Dissertations / Theses on the topic 'Biosensing platform'

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

Photonic crystals have been shown to be a promising technology for improving the performance of light emitting diodes, solar cells and optical communication components. More recently there has been interest in the application of photonic crystals for bio‐chemical sensing since they provide the potential benefits of high sensitivity, label free, real time detection with low limit of detection. Optical sensing mechanisms such as Surface Plasmon Resonance (SPR), and Evanescent Field (EF) sensing methods are currently popular. These are all sensitive to small changes in refractive index (RI) of part of the device. To date SPR methods provide the highest level of sensitivity but have the disadvantage of requiring an expensive gold coating. AROMA Sensor: As a high sensitivity, low cost alternative to conventional SPR methods, this thesis investigates a new concept for bio‐chemical sensing recently developed at Southampton, which uses vertical projection of leaky transmitted modes of a photonic crystal as the sensing method. We call this Angle Resolved Out‐coupled Mode Analysis (AROMA). This method is highly sensitive to small changes in refractive index at the sidewalls of the holes of a photonic crystal resulting in a strong angular shift of an out coupled beam of light. Changes in RI causes a shift in the projected spot position that can be recorded by a CCD/ CMOS camera. Sensor performance is shown to far exceed normal SPR. Simulation and experimental results demonstrate a sensitivity of 10 degree/RIU from a non‐optimised sensor and simulation results indicate an improved sensitivity of 6500 degree/RIU by optimising the sensor operating point. Responsivity of the sensor was investigated by sequentially depositing a series of sub nm ZnO layers, and was found to be highly linear. Photonic crystal coupler and system integration: Apart from the sensor, a new concept for light coupling is developed and optimised. We extend photonic crystal technology to create a combined light coupler/splitter component allowing arbitrary N‐channel light coupling to a simple slab waveguide device. The coupler is combined with multiple sensors to make a fully functional multi‐channel (4‐12 channels) sensor operating at 785nm. This is integrated into a high refractive index (n=1.7) Silicon Oxynitride (SiON) slab waveguide deposited onto a transparent borosilicate glass substrate. The aim for the slab waveguide was to mimic the refractive index of available polymer materials so that the entire system could eventually be fabricated on a flexible polymer substrate by nanoimprint lithography. Design and modelling: This thesis describes the design and optimisation of each component (sensor, coupler and slab waveguide), presenting in depth background physics and rigorous design methods for each component. 3D models were developed based on Rigorous Coupled Wave Analysis (RCWA) and Finite‐Difference Time‐Domain (FDTD) methods. RCWA models allowed accurate prediction and optimisation of light coupling and projection angles for any selected operating wavelength. FDTD methods allowed careful analysis of the interaction between the light field in the slab waveguide and materials placed in the holes. It also predicts the far‐field projected beam pattern for the sensor. Applications: Capability to detect (dry) monolayer coatings was proven for a simple self‐assembled monolayer molecule coating (p‐tolyltrichlorosilane (TTCS)) and also deoxyribonucleic acid (DNA) was successfully detected, close to physiological levels. To achieve this a complex hybridisation process was developed. Sensor response as a function of self‐assemble molecule (SAM) length and distance from the sidewalls was investigated in detail by using reversible chains of long chain charged molecules (lysine, poly‐lysine, bovine serum albumin protein). A detector surface with a layer of poly‐lysine‐g‐PEG was successfully replaced by a poly‐lysine molecule with larger molecule weight. Sequentially additional bovine serum albumin protein binding with the Polylysine was detected. Capability to detect biomolecules in an aqueous environment is intrinsically difficult for most bio‐sensors. By fabricating the device on a transparent glass substrate, and designing the device to project light backwards through the substrate, it became possible to detect small changes in refractive index for liquids placed on the exposed top surface with no detriment to the readout method. The bulk sensitivity of the sensor for liquids was evaluated by measuring a sequence of glucose solutions with increasing concentrations. A highly linear response was again observed.

[eng] Functional diversification in the More than Moore era has attracted an increasing attention on MEMS resonators as sensing devices for system-on-chip applications thanks to its miniaturization capabilities, simple readout schemes, and integration with current ICs fabrication technologies. These advantages make them the perfect candidates to develop biosensing applications in the chemical and biological domains provided its portability, high-throughput, reduced footprint, and minimal processing time. This thesis focuses on analyzing, designing, and developing MEMS resonators oriented and optimized for VOCs detection and calorimetric sensing that are monolithically integrated with a commercial 0-35-μm CMOS technology with on-chip readout via a CMOS-MEMS fabrication approach. An electrostatic actuation scheme and a capacitive readout allow the MEMS structures to operate as a self-sustained oscillator when coupled to a specific amplifier providing a quasidigital output signal. The MEMS resonators are defined using the available CMOS layers. The adopted fabrication approach uses an intra-CMOS post-fabrication wet-etching step to release the mechanical moving parts by removing the surrounding sacrificial oxide, while the readout circuit is protected thanks to the passivation layer. Extensive analytical studies and FEM simulations, together with experimental characterization in open-loop and closedloop configuration of the fabricated CMOS-MEMS devices are addressed, offering an invaluable source of information for design optimization. Four-anchored plate resonators operating in the MHz range are designed and evaluated to work as a gas sensor with specific surface functionalization (via dip-cast immersion and inkjet polymer deposition) achieving a mass resolution per unit area as low as 213 pg·cm-2·Hz-1 and an Allan deviation below 0.5 ppm. Tolerance against environment perturbations such as temperature, humidity and fluid flow are discussed in detail for multiple anchor topologies, providing an optimum folded flexure alternative that alleviates such disturbances by 20-times. After P4V polymer coating, the fabricated system demonstrated acetone detection with a resolution down to 20 ppb that directly points towards non-invasive exhaled breath diagnosis for diabetic patients. In the case of calorimetric sensing, CC-Beams resonators in the MHz range have been designed as the temperature sensor providing extremely high temperature sensitivity up to -7900 ppm·ºC-1 that, together with a fair sub-ppm Allan deviation, achieves an outstanding temperature resolution of 300 μK. In this line, a cointegration of the resonator within a microfluidics PDMS-based platform has been developed that enables a Lab-on-Chip CMOS-compatible system capable of routing a fluid of interest to interact with a CMOS electrode. A conformal 2D planarization process is proposed to increase the IC active surface, combined with a standard wire bonding technique for socket connection. The obtained results confirm micro-calorimetric operation with an experimentally measured thermal efficiency from sample to resonator of 20%, conducting to an energy and heat resolutions as low as of 150 pJ and 630 nW, respectively.
[cat] La diversificació de funcionalitats en un mateix sistema integrat intrínsec a l’aproximació “More than Moore” ha evidenciat un interès creixent en els dispositius basats en ressonadors MEMS per aplicacions tipus “System-on-Chip” gràcies a la seva capacitat de miniaturització, a la implementació directa d’esquemes de lectura elèctrics i a la seva possible integració amb les tecnologies actuals de fabricació de circuits integrats. Aquests avantatges els fan candidats idonis pel desenvolupament d’aplicacions de bio-sensat en l’àmbit de la química i de la biologia donada la seva portabilitat, elevat rendiment, dimensions reduïdes i temps mínim de processat. Aquest treball de tesi es focalitza en l’anàlisi, disseny i desenvolupament de ressonadors MEMS orientats i optimitzats per a la detecció de COVs i pel sensat calorimètric, integrats monolíticament amb circuits CMOS de lectura mitjançant l’ús d’una tecnologia comercial CMOS de 0.35-μm i d’una estratègia de fabricació CMOS-MEMS. Els ressonadors MEMS desenvolupats poden operar en mode oscil·lador, gràcies a la implementació d’un esquema elèctric amb actuació electrostàtica i lectura capacitiva, que permet la seva connexió directa a un circuit amplificador específic obtenint-se així un senyal de sortida quasi digital. Els dispositius MEMS s’han mecanitzat emprant les capes disponibles de la tecnologia CMOS mitjançant una aproximació intra-CMOS per la seva fabricació. Aquesta requereix d’una etapa posterior de gravat humit per a l’alliberació de les parts mecàniques mòbils dels ressonadors basada en l’eliminació de l’òxid de sacrifici tot mantenint-se protegit la resta del circuit integrat gràcies a la capa de passivació. En aquest treball es presenten un gran nombre d’estudis analítics i simulacions FEM que, juntament amb la caracterització experimental tant en llaç obert com en llaç tancat dels diferents dispositius CMOS-MEMS fabricats, suposen una important font d’informació per a l’optimització dels dissenys. S’han dissenyat ressonadors tipus plataforma amb quatre ancoratges que operen dins el rang dels MHz i seguidament, s’han avaluat per la seva operació com a sensors de gas gravimètrics mitjançant una funcionalització superficial específica (ja sigui a través d’immersió en dissolució o bé per deposició d’un polímer emprant impressió de tinta) amb els que s’ha obtingut una resolució en massa per unitat de superfície de 213 pg·cm-2·Hz-1 així com una desviació d’Allan inferior als 0.5 ppm. Addicionalment s’ha analitzat la tolerància que presenten varies topologies de suport front a pertorbacions ambientals com la temperatura, la humitat i el flux d’un gas constatant que les estructures tipus “U” minimitzen aquests efectes fins 20 vegades. Mitjançant la deposició del polímer P4V sobre els sistemes fabricats, s’ha demostrat la capacitat de detectar acetona amb una resolució de 20 ppb el que capacita per la seva potencial aplicació en el diagnosi clínic de pacients diabètics de forma no invasiva per mitjà de l’exhalat. En el cas dels sensors calorimètrics, s’han dissenyat ressonadors tipus pont amb dos ancoratges que operen dins el rang dels MHz i funcionen com a sensors de temperatura de molt alta sensibilitat assolint un valors de fins a -7900 ppm·ºC-1 que, juntament amb una desviació Allan de l’oscil·lador inferior a ppm, s’assoleix una excel·lent resolució tèrmica de 300 μK. En aquesta línia d’actuació, s’ha desenvolupat tot un procés de co-integració dels ressonadors juntament amb una plataforma de microfluídica basada en PDMS amb el que s’obté un sistema tipus “Lab-on-Chip” compatible amb circuits integrats i que permet dirigir un fluid d’interès per a la seva interacció sobre un elèctrode CMOS. Es presenta un procés de planarització 2D, que permet incrementar la superfície efectiva del xip, juntament amb la utilització de la tècnica “wire bonding” estàndard per a la connexió elèctrica amb l’encapsulat. Els resultats experimentals confirmen l’operació del sistema com a micro-calorímetre obtenint una eficiència tèrmica de la mostra cap al ressonador del 20%, que esdevé en una resolució d’energia i calor de 150 pJ i 630 nW, respectivament.
[spa] La diversificación de funcionalidades en un mismo sistema integrado intrínseca a la aproximación “More than Moore” ha evidenciado un interés creciente en los dispositivos basados en resonadores MEMS para aplicaciones tipo “System-on-Chip” gracias a su capacidad de miniaturización, a la implementación directa de esquemas de lectura eléctricos y a su posible integración con las tecnologías actuales de fabricación de circuitos integrados. Estas ventajas los hacen candidatos idóneos para el desarrollo de aplicaciones de bio-sensado en el ámbito de la química y de la biología dada su portabilidad, elevado rendimiento, tamaño reducido y tiempo mínimo de procesado. Este trabajo de tesis se focaliza en el análisis, diseño y desarrollo de resonadores MEMS, orientados y optimizados para la detección de COVs y para sensado calorimétrico, integrados monolíticamente con circuitos CMOS de lectura mediante el uso de una tecnología comercial CMOS de 0.35-μm y de una estrategia de fabricación CMOS-MEMS. Los resonadores MEMS desarrollados pueden operar como oscilador, gracias a la implementación de un esquema eléctrico con actuación electrostática y lectura capacitiva, que permite su conexión directa a un circuito amplificador específico obteniéndose así una señal de salida casi digital. Los dispositivos MEMS se han mecanizado utilizando las capas disponibles de la tecnología CMOS mediante una aproximación intra-CMOS para su fabricación. Esta requiere de una etapa posterior de gravado húmedo para la liberación de las partes mecánicas móviles de los resonadores basada en la eliminación del óxido de sacrificio manteniéndose protegido el resto del circuito integrado gracias a una capa de pasivación. En este trabajo se presentan un gran número de estudios analíticos y simulaciones FEM que, junto con la caracterización experimental tanto en lazo abierto como lazo cerrado de los diferentes dispositivos CMOSMEMS fabricados, suponen una importante fuente de información para la optimización de los diseños. Se han diseñado resonadores tipo plataforma con cuatro anclajes que operan en el rango de los MHz y seguidamente, se han evaluado para su operación como sensores de gas gravimétricos mediante una funcionalización superficial específica (ya sea a través de inmersión en disolución o bien por deposición de un polímero usando impresión de tinta) con los que se ha obtenido una resolución en masa por unidad de superficie de 213 pg·cm-2·Hz-1 así como una desviación Allan inferior a los 0.5 ppm. Adicionalmente se ha analizado la tolerancia que presentan varias topologías de anclaje frente a perturbaciones ambientales como la temperatura, la humedad y el flujo de un gas constatándose que las estructuras tipo “U” minimizan estos efectos hasta 20 veces. Mediante la deposición del polímero P4V sobre los sistemas fabricados, se ha demostrado la capacidad de detectar acetona con una resolución de 20 ppb lo que capacita para su potencial aplicación en el diagnóstico clínico de pacientes diabéticos de forma no invasiva a través del exhalado. En el caso de los sensores calorimétricos, se han diseñado resonadores tipo puente de doble anclaje que operan en el rango de los MHz y funcionan como sensores de temperatura de muy alta sensibilidad alcanzando valores de hasta -7900 ppm·ºC-1 que, junto con una desviación Allan del oscilador inferior a ppm, se consigue una excelente resolución térmica de hasta 300 μK. En esta línea de actuación, se ha desarrollado todo un proceso de co-integración de los resonadores junto con una plataforma de microfluídica basada en PDMS con lo que se obtiene un sistema tipo “Lab-on-Chip” compatible con circuitos integrados y que permite dirigir un fluido de interés para su interacción sobre un electrodo CMOS. Se presenta un proceso de planarización 2D, que permite incrementar la superficie efectiva del chip, junto con la utilización de la técnica “wire bonding” estándar para la conexión eléctrica con el encapsulado. Los resultados experimentales confirman la operación del sistema como micro-calorímetro obteniendo una eficiencia térmica de la muestra hacia el resonador del 20%, lo que supone una resolución de energía y calor de 150 pJ y 630 nW, respectivamente.

