Non-labelled surface sensitive techniques as platforms for pharmaceutical nanotechnology research

Insufficient delivery of drugs to the target sites like tumors and cells has been a barrier for achieving satisfying therapeutic effects in many diseases. Distribution and exposure of drugs to normal and healthy tissues may enhance the possibility of side effects and toxicity in vivo. Nanoparticle (NP) drug delivery systems have been developed to enable targeting of drugs to target sites and at the same time also reduce or even eliminate the distribution and exposure of drugs to non-targeted sites (normal and healthy tissues). The interactions of ligand attached NPs with specific receptors on the cell surface enable intracellular delivery of drugs. Knowledge of the molecular mechanisms (kinetics and affinity) of specific NP surface interactions is vital for designing and optimizing NPs based targeted drug delivery systems. Biophysical non-labelled surface sensitive detection techniques allow the characterization of the specific NP-cell interactions in vitro at the molecular levels. In this work, surface sensitive non-labelled surface plasmon resonance (SPR) and quartz crystal microbalance (QCM) biosensors were optimized, utilized and further developed as platforms for in vitro characterization and evaluation of the targeting of NP drug delivery systems. A multi-parameter SPR (MP-SPR) prototype was modified, improved and optimized for characterizing molecular surface interactions and phospholipid based thin film properties. The methodologies to extract simultaneously the thickness and the optical properties of thin films were developed by using the multi-wavelength SPR technique. The methodologies were extended to cover the film thickness from few nanometers to micrometers by combining the SPR wavelength and the waveguide mode analysis. These methods were successfully utilized for analyzing LB mono- and multilayers and further for the polyelectrolyte multilayer films. In order to enable the combined use of SPR and QCM techniques for drug and NP interaction studies, these two devices were synchronized to achieve consistent hydrodynamic conditions in the flow channels by computational fluid dynamics (CFD) modelling. The flow channels and the device synchronization were verified by the streptavidin-biotin and liposome-surface interactions. The synchronized SPR and QCM devices were further utilized for the examination of the targeting properties via the streptavidin-biotin liposome interactions under different shear flows. The effect of the flow rate and shear stress on the targeted liposome with the target surface was investigated. The results from SPR and QCM measurements were compared, showing that the binding of the targeted liposome was flow rate and shear stress regulated. According to the SPR measurements, high flow rates improved the binding of liposomes to the target surface. However, the results obtained from the QCM measurements were somehow different. They gave additional information about the liposome binding behavior, indicating deformation or rupture of the bound liposomes at high flow rates and shear stresses. In conclusion, SPR and QCM, the two label free surface sensitive techniques, are excellent platforms for pharmaceutical nanotechnology research. These allow for both the nanoparticle interaction studies and the characterization of nanoscale thin films. Especially, the combined use of the synchronized SPR and QCM techniques forms a powerful platform for the qualitative and quantitative characterization of NP-surface interactions for obtaining in-depth understanding of the targeting behavior of NP drug delivery systems. The results obtained provides the basis for developing new complementary in vitro platforms to traditional cell based in vitro assays for optimizing and screening of NP based targeted drug delivery systems.Lääkeannostelu on keskeinen alue lääketutkimuksessa. Lääkeaineiden riittämätön jakelu kohteeseen estää monien sairauksien kohdalla tyydyttävän terapeuttisen vaikutuksen saavuttamisen. Lääkeaineiden leviäminen ja vaikutus terveisiin kudoksiin saattaa lisätä sivuvaikutuksien ja toksisuuden mahdollisuutta in vivo. On kehitetty nanopartikkelipohjaisia lääkeannostelumenetelmiä, jotta lääkeaineita voitaisiin kohdentaa paremmin ja samalla vähentää tai jopa kokonaan välttää lääkeaineiden leviämistä ja vaikutusta kohdealueen ulkopuolelle. Ligandilla funktionalisoitujen nanopartikkeleiden vuorovaikutus solupinnalla olevien spesifisten reseptorien kanssa mahdollistaa lääkeaineiden kuljettamisen solun sisään. Spesifisten nanopartikkeleiden pintavuorovaikutukset ja niiden molekulaaristen mekanismien tuntemus (kinetiikka ja affiniteetti) ovat keskeisiä kehitettäessä ja optimoitaessa nanopartikkelipohjaisia lääkeannostelumenetelmiä. Biofysikaaliset pintaherkät mittausmenetelmät tarjoavat mahdollisuuden karakterisoida in vitro spesifisiä nanopartikkelin ja solun välisiä vuorovaikutuksia molekyylitasolla ilman leimoja. Pintaherkät ja leimattomat pintaplasmonresonanssi- (SPR) ja kvartsikidemikrovaaka- (QCM) biosensorit optimoitiin ja niitä hyödynnettiin ja kehitettiin edelleen alustoiksi kohdennettujen nanopartikkelipohjaisten lääkeannostelumenetelmien in vitro -karakterisointia ja arviointia varten. Moniparametristä SPR- (MP-SPR) prototyyppilaitetta muokattiin, paranneltiin ja optimoitiin molekulaaristen pintavuorovaikutusten ja fosfolipidipohjaisten ohutkalvojen karakterisointia varten. Moniaallonpituus-SPR-menetelmää hyödyntäen kehitettiin metodologia, jolla voi samanaikaisesti määrittää ohutkalvojen paksuuden ja sen optiset ominaisuudet. Lisäksi tämä metodologia laajennettiin kattamaan kalvojen paksuudet muutamasta nanometristä mikrometreihin yhdistämällä SPR-aallonpituus- ja aaltojohdinanalyysit. SPR- ja QCM-laitteiden hydrodynaamiset ominaisuudet synkronisoitiin virtauslaskentamallinusten (CFD) kautta, mikä mahdollisti SPR- ja QCM-laitteiden yhteiskäytön lääkeaineiden ja nanopartikkeleiden vuorovaikutuksien tutkimiseen. Virtauskanavien ja laitteiden synkronisointi todennettiin tutkimalla biomolekulaarista vuorovaikutusta ja nanopartikkeli-pinta-vuorovaikutusta. Synkronoituja SPR- ja QCM-laitteita hyödynnettiin edelleen kohdennuksen ominaisuuksien tutkimiseen mittaamalla streptavidin-biotinyloitujen liposomien vuorovaikutuksia eri virtausnopeuksilla. Virtausnopeuden ja leikkausjännitteen vaikutus kohdennetun liposomin käyttäytymiseen kohdepinnan kanssa tutkittiin. SPR- ja QCM-mittauksia verrattiin keskenään. Mittaukset osoittivat, että kohdennetun liposomin sitoutumista pintaan säätelivät virtausnopeus ja leikkausjännite. QCM antoi lisätietoa liposomien sitoutumiskäyttäytymisestä ja osoitti, että pinnalle sitoutuneet liposomit muuttivat muotoaan tai hajosivat korkeissa virtaunopeuksissa tai leikkausjänitteissä. Päätelmänä voidaan todeta, että, SPR ja QCM ovat erinomaisia menetelmiä farmaseuttisen nanoteknologian tutkimukseen. Nämä menetelmät mahdollistavat nanopartikkeleiden vuorovaikutuksien tutkimisen sekä ohutkalvojen karakterisoimisen. Synkronoitujen SPR- ja QCM-menetelmien yhteiskäyttö muodostaa tehokkaan alustan kvalitatiiviselle ja kvantitatiiviselle nanopartikkeli-pinta-vuorovaikutuksien karakterisoinnille, joka auttaa paremmin ymmärtämään nanopartikkelipohjaisten lääkeannostelumenetelmien kohdennuskäyttäytymistä. Tulokset tarjoavat perustan uusien täydentävien in vitro -alustojen kehittämiselle perinteisten solupohjaisten in vitro -menetelmien rinnalle nanopartikkelipohjaisten lääkeannostelumenetelmien optimointiin ja seulontaan

Detection of Alzheimer's disease biomarkers and mycotoxins using spectroscopic ellipsometry.

Neurological diseases such as Alzheimer's, Parkinson's, MS, which are common around the world and particularly in developed countries with high proportion of elderly, stimulate the development of bio-sensors for early diagnostics of such diseases. For instance the treatment of Alzheimer's patients constitutes a substantial proportion of NHS budget nowadays. Therefore, the development of highly sensitive optical devices for early diagnoses of Alzheimer's disease (AD) will be beneficial to society. This PhD is mainly dedicated to the application of the method of Spectroscopic Ellipsometry for AD diagnostics. Total Internal Reflection Ellipsometry (TIRE) was utilized in this work as an immunosensor for detection of Amyloid Precursor Protein 770 (APP[770]) and beta amyloid peptide (Abeta[1-16)]. The detection of low concentrations of APP[770] in a complex medium containing other proteins, salts, and amino acids was achieved using the method of TIRE in direct immuno assay with monoclonal DE2 antibodies. The calibration of TIRE with a complementary QCM measurements in air allowed the evaluation of (originally unknown) concentration of APP[770] as 121 pmol/l. The immune reaction between APP[770] and DE2 antibodies was also tested using QCM technique operating in liquid. The application of TIRE was extended to the detection of much smaller peptide Abeta[1-16] which bind to the same DE2 antibody. The results were very encouraging since low concentrations (0.05ng/ml) of Abeta[1-6] were detected; this showed good prospects for detection of Abeta[40-42], an actual marker of AD.The second part of this work was dedicated to detection of mycotoxins, a hazardous contaminant in agriculture products (grains) and associated food and feed. Two mycotoxins, namely Aflatoxins B1 and Zearalenone, were detected in TIRE direct immunoassay. Zearalenone was also detected using TIRE competitive assay. The obtained limits of detection of 0.04 ng/ml for Aflatoxin and 0.1 ng/ml for Zearalenone are well below the legislation limit. Such remarkable results are due to a combination of high sensitivity of TIRE method and the aggregation of hydrophobic molecules of mycotoxins in aqueous solutions. A new method of purification of substances contaminated with mycotoxins based on the use of polyelectrolyte microcapsules functionalized with specific antibodies was successfully tested in this work

Storage of water molecules into biomimetic heterostructures: the role of roughness

The development of devices based on heterostructured thin films of biomolecules conveys a huge contribution on biomedical field. However, to achieve high efficiency of these devices, the storage of water molecules into these heterostructures, in order to maintain the biological molecules hydrated, is mandatory. Such hydrated environment may be achieved with lipids molecules which have the ability to rearrange spontaneously into vesicles creating a stable barrier between two aqueous compartments. Yet it is necessary to find conditions that lead to the immobilization of whole vesicles on the heterostructures. In this work, the conditions that govern the deposition of open and closed liposomes of 1.2-dipalmitoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)] (sodium Salt) (DPPG) onto polyelectrolytes cushions prepared by the layer-by-layer (LbL) method were analyzed. Electronic transitions of DPPG molecules as well as absorption coefficients were obtained by vacuum ultraviolet spectroscopy, while the elemental composition of the heterostructures was characterized by x-ray photoelectron spectroscopy (XPS). The presence of water molecules in the films was inferred by XPS and infrared spectroscopy. Quartz crystal microbalance (QCM) data analysis allowed to conclude that, in certain cases, the DPPG adsorbed amount is dependent of the bilayers number already adsorbed. Moreover, the adsorption kinetics curves of both adsorbed amount and surface roughness allowed to determine the kinetics parameters that are related with adsorption processes namely, electrostatic forces, liposomes diffusion and lipids re-organization on surface. Scaling exponents attained from atomic force microscopy images statistical analysis demonstrate that DPPG vesicles adsorption mechanism is ruled by the diffusion Villain model confirming that adsorption is governed by electrostatic forces. The power spectral density treatment enabled a thorough description of the accessible surface of the samples as well as of its inner structural properties. These outcomes proved that surface roughness influences the adsorption of DPPG liposomes onto surfaces covered by a polyelectrolyte layer. Thus, low roughness was shown to induce liposome rupture creating a lipid bilayer while high roughness allows the adsorption of whole liposomes. In addition, the fraction of open liposomes calculated from the normalized maximum adsorbed amounts decreases with the cushion roughness increase, allowing us to conclude that the surface roughness is a crucial variable that governs the adsorption of open or whole liposomes. This conclusion is fundamental for the development of well-designed sensors based on functional biomolecules incorporated in liposomes. Indeed, LbL films composed of polyelectrolytes and liposomes with and without melanin encapsulated were successfully applied to sensors of olive oil.Fundação para a Ciência e a Tecnologia (FCT)- PEst-OE/FIS/UI0068/2011 ; SFRH/BD/62229/200

Biomimetic Interfaces for Surface Sensitive Drug Discovery Techniques

For the last few decades, the expences of pharmaceutical development and drug discovery have been constantly increasing whereas the amount of new pharmaceutical products reaching the market has been diminishing. The drug discovery methods today rely heavily on different screening technologies in the early discovery phase. High-throughput screening is usually the dominant approach along with different computational methods, but these methods lack the ability to monitor the interactions between drugs and cells in real-time. The ability to measure drug-cell interactions and cell responses during drug stimulation in real-time could provide complementary kinetic information to traditional methods already used in drug discovery. This time-resolved information should help to build a better mechanistic understanding of the effect of drug formulation design on the drug release actions, the drug delivery process and the efficacy of the drug, especially when it comes to new biological drugs and nanoparticle formulations. This dissertation addresses challenges in developing functional surfaces and analysis methods based on the surface plasmon resonance technique for pharmaceutical research purposes. The research in this thesis spans from traditional drug-protein interaction studies and preparation of cell model surfaces to interaction studies with living cells. An approach where proteins were immobilized in a hydrogel was used for studying the interaction kinetics between protein kinase C ε and both an activating and an inhibiting single-chain antibody. The affinities determined for the interactions were able to predict the level of activation or inhibition in subsequent cell culture assays. This thesis also presents two types of new analysis methods, i.e. label-enhanced and multi-wavelength surface plasmon resonance (SPR) methods were developed in order to improve the sensitivity of bioassays and accuracy for characterizing ultra-thin films, respectively. The label-enhanced SPR method was shown to improve assay sensitivity up to 100-fold, whereas the multi-wavelength SPR analysis provided the means to characterize organic layers in the range from a few nanometers to hundreds of nanometers, i.e. layer thicknesses of relevance to biological membranes and hydrogels. New surface coating chemistries based on dextran and thiol-PEG were also developed in this thesis in order to enable the preparation of robust biomimetic membranes by vesicle spreading or adsorption. The dextran-based and PEG-based coatings promoted supported lipid bilayer and adsorbed vesicle layer formation, respectively. The new analysis approaches developed in this thesis were further utilized in order to characterize the optical properties of the formed lipid layers on the dextran- and PEG-based coatings. Finally, a new analytical approach for signal processing of the real-time and label-free SPR measurements performed together with living cells is introduced which provides the mean to differentiate between para- and transcellular cell absorption routes of drug molecules. This dissertation contributes to the pharmaceutical research field by introducing new measuring tools, improved in vitro biomimetic models and new approaches for processing of the signal from label-free measurements in order to provide relevant real-time and complementary information to traditional drug development and discovery tools. This will hopefully benefit the pharmaceutical research field and possibly enable a more efficient development of new pharmaceuticals and therapies in the future.Uusien lääkkeiden kehitys on yleisesti ottaen hidasta, ja lääkekehityksen vaatima rahallinen panostus on viime aikoina vain kasvanut entisestään. Tämän lisäksi myös uusien markkinoille saapuvien lääkevalmisteiden määrä on vuosia jatkuvasti laskenut. Vaikka lääkekehitysmenetelmät ovat kehittyneet viime vuosina merkittävästi varsinkin laskennallisten menetelmien osalta, esikliinisen vaiheen tutkimusten ennustavuus lääkevalmisteen käyttäytymisestä elävässä eläimessä tai ihmisessä ei ole vielä toivottavalla tasolla. Paremmilla aikaisen vaiheen tutkimusmenetelmillä voitaisiin nopeuttaa ja vähentää lääkekehityksen kustannuksia, ja mahdollisesti myös kehittää tehokkaampia ja turvallisempia lääkeaineita. Tämän väitöstutkimuksen tärkeimpänä tavoitteena on kehittää uusia tutkimusmenetelmiä perustuen biologiaa matkiviin pinnoitteisiin ja näiden käyttämiseen lääkeaineiden fysikaalisten ja kemiallisten ominaisuuksien määrittämiseen. Väitöksessä käytetyt tutkimusmenetelmät perustuvat mittausmenetelmiin, joissa ei käytetä leimaus- tai merkkiaineita (ns. leimavapaa menetelmä) ja joiden avulla voidaan mitata nanometri kokoluokan pintarakenteita ja vuorovaikutuksia erilaisten aineiden välillä. Väitöstutkimuksen kuluessa on kehitetty mittateknologiaan uusia laskentatyökaluja, mittausten herkkyyttä parantavia fysikaalis-kemiallisia menetelmiä, elävien solujen käyttöä mittauksissa sekä kemiallisia menetelmiä muokata pintoja soveltuvaksi mittauksiin. Erityisesti elävien solujen pintarakennetta matkivien pinnoitteiden kehitys on ollut tärkeässä roolissa väitöstutkimuksessa. Väitöstutkimuksessa kehitettyjen menetelmillä voidaan tulevaisuudessa täydentää lääkekehityksessä käytettyjen menetelmien työkalupakkia . Tutkimuksen menetelmät voivat siis tulevaisuudessa auttaa kehittämään tehokkaampia lääkehoitoja, nykyistä nopeammin ja nykyistä pienemmillä kustannuksilla

Electronic tongue technology applied to the analysis of grapes and wines

El desarrollo de nuevos métodos de análisis para caracterizar los alimentos es de vital importancia para mejorar los actuales sistemas de control de calidad de los productos alimenticios. Dentro de este campo, el concepto de lengua electrónica (ETs o e-tongues) ha crecido rápidamente en los últimos años debido a su gran potencial. Estos dispositivos se basan en sensores electroquímicos combinados con análisis de datos multivariantes. De acuerdo con la IUPAC (Unión Internacional de Química Pura y Aplicada), una lengua electrónica es un sistema multisensor, que consiste en un número de sensores de baja selectividad y utiliza procedimientos matemáticos avanzados para el procesamiento de señales basados en el reconocimiento de patrones (PARC) y/o análisis multivariante [redes neuronales artificiales (RNA), análisis de componentes principales (PCA), etc.]. Por lo tanto, las ETs son sistemas holísticos que proporcionan información global y cualitativa acerca de la muestra en lugar de datos cuantitativos acerca de compuestos específicos. Sin embargo, si la matriz de datos obtenida por estos sistemas se analiza con herramientas de procesamiento quimiométrico adecuadas, se podría extraer información descriptiva o predictiva de parámetros específicos. Existe un término más reciente en el campo de las lenguas electrónicas, ampliamente denominado lengua bioelectrónica (bioET), que incluye el uso de uno o varios biosensores implementados en las ETs. Durante esta investigación se han aplicado ETs y bioETs para estudiar las uvas tintas y los vinos con el fin de predecir mejor el momento óptimo de la vendimia de uvas, así como los parámetros de calidad de interés en los vinos.Departamento de Química Física y Química InorgánicaDoctorado en Físic

Fabrication and characterization of multilayered assemblies based on polyelectrolytes and hybrid systems with carbon nanomaterials for applications in nanofiltration and as smart surfaces.

216 p.The main goal of this thesis was the study of multilayered assembled structures of polyelectrolytes solely (i) or combined with carbon nanomaterials (ii) in order to use them for specific applications such as nanofiltration membranes and smart surfaces; (i) Physico-chemical studies of PEMs stability in different media, assembly of the multilayers in extreme ionic strength conditions and studies of different growth regimes by choosing different pairs of polycations and polyanions have been performed (ii) In combination with carbon nanomaterials hybrid materials have been also created. On the one hand, together with carbon nanotubes developing an intermediate layer acting as a spacer between commercial porous supports and PEMs with nanofiltration properties in water filtration treatments was developed. On the other hand, a new approach was meant to combine polyelectrolytes with graphene oxide producing capsules using erythrocytes as templates with a tuneable content in carbon

Nanoparticle Chemical Sensors: A Study on Optical Humidity Sensor Design

The need for sensors capable of operating in harsh environments such as those containing flammable, corrosive or reactive vapors is a niche which thin-film optical devices, with their robustness and ease of maintenance may effectively fill. Two such systems were developed using spin-coating techniques and evaluated for applicability as humidity sensors. The first is based on aggregated silica nano particles. The second is a poly electrolyte multi-layer film impregnated with silver nano particles which exhibited strong surface plasmon response. Ellipsometric experiments performed using a sealed test cell with constant humidity maintained using saturated salt solutions showed that the former responded strongly to changing humidity. The latter possessed interesting hysteresis behavior as analyzed in a climate-controlled glovebox via reflectometry, but proved insufficiently responsive to changing humidity. The silica nano particle substrate was found to be a simple, tunable sensor platform which may be viable for the detection of a wide variety of vapor-phase chemical species

A Label Free CMOS-Based Smart Petri Dish for Cellular Analysis

RÉSUMÉ Le dépistage de culture cellulaire à haut débit est le principal défi pour une variété d’applications des sciences de la vie, y compris la découverte de nouveaux médicaments et le suivi de la cytotoxicité. L’analyse classique de culture cellulaire est généralement réalisée à l’aide de techniques microscopiques non-intégrées avec le système de culture cellulaire. Celles-ci sont laborieuses spécialement dans le cas des données recueillies en temps réel ou à des fins de surveillance continue. Récemment, les micro-réseaux cellulaires in-vitro ont prouvé de nombreux avantages dans le domaine de surveillance des cellules en réduisant les coûts, le temps et la nécessité d’études sur des modèles animaux. Les microtechniques, y compris la microélectronique et la microfluidique,ont été récemment utilisé dans la biotechnologie pour la miniaturisation des systèmes biologiques et analytiques. Malgré les nombreux efforts consacrés au développement de dispositifs microfluidiques basés sur les techniques de microscopie optique, le développement de capteurs intégrés couplés à des micropuits pour le suivi des paramètres cellulaires tel que la viabilité, le taux de croissance et cytotoxicité a été limité. Parmi les différentes méthodes de détection disponibles, les techniques capacitives offrent une plateforme de faible complexité. Celles-ci ont été considérablement utilisées afin d’étudier l’interaction cellule-surface. Ce type d’interaction est le plus considéré dans la majorité des études biologiques. L’objectif de cette thèse est de trouver des nouvelles approches pour le suivi de la croissance cellulaire et la surveillance de la cytotoxicité à l’aide d’un réseau de capteurs capacitifs entièrement intégré. Une plateforme hybride combinant un circuit microélectronique et une structure microfluidique est proposée pour des applications de détection de cellules et de découverte de nouveaux médicaments. Les techniques biologiques et chimiques nécessaires au fonctionnement de cette plateforme sont aussi proposées. La technologie submicroniques Standard complementary metal-oxide-Semiconductor (CMOS) (TSMC 0.35 μm) est utilisée pour la conception du circuit microélectronique de cette plateforme. En outre, les électrodes sont fabriquées selon le processus CMOS standard sans la nécessité d’étapes de post-traitement supplémentaires. Ceci rend la plateforme proposée unique par rapport aux plateformes de dépistage de culture cellulaire à haut débit existantes. Plusieurs défis ont été identifiés durant le développement de cette plateforme comme la sensibilité, la bio-compatibilité et la stabilité et les solutions correspondantes sont fournies.----------ABSTRACT High throughput cell culture screening is a key challenge for a variety of life science applications, including drug discovery and cytotoxicity monitoring. Conventional cell culture analysis is widely performed using microscopic techniques that are not integrated into the target cell culture system. Additionally, these techniques are too laborious in particular to be used for real-time and continuous monitoring purposes. Recently, it has been proved that invitro cell microarrays offer great advantages for cell monitoring applications by reducing cost, time, and the need for animal model studies. Microtechnologies, including microelectronics and microfluidics, have been recently used in biotechnology for miniaturization of biological and analytical systems. Despite many efforts in developing microfluidic devices using optical microscopy techniques, less attention have been paid on developing fully integrated sensors for monitoring cell parameters such as viability, growth rate, and cytotoxicity. Among various available sensing methods, capacitive techniques offer low complexity platforms. This technique has significantly attracted attentions for the study of cell-surface interaction which is widely considered in biological studies. This thesis focuses on new approaches for cell growth and cytotoxicity monitoring using a fully integrated capacitive sensor array. A hybrid platform combining microelectronic circuitry and microfluidic structure is proposed along with other required biological and chemical techniques for single cell detection and drug discovery applications. Standard submicron complementary metal–oxide–semiconductor (CMOS) technology (TSMC 0.35 μm) is used to develop the microelectronic part of this platform. Also, the sensing electrodes are fabricated in standard CMOS process without the need for any additional post processing step, which makes the proposed platform unique compared to other state of the art high throughput cell assays. Several challenges in implementing this platform such as sensitivity, bio-compatibility, and stability are discussed and corresponding solutions are provided. Specifically, a new surface functionalization method based on polyelectrolyte multilayers deposition is proposed to enhance cell-electrode adherence and to increase sensing electrodes’ life time. In addition, a novel technique for microwell fabrication and its integration with the CMOS chip is proposed to allow parallel screening of cells. With the potential to perform inexpensive, fast, and real-time cell analyses, the proposed platform opens up the possibility to transform from passive traditional cell assays to a smart on-line monitoring system

Microcapsule biosensors based on competitive binding and fluorescence resonance energy transfer assays

Fluorescent sensing systems offer the potential for minimally invasive monitoring with implantable devices, but they require carrier technologies that provide suitable immobilization, accessibility, and biocompatibility while maintaining adequate response characteristics. Towards the development of this goal, a general design of a biosensor with the capability of detecting different metabolites was investigated. The approach is based on the encapsulation of a competitive binding assay in microcapsules and monitoring the changes in fluorescence resonance energy transfer (FRET) in the presence of analyte. To experimentally demonstrate this type of sensing system, glucose was chosen as the model target analyte. The design, fabrication, and characterization of several embodiments of a non-consuming fluorescence affinity glucose sensor are described in this dissertation. The novel feature of this system used through out the work is the employment of microcapsules for entrapping the sensing assay, which allows for the free movement of sensing elements while maintaining their constant concentrations with continuously-varying analyte concentration. Initially, a FRET based glucose sensor was demonstrated by encapsulating multilayers of Concanavalin A (Con A)/dextran in microcapsules. Even though microcapsules comprised of Con A/dextran complexes showed reasonable glucose sensitivity, there are some significant obstacles to practical use of this system due to toxicity, aggregation, and irreversible binding. Therefore, to overcome the limitations of Con A, an improved FRET assay was developed by replacing Con A with apo-glucose oxidase (apo-GOx). Apo-GOx is highly specific toward β-D-glucose, reduces the concern over aggregation as it can only bind to one glucose molecule (whereas, Con A binds to four glucose molecules), and also could be more biocompatible than Con A by recombinant production. The first attempt at the apo-GOx/dextran assay encapsulated in microcapsules used a blue-light-excited FRET pair (FITC/TRITC). The assay elements were encapsulated in microcapsules using photosensitive polymers (poly(styrene sulfonate) and diazoresin) in the shell structure. The results of glucose sensitivity experiments showed a controllable and reversible sensor response with sensitivity in the range of 2--6%/mM over the range of 0--40 mM glucose. In spite of the advantages of this system, it is not ideal for in vivo studies, as the short-wavelength dyes will be difficult to interrogate transdermally due to high tissue scattering. Additionally, diazoresin contains formaldehyde groups that could prove to be toxic. (Abstract shortened by UMI.