty in detection. Additionally, they are time, labor and cost intensive. These technical limitations are unfavorable towards the development of a biosensor technology for continuous detection of toxins. Federal agencies including the Departments of Homeland Security, Agriculture, Defense and others have urged the development of a detect-to-protect class of advanced biosensors, which enable environmental surveillance of toxins in resource-limited settings. In this study a Surface-Enhanced Raman Spectroscopy (SERS) immunosensor, aka a SERS-linked immunosensor assay (SLISA), has been developed. Colloidal silver nanoparticles (Ag NPs) were used to design a flexible SERS immunosensor. The SLISA proof-of-concept biosensor was validated by the measurement of a dose dependent expression of RAD54 and HSP70 proteins in response to H2O2 and UV. A prototype microchip, best suited for SERS acquisition, was fabricated using an on-chip SLISA to detect RAD54 expression in response to H2O2. A dose-response relationship between H2O2 and RAD54 is established and correlated with EPA databases, which are established for human health risk assessment in the events of chemical exposure. SLISA outperformed ELISA by allowing RISE (rapid, inexpensive, simple and effective) detection of proteins within 2 hours and 3 steps. It did not require any label and provided qualitative information on antigen-antibody binding. SLISA can easily be translated to a portable assay using a handheld Raman spectrometer and it can be used in resource-limited settings. Additionally, this is the first report to deliver Ag NPs using TATHA2, a fusogenic peptide with cell permeability and endosomal rupture release properties, for rapid and high levels of Ag NPs uptake into yeast without significant toxicity, prerequisites for the development of the first intracellular SERS immunosensor.

2009/2010
To reach new and relevant insights in biomolecular sciences, new tools for fine and precise measurement are needed. Nowadays advances in the field of micro-electro-mechanical systems (MEMS) offer unique opportunities in the design of ultrasensitive analytical devices to support the molecular sensing investigations. Among them Microcantilever (MC) biosensors are label-free platforms that combine a biologically sensitive with a physical transducer in order to selectively and quantitatively detect the presence of specific compounds in a given external environment. Since they can be operated either as nanomechanical resonator or as surface stress sensor, MCs - activated with DNA probes or antibodies for molecular recognition - enable the measurement of mass with extraordinary sensitivity. In particular, the development of mass detector biosensor based on MC systems would permit to shift from qualitative data to quantitative measurements of key molecules involved in physiological processes. This can lead crucial informations to characterize complex mechanisms such as angiogenesis and tumor progression and to the quantification of small amounts of cancer markers, such as Angiopoietin-1 (ANG-1), and their modulation during the early stages of tumor development.
XXIII Ciclo
1979

Use of DNA-based molecular probes on a biosensing surface enables unprecedented designs, with nely tuned responsive structures. Furthermore, cost and production of DNA, in addition to its overall ability to interact with various biological molecules, make DNA the ideal candidate for biosensing surface functionalization. However, all DNA based biosensing relies at some point on the pairing of to single stranded DNA molecules, namely DNA hybridization. I utilized a label-free, optical, multiplexed, biosensing platform to study probing capabilities of surface grafted DNA, from simple short sequences to highly ordered complex nanostructures. My PhD research started with the study of kinetics of simple oligo hybridization on surface. DNA hybridization on surface is usually treated under the well established Langmuir model, used to treat majority of surface adsorbtion phenomena. However, electrostatics of DNA phosphate backbone in addition to high denstiy of DNA monolayer on sensing surface, prevents this model to be applied in all it's potency. We observed strong suppression in binding kinetics even at concentrations below the KD. Moreover, this suppression correlated positively with DNA probe density, indicating a possible electrostatic in uence. Based on the electrostatic theory of DNA monolayers I developed a simple model accounting for the electrostatic penalty associated with entry of DNA molecule into charged DNA monolayer. Moreover, in addition to electrostatic repulsion hampering, steric eects arising from such high density areas also aect the overall sensitivity and rapidity of surface nucleic acids sensing with DNA. We proposed dierent strategies to combat this obstacles, including varying salt concentrations to counter electrostatic repulsion, grafting DNA probes on hydrogel, eectively reducing charge density. Use of multiple strands was also proposed considering the observed high anity towards DNA hybridization to partially double stranded probes, that provide further stacking stabilization, and decrease the dissociation events. DNA as a sensing probe was also successfully utilized in sensing micro-RNA (miRNA), a widely acknowledged and used biomarker for early-disease diagnostics. MiRNA molecules, like any other RNA molecule, readily hybridizes with DNA, forming a RNA/DNA hybrid, however, miRNAs specically are very scarcely and non-uniformly distributed in sera, blood and tissue, this inhibits the availability of miRNAs, despite their potential as biomarkers. We grafted various DNA strands with complementary sequences to 5 dierent known miRNAs. Our multiplexed assay was able to detect as low as 0.5 pM miRNA while yielding 30x fold mass amplication 90 minutes after the sample injection. Amplication was achieved by a specic antibody (Ab1) targeting DNA-RNA hybrids, polyclonal secondary antibody (Ab2) targeting primary antibody and careful system optimization. A simple numeric model was developed accounting the formation of DNA/RNA hybrids, Ab1 binding to hybrid and formation of Ab1-Ab2. Last interaction was treated as a competitive system, since the Ab pairs also tend to form in solution as well as on surface. System was optimized for amplication factor while keeping the miRNA concentration and total assay duration xed. The amplication signal was shown to be sequence dependent, since the kinetics of DNA/RNA hybridization depends on the sequence and predictive models are not available. DNA microarrays are often employed to study the binding and kinetics of various transcription factors. We investigated binding yeast gene regulator Gal4, on spots containing consensus and non-consensus sequence both in the form of simple DNA strand and DNA hairpin structure. Through the experimental observations, we found that the initial binding step is charge mediated and can be therefore ne tuned through ionic conditions. A Two-step nested well model was built, to explain these observations. First encounter is a non-specic interaction, followed by conformational adjustment until the consensus sequence is reached. Finally, potential of DNA to self-assemble into structure with higher complexity and perform specic functions was explored. Hybridization Chain Reaction (HCR) is nowadays well established isothermal reaction based on self-assembly properties of DNA. This nucleic acid triggered isothermal reaction shows great promise in biosensing applications, primarily as a means for enzyme-free signal amplication. We observed the formation of HCR laments in real time by grafting trigger DNA sequence on surface and releasing interacting hairpins in solution. Observed binding curves were cleary dierent from simple DNA hybridization, indicating multiple reaction occuring on the surface. We modelled this behaviour similarly to protein-DNA binding. Binding of rst hairpin is fast, due to oligo-like hybridization by the hairpin overhang. This recognition step is followed by slow hairpin opening which ends in exposition of overhang, or binding site for second hairpin. Two relaxation model thus accounts for rst hairpin binding to trigger strand on surface and to hairpin unzipping. Fluorescence confocal microscopy investigation was performed on HCR spots with dye-conjugated rst hairpin. Direct visual comparison with DNA monolayer shows dramatic dierence in intensity prole of the spot, which conrms the presence of HCR laments on surface. In the last part of the thesis work I explored the functionalization of the biosensor surface with large-scale a complex structure enabling control of probes with nanometer precision. Highly ordered DNA origami rectangles were developed and functionalized with sticky ends (tethers) for adsorbtion on DNA grafted surface. Investigation was carried out on the number of 40bp long sticky ends required to bring the structure to the surface, as well the kinetics based on the number of tethers. For this purpose, 2, 4 and 6 legged rectangles were produced, their stability and proper folding was veried directly via AFM. Observations of origami binding revealed: despite the large size and net charge of the origami, rectangles with at least 4 tethers complementary to DNA probes readily bind to the biosensor surface; number of tethers primarily aects the stability through lowering the dissociation rate, which was interpreted as probability of all available tethers being simultaneously detached - which directly correlates with number of tethers.

Optics and photonics enable important technological solutions for critical areas such as health, communications, energy, and manufacturing. Novel nanofabrication techniques, on the other hand, have enabled the realization of ever shirking devices. On-chip photonic micro-resonators, the fabrication of which was made possible in the recent decade thanks to the progress in nanofabrication, provide a sensitive and scalable transduction mechanism that can be used for biochemical sensing applications. The recognition and quantification of biological molecules is of great interest for a wide range of applications from environmental monitoring and hazard detection to early diagnosis of diseases such as cancer and heart failure. A sensitive and scalable biosensor platform based on an optimized array of silicon nitride microring resonators is proposed for multiplexed, rapid, and label-free detection of biomolecules. The miniature dimension of the proposed sensor allows for the realization of handheld detection devices for limited-resource and point-of-care applications. To realize these sensors, the design, fabrication, stabilization, and integration challenges are addressed. Especially, the focus is placed on solving a major problem in using resonancebased integrated photonic sensors (i.e., the insufficiency of wavelength scan accuracy in typical tunable lasers available) by using an interferometric referencing technique for accurate resonance tracking. This technique can improve the limit of detection of the proposed sensor by more than one order of magnitude. The method does not require any temperature control or cooling, and the biosensor platform does not require narrow linewidths necessary for the biosensors based on ultrahigh quality factor resonators, thus enabling low-cost and reliable integration on the biosensor platform.

The development of a highly integrated optical biosensor is expected to significantly impact on the performances and on the throughput of biochemical assays, with applications in the field of pharmaceutical research, point-of-care diagnostic, food-borne pathogens screening and safety. This dissertation studies the development of a label-free on-chip biosensor for the selective detection of Aflatoxin-M1 from milk content. We detail the design and the realization of two types of multiplexed sensors. They are based on the silicon photonics technology and operates in liquid ambient at wavelengths in the near-visible and near-infrared spectra. Most of this work is focused on the first type of sensor,which is based on a whispering-gallery-mode resonator. In particular, we analyze microdisk, microring and wedge resonator structures, studying the sensitivity and the quality factor of each. The appeal of these structure is given by the low detection limit that can be achieved in a footprint of few tens of microns per side. The second sensor type is based on a spectrally resolved asymmetric Mach-Zehnder interferometer. In this case, the high level of folding permitted by the use of high refractive-index-contrast materials enables the fabrication of sensitive interferometers in a reasonable footprint. The experimental characterization of the bulk refractometric sensing of the devices is performed in continuous flow, using a dedicated microfluidic flow-cell in PDMS. This characterization assesses the high resolution of both device types, which are able to resolve variations in the refractive index of the liquids with a limit of detection down to 10^-ô€€€6 refractive index units (RIU). The selective superficial sensing is also evaluated, implementing a biorecognition functional layer with DNA-aptamers. The assay of buffered solutions containing Aflatoxin-M1 molecules confirms that the devices under test are suitable biosensors, with specific detection limits down to about 100 pg/mlô€€€ for the Mach-Zehnder interferometer, and slightly larger for the microring resonator. A procedure for the regeneration of the sensor has been optimized, enabling reproducible sensing up to nine times. The detection of the receptor-ligand binding in real-time enabled the study of the kinetics of the binding reaction, and we measured for the first time the kinetic rate constants of the antiaflatoxin aptamers of our sensors.

The development of a highly integrated optical biosensor is expected to significantly impact on the performances and on the throughput of biochemical assays, with applications in the field of pharmaceutical research, point-of-care diagnostic, food-borne pathogens screening and safety. This dissertation studies the development of a label-free on-chip biosensor for the selective detection of Aflatoxin-M1 from milk content. We detail the design and the realization of two types of multiplexed sensors. They are based on the silicon photonics technology and operates in liquid ambient at wavelengths in the near-visible and near-infrared spectra. Most of this work is focused on the first type of sensor,which is based on a whispering-gallery-mode resonator. In particular, we analyze microdisk, microring and wedge resonator structures, studying the sensitivity and the quality factor of each. The appeal of these structure is given by the low detection limit that can be achieved in a footprint of few tens of microns per side. The second sensor type is based on a spectrally resolved asymmetric Mach-Zehnder interferometer. In this case, the high level of folding permitted by the use of high refractive-index-contrast materials enables the fabrication of sensitive interferometers in a reasonable footprint. The experimental characterization of the bulk refractometric sensing of the devices is performed in continuous flow, using a dedicated microfluidic flow-cell in PDMS. This characterization assesses the high resolution of both device types, which are able to resolve variations in the refractive index of the liquids with a limit of detection down to 10^-􀀀6 refractive index units (RIU). The selective superficial sensing is also evaluated, implementing a biorecognition functional layer with DNA-aptamers. The assay of buffered solutions containing Aflatoxin-M1 molecules confirms that the devices under test are suitable biosensors, with specific detection limits down to about 100 pg/ml􀀀 for the Mach-Zehnder interferometer, and slightly larger for the microring resonator. A procedure for the regeneration of the sensor has been optimized, enabling reproducible sensing up to nine times. The detection of the receptor-ligand binding in real-time enabled the study of the kinetics of the binding reaction, and we measured for the first time the kinetic rate constants of the antiaflatoxin aptamers of our sensors.

This thesis treats the development of an integrated optical sensor array. The sensors are slot-waveguide ring resonators, integrated with on-chip surface grating couplers and light splitters, for alignment tolerant, real-time, refractive index sensing, and label-free biosensing. The work includes: the design of components and system layouts, the development of fabrication methods, the fabrication of sensor chips, the characterization of the chips, and the development of physical system models for accurate extraction of resonance wavelengths in measured spectra. The main scientific achievements include: The evaluation of a novel type of nano-structured optical waveguide for biochemical sensing. The realization of an array of such slot-waveguide sensors, integrated with microfluidic sample handling, for multiplex assays. The first study of the thermal behavior of slot-waveguide sensors and the discovery of unique temperature compensation capabilities. From an application perspective, the use of alignment tolerant surface gratings to couple light into the optical chip enables quick replacement of cartridges in the read-out instrument. Furthermore, the fabrication sequence avoids polishing of individual chips, and thus ensures that the cost benefits of silicon batch micro-fabrication can be leveraged in mass production. The high sensitivity of the slot waveguide resonators, combined with on-chip referencing and physical modeling, yields low limits of detection. The obtained volume refractive index detection limit of 5 × 10−6 refractive index units (RIU), and the surface mass density detection limit of 0.9 pg/mm2, shows that performance comparable to that of commercial non-integrated surface plasmon resonance sensors, made from bulk optical components, canbe achieved in a compact cartridge.
Qc20100715
SABIO

There is a significant need for the development and use of numerical methods to simulate advance and complex optical biosensor structures. Finite Element Method (FEM) has been established as one of the most powerful and versatile numerical method and has been implemented in this thesis to characterize, analyse and optimise label-free optical biosensors for the detection of micron size biological objects like bacteria such as E.coli and nanometre size biomolecules such as antibody, nucleic acids and proteins. These sensors are all suitable for deep-probe sensing as large evanescent field can be excited in the sensing medium with substantial penetration depth achieved by techniques like Surface Plasmon Resonance (SPR) and sensor architectures based on nanowires and slot waveguides. This thesis presents three different architectures of label-free optical biosensors. First, a fiber optic surface plasmon resonance (SPR) biosensor for the detection of E.Coli is optically modeled by using the finite-element approach in conjunction with the perturbation technique which is computationally more efficient and can be used for waveguides with low or medium loss values. The same sensing architecture is used when surrounding index is varied from 1.30 -1.44 to cover most of the biological elements that are used in the biosensing applications. Second one is based on evanescent-wave guiding properties of nanowire waveguides a theoretical investigation of silica nanowires employing a wire assembled Mach-Zehnder structure to detect the presence of E.Coli is studied second. Finally, a slot-waveguide based micro-ring resonator is investigated for the detection of DNA Hybridization using H-field FEM based full-vector formulation. It is found that all of the numerical methods provide good agreement with the experimental sensitivities and detection limits.

The development of a highly sensitive, label-free, multiplexed biosensor platform for point-of-care diagnostics is presented. The sensor surface of a non-faradaic electrochemical impedance spectroscopy (EIS) immuno-sensor platform was developed and fully characterised. Optimisation of the binding of monoclonal antibodies (mAb) towards the model target human chorionic gonadotropin (hCG) to the OEG self-assembled monolayers (SAMs) was carried out. Optimal conditions for immobilisation were found for buffer pH approximately one unit below the pI of the antibody. The same condition resulted in both higher antibody density on the sensor surface as well as higher response to the antigen. At the same time the surface showed good resistance to non-specific adsorption of proteins. Based on these principles, a biosensor to detect hCG in full serum was demonstrated. By using the phase of the impedance at 100 mHz as the sensor response, a linear relationship of the phase shift vs the logarithm of hCG concentration was established between 2.6 x 10�14 M and 2.6 x 10�10 M with a sensitivity of 0.6 degree per decade, which is a significant improvement over current state-of-the-art biosensor systems. Finally, The dielectric properties of COOH-terminated hexa(ethylene glycol)undecanethiol (OEG) and 11-mercaptoundecanol (MUD) and mixed MUD:OEG SAMs, at different ratios, were studied by means of EIS. The study demonstrates that small amounts of MUD in the mixed MUD:OEG SAMs lead to a considerable decrease of the phase of the impedance as well as a significant increase in the resistivity of the SAM at low frequencies, indicating a significant improvement of the dielectric properties. Furthermore, a considerable change in the formation of clusters of OEG molecules for mixed MUD:OEG SAMs with increasing MUD content was shown by AFM imaging.

This thesis focused on developing an inexpensive and user friendly “point- of- care” (POC) device for early disease diagnosis. Proteomics research has elucidated many new proteins as biomarkers that have the potential to greatly improve disease diagnosis. A combination of several biomarkers has been determined to provide the information necessary for robust diagnosis of a disease in any person within a population. This technology was employed in a clinical application to identify two disease states: (i) vulnerable coronary plaque rupture, which is the cause of acute coronary syndromes stroke; and (ii) neurodegenerative diseases, which are some of the leading causes of death and debilitation worldwide. In this thesis, nanomaterials were utilized to generate high surface-area-to-volume structures in developing a biosensor platform for early disease diagnosis. These devices are known as nanomonitors. The protein-specific capacitance measurement method was employed as the basis for protein biomarker detection in a preoperative state. Troponin T and alpha-synuclein were employed as the target protein biomarkers because they have been identified to be clinically relevant in identifying vulnerable coronary vascular plaque rupture and neurodegenerative disease states, respectively. The primary purpose of this thesis was to measure performance parameters such as limit of detection, specificity, dynamic range, and detection speed of nanomonitor devices for protein biomarker-based disease detection with accuracy greater than 95 percent.
Thesis (M.S.)--Wichita State University, College of Engineering, Dept. of Electrical Engineering and Computer Science

[ES] Los biosensores son dispositivos analíticos con aplicabilidad en diferentes campos y con numerosas ventajas frente a otros métodos analíticos convencionales, como son el uso de pequeños volúmenes de muestra y reactivos, su sensibilidad y su rápida respuesta, sin necesidad de pretratamiento de la muestra, equipos caros o personal especializado. Sin embargo, se trata de un campo de investigación relativamente nuevo en el que todavía queda mucho camino por andar. Esta Tesis doctoral pretende aportar un granito de arena a este campo de conocimiento mediante el estudio del potencial de diferentes materiales porosos como transductores para el desarrollo de biosensores ópticos con respuesta en tiempo real y sin marcajes. Los materiales propuestos van desde aquellos artificialmente sintetizados, como silicio poroso (SiP), nanofibras (NFs) poliméricas o membranas poliméricas comerciales, hasta materiales naturales con propiedades fotónicas que todavía no habían sido explotadas para el sensado, como son los exoesqueletos de biosílice de diatomeas. Todos ellos tienen en común la simplicidad en su obtención, evitando costosos y laboriosos procesos de nanofabricación. Para su estudio, se analizará su respuesta óptica y, en aquellos casos en los que ésta permita llevar a cabo experimentos de detección, se desarrollarán estrategias para su biofuncionalización y su implementación en experimentos de biosensado. En el caso del SiP y las NFs se han optimizado los parámetros de fabricación para obtener una respuesta óptica adecuada que permita su interrogación. A continuación, se ha llevado a cabo su biofuncionalización empleando métodos covalentes y no covalentes, así como diferentes bioreceptores (aptámeros de ADN y anticuerpos) para estudiar su potencial y sus limitaciones como biosensores. En el caso de las membranas comerciales y el exoesqueleto de sílice de diatomeas, se ha caracterizado su respuesta óptica y se han llevado a cabo experimentos de sensado de índice de refracción para estudiar su sensibilidad. Así mismo, se ha desarrollado un método de funcionalización de la superficie del exoesqueleto de diatomeas basado en el uso de polielectrolitos catiónicos. Como resultado, se ha demostrado el potencial tanto de NFs para el desarrollo de biosensores, como el de membranas comerciales para sensores cuya aplicación no requiera una elevada sensibilidad pero sí un bajo coste. Además, se ha puesto de manifiesto el gran potencial del exoesqueleto de diatomeas para el desarrollo de sensores basados en su respuesta óptica. Por el contrario, las limitaciones encontradas en el desarrollo de biosensores basados en SiP han evidenciado la necesidad de un estudio riguroso y la optimización de la estructura de materiales porosos previamente a ser usados en (bio)sensado.
[CA] Els biosensors són dispositius analítics amb aplicabilitat en diferents camps i amb nombrosos avantatges enfront d'altres mètodes analítics convencionals, com són l'ús de xicotets volums de mostra i reactius, la seua sensibilitat i la seua ràpida resposta, sense necessitat de pretractament de la mostra, equips cars o personal especialitzat. No obstant això, es tracta d'un camp d'investigació relativament nou en el qual encara queda molt camí per fer. Aquesta Tesi doctoral pretén aportar el seu òbol a aquest camp de coneixement mitjançant l'estudi del potencial de diferents materials porosos com a transductors per al desenvolupament de biosensors òptics amb resposta en temps real i sense marcatges. Els materials proposats van des d'aquells artificialment sintetitzats, com a silici porós (SiP), nanofibras (NFs) polimèriques o membranes polimèriques comercials, fins a materials naturals amb propietats fotòniques que encara no havien sigut explotades per al sensat, com són els exoesquelets de biosílice de diatomees. Tots ells tenen en comú la simplicitat en la seua obtenció, evitant costosos i laboriosos processos de nanofabricació. Per al seu estudi, s'analitzarà la seua resposta òptica i, en aquells casos en els quals aquesta permeta dur a terme experiments de detecció, es desenvoluparan estratègies per a la seua biofuncionalizació i la seua implementació en experiments de biosensat. En el cas del SiP i les NFs s'han optimitzat els paràmetres de fabricació per a obtenir una resposta òptica adequada que permeta la seua interrogació. A continuació, s'ha dut a terme la seua biofuncionalizació emprant mètodes covalents i no covalents, així com diferents bioreceptors (aptàmers d'ADN i anticossos) per a estudiar el seu potencial i les seues limitacions com a biosensors. En el cas de les membranes comercials i l'exoesquelet de sílice de diatomees, s'ha caracteritzat la seua resposta òptica i s'han dut a terme experiments de sensat d'índex de refracció per a estudiar la seua sensibilitat. Així mateix, s'ha desenvolupat un mètode de funcionalizació de la superfície de l'exoesquelet de diatomees basat en l'ús de polielectròlits catiònics. Com a resultat, s'ha demostrat el potencial tant de NFs per al desenvolupament de biosensors, com el de membranes comercials per a sensors amb una aplicació que no requerisca una elevada sensibilitat però sí un baix cost. A més, s'ha posat de manifest el gran potencial de l'exoesquelet de diatomees per al desenvolupament de sensors basats en la seua resposta òptica. Per contra, les limitacions trobades en el desenvolupament de biosensors basats en SiP han evidenciat la necessitat d'un estudi rigorós i l'optimització de l'estructura dels materials porosos prèviament a ser usats en (bio)sensat.
[EN] Biosensors are analytical devices with application in diverse fields and with several advantages relative to other conventional methods, such as the use of small volumes of sample and reagents, their sensitivity and their fast response, without the need of the sample pretreatment, expensive equipments or specialised technicians. Nevertheless, this is a relatively new research field in which there is a long way to go yet. This doctoral Thesis aims at doing its bit to this field of knowledge by studying the potential of different porous materials as transducers for the development of real-time and label-free optical biosensors. The proposed materials range from those artificially synthesised, such as porous silicon (pSi), polymeric nanofibres (NFs) or commercial polymeric membranes, to natural materials with photonic properties that had not been exploited for sensing yet, such as biosilica exoskeletons of diatoms. All of them have in common its simple production, avoiding expensive and laborious nanofabrication processes. For their study, their optical response will be analysed and, in those cases in which such optical response allows performing detection experiments, strategies for their biofunctionalisation and their implementation in biosensing experiments will be developed as well. Regarding pSi and NFs, the fabrication parameters were optimised to get a suitable optical response for their interrogation. Afterwards, their surface functionalisation was carried out by covalent and non-covalent methods, as well as different bioreceptors (DNA aptamers and antibodies), to study their potential and their constraints as biosensors. Concerning commercial membranes and the biosilica exoskeleton of diatoms, their optical response was characterised and refractive index sensing experiments were carried out to study their sensitivity. Additionally, a biofunctionalisation method for the surface of the diatoms exoskeleton was developed based on the use of cationic polyelectrolytes. As a result, it was demonstrated the potential of NFs for the development of biosensors, as well as the potential of commercial membranes for developing sensors for an application that does not require a high sensitivity but a low cost. Furthermore, the great potential of biosilica exoskeleton of diatoms for the development of sensors based on their optical response has been revealed. By contrast, the constraints found in the development of pSi illustrate the importance of an accurate study and optimisation of porous materials structure before using them for (bio)sensing.
Martínez Pérez, P. (2021). Development and Optimization of Experimental Biosensing Protocols Using Porous Optical Transducers [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/172541
TESIS

L‐glutamate is associated with several neurological disorders; thus, monitoring fast dynamics of L‐glutamate is of great importance in the field of neuroscience. Electrode miniaturization demanded by many applications leads to reduced surface area and decreased amounts of immobilized enzymes on coated electrodes. As a result, lower signal‐to‐noise ratios are observed for oxidase‐enzyme based sensors. To increase the signal‐to‐noise ratio we have developed a process to fabricate micro‐ and nano‐ structures on the microelectrode surface. Localized surface‐plasmon resonances (SPR) has been extensively used to design label‐free biosensors that can monitor receptor‐ligand interactions. A major challenge with localized SPR sensors is that they remain highly susceptible to interference because they respond to both solution refractive index changes and surface binding of the target analyte. The key concept introduced in the present work is the exploitation of transverse and longitudinal resonance modes of nanorod arrays to differentiate between bulk refractive index changes and surface interactions. The transverse bulk sensitivity of the localized SPR sensor (107 nm/RIU) remains competitive with typical single mode gold nanosphere SPR sensors. The figure of merit for the device’s cross‐sensitivity (1.99) is comparable to that of typical wavelength‐interrogated propagating SPR sensors with self referencing.

Cette thèse présente la conception et la mise en œuvre d'un imageur CMOS pour être utilisé dans biocapteurs intégrés basés sur Résonance Plasmonique de Surface (SPR). Tout d'abord, les conditions optimales pour la résonance plasmon dans une interface compatible CMOS / post-CMOS sont obtenus par modélisation avec COMSOL. Deuxièmement, un imageur CMOS de Colonne Actif (CMOS-ACS) du 32x32 pixels est mis en œuvre sur une technologie CMOS 0,35 um. Dans une interface d'or-eau avec une excitation de prisme, on constate que pour les prismes avec des indices de réfraction de 1,55 et 1,46, le couplage optimal avec le plasmon est obtenu pour des films d'or d'une épaisseur de 50 et 45 nm, respectivement. Dans ces conditions, environ 99,19% et 99,99% de l'énergie de la lumière incidente est transférée à le surface plasmon pour les deux prismes respectivement, à condition que la lumière incidente, avec une longueur d'onde de 633 nm, arrive avec un angle d'incidence de 68,45° et 79,05° respectivement. Il est également obtenu qu'un changement de RIU 10-4 de l'indice de réfraction du milieu diélectrique, produit un changement de 0,01 ° dans l'angle de résonance de plasmons qui, dans un schéma de modulation d'intensité de lumière produit une variation de 0,08% dans la lumière réfléchie au photodétecteur. En ce qui concerne le imageur CMOS, une photodiode n-well/p-substrate est choisi comme l'élément de photodétection, en raison de sa faible capacité de jonction, ce qui conduit à un rendement élevé et le gain de conversion élevé comparativement à une photodiode n-diff/p-substrate. Des simulations sur ordinateur avec Cadence et Silvaco produit une capacité de jonction de 31 FF et 135 fF respectivement. Le pixel de l'imageur est basé sur une configuration à trois transistors (3T) et présente un facteur de remplissage de 61%. Le circuit de lecture utilise une technique de capteur de colonne actif (ACS) pour réduire le bruit à motif fixe (Fixed Pattern Noise ou FPN en anglais) liée au le Capteur à Pixels Actif (APS) traditionnelle. En outre, Non-Corrélés Echantillonnage Double (Non-Correlated Double Sampling ou NCDS en anglais) et Delta double échantillonnage (DDS) sont utilisés comme techniques de réduction du bruit. Un montage optique expérimental est utilisé pour caractériser les performances de l'imageur, et nous avons obtenu un gain en conversion de 7,3 uV/e-, une capacité de jonction de la photodiode de 22 fF, un bruit de lecture de 324,5 uV, ce qui équivaut à 45 électrons, et une gamme dynamique de 50,5 dB. Les avantages de l'ACS et NCDS-DDS sont observées dans le niveau faible de FPN du pixel et de la colonne, avec une valeur de 0,09% et 0,06% respectivement. Le travail présenté dans cette thèse est une première étape vers l'objectif de développer une plateforme entièrement intégrée SPR pour biocapteurs, incorporant source de lumière, l'interface SPR, canal microfluidique, les éléments d'optique et imageur CMOS
This dissertation presents the design and implementation of a CMOS imager for use in integrated biosensors based on Surface Plasmon Resonance. First, the optimal conditions for plasmon resonance in a CMOS/Post-CMOS compatible interface are obtained by COMSOL modelling. Second, a 32x32-pixel CMOS-Active Column Sensor (CMOS-ACS) is implemented on 0.35 um CMOS technology. In a gold-water interface with prism excitation, it is found that for prisms showing refractive indexes of 1.55 and 1.46, optimal plasmon coupling is obtained for gold films with thicknesses of 50 and 45 nm respectively. Under these conditions, approximately 99.19% and 99.99% of the incident light's energy is transferred to the surface plasmon for both prism respectively, provided that the incident light, with a wavelength of 633 nm, arrives with incidence angles of 68.45° and 79.05° respectively. It is also obtained that a change of 10-4 RIU in the refractive index of the dielectric medium, produces a change of 0.01° in the plasmon resonance angle, which under a light intensity modulation scheme produces a change of 0.08% in the reflected light's energy reaching the photodetector. Concerning the CMOS imager, a n-well/p-substrate photodiode is selected as the photosensing element, due to its low junction capacitance, which results in high efficiency and high conversion gain compared to the n-diff/p-substrate photodiode. Computer simulations with Cadence and Silvaco produced a junction capacitance of 31 fF and 135 fF respectively. The imager's pixel is based on a three-transistor (3T) configuration and shows a fill factor of 61%. The readout circuitry employs an Active Column Sensor (ACS) technique to reduce the Fixed Pattern Noise (FPN) associated with traditional Active Pixel Sensors (APS). Additionally, Non-Correlated Double Sampling (NCDS) and Delta Double Sampling (DDS) are used as noise reduction techniques. An experimental optical setup is used to characterize the performance of the imager, obtaining a conversion gain of 7.3 uV/e-, a photodiode junction capacitance of 21.9 fF, a read noise of 324.5 uV, equivalent to ~45 e- and a dynamic range of 50.5 dB. The benefits of ACS and NCDS-DDS are observed in the low pixel and column FPN of 0.09% and 0.06% respectively. The work presented in this thesis is a first step towards the goal of developing a fully integrated SPR-biosensing platform incorporating light source, SPR interface, microfluidic channel, optical elements and CMOS imager

The idea to have highly effective autonomous sensors able to measure and share information about the quality of our environment, and particularly water, in our lakes and rivers, our water supply system and the outputs of municipal and industrial wastewater treatment systems is revolutionary and fascinating. These sensors could be densely deployed at multiple locations, and the information may be available to citizens through the Internet. This idyllic vision, nowadays, is far away from being reality, despite the huge effort made to develop innovative molecular sensors. The main challenges related to the realization of these autonomous sensors network are the biofouling, power supply and compactness. In fact, despite thousands of papers in literature about development of novel nanostractured materials for sensing, for instance, there is still not a single example of any of these device being used in direct contact with water for long-term environmental monitoring. The work presented in this thesis proposes a new kind of optical sensor that combines a fast and low cost method to detect water pollutant with good performance and robustness. In particular, this work is focused on the detection of small molecular pollutants, as oils compounds and surfactants. An innovative aspect of the proposed approach relies on the use of a novel class of materials as sensing substrate which have peculiar and fascinating optical properties: these are amorphous perfluorinated polymers with refractive index similar to that of water. When immersed in aqueous solutions, they provide extremely low reflection or scattering of light, hence they become barely visible. For this reason, this class of materials is called phantom. In this limit, when a thin molecular layer spontaneously adsorbs on the surfaces of these materials, the reflected or scattered light increases, providing the basis for optical detection of molecules. In this work, three different phantom materials made of perfluorinated polymers are exploited in the framework of the detection of water contaminants: a prism, microporous membranes and micro-beads, that represent the building blocks for the assembly of an invisible chromatography column. The membrane and the micro-beads were produced for the first time during this work. The use of fluoropolymer prism substrate for molecular detection was already proposed in recent works to realize label-free biosensors based on the functionalization of the surface with antibodies. Here I extend the exploitation of this system to the detection of molecular pollutant through their adsorption on the bare surface of the fluoropolymer materials, without the need of any surface treatment. Despite the lack of surface functionalization, a selectivity in the adsorption of various classes of molecules is demonstrated.

Thesis (Ph.D.)--Boston University PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you.
Microarray technologies have provided powerful tools in modem biotechnology to decipher the complex interconnectivity of genetic and regulatory network. Label-free biosensors have emerged as competitive technologies because they offer more economic and simple procedures, allow molecular interactions in their native state, and provide interaction kinetics, compared to the traditional luminescence-based sensors. While these advantages enable better understandings of the intricate biomolecular interactions, label-free biosensors have yet to be widely adapted in biological and medical research. We present two approaches to develop successful, high-throughput, label-free biosensors by using Interferometric Reflectance Imaging Sensor (IRIS). First, we offer strategies to improve the performance of label-free biosensors by increasing sensitivity and accuracy. Sensitivity enhancement is achieved by utilizing mass tags. Accuracy improvement is accomplished through extensive characterization of stability of DNA and protein microarrays. Following stability characterization of the surface chemistry, the substrate, and the immobilized biomolecules, microarray fabrication methods and normalization techniques are developed to reduce error, hence, increasing the accuracy of quantitative analysis. The degradations of the sensor surface that we discover from stability characterization are susceptible to other label-free biosensors. Thus, the correction strategies that we present can be utilized for accurate quantitative analysis of a variety of label-free biosensors. Second, we develop four applications for IRIS. 1) We present applications for quantitative gene expression analysis and disease screening using DNA microarrays by demonstrating quantitative analysis of DNA hybridization and successful detection of single nucleotide polymorphism. 2) We present an application for quantitative analysis of transcription factors using ssDNA and dsDNA microarrays. We discover a new binding motif for TATA-binding protein and propose alternative models for eukaryotic transcription initiation. 3) We present an application to study immune response using antibody microarrays by demonstrating dynamic detection interleukin-6 with ~ ng/mL sensitivity and successfully detecting the small macromolecule in biologically complex fluid. 4) We propose an application to investigate cellular response to external stimuli, such as drugs, toxins, and pathogens, using patterned cell-protein microarrays. We present strategies of fabricating cell-protein microarrays and demonstrate interleukin-8 detection with ~ pg/mL sensitivity using gold nanoparticles. Improved performance and diverse biological applications of IRIS will help successful implementations of high-throughput label-free biosensors in healthcare.

The search for analytical tools suitable for wide-scale application of DNA analysis is an hot research topic, although thanks to well-established microarray based technology, analysis of DNA sequences and SNP detection can be worked out through a fairly laboratory routine. DNA analysis has nowadays become of increasing interest for several different purposes, mainly thanks to the successful employment of microarray technology, characterized by high sensitiveness and high-throughput analysis, which rapidly advanced genetics leading to devel- opment of many fields of application of DNA analysis, which keeps high the trend in alternative technologies, which could overcome inherent limitations of microarrays technology. In this regard, many efforts have been spent to study electrochemical/electrical based detection strategies by means of which it could be possible to accomplish sensitive analysis by using portable equipments, cheaper and more practical than optical ones, and with scalable-devices compatibles with standard microelectronic processing. Emerging nano-probes with increased chemical-physical properties are considered with growing interest in DNA biosensors as ideal candidates to enhance electrochemical/electrical based detection systems. Among these, nano-gaps adjusted to fit DNA, or in general analyte molecules sizes, are very promising because they can enable direct electrical detec- tion schemes, thus providing a straightforward electronic analogue of the successful DNA microarray standard. Electrical properties of DNA have been the principal focus of many experimental and theoretical research, since early experimental founding confirmed an old hypothesis, for its relevance in the biological function of DNA, being related both with damage and base repair, but also for the appealing potential for biosensor and, in general, in bioelectronic applications. Thanks to its peculiar interactions, it allows versatile manipulations of the structure, compared to other organic and synthetic polymers which have been considered for such purposes. Even though many questions still are open on electrical properties of DNA, it is generally accepted that DNA's conductivity is intimately linked with details of the sequences involved, its length and the overall environment in which molecule is found. The sensitivity to structure's alteration, as that induced by the presence of a mutation, confirmed by experimental and theoretical works allows to exploit DNA electrical properties for biosensor applications. Relying on this agreed description of DNA electrical properties features, the general aim of this thesis was to explore the possibility of developing a platform for the direct transduction of DNA hybridization event based on a nano-gap device and electrical signaling enabled by long range electron transport through DNA molecules.
La detección de hibridación de cadenas de ADN es un reto relevante científicamente y tecnológicamente, que puede aprovechar de las posibilidades proporcionadas por los alcances en los procesos de nano fabricación y caracterización, inspiradores de la idea de una medicina en el punto de atención. El propósito de este trabajo es de establecer un sistema de detección de hibridación de ADN, y polimorfismo de un solo nucleótido (SNP), basado en la medida eléctrica de la reasistencia de un nano-gap funcional izado con el ADN diana. El desarrollo y test del sistema se ha llevado a cabo fijando diferentes objetivos. Un estudio preliminar de la literatura relacionada con las propiedades eléctricas del ADN se ha conducido con la finalidad de establecer el marco de factibilidad del proyecto. De acuerdo con los resultados de este estudio ha sido posible idear el sistema y optimizar su eficacia respeto a las experiencias reportadas. Fijar una estrategia de fabricación de los dispositivos capaz de proveer nano-gaps aptos a la medida de conductividad muy baja, según una rutina de fácil implementación y con alta reproducibilidad de los resultados. Estos se han caracterizados mediante el utilizo de diferentes técnicas basadas primariamente en métodos de detección Óptica y Eléctrica/Electro-química. Obtener la bio-funcionalización selectiva de los electrodos en el nano-gap testando y caracterizando métodos diferentes. Probar el principio de funcionamiento del sistema a través de la medida de la conductividad en los nano-gap durante las diferentes etapas de funcionalización con los bio-receptores y el DNA target. Optimizar el sensor testando su selectividad respeto a la presencia de mutaciones, la sensibilidad a medir diferentes concentraciones del target, y finalmente la posibilidad de regeneración del dispositivo después desnaturalización del ADN hibridado

To truly realize and exploit the extremely powerful information given from surface-enhanced Raman scattering (SERS) spectroscopy, it is critical to develop an understanding of how to design highly sensitive and selective substrates, produce specific and label-free spectra of target analytes, and fabricate long-lasting and in-the-field ready platforms for trace detection applications. The study presented in this dissertation investigated the application of two- and three-dimensional substrates composed of highly-ordered metal nanostructures. These systems were designed to specifically detect target analytes that would enable the trace, label-free, and real-time detection of chemicals and biomolecules. Specifically, this work provides new insight into the required properties for maximizing electromagnetic and chemical Raman enhancement in three-dimensional porous alumina substrates by designing metal nanostructure shape, density, aggregated state, and most importantly aligning the substrate pore size with the excitation wavelength used for plasmonic enhancement leading to the ppb detection of vapor phase hazardous chemicals. A new micropatterned silver nanoparticle substrate fabricated via soft lithography with specific functionalization was developed, which allows the simultaneous analyte and background detection for trace concentrations of the target biomolecule, immunoglobulin G. Also, a novel functionalized SERS hot spot fabrication technique, which utilizes highly specific aptamers as both the mediator for electrostatic assembly of gold nanoframe dimers as well as the biorecognition element for the target, riboflavin, to properly locate the tethered biomolecule within the enhanced region for trace detection, was demonstrated. We suggest that the understanding of SERS phenomena that occur at the interface of nanostructures and target molecules combined with the active functionalization and organization of metal nanostructures and trace detection of analytes discussed in this study can provide important insight for addressing some of the challenges facing the field of SERS sensor design such as high sensitivity and selectivity, reliable and repeatable label-free identification of spectral peaks, and the well-controlled assembly of functional metal nanostructures. This research will have a direct impact on the future application of SERS sensors for the trace detection of target species in chemical, environmental, and biomedical fields through the development of specific design criteria and fabrication processes.

El treball presentat en aquesta tesi doctoral te com objectiu principal la contribució en el camp de la microbiologia per entendre el biofilms i el possible control de desenvolupament mitjançant l’ús de mètodes i enfoc multidisciplinari. Els biofilms estan definits com comunitats de microorganismes que creixen envoltats en una matriu exopolisacárida i s’adhereixen a una superfície inert o teixit viu. La formació dels biofilms bacterians tenen un gran interès en microbiologia clínica degut al desenvolupament d’infeccions que son causades pel contacte directe o per colonització de dispositius mèdics implantats i pròtesis. Actualment es consideren causa de més del 60 % de les infeccions bacterianes. El problema dels biofilms bacterians a nivell clínic es que mostren millor resistència a antibiòtics arribant inclús a ser de 500 a 5000 cops més resistents a agents antimicrobians comparant amb la mateixa bactèria planctònica (bactèria en suspensió). Hi ha hagut moltes temptatives d’adaptar mètodes a laboratoris clínics on es reprodueixen les condicions pel desenvolupament de biofilms, però encara no s’ha arribat a obtenir òptims protocols estàndard per a aquest propòsit de monitoritzar la formació i toxicitat a temps real. Ha crescut l’interès en disseny, desenvolupament i utilització de dispositius de microfluídica que poden emular els fenòmens biològics que ocorren amb diferents geometries, dinàmica de fluids i restriccions de transport de biomassa en microambients fisiològics. La recerca descrita en aquesta tesis s’ha dut a terme amb diferents mètodes “label-free” basats en la variació acústica y/o propietats elèctriques per a la monitorització de biofilms. El treball presentat en la monografia descriu un dispositiu “custom-made” per a la utilització d’Espectroscòpia de impedància electroquímica com a eina útil per a l’obtenció d’informació d’adherència i formació de biofilms. El fet d’afegir nanopartícules com a segon biosensor permet la correlació de biofilm amb la seva toxicitat a temps real per a la detecció del punt òptim de tractament de biofilms. Finalment el disseny d’aquesta tecnologia s’utilitza per l’assaig de la resposta de biofilms a antibiòtics com a model in vitro d’infeccions causades per biofilms.
El trabajo presentado en esta tesis doctoral tiene como principal objetivo la contribución en el campo de la microbiología para entender los biofilms y el posible control de desarrollo mediante el uso de métodos y enfoque multidisciplinar. Los biofilms están definidos como comunidades de microorganismos que crecen embebidos en una matriz exopolisacárida y se adhieren a una superficie inerte o tejido vivo. La formación de los biofilms bacterianos tiene un gran interés en microbiología clínica debido al desarrollo de infecciones que son causadas por contacto directo o por colonización de dispositivos médicos implantados y prótesis. Actualmente se consideran la causa de más del 60 % de las infecciones bacterianas. El problema de los biofilms bacterianos a nivel clínico es que muestran mejor resistencia a antibióticos llegando incluso a ser de 500 a 5000 veces más resistentes a agentes antimicrobianos comparado a la misma bacteria planctónica (bacteria en suspensión). Ha habido muchas tentativas de adaptar métodos a laboratorios clínicos donde se reproducen las condiciones para el desarrollo de biofilms, pero aún no se ha llegado a obtener óptimos protocolos estándar para este propósito de monitorizar la formación y toxicidad en tiempo real. Ha crecido el interés en diseño, desarrollo y utilización de dispositivos de microfluídica que puedan emular los fenómenos biológicos que ocurren con diferentes geometrías, dinámica de fluidos y restricciones de transporte de biomasa en microambientes fisiológicos. La investigación descrita en esta tesis se lleva a cabo con diferentes métodos “label-free” basados en variación acústica y/o propiedades eléctricas para la monitorización de biofilms. El trabajo presentado en esta monografía describe un dispositivo “custom-made” para la utilización de Espectroscopia de impedancia electroquímica como herramienta útil para obtener información de adherencia y formación de biofilms. El hecho de añadir nanopartículas como segundo biosensor permite la correlación de biofilm con su toxicidad en tiempo real para la detección del punto óptimo del tratamiento de biofilms. Finalmente el diseño de esta tecnología es usada para el ensayo de la respuesta de biofilms a antibióticos como modelo in vitro de infecciones causadas por biofilms.
The work presented in this thesis has the main aim to contribute in the field of clinical microbiology to understand the biofilms and the possible of development through the use of methods with multidisciplinary approach. Biofilms are defined as communities of microorganisms that grow embedded in a matrix of exopolysaccharides and adhering to an inert surface or living tissue. The formation of bacterial biofilms has an interest in clinical microbiology with the development of infections that usually arise from either direct contact or the colonization of implanted medical devices and prostheses. Currently they are considered the cause of over 60% of bacterial infections. The problem of bacterial biofilms at clinical level is showing great resistance to antibiotics, so that the biofilm bacteria are 500 to 5000 times more resistant to antimicrobial agents that the same bacteria grown in planktonic cultures (bacteria in suspension). There have been attempts to adapt methods to clinical laboratories where they reproduce the conditions of biofilms, but have not yet adopted an optimal standard protocol for this purpose to follow-up the formation and toxicity in real-time. There has been a growing interest in design, development and utilization of microfluidic devices that can emulate biological phenomena that occur in different geometries, fluid dynamics and mass transport restrictions in physiological microenvironments. The research described in this thesis deals with different label-free methods based on variation of acoustic and electric properties for biofilm monitoring. The work presented in this monograph describe a custom-made device for using electrochemical impedance spectroscopy (EIS) as useful tool to obtain information of adherence and formation of biofilms. The addition of nanoparticles as toxicity biomarker allows the correlation of biofilm formation with its toxicity in real-time for detention of the optimal point for biofilm treatment. Finally the design of this technology is used for testing the biofilm response to antibiotic as in vitro model of biofilm-related infection.