Rapid methods to identify bacteria in biological samples are important for prompt antimicrobial therapy. The current detection methods are classical biological sample cultures and biochemical tests, which are however, time-consuming and not highly sensitive. A novel and highly performing approach is offered by aptamers acting as recognition elements able to detect epitopes on the surface of a bacterium. Aptamers interacting with specific bacteria are known and then could provide a solid base for developing promising solutions for this issue. With this PhD work I intended to tackle one drawback of aptamer-based biosensor: the lack of platforms for high density aptamers immobilization. Cluster-assembled thin films, have been optimized as supports to demonstrate that aptamers, targeting Staphylococcus aureus, well adhere on these substrates and keep their functionality. Moreover, the characteristics of the nanostructured zirconium oxide thin film: thermal stability, good reactivity towards -OH and -COOH groups and nano-morphology, make this material a suitable candidate for the realization of platforms for general screening and biosensing applications. This strategy will offer a promising way for the development of an user-friendly aptamer-based biosensors for screening biological samples. Furthermore, I focused on a technological problem, regarding the need of substrates to perform correlative light-electron microscopy(CLEM), designing, developing and testing a device which improve the feasibility of correlative fluorescence/confocal and scanning electron microscopy.

Thesis (Ph.D.)--Boston University
Label-free optical biosensors have been established as proven tools for monitoring specific biomolecular interactions. However, compact and robust embodiments of such instruments have yet to be introduced in order to provide sensitive, quantitative, and high-throughput biosensing for low-cost research and clinical applications. Here we present the interferometric reflectance-imaging sensor (IRIS). IRIS allows sensitive label free analysis using an inexpensive and durable multi-color LED illumination source on a silicon based surface. IRIS monitors biomolecular interaction through measurement of biomass addition to the sensor's surface. We demonstrate the capability of this system to dynamically monitor antigen-antibody interactions with a noise floor of 5.2 pg/mm^2 and DNA single mismatch detection under isothermal melting conditions in an array format. Ensemble detection of binding events using IRIS did not provide the sensitivity needed for detection of infectious disease and biomarkers at clinically relevant concentrations. Therefore, a new approach was adapted to the IRIS platform that allowed the detection and identification of individual nanoparticles on the sensor's surface. The new detection method was te1med single-particle IRIS (SP-IRIS). We developed two detection modalities for SP-IRIS. The first modality is when the target is a nanoparticle such as a virus. We verified that SP-IRIS can accurately detect and size individual viral particles. Then we demonstrated that single nanoparticle counting and sizing methodology on SP-IRIS leads to a specific and sensitive virus sensor that can be multiplexed. Finally, we developed an assay for the detection of Ebola and Marburg. A detection limit of 3 x 10^3 PFU/ml was demonstrated for vesicular stomatitis virus (VSV) pseudotyped with Ebola or Marburg virus glycoprotein. We have demonstrated that virus detection can be done in human whole blood directly without the need for sample preparation. The second modality of SP-IRIS we developed was single molecule counting of biomarkers utilizing a sandwich assay with detection probes labeled with gold nanoparticles. We demonstrated the use of single molecule counting in a nucleic acid assay for melanoma biomarker detection. We showed that a single molecule counting assay can lead to detection limits in the attomolar range. The improved sensitivity of IRIS utilizing single nanoparticle detection holds promise for a simple and low-cost technology for rapid virus detection and multiplexed molecular screening for clinical applications.

Solid-supported membranes present a biomimetic platform that can be adapted for bio-physical, biochemical and electrophysiological studies. In addition to this they offer an environment to host membrane proteins for the purposes of biosensing. This thesis examines the use of such a system and the possibilities it presents for the studies of ion channels and their potential applications in biosensing. Electrochemical impedance spectroscopy (EIS) is a powerful technique in the study of solid-supported membranes giving access to capacitance and resistance data, and as such was employed as the main method of characterisation. Electrodes were designed for this purpose in conjunction with Philips Research, and the suitability of the surface for the formation of insulating tethered bilayer lipid membranes (tBLMs). The development of these electrodes led to the incorporation of a SiO2 insulating layer, however its addition resulted in diculties with the formation of self-assembled monolayers (SAMs). However, further refinement of the manufacturing process should resolve these issues. Despite these diffculties, studies were performed using first generation electrodes (P1). Two ionophores, valinomycin and gramicidin, were employed in the characterisation of the ion transport properties of the tBLM system. These studies yielded important information about the structure of the tBLM system under investigation, as well as the ways ion transport can be presented in EIS. Using the work on ionophores as a foundation, an investigation into the incorporation and characterisation of ion channels in tBLM was conducted. Three channels were studied - a ligand-gated eukaryotic Ca2+-permeant channel (TRPC5), a voltage-gated prokaryotic Na+-permeant channel (NavCbt), and a pH-gated K+-permeant channel (KcsA). The success of these studies varied, but provided strong evidence that ion channel incorporation is possible. Further investigation of channel function in the tBLM is required as measured activity is lower than that suggested by literature.

Au cours de ma thèse, j'ai développé un procédé de fabrication spécifique capable de produire un biocapteur qui combine deux techniques de biodétection différentes, la réponse plasmonique basée sur la résonance de plasmon de surface localisée (LSPR) et la réponse électrochimique. Les méthodes et les résultats qui sont présentés dans ce manuscrit ont été définis pour converger vers un dispositif fluidique unique combinant ces deux approches de détection différentes. Afin de trouver la configuration permettant l'excitation des résonances plasmoniques, la géométrie des nanocavités MIM (métal/isolant/métal) en réseau de lignes interdigitées a été optimisée par des simulations électromagnétiques. La fabrication par nanoimpression douce assistée UV (SoftUV-NIL) a été optimisée et, finalement, la caractérisation optique de ces nanocavités a été comparée avec succès aux simulations théoriques. Parallèlement à la réalisation de ce dispositif nanostructuré, des dispositifs électrochimiques fluidiques plus simples qui intègrent des microélectrodes classiques ont également été développés. L'objectif était d'abord de développer une chimie innovante pour le couple « biotine/streptavidine » et de comprendre ensuite comment les paramètres fluidiques peuvent affecter l'efficacité de capture des biomolécules. Ce manuscrit se termine par une discussion sur le rôle des paramètres fluidiques concernant l’efficacité de la biodétection sur la base de la théorie de Squires
During my thesis, I worked on the development of a specific fabrication process able to produce a device that combines two different biodetection techniques, plasmonic response based on Localized Surface Plasmon Resonance (LSPR) and electrochemical response. Methods and results that are presented in this manuscript were defined to converge towards a unique fluidic device combining these two different sensing approaches. This device integrates interdigitated array of MIM nanocavities. In order to find the easier working configuration allowing the excitation of plasmonic resonances, their geometry has been optimized through electromagnetic simulations. The fabrication of these dual devices has been optimized based on Soft-UV NIL and, finally, optical characterization of these nanocavities has been successfully compared with theoretical simulations. In parallel to this challenging goal, simpler fluidic electrochemical devices that integrate conventional microelectrodes have also been developed. The goal was first to develop an innovative chemistry for the couple biotin/streptavidin and secondly to learn how fluidic parameters can affect the capture efficiency of molecules. This manuscript ends with a discussion on the role of the fluidic parameters on the biodetection efficiency based on the theory of Squires

The purpose of this study is to demonstrate the ability of surface enhanced Raman scattering (SERS) active particles to enable multiplexed immunoassays in a lateral flow format for point of care (POC) testing. The SERS particles used for this study are chemically active glass coated gold particles, containing tracer molecules which in principle can be chosen to provide Raman Spectra with unique features allowing multiple tracers to be simultaneously measured and distinguished without interference between each other. Lateral flow immunoassay technology is the important part of this study and can be conveniently packaged for the use of other than highly skilled technicians outside of the laboratory. A well-known (single channel - simplex) device for the pregnancy test is a typical example of the lateral flow assay. Similar formats have been/are being developed by others for a range of POC applications – but most diagnostic applications require simultaneous determination of a range of biomarkers and multiplexed assays are difficult to achieve without significant interference between the individual assays. This is where SERS particles may provide some advantages over existing techniques. Cardiac markers are the growing market for point of care technology therefore biomarkers of cardiac injury (Troponin, myoglobin and CRP) have been chosen as a model. The object of the study is to establish the proof of concept multiplexing assay using these chosen biomarkers. Thus, initially all different particles were characterised in single and mixture form. Also development of conjugate chemistry between antibodies for each analyte that have been purchased from commercial sources and SERS particles were analysed using different conditions like buffer, pH and antibody loading concentration to get the optimum intensity. The selected SERS particles and their conjugates were tested for size, aggregation and immune quality using a range of techniques: ultraviolet-visible (UV/Vis) absorption spectroscopy, dynamic light scattering (DLS) and lateral flow assay. These characterisations methodologies gave the understanding of optimum conditions of the each conjugates and individual’s behaviour in mixture conditions as well. After the characterisation all conjugates were tested singularly on the lateral flow assay using buffers and serum. The results of this single analyte immunoassay explained the individual’s bioactivity on the lateral flow strip. Further in study, multiplex assay have been demonstrated in serum. These outcomes have described each candidate characteristic in a mixture form on the lateral flow strip. In order to get the optimum Raman intensity from multiplex assay, the detection and capture antibodies loading concentrations were tuned in the assay. Later on different combinations (high, medium and low concentrations) of all three analytes were analysed and has found some interferences in multiplex assay. To investigate these issues various aspect were considered. First of all, different possibilities of non-specific interactions between the co-analytes and antibodies were tested. In addition, steric hindrance and optical interference investigations were performed via several assays and analysis using Scanning electron microscopy. The outcomes have confirmed related optical interferences. Therefore other assay (wound biomarkers) established to eliminate the interferences. In summary, the works reported here have built and test the equipment and necessary reagents for individual assays before moving on the more complicated task. In addition, the entire study has given a deep knowledge of multiplex assay on a single test line including the investigation of the issues for selected cardiac biomarkers and their applications in the future.

Microcantilever sensor system as a promising field attracted much attention recently. This system has the potential to be applied for a biosensing technology which is parallel reference, label free, sensitive and real time. In this thesis, polyimide has been selected as a material to fabricate cantilever due to its excellent physical, electrical and mechanical properties, on top of its cost advantage. Importantly, we showed it is feasible to microfabricate large array of microcantilever sensors with high-power UV laser directly. It is low cost and rapid, the parameters for laser direct writing fabrication has been studied. The thesis also shows that it is possible to functionalise the polyimide film first and subsequently cut it to functionalised cantilever sensor array. The unique fabrication and functionalisation process can solve the problem of high-cost microfabrication using silicon and low-efficient functionalisation using capillary tubing all together. In addition, the fabrication process has been further developed to avoid the problem of the cross contamination from receptors on both sides. With this improvement, we developed an internally referenced microcantilever biosensors system for DNA hybridization detection. Different receptors can be coated on each side of the polymer film before fabricating to cantilever biosensors This newly developed capability enables us to coat receptors with similar but slightly different biological properties on each side of the cantilever sensor, a process which is extremely difficult by using conventional capillary tubing methods due to the possibility of thiol exchange on surfaces and hence cross-contamination. A polyimide microcantilever sensor with embedded microfluidic channel has been developed in this thesis. Photoresist material is used to form the precise microfluidic channel within the microcantilever device. The multilayer polymer film device is still soft enough to operate in static mode. The main advantage of the system presented here is that since the device is made entirely of polymer materials, the fabrication process is simple and low-cost. The magnetic beads have been used to amplify the signal of the biosensing processing; the application of polyimide microfluidic microcantilevers to the detection of Cryptosporidium and thrombin is reported in this thesis. Paper based autonomous micocantilever system has also been investigated in this thesis. We build a cantilever system without external pump or force with paper and magnetic field. The limitation of the system is that it takes too much time to pump magnetic beads through the cantilever with capillary. However, we found that it has the potential to develop a long time range timer based on the slowest property. Different methods have been investigated to slow down the speed, when liquid pass through the paper microfluidic. Finally, we demonstrate some timer devices whose ranges are from minutes to month. The devices have the potential to be used as time-based diagnostic assays, food label, etc.

Biosensors are instrumental in the detection of analytes in a wide range of areas including enzyme kinetics and disease diagnosis. A proof-of-principle upconversion nanophosphor (UCNP) based biosensor based on luminescence energy transfer between UCNPs, acting as the energy transfer donor, and enzymes and biologically relevant proteins, the energy transfer acceptor is reported here. Analyte detection has been performed by ratiometric sensing by monitoring the change in the multiple emission bands of the UCNPs. Chapter 1 is an introduction into the emerging field of UCNPs as biosensing agents. These nanoparticles offer numerous advantages over current biosensing agents (namely organic dyes and quantum dots) including resistance to photobleaching and photoblinking, long emissive lifetimes, a large anti-Stokes' shift and near infrared (nIR) excitation to eliminate autofluoresence, and multiple characteristic emission bands for sensing multiple analytes. Chapter 2 describes the synthesis and characterisation of Yb3+/Tm3+ and Yb3+/Er3+ co-doped UCNPs via a range of different preparative methods; thermal decomposition, microwave irradiation and a one-step solvothermal process to produce hydrophilic UNCPs. In addition, commercial UCNPs, kindly donated by Phosphor Technology, were also characterised and used as a benchmark for characterisation of the newly synthesised UCNPs. Chapter 3 describes the detection of the enzyme pentaerythritol tetranitrate reductase (PETNR), through energy transfer between the commercial Yb3+/Tm3+ doped UCNPs and the enzyme using ratiometric sensing. These proof-of-principle results were published in Dalton Transactions. In addition, ratiometric change of the UCNP emission bands was able to monitor the enzyme-substrate turnover in a two electron redox reaction. Chapter 4 describes techniques for increasing the scope and sensitivity of the proof-of-principle UCNP-enzyme biosensing system. Small, hydrophilic Yb3+/Tm3+ and Yb3+/Er3+ doped UCNPs, synthesised in chapter 2, were able to detect glucose oxidase and cytochrome c, in addition to PETNR. Covalent attachment of PETNR to Yb3+/Tm3+ doped UCNPs was additionally achieved. Chapter 5 describes the incorporation of UCNPs into optical ring resonators (ORRs) in order to develop a lost cost, label-free, rapid response biosensor. Drop casting and inkjet printing methods for the deposition of UCNPs onto these devices were investigated and emission of UCNPs was achieved, for the first time, by ORR excitation.

L'objectiu d'aquesta tesi ha estat el desenvolupament i l'aplicació de plataformes innovadores de biosensado en el punt d'atenció. La tesi es divideix en cinc capítols seguits d'una secció de conclusions generals. El capítol 1 comença amb una introducció general als conceptes de biosensors i biosensado en el punt d'atenció. Després, s'enfoca en una de les proves de punt d'atenció més reeixides: l'assaig de flux lateral (LFA). Els aspectes clau de l'assaig, com els components i reactius, els procediments de fabricació i operació estan coberts en profunditat. El capítol continua amb una revisió dels desafiaments actuals de LFA ha enfrontat i les millores més rellevants reportades en els últims anys. El Capítol 2 descriu breument els objectius que van motivar aquest treball. El capítol 3 presenta una nova plataforma de detecció (PEB) que combina les característiques clau d'un assaig de flux lateral, la prova de punt d'atenció més utilitzada, amb les capacitats de tractament de mostres de l'electroforesi. En particular, es demostra la capacitat de PEB per separar diferents tipus de partícules i detectar anticossos IgG humans en mostres de sang no tractades. Finalment, per fer que la plataforma sigui aplicable en el punt d'atenció, PEB es combina amb un telèfon intel·ligent que controla l'electroforesi i llegeix el senyal òptica generada. El Capítol 4 explica una estratègia simple i de baix cost per millorar el rendiment analític dels LFA. Mitjançant l'ús de barreres de cera solubles, les nanopartícules s'acumulen temporalment a la part superior de la línia de detecció (TL). Aquest pas estès d'incubació interna promou la formació de l'inmunocomplejo, generant una millora de sensibilitat i de senyal en comparació amb la detecció convencional de LFA per IgG humana (H-IgG). El capítol 5 presenta una plataforma de detecció en el punt d'atenció que consisteix en un microtub i dues peces de fibra de vidre. El principi de detecció es basa en la transferència d'energia de ressonància de Förster utilitzant nanoclústers d'or com a indicador de senyal i nanopartícules d'or conjugades amb anticossos com un desactivador. La plataforma ha estat validada per a la detecció d'Escherichia coli O157: H7 en aigua de riu i de l'aixeta, demostrant una elevada sensibilitat.
El objetivo de esta tesis ha sido el desarrollo y la aplicación de plataformas innovadoras de biosensado en el punto de atención. La tesis se divide en cinco capítulos seguidos de una sección de conclusiones generales. El Capítulo 1 comienza con una introducción general a los conceptos de biosensores y biosensado en el punto de atención. Luego, se enfoca en una de las pruebas de punto de atención más exitosas: el ensayo de flujo lateral (LFA). Los aspectos clave del ensayo, como los componentes y reactivos, los procedimientos de fabricación y operación están cubiertos en profundidad. El capítulo continúa con una revisión de los desafíos actuales de LFA ha enfrentado y las mejoras más relevantes reportadas en los últimos años. El Capítulo 2 describe brevemente los objetivos que motivaron este trabajo. El Capítulo 3 presenta una nueva plataforma de detección (PEB) que combina las características clave de un ensayo de flujo lateral, la prueba de punto de atención más utilizada, con las capacidades de tratamiento de muestras de la electroforesis. En particular, se demuestra la capacidad de PEB para separar diferentes tipos de partículas y detectar anticuerpos IgG humanos en muestras de sangre no tratadas. Finalmente, para hacer que la plataforma sea aplicable en el punto de atención, PEB se combina con un teléfono inteligente que controla la electroforesis y lee la señal óptica generada. El Capítulo 4 explica una estrategia simple y de bajo costo para mejorar el rendimiento analítico de los LFA. Mediante el uso de barreras de cera solubles, las nanopartículas se acumulan temporalmente en la parte superior de la línea de detección (TL). Este paso extendido de incubación interna promueve la formación del inmunocomplejo, generando una mejora de sensibilidad y de señal en comparación con la detección convencional de LFA para IgG humana (H-IgG). El Capítulo 5 presenta una plataforma de detección en el punto de atención que consiste en un microtubo y dos piezas de fibra de vidrio. El principio de detección se basa en la transferencia de energía de resonancia de Förster utilizando nanoclusters de oro como indicador de señal y nanopartículas de oro conjugadas con anticuerpos como un desactivador. La plataforma ha sido validada para la detección de Escherichia coli O157: H7 en agua de río y del grifo, demostrando una elevada sensibilidad.
The objective of this thesis has been the development and application of innovative biosensing platforms at the point of care. The thesis is divided into five chapters followed by a section of general conclusions. Chapter 1 begins with a general introduction to the concepts of biosensors and point-of-care biosensing. Then, it focuses on one of the most successful point-of-care tests: the lateral flow test (LFA). Key aspects of the assay, such as components and reagents, manufacturing and operating procedures are covered in depth. The chapter continues with a review of the current challenges LFA has faced and the most relevant improvements reported in recent years. Chapter 2 briefly describes the objectives that motivated this work. Chapter 3 introduces a new detection platform (PEB) that combines the key features of a lateral flow assay, the most widely used point-of-care test, with the capabilities of electrophoresis sample treatment. In particular, the ability of PEB to separate different types of particles and detect human IgG antibodies in untreated blood samples is demonstrated. Finally, to make the platform applicable at the point of care, PEB is combined with a smartphone that controls the electrophoresis and reads the generated optical signal. Chapter 4 explains a simple, low-cost strategy to improve the analytical performance of LFAs. By using soluble wax barriers, nanoparticles temporarily accumulate at the top of the detection line (TL). This extended internal incubation step promotes immunocomplex formation, generating improved sensitivity and signal compared to conventional LFA detection for human IgG (H-IgG). Chapter 5 presents a point-of-care detection platform consisting of a microtube and two pieces of fiberglass. The detection principle is based on Förster resonance energy transfer using gold nanoclusters as a signal indicator and antibody-conjugated gold nanoparticles as a quencher. The platform has been validated for the detection of Escherichia coli O157: H7 in river and tap water, demonstrating high sensitivity.

Metal nanomaterials, such as gold nanoparticles (Au NPs), exhibit unique localised surface plasmon resonance, which can be exploited for probing biochemical and biophysical phenomena at the nanoscale and molecular level. Furthermore, the ability to control the synthesis and growth of such nanomaterials using organic and biomimetic molecules, such as nucleic acids and small molecules, facilitates deeper understanding of the interactions between biomolecules and nanomaterials. This thesis described the development of various highly sensitive and novel diagnostic platforms for detecting micro-RNA (miRNA), small molecule and protein biomarkers, by utilising the unique plasmonic properties of Au NPs, as well as modulating the morphology and size of various gold nanostructures. Au NP-conjugated nucleic acid probes, together with a poly(ethylene glycol)-functionalised microarray, enabled highly sensitive and multiplexed detection of miRNAs, conveniently under an optical microscope. Also, colorimetric detection of small molecules using the naked eye was achieved via the controlled growth of aptamer-functionalised Au NPs into various distinct nanostructures, which were dependent on aptamer–target interactions and aptamer-mediated NP growth. Lastly, the interactions between small molecules and Au seeds, and the effect on the size and aspect ratios of grown gold nanorods were investigated and elucidated. The size-modulating mechanism was further incorporated in an immunoassay for the sensitive detection of a protein biomarker, enabling its application in clinical diagnostics. The platforms developed in this thesis could serve as a basis for future development of new biosensing strategies that utilise plasmonic nanomaterials.

Las plataformas Micro / Nanofluídicas, simples y miniaturizadas son especialmente interesantes debido a sus ventajas como la reducción de los volúmenes de muestra y reactivos, la disminución del tiempo de análisis, la posibilidad de portabilidad y la integración de técnicas analíticas convencionales. Además, es importante señalar el papel que pueden jugar los nanomateriales en términos de mejora de las propiedades electroquímicas después de ser integrados en plataformas microfluídicas, o incluso modificaciones superficiales de los transductores. Así, la combinación de la nanotecnología, la electroquímica y la microfluícia, podría proporcionar una plataforma de detección muy potente, por lo que, en esta Tesis se estudian diferentes dispositivos microfluídicos con transductores electroquímicos integrados para aplicaciones bioanalíticas. En esta Tesis se exponen también los aspectos generales y los resultados experimentales, a partir de una introducción general, la cual presenta los trabajos más recientes relacionados con el uso de nanomateriales y tecnologías lab-on-a-chip como una sinergia prometedora para una amplia gama de aplicaciones. Después se presenta la detección electroquímica de proteínas mediante el uso de puntos cuánticos como marcadores. En primera instancia, se describe un chip microfluídico híbrido compuesto por una canal de polidimetilsiloxano flexible (PDMS) y policarbonato (PC) como substrato. Este substrato a su vez tiene impreso electrodos serigrafiados integrados de carbono (SCPE). El dispositivo desarrollado combina las ventajas de los chips microfluídicos flexibles, tales como su bajo coste, la posibilidad de ser desechables y la susceptibilidad de ser producidos en masa con las ventajas de la electroquímica por su facilidad de integración y la posibilidad de ser miniaturizables. En la segunda parte, se realizó la detección electroquímica de puntos cuánticos como marcadores en un ensayo para la determinación de un biomarcador de la enfermedad de Alzheimer: Apolipoproteína E (ApoE). El inmunocomplejo se llevó a cabo mediante el uso de partículas magnéticas tosilactivadas, las que fueron a su vez utilizadas como plataforma de preconcentración de muestra dentro de un canal microfluídico. Debido a la necesidad de lograr límites inferiores de detección en inmunoensayos, en esta Tesis se han propuesto diferentes estrategias para mejorar la sensibilidad de los dispositivos. La primera de ellas es el uso de un campo magnético para inmovilizar una mayor cantidad de partículas magnéticas en una disposición controlable dentro de un canal microfluídico con el fin de obtener una zona de precocentración, donde se lleva a cabo el inmunoensayo. La segunda estrategia que se presenta en esta Tesis es el uso de un sistema de reciclaje de fabricación propia. En esta parte, el incremento de la señal de los puntos cuánticos se demuestra mediante el uso de una bomba peristáltica externa conectada a un chip microfluídico que forma un sistema cerrado. Después de esta demostración, se propuso una micro-bomba peristáltica con válvulas integradas. Todas las etapas de fabricación se optimizaron así como también se desarrolló un software para su control. Por último, el bismuto, que es un material bien conocido para aglomerar los metales pesados, fue usado para aglomerar los puntos cuánticos cuyo núcleo está formado por cadmio II, de esta forma se pudo mejorar la señal electroquímica mediante la reducción de los QDs junto con el Bismuto III. Diferentes optimizaciones fueron hechas usando canales microfluídicos. Adicionalmente, se presentan otras nueva plataforma basada en diamante dopado con boro, transductor utilizado para la determinación electroquímica de la atrazina basado en el desarrollo de un magneto-inmunoensayo. Este inmunoensayo se realizó mediante un ensayo competitivo con peroxidasa de rábano silvestre (HRP) como marcador enzimático y micropartículas magnéticas como plataforma de preconcentración. Otra plataforma propuesta es el transistor orgánico de efecto campo de doble puerta, como transductor para biosensores, desarrollado por la tecnología de inyección de tinta sobre un substrato flexible. Este tipo de transistores orgánicos tiene ventajas importantes para biosensores en términos de coste de fabricación y biocompatibilidad, así como su posibilidad de integración en microcanales. Para demostrar la aplicabilidad de este dispositivo en el campo biológico, se ha funcionalizado su capa externa con un anticuerpo de captura que detecta una proteína modelo sin ningún marcador. Se realiza la fabricación del dispositivo, teniendo en cuenta su estructura, los materiales que lo componen, sus características eléctricas y posibles aplicaciones. Por último, se exponen las conclusiones generales y futuras propuestas.
Simple and miniaturized micro / nanofluidic platforms are especially interesting due to their advantages like the reduction of sample and reagent volumes, the decrease of the analysis time, the possibility of portability and the integration of conventional analytical techniques. Furthermore it is important to point out the role that nanomaterials can play in terms of enhancing electrochemical properties after being integrated into the microfluidic platform or even in the electrode, where the detection event will be performed. Combined together, nanotechnology, electrochemistry and microfluidics could provide a really powerful biosensor platform, thus in the present Thesis different microfluidic platforms with integrated electrodes as transducers in biosensing applications were evaluated. General aspects and experimental results are exposed, starting from a General Introduction that describes various aspects related with the use of nanomaterials and lab-on-a-chip technologies as a promising synergy for a wide range of applications. The electrochemical detection of proteins (ex. Apolipoprotein-E, ApoE) by using CdS or CdSe@ZnS Quantum Dots (QDs) as labels has been one of the main objectives of this Thesis. The immunocomplex was performed by using tosylactivated magnetic beads as preconcentration platform into the same microfluidic system. Due to the need to achieve a lower limit of detection of the immunoassays, different strategies for electrochemical signal enhancing are proposed. The first one is the use of a magnetic field to immobilize magnetic beads in a controllable way into a microfluidic channel in order to obtain a stable magnetic plug where the immunoassay is performed. The second strategy is the use of a home-made recycling system. In this part, the increasing signal of QDs is demonstrated by using an external peristaltic pump connected to a microfluidic chip forming a loop system. After this demonstration, a micro-peristaltic pump with integrated valves is also proposed. All the fabrication steps have been optimized and the software for sequential control of the valves also has been developed. Finally, bismuth is used as it is a well-known material that agglomerates with heavy metals. We took advantages of this property for improving the electrochemical signal of QDs, due to the cadmium content that QDs have in their core. Optimization of the bismuth concentration has been done in order to achieve the highest signal. This detection has been performed in batch system as well as in microfluidic mode. In addition, another novel platform for electrochemical determination of a pesticide (atrazine) based on magneto-immunoassay using boron-doped diamond (BDD) electrode is presented. BDD electrode has been modified by electroreduction of potassium tetrachloroplatinate (K2PtCl4) in order to grow platinum nanoparticles (Pt-NPs) onto the electrode surface. The immunoassay was based on a direct competitive assay using horseradish peroxidase (HRP) as enzymatic label and magnetic microparticles as preconcentration platform. A flexible organic double gate Bio-Field Effect Transistor (Bio-FET) developed by inkjet technology onto a flexible substrate is also presented. This kind of organic transducers has important advantages for biosensors in terms of fabrication cost and biocompatibility as well as their integration into microchannels. To demonstrate the applicability of this device in the biological field, its functionalization with a capture antibody, in order to detect a model protein in a label-free mode was performed. The device fabrication, its structure, materials composition optimization, electrical characteristics and other functionalities are also discussed. Finally, the general conclusions are exposed including some opinions / recommendations for further continuation of the research in the field.

My PhD Thesis work, developed in Veneto Nanotech Laboratories (Nanofab in Marghera, LaNN in Padova and ECSIN in Rovigo), was aimed at the exploitation of the Surface Plasmon Resonance (SPR) phenomenon for the set-up of biosensing platforms for clinical and environmental applications. In particular, two types of SPR-based platforms were set-up and optimised: the first one was an oligonucleotide-based platform for the detection of Cystic Fibrosis (CF) causing mutations while the second one was an antibody-based platform for the detection of Legionella pneumophila whole cells. Both sensors are based on the same detection strategy, exploiting the advantages of using a highly sensitive Grating Coupled - Surface Plasmon Resonance (GC-SPR) enhanced spectroscopy method, designed using a conical illumination configuration for label-free molecular detection. Concerning DNA platform for Cystic Fibrosis, a strategy for the detection of some of the most frequent mutations responsible for CF among the Italian population is investigated. For the detection of the CF mutations, gold sinusoidal gratings are used as sensing surfaces, and the specific biodetection is achieved through the usage of allele specific oligonucleotide (ASO) DNA hairpin probes, designed for single nucleotide discrimination. Substrates were used to test unlabeled PCR amplified homozygous wild type (wt) and heterozygous samples (wt/mut) - deriving from clinical samples - for the screened mutations. Hybridisation conditions were optimised to obtain the maximum discrimination ratio (DR) between the homozygous wild type and the heterozygous samples. SPR signals obtained from hybridising wild type and heterozygous samples showed DRs able to identify univocally the correct genotypes, as confirmed by fluorescence microarray experiments run in parallel. Furthermore, SPR genotyping was not impaired in samples containing unrelated DNA, allowing the platform to be used for the parallel discrimination of several alleles also scalable for a high throughput screening setting. Concerning antibody platform for Legionella pneumophila bacteria detection, a strategy for the exploitation of the SPR phenomenon to develop a fully automated platform for fast optical detection of Legionella pneumophila pathogens was investigated. The legal limit of L. pneumophila in a high-risk hospital environment in Italy is 102 CFU/L, and the gold standard for its identification is a time consuming microbiological culture method, that requires up to 7 days. Starting from these considerations a sensitive GC-SPR system was applied to the detection of L. pneumophila to test the detection limit of the developed sensing device in term of detectable bacterium CFU. The detection was accurately set up and precisely optimised firstly through the usage of flat gold functionalised slides to be then translated to sinusoidal gold gratings for label-free GC-SPR detection using ellipsometer, in order to ensure a reproducible and precise identification of bacteria. Through azimuthally-controlled GC-SPR, 10 CFU were detected, while in the case of fluorescence analysis results, a negative readout is obtained if incubating less than 104 CFU. Successful results were obtained when incubating environmental derived samples. This detection platform could be implemented as a prototype in which water and air samples will be sequentially concentrated, injected into a microfluidic system, and delivered to the SPR sensor for analysis. The peculiar Grating Coupled - Surface Plasmon Resonance method applied for this work has therefore revealed to be an accurate and highly sensitive strategy – with multiplexing possibility - for the sensing and detecting of different kind of biomolecules, from DNA fragments to whole bacteria cell.
Il mio lavoro di Tesi di Dottorato, sviluppato presso i laboratori Veneto Nanotech (Nanofab a Marghera, LaNN a Padova ed ECSIN a Rovigo), ha avuto come obiettivo l’utilizzo della tecnologia di risonanza plasmonica di superficie (SPR – Surface Plasmon Resonance) per lo sviluppo di piattaforme biosensoristiche per applicazioni clinica ed ambientali. In particolare, durante il lavoro di Dottorato sono state messe a punto due piattaforme SPR: la prima piattaforma utilizza sonde oligonucleotidiche a DNA per l'individuazione di mutazioni causanti fibrosi cistica (CF) mentre la seconda utilizza anticorpi per il rilevamento di cellule di Legionella pneumophila. Entrambi i sensori sono basati sulla stessa strategia di rilevamento, ovvero l’utilizzo di una metodologia Grating Coupled – Surface Plasmon Resonance (GC-SPR) progettata utilizzando una configurazione conica di illuminazione ad azimut rotato per la rilevazione diretta – senza passaggi di marcatura, label-free – dell’analita in esame. Per quanto riguarda la piattaforma a DNA per la fibrosi cistica, si è sviluppata una strategia per l'individuazione di alcune delle mutazioni più frequenti responsabili CF tra la popolazione italiana. Per la rilevazione di tali mutazioni le superfici di analisi utilizzate sono grigliati sinusoidali, e la rilevazione specifica delle sequenze di interesse si ottiene attraverso l'utilizzo di oligonucleotidi allele-specifici (ASO – allele specific oligonucleotide) con struttura ad hairpin, disegnati per la discriminazione di un singolo nucleotide. I substrati plasmonici sono stati utilizzati per testare campioni wild-type ed eterozigoti (wt/mut) per le mutazioni in esame, amplificati tramite PCR a partire da campioni clinici. Le condizioni di ibridazione sono state ottimizzate per ottenere il rapporto di discriminazione (DR – discrimination ratio) massimo tra campioni wild-type ed eterozigoti. I segnali SPR ottenuti ibridando campioni wild-type e campioni eterozigoti hanno mostrato DR in grado di identificare univocamente i genotipi corretti, come confermato da esperimenti di fluorescenza in microarray eseguiti in parallelo. Inoltre la genotipizzazione ottenuta tramite SPR non è stata inficiata in campioni contenenti DNA interferente, consentendo quindi di utilizzare la piattaforma per la discriminazione in parallelo dei diversi alleli, e la possibilità futura di scalare il sistema con un approccio di high throughput screening. Per quanto riguarda la piattaforma ad anticorpi per la rilevazione di Legionella pneumophila, la medesima strategia basata su GC-SPR è stata messa a punto per ottenere una rilevazione rapida e sensibile di tale patogeno. Il limite legale di L. pneumophila in ambienti ospedalieri ad alto rischio in Italia è di 102 UFC/L (unità formanti colonia) e la metodologia di riferimento per la sua identificazione è una tecnica di coltura microbiologica che richiede tempi di attesa fino a 7 giorni. Partendo da tali considerazioni un sistema GC-SPR altamente sensibile è stato sviluppato ed applicato per la rivelazione di L. pneumophila: la rivelazione è stata accuratamente impostata ed ottimizzata con un ceppo standard del battere, prima attraverso l'utilizzo di superfici d’oro non nanostrutturate (flat) opportunamente funzionalizzate ed analizzate tramite fluorescenza, e successivamente attraverso reticoli sinusoidali (grating) d’oro analizzati tramite elissometria GC-SPR. Attraverso la metodologia GC-SPR ad azimut rotato è stato possibile rilevare fino a 10 UFC, mentre con l’analisi in fluorescenza non è stato possibile identificare quantitativi di battere inferiori a 104 UFC. Risultati positivi sono stati ottenuti anche incubando campioni di L. pneumophila isolati direttamente dall’ambiente ospedaliero. Questa piattaforma di rilevazione potrà essere implementata come prototipo in cui campioni di acqua e aria potranno venir sequenzialmente concentrati, iniettati in un sistema di microfluidica, ed incubati sulla superficie del sensore SPR per l'analisi, obiettivi questi del progetto POSEIDON (Horizon2020) attualmente in corso. La particolare metodologia GC-SPR ad azimut rotato applicata in questo lavoro di Tesi si è dimostrata essere una strategia accurata e altamente sensibile - con possibilità di multiplexing - per la rilevazione di diversi tipi di biomolecole, a partire da frammenti di DNA fino ad intere cellule batteriche.

Point-of-care (POC) devices are compact, mobile and fast detection platforms expected to advance early diagnosis, treatment monitoring and personalized healthcare, and revolutionize today’s healthcare system, especially in remote areas. The need for POC devices strongly drives the development of novel biosensor technology. Building a small, fast, simple, and sensitive platform for biomolecule detection is a challenge that relies on the integration of multiple fields of expertise and engineering. Optical nanoresonators have shown great promise as label-free biosensors because of direct light coupling and sub-wavelength sensing modes. Metallic nanoresonators with localized surface plasmon resonances (LSPR) are already well studied and were proven a solid alternative to the commercialized surface plasmon resonance (SPR) sensors. More recently, dielectric nanoresonators have also gained traction due to the reduced losses and the ability to manipulate both the electric and magnetic components of the incident light. In this thesis, we advance the field of biosensing and use optical nanoresonators as operative platforms relevant for disease diagnosis and treatment monitoring. By combining different optimized optical nanoresonators, both metallic and dielectric, with state-of-the-art microfluidics and surface chemistry, we have developed and tested several detection platforms. We first focused on developing a microfluidic lab-on-chip device for multiplexed biosensing utilizing the LSPR of gold nanoresonator arrays. By simultaneously tracking the extinction of 32 sensor arrays, we demonstrated multiplexed quantitative detection of four breast cancer markers in human serum. We showed that with well-optimized immunoassays, a low limit of detection (LOD) can be reached, paving the way towards clinically-relevant POC devices. Additionally, we implemented silicon nanoresonators supporting Mie resonances into functional and clinically-relevant applications. By integrating several arrays of Si nanoresonators with state-of-the-art microfluidics, we demonstrated their ability to detect cancer markers in human serum with high sensitivity and high specificity. Furthermore, we showed that the fabrication of Si nanoresonator array using low cost and scalable projection lithography leads to sufficiently low limits of detection, while enabling cheaper and faster sensor production for future POC applications. We also investigated the respective role of electric and magnetic dipole resonances and showed that they are associated with two different transduction mechanisms: resonance redshift and extinction decrease. Our work advances the development of future point-of-care sensing platforms for fast and low cost health monitoring at the molecular scale.
La instrumentación Point-of-care (POC) es compacta, móvil y permite una detección rápida, razón por la que se prevé que sean de gran ayuda en áreas como el diagnostico precoz, la monitorización de tratamientos y la medicina personalizada, revolucionando los modelos sanitarios, especialmente en las zonas de difícil acceso y con menos recursos. La necesidad de este tipo de dispositivos impulsa el desarrollo de novedosas tecnologías en el campo de los bio-sensores. Diseñar equipos para la detección de bio-moléculas que sean rápidos, pequeños y sencillos es un reto que requiere la integración de múltiples campos de la ciencia y la ingeniería. Los nano-resonadores ópticos muestran un gran potencial como bio-sensores sin necesidad de marcaje, gracias a su capacidad de acoplase directamente con la luz en modos menores que la longitud de onda. Los nano-resonadores metálicos basados en resonancias plasmónicas superficiales localizadas (LSPR) han sido estudiados y han demostrado ser una firme alternativa a los ya comerciales basados en resonancias plasmónicas superficiales (SPR). Los nano-resonadores dieléctricos han sido recientemente objeto de atención debido a sus bajas perdidas y la capacidad de manipular los componentes eléctricos y magnéticos de la luz. En esta tesis presentamos avances en el campo de la bio-detección y en el uso de los nano-resonadores ópticos como potenciales herramientas para la detección de enfermedades y monitorización de los tratamientos. Hemos desarrollado y evaluado distintas plataformas de detección combinando los nano-resonadores ópticos, tanto metálicos como dieléctricos, con las más avanzadas técnicas de microfluídica y química de superficies. En primer lugar, nos centramos en el desarrollo de un dispositivo microfluídico basado en sensores LSPR de oro que permite multiplexar 32 canales. Los 32 sensores se monitorizan en tiempo real para demostrar la cuantificación de 4 marcadores de cáncer de mama en suero sanguíneo humano. Demostramos que mediante la optimización de los ensayos se pueden alcanzar bajos límites de detección (LOD), lo que allana el camino hacia dispositivos POC de uso clínico. Por otro lado, hemos utilizado los nano-resonadores de silicio integrados con la microfluídica para también detectar marcadores de cáncer en suero. Estos sensores, cuyo principio de funcionamiento se basa en resonancias de MIE, han demostrado ser una alternativa razonable a los sensores de oro. Además, demostramos que un proceso de fabricación de nano-resonadores de silicio rápido, escalable y de bajo coste da lugar a límites de detección suficientes para la producción de futuras POC. También realizamos un minucioso estudio del rol de las resonancias eléctricas y magnéticas en dichos sensores y su relación con el desplazamiento y el cambio magnitud de la resonancia del sensor global. Nuestro trabajo es un avance en el desarrollo de futuros instrumentos POC rápidos y baratos en el ámbito de la salud a escala molecular.

Natural vesicles produced from genetically engineered cells with tailored membrane receptor composition are promising building blocks for sensing biodevices. This is particularly true for the case of G-protein coupled receptors (GPCRs) present in many sensing processes in cells, whose functionality crucially depends on their lipid environment. Membrane receptors are involved in a variety of biochemical pathways and therefore constitute important targets for therapy and development of new drugs. Bioanalytical platforms and binding assays, using these transmembrane receptors, for drug screening or diagnostic require building well-characterized lipid membrane arrays, acting as supports to prevent protein denaturation during biochip processing. The controlled production of natural vesicles containing GPCRs, their characterization and their reproducible deposition on surfaces are among the outstanding challenges in the road map to realize practical biomolecular devices based on GPCRs. In addition, quantification of the protein receptors in such lipid membrane arrays is a key issue in order to produce reproducible and well-characterized chips. In this thesis we present the production and characterization of membrane nanovesicles (NV) from Saccaromyces Cerevisiae containing heterologously expressed olfactory receptors - a member of the family of GPCRs. We have demonstrated that membrane fractions from yeast cells spontaneously form closed spherical nanovesicles in solution. A simple method to homogenize the size of the nanovesicles to a diameter of around 100 nm at a concentration of more than 1010 nanovesicles mL-1 is also presented. It is also showed that after a genetic engineering process the olfactory receptors of interest were well expressed in the yeast membrane. Furthermore, we report for the first time a novel immunochemical analytical approach for the quantification of transmembrane proteins (i.e. GPCR) in their natural lipid environment. The procedure allows direct determination of tagged receptors (i.e. c-myc tag) without any previous protein purification or extraction steps. The proposed approach uses monoclonal antibodies addressed against the c-myc tag, frequently used in protein expression, on a microplate-based ELISA format with high detectability. The immunochemical method quantifies this tag on proteins or bioreceptors embedded in nanovesicles with detectability in the picomolar range, using protein bioconjugates as reference standards. The applicability of the method is demonstrated through the quantification of the c-myc-olfactory receptors (ORs, c-myc-OR1740 and c-myc-OR7D4) in plasma membrane nanovesicles (NVs). We also show by direct observation with Atomic Force Microscopy that nanovesicles deposit and flatten without rupturing on glass and gold substrates following approximately a diffusive law. We show that on glass surface coverages larger than 20-25% of the substrate can be reproducibly achieved under practical nanovesicle concentrations and reasonable time scales, while keeping to the minimum the presence of background residuals coming from the nanovesicles production process. On the other hand, on functionalized gold substrates surface coverages around 10-15% were achieved. Then, the role of surface chemistry was studied showing that modification of gold substrates indicates a higher affinity of natural nanovesicles for acid modified surfaces as compared to amino or alcohol modified surfaces. Nanovesicles deposition in acid modified gold surfaces and glass have been exploited for the generation of an array of multiple nanovesicles. Present results constitute an important step in the practical realization of biosensor devices based on natural nanovesicles integrating G-protein coupled membrane receptors. When olfactory receptors are genetically expressed in closed vesicles from natural yeast membrane fractions the verification of their capability for capturing specific odorant molecules are critical for the design of artificial noses. Thus, we demonstrated by Surface Plasmon Resonance (SPR) measurements on L1 Biacore chips that the receptors were functional. Despite the fact that the expression of olfactory receptors in nanovesicles is low, a fact that is coherent with the general expression level of GPCRs proteins in cells, the integration in nanovesicles together with a careful choice of the SPR experimental conditions and data analysis allowed us to obtain a concentration-dependent SPR response vs. odorant concentration with a sensitivity of 0.5-1.8RU/micromolar. The selectivity of OR carrying NV towards its specific odorant was proved in cross-check experiments with unspecific odorant molecules and control receptors. These results constitute a proof of concept that ORs embedded in nanovesicles properly respond to odorants and definitely open the perspective to use the surface plasmon resonance technique for the detection of small odorants at concentration in the micromolar range.
Vesícules naturals produïes a partir de cèl·lules modificades genèticament són prometedors components de sensat per utilitzar com a detectors en biodispositius. Això és particularment cert en el cas de receptors adjuntats a proteïna G (GPCRs) presents en molts processos cel·lulars, on la seva funcionalitat depèn estrictament del seu entorn lipídic. Els receptors de membrana estan involucrats en una gran varietat de vies bioquímiques i per tant són objectiu d’estudi per teràpia i desenvolupament de nous fàrmacs. Per tant, plataformes bioanalítiques i assajos d’unió receptor-lligand, utilitzant receptors transmembrana, requereixen la construcció de matrius de membranes lipídiques ben caracteritzades, actuant com a suport per evitar la desnaturalització de proteïnes durant el processament del bioxip. En aquesta tesi es presenta la producció i caracterització de nanovesícules de membrana (NV) provinents de cèl·lules de llevat Saccharomyces cerevisiae que contenen receptors olfactius (un membre de la família de GPCRs) heteròlogament expressats a la membrana. Hem demostrat que les fraccions de membrana, a partir de cèl·lules de llevat, en solució formen espontàniament nanovesícules esfèriques tancades. També s’ha demostrat, que després d’un procés de enginyeria genètica els receptors olfactius van ser expressats correctament a la membrana del llevat. També s’ha presentat un mètode simple per homogeneïtzar la mida de les nanovesícules. A més a més, es presenta per primer cop un nou mètode immunoquímic per la quantificació directa de les proteïnes transmembrana (GPCR) en el seu ambient lipídic natural. El mètode utilitza anticossos monoclonals en un assaig basat en ELISA amb alta detectabilitat. L’aplicació del mètode es demostra a través de la quantificació del receptors olfactius OR1740 i OR7D4 expressats en nanovesícules de membrana plasmàtica. També es presenta, mitjançant observació directa amb AFM, com les nanovesícules es depositen i s’aplanen sense trencar-se sobre substrats de vidre i or seguint la llei de difusió. Es demostra com en el cas del vidre els màxims recobriments superficials obtinguts són del 20-25% i en el cas del or funcionalitzat del 10-15%, controlant la concentració de nanovesícules, el temps de depòsit, la presència de residus procedents del procés de producció de les nanovesícules, la química de la superfície, la força iònica del medi, etc. Finalment, s’ha demostrat per SPR que els receptors expressats eren funcionals i que aquesta tècnica òptica permet la detecció de petites molècules, com són els odorants, a les concentracions en el rang micromolar. Els resultats presentats en aquesta tesis contribueixen donant un pas important a la realització de dispositius biosensors basats en nanovesícules naturals que integren receptors de membrana adjuntats a proteïna G.

Over the past two decades, digital microfluidics (DMF) has grown significantly as a powerful tool for lab-on-a-chip (LOC) applications. Since the introduction of DMF with its primary capabilities in sample manipulation (transport, splitting and mixing) in the droplet format, many advances have been made towards the development of platforms capable of performing entire processes of biochemical assays in an automated fashion. These advances include the progress made towards the fabrication methods, and the development of techniques for sample manipulation and integration of biosensors. Despite the general success in such developments, DMF still lacks capabilities in sample manipulation (including the droplets and their contents, e.g. cells and microbeads), specifically for biosensing, in which sample preparation and post-sensing removal of the sample are required. Therefore, in majority of the applications, DMF has been used for processing parts of the entire assay, and after biosensing, the recovery of the chip was hindered due to the contamination of the biosensor for its hydrophilic behavior. This thesis aims at the development of advanced operators for accurate pre-sensing sample preparation, biosensing and post sensing sample removal. For this purpose, an unequal droplet splitting method is developed based on the geometrical modification of one actuating electrode, which enables dispensing/splitting droplets with a wide range of volumes and with an accuracy of over 99%. An electrohydrodynamic technique is developed for rapid mixing inside the stationary droplets, enhancing the mixing time and eliminating the need for frequent and cyclic transport of the droplet on the chip. A dielectrophoretic-gravity driven technique is developed for concentrating and focusing the particles and cells inside the sample droplet. Also, a systematic study has been performed on the surface properties and geometry of the biosensors to optimize their geometry and configuration on DMF devices for complete sample removal after biosensing. Finally, the application of the developed techniques for enhanced on-chip biosensing is shown for detection of Cryptosporidium as a proof of concept.
Applied Science, Faculty of
Engineering, School of (Okanagan)
Graduate

Surface Enhanced Raman Scattering (SERS) technique merges an excellent sensitivity and a highly specific label free detection which can be exploited in miniaturized devices with a multiplexed approach. The development of plasmonic nanostructures, aimed to SERS analysis, satisfies therefore the need for point-of-care multianalyte sensing and biosensing platforms, both in the framework of diagnostics and therapy monitoring. In this thesis, SERS active metal-dielectric nanostructures based on silver-coated porous silicon (Ag-pSi) are carefully optimized for biodetection purposes. The thesis is organized in two parts. Basic concepts, necessary to the understanding of the experimental work are provided in the “Background” section, dealing with fundamentals of SERS spectroscopy (Chapter 1), SERS substrates fabrication aimed to biosensing applications (Chapter 2) and the main techniques devoted to the substrates characterization (Chapter 3). On the other side, the “Experimental” part includes the applied materials and methods (Chapter 4) and the presentation and discussion of the experimental results. (Chapters 5-8). In detail, a reliable SERS sensing requires a deep characterization of the optical and SERS response of the substrate providing the Raman enhancement. Theoretical and experimental techniques (FEM simulations and multi-wavelength Raman mapping) are systematically applied to get new insight into the fundamental and applicative SERS properties of Ag-pSi (Chapter 5). Two different approaches for the fabrication of Ag-pSi multianalyte platforms are then presented and discussed. Chapter 6 deals with the in situ synthesis of silver nanoparticles (NPs) patterns synthesized by ink-jet printing. The correlation between the growth parameters, morphology and SERS response is studied in order to optimize the SERS signal efficiency and uniformity of the Ag-pSi printed nanostructures. On the other hand, Chapter 7 concerns with the fabrication of multichamber Ag-pSi-PDMS microfluidic chips, which can be applied as portable SERS multiplexing devices. An all-microfluidic in-flow synthesis of silver NPs is performed, integrating the preparation of the SERS active substrate and the detection step on the same chip. Finally, a biofunctionalization protocol developed for the detection of miRNA222 (a recognized tumor marker) is optimized for the application to the Ag-pSi SERS substrates, assessing their compatibility to bioassays and suggesting the Ag-pSi nanostructures integrated in elastomeric chips as advantageous platforms for miRNA profiling, as well as for several other bioanalytical applications (Chapter 8).

Cell-free protein synthesis (CFPS) is a powerful protein expression platform where protein synthesis machinery is borrowed from living organisms. Target proteins are synthesized in a reaction tube together with cell extract, amino acids, energy source, and DNA. This reaction is versatile, and dynamic optimizations of the reaction conditions can be performed. The "œopen" nature of CFPS makes it a compelling candidate for many technologies and applications. This dissertation reports new and innovative applications of CFPS including 1) enzyme encapsulation in a virus-like particle, 2) detection of endocrine disrupting chemicals in the presence of blood and urine, and 3) expression of a multi-disulfide bond therapeutic protein. Two major limitations of enzymes are their instability and recycling difficulty. To overcome these limitations, we report the first enzyme encapsulation in the CFPS by immobilizing in a virus-like particle using an RNA aptamer. This technique allows simple and fast enzyme production and encapsulation We demonstrate, for the first time, the Rapid Adaptable Portable In vitro Detection biosensor platform (RAPID) for detecting endocrine disrupting chemicals (EDCs) in human blood and urine samples. Current living cell-based assays can take a week to detect EDCs, but RAPID requires only 2 hours. It utilizes the versatile nature of CFPS for biosensor protein complex production and EDC detection. Biotherapeutic protein expression in E. coli suffers from inclusion body formation, insolubility, and mis-folding. Since CFPS is not restricted by a cell wall, dynamic optimization can take place during the protein synthesis process. We report the first expression of full-length tissue plasminogen activator (tPA) using CFPS. These research works demonstrate the powerful and versatile nature of the CFPS.

博士
國立交通大學
生物科技學系
100
Real-time surveillance of the biomarker is critical for improvements in illness management and is especially important for early detection, rapid intervention, and a possible reduction of the disease occurrence. Enhanced surveillance requires rapid, robust, and inexpensive analytical techniques capable of providing a detailed analysis of biological molecules. A simple and low-cost method to fabricate poly-crystalline silicon nanowire field-effect transistor (poly-SiNW FET) for bio-sensing application was demonstrated. The poly-silicon nanowire (poly-SiNW) channel was fabricated by employing the poly-silicon (poly-Si) sidewall spacer technique, which approach was comparable with current commercial semiconductor process and forsaken expensive electron beam (E-beam) lithography tools for large-scale production. The electronic properties of the poly-SiNW FET in aqueous solution were found to be similar to those of single-crystal SiNW FETs reported in the literature. Functionalized poly-SiNW FETs were used as the biosensors for specific and ultrasensitive detection of neurotransmitter dopamine and high pathogenic avian influenza virus DNA in this study. Specific electric changes were observed for dopamine and DNA sensing when nanowire surface of poly-SiNW FETs was modified with specific recognition capturers and those biological molecules at fM to pM range could be distinguished. We further demonstrated that specific detection, confirmation and recovery of DNA probe on the nanowire surface could be achieved with SiNW-FETs using hemagglutinin DNA as the diagnostic target. With its characteristics (ultrasensitive, label-free, and real-time detection) and advantages (potential for mass commercial production and integration with microfluidic system and circuit), poly-SiNW FET can be developed to become a portable biosensor for field use and point-of-care diagnoses.

碩士
國立交通大學
機械工程系所
104
The goal of this project is to develop a novel Lab-on-a-chip (LOC) detection system. The proposed system consists of a microfluidic subsystem and a label-free (LF) detection subsystem. The system can facilitate sample preparation and provide immediate detections. The microfluidic subsystem consists of several components including two liquid inlet which can inject the samples and reagents, and filters for cell debris purification after lysis. All these components will be fabricated on a silicon chip simultaneously. Regarding the detection subsystem, we developed a sensitive photonic crystal (PC) biosensor for LF detection of proteins. The PC functions as an optical filter where only a particular combination of wavelength/illumination angle can excite the structure resonance, resulting in a strong reflection, while other combinations of wavelengths and illumination angles are transmitted through. Through monitoring the shift of the reflected wavelength, the concentration of the analyte can be determined. The designed LOC system was demonstrated to detect the Beta-actin proteins inside the cell and simultaneously to filter the cell debris. The result indicates the detection limit of 160 ng/ml can be achieved without any prefiltering procedure.

碩士
國立中正大學
化學暨生物化學研究所
102
The objective of this work is to develop a novel chemical and biochemical sensing platform, namely, a reflection-based tubular waveguide particle plasmon resonance (RTW-PPR) biosensing platform, which is based on the tubular waveguide particle plasmon resonance (TW-PPR) biosensor. The working principle of this invention is given by the following processes: 1. a light emitting diode (LED) emits light with an excitation wavelength corresponds to the particle plasmon resonance (PPR) of gold nanoparticle; 2. the light is transmitted by optical fibers and coupled into a glass tube; 3. the light traveled in the tube wall by multiple total internal reflections (TIRs); 4.The evanescent wave excites the PPR of gold nanoparticles on the surface of the tube wall. When the refractive index of the medium surrounding the nanoparticles changes (eg. adsorption of biomolecules on nanoparticle surface), the peak wavelength and extinction cross-section of the particle plasmon resonance (PPR) band changes. To construct the sensor tube, the bottom of the tube will be modified with a reflective layer. When the incident light reaches the reflective layer, the light will travel to opposite direction. The PPR effect increases through an increase of optical path length by reflection, when the sensor length is the same, thus effectively enhances the sensitivity of the sensing system. The light is finally detected by a photodiode (PD) through fiber optics. This system does not require labeling (eg. modified fluorescent molecular). Hence, the RTW-PPR biosensing platform can achieve label-free and real-time detection with high sensitivity. In this work, we optimized the optical and sensing elements of the system. System stability, reproducibility, sensor sensitivity and sensor resolution (SR) will be tested by a series of refractive index (RI) experiments and biochemical detection experiments. In the RI experiment, using different weight percents of sucrose in pure water, a refractive index resolution of 2.21x10-5 RIU and a sensor sensitivity of -6.17 RIU-1 have been achieved by the sensor. In the biochemical detection experiments, OVA were used to functionalize the gold nanoparticle in order to detect anti-OVA antibody. Results show that the calibration curve is linear (R2>0.99) and the limit of detection (LOD) is about 5.71x10-7 g/mL (3.81x10-9 M). In summary, we have developed a novel RTW-PPR biosensing platform successfully, and its feasibility in biosensing has been demonstrated.

Tetracyclines are a type of antibiotic that exhibits activity against most gram-positive and gram-negative bacteria. These antibiotics are often added at subtherapeutic levels to feed to act as growth promoters. Due to its low bioavailability, only a fraction of the antibiotic is metabolized in animals, causing waste. These residues can enter human bodies through the food chain and lead to increased antimicrobial resistance, causing allergic or toxic reactions, which led several countries to implement a maximum level of residues for this type of antibiotic. Most methods of detecting TCs are time consuming or inadequate for field analysis. In this way, an inexpensive, easy-to-execute and fast analytical method is required. In this sense, in the present work a colorimetric biosensor was developed in paper for the detection of four type of tetracyclines, presenting an alternative in the performance of point-of-care tests. Paper is low-cost, abundant, biodegradable and easy to dispose of by incineration. The construction of the sensors was performed using Lab-on-Paper technology and is based on the synthesis of gold nanoparticles by reducing a gold salt in which tetracyclines constitute the reducing agent itself. Different concentrations of TCs result in the formation of different colour intensities. Different concentrations of tetracyclines were tested and analysed using ImageJ software, allowing linear calibration lines to be obtained, that relate the concentration of antibiotics in a range between 0.1 and 10 μg/mL and the arithmetic mean of the RGB channels. Validation tests of the sensors developed with TC in milk were also performed. It was observed that it is possible to detect this type of antibiotic in pre-treated milk, and four forms of milk treatment were studied.

碩士
國立中興大學
生醫工程研究所
101
A fiber-optical biosensing platform in coordination with localized fluidic delivery to perform topical cell-scale assay inside living tissue or organs in vivo. The deviation from the cell density and spatial configuration in the vicinity of sensor probe can cause error in threshold value determination, which is difficult for the conventional extrinsic catheter fiber-optic designs to predict or calibrate for in vivo applications. The strategy for correcting / calibrating the difference from the spatial issue is to stain the cells with two fluorescent agents. The 1st fluorophore (indicator) will has a known effect on all cells which is not affected by the conditions (“normal / control” or “treated” with desired physiological changes) of the cells; the signal from the indicator will be considered as “baseline” reflecting each independent measurement with specific density and configuration of the cells in the vicinity of sensor tip. The 2nd fluorophore (reporter) will exhibit level of physiological change on “treated” cells. The significance of the physiological changes on individual cell will be evaluated by the ratio of the two fluorescent signals (reporter / indictor) to report the normalized deviation between “control” and “treated” cell population. In this research, we applied the fiber-optic sensor platform in monitoring chemotherapeutic, cyclophosphamide (CPA), induced 3D-distributed MCF-7 cell (human breast carcinoma cell line) apoptosis for verifying the feasibility and capability of the system in monitoring cell population or tissue level activities in vivo. In the 1st stage of the development, 25mM CPA was found to enhance apoptosis of cancer cells in cell viability (MTT) assay under 2D cell culture, increase the adsorption amount of fluorophore FM 1-43 on apoptotic cells, but had no effect on the adsorption amount of indocyanine derivatives on apoptotic cells. In the 2nd stage of study, the fiber-optic sensing platform monitored the kinetics of fluorescence changes around the micro-environment of sensor tip with a 200μm i.d. optical fiber, conveying the excitation and returning emission, and a 325μm i.d. microcapillary initially delivering fluorophore, 750nl 300 μm naphthalene asymmetric indocyanine derivative (Cpd.B), in indicating the spatial distribution (density) of cells in the tissue-mimic system, following by delivering fluorophore, 750nl 160μM FM1-43, in demonstrating the apoptotic activity induced by CPA after calibrated with the previous Cpd.B reported cell distribution indicating signal. When cell density exceeded 107cells/ml, the increase percentage of peak values in dynamic fluorescent change pattern of Cpd.B were in proportion to cell densities, but not correlated statistically with CPA-induced apoptotic activities. However, the increase percentages of peak values in dynamic fluorescent change pattern of FM 1-43 were in proportion to both cell densities and CPA-induced apoptotic activities. In the cell density between 107-108cells/ml, the increase percentage of peak values in dynamic fluorescent change pattern from FM 1-43 interacted with CPA-induced apoptotic cells were 2.1-3.3 folds of peak values from FM 1-43 interacted with control cell. When sequentially interacted with Cpd.B and FM 1-43, the ratios from increase percentage of peak values of FM 1-43 divided by increase percentage of peak values of Cpd.B were around 0.71-1.58 in the control cells with different densities, while the ratios from CPA-induced apoptotic cells were around 3.62-10.68. The value of ratio could be applied as indication of apoptotic activity without interference of spatial distribution of cells. The preliminary result verified the feasibility and capability of the system in monitoring cell population or tissue level activities in vivo.

碩士
國立交通大學
材料科學與工程學系奈米科技碩博士班
101
In this thesis, a biosensing platform integrating zinc oxide nanowires and blood plasma separation microfluidic device was demonstrated and investigated; Plasma microfluidic system utilizing gravity separation was prepared for effective filtering of the blood cells that usually caused signal interference in fluorescence intensity identification. The ZnO nanowire with biocompatibility nanopillar structure was optimized for the increase of the surface area in capture of target molecules. Prior to sensing, both Amino Propyl Triethoxy Silane (APTES) and Biotin were modified on the ZnO nanowire via self-assembly technique for Streptavidin detection. Whole blood samples spiked with various concentrations of Streptavidin were adopted to characterize the efficiency of the proposed system and the limit of detection (LOD). Fluoresence images confirmed the enhancement of the proposed sensing platform; with ZnO nanowire the LOD was enhanced from 42 pM to 4.2 pM. Finally, an automatic fluid flow mechanism was demonstrated to increase the feasibility of the proposed system. It is believed that by integrating these features the proposed platform can be beneficial to future point-of-care applications.

碩士
國立中興大學
生醫工程研究所
102
A fiber-optical biosensing platform is developed to providing real-time evaluation of chemotherapy efficacy in vivo. It was verified by monitoring the apoptotic response of MDA-MB-231 xenografts in nu/nu mice induced by the maximal tolerance dosage of cyclophosphamide (CPA). Percutaneous 200μm i.d. optical fibers were applied to convey excitation and detect fluorescent emission from topically delivered FM1-43 (indicating apoptotic activities), and phospholipid modified marina blue (Mb, calibrating spatial distribution effect of cells).

The generation of new nanoscale fabrication techniques is both novel and necessary for the generation of new devices and new materials. Graphene, a heavily studied and versatile material, provides new avenues to generate these techniques. Graphene’s 2-dimensional form remains both robust and uncommonly manipulable. In this project we show that graphene can be combined with the yeast cell, Saccharomyces cerevisiae, arguably the most studied and utilized organism on the planet, to generate these new techniques and devices. Graphene oxide will be used to encapsulate yeast cells and we report on the development of a method to electrically read the behaviour of these yeast cells. The advantage of an encapsulation process for a cell sensor is the ability to create a system that can electrically show both changes in ion flow into and out of the cell and mechanical changes in the cell surface. Since the graphene sheets are mechanically linked to the surface of the cell, stresses imparted to the sheets by changes in the cell wall or cell size would also be detectable. The development process for the encapsulation will be refined to eradicate excess gold on the yeast cells as well as to minimize the amount of stray, unattached graphene in the samples. The graphene oxide encapsulation process will also be shown to generate a robust substrate for material synthesis. With regards to cell sensing applications, sources of noise will be examined and refinements to the device setup and testing apparatus explored in order to magnify the relevant electrical signal. The spherical topography of an encapsulated yeast cell will be shown to be an advantageous substrate for material growth. Zinc oxide, as a sample material being investigated for its own applications for photovoltaics, will be grown on these substrates. The spherical nature of the encapsulated cell allows for radial material growth and a larger photo-active area resulting in a device with increased efficiency over a planar complement. The zinc oxide nanorods are grown via an electrochemical growth process which also reduces the graphene oxide sheets to electrochemically reduced graphene. XRD analysis confirms that the material synthesized is infact zinc oxide. The nanorods synthesized are 200nm to 400nm in width and 1µm in length. The increase efficiency of the non-planar device and the effectiveness of the encapsulated cell as a growth substrate indicate encapsulated cells as a research avenue with significant potential.

碩士
國立交通大學
應用化學系碩博士班
106
Single chain fragment variable (scFv), consists of heavy chain and light chain of fragment variable linked with flexible peptide, is the smallest unit of antibody with antigen-binding activities. Reduced size of protein facilitates the expression in E. coli. Phage display technology enables screening of scFv to against interested antigen. With the scFv expressed in E. coli, it has the potential to replace the use of antibodies produced by animal and consequently reduces the cost of diagnosis. 　　C-reactive protein (CRP), the target antigens of this study, is recognized as a mediator of the acute-phase response, associated with various chronic inflammatory mechanism. CRP is a common biomarker in current assays. In this study, Maltose-binding protein fused CRP (MBP-CRP) expressed in E. coli was employed for scFv screening using a phage display library. Simultaneously, human cell produced CRP (CRPcell) served as the other target antigen for scFv screening. The scFv obtained from the screening, scFvCRP, was further inserted with his-tag at the N-terminus genetically to facilitate the process of protein purification. The affinity of scFvCRP was further characterized with indirect enzyme-linked immunosorbent assay (ELISA) and microscale thermophoresis (MST). Kd value was estimated to be 105 nM by indirect ELISA, whereas the Kd is 0.43 ± 0.06 nM obtained from MST analysis. The discrepancy between the two measurements becomes an interesting issue to be discussed. A detection platform was further established with the application of a NiO-coated chip on quartz crystal microbalance (QCM). Valid detection concentration was achieved below 100 nM (2.5 mg/L) of CRP.

The genome sequencing programs have identified hundreds of thousands of genetic and proteomic targets for which there are presently no ascribed functions. The challenge for researchers now is to characterize them, as well as identify and characterize their natural variants. Historically, this has meant studying each individual target separately. However, due to the recent development of multi-analyte microarray devices, these characterizations can be performed in a combinatorial manner in which a single experiment provides information on thousands of targets at a time. In the past decade, microarray technology has settled in on two major designs. The first entails spotting individual receptor types onto a functionalized glass substrate. This is a simple and inexpensive process; however, due to the limited resolution of the mechanical devices used to do the spotting, the densities of these arrays are relatively low. Moreover, receptor preparation requires substantial time and effort. The second variety of microarray uses photolithographic techniques adapted from the semi-conductor industry to chemically synthesize the receptor elements in situ on the sensing surface. Because lithographic patterning is spatially very precise, these arrays achieve very high densities, with as many as one million features per square centimeter. Although these arrays obviate the necessity for laborious "off chip" probe preparation, they are expensive to produce and are limited to two types of receptors (oligonucleotides and peptides). This dissertation presents the development work performed on a hydrogel-based biosensor platform which provides a high density and low cost alternative to the two aforementioned designs. The array features are fabricated lithographically from a liquid pre-polymer doped with biologically active sensing elements at sizes as small as 50[micrometer]. Each of the feature types is uniquely shaped, which enables the features to be mass-produced in batches, pooled together and then assembled into randomly ordered arrays using highly-parallelized self-assembly techniques. The three-dimensional hydrogel features accommodate a wide variety of sensing elements, such as enzymes, antibodies and cells, which cannot be deployed using the traditional designs. This dissertation presents methods developed to integrate cellular and oligonucleotide sensing elements into the hydrogel features which preserve their biological activity and optimize the sensor's performance.
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碩士
國立交通大學
分子醫學與生物工程研究所
106
Protein tyrosine kinases are highly related to many human diseases, including neuron degenerative diseases, autoimmunity, diabetes, and cancers. Previously, a tyrosinase-based tyrosine kinase biosensor was developed and characterized by using Src as a model in our lab. In this study two other tyrosine kinases, Hck and Her2, were further explored and studied on a novel electrochemical detection setup with the previously developed tyrosine kinase sensing protocol. Hck protein kinase has been shown to be related to various immune disorders, such as chronic myelogenous leukemia, multiple myeloma and acute lymphatic leukemia. The dysfunction of Her2 protein kinase is strongly associated with the pathogenesis of various human cancers, including non-small cell lung cancer, ovarian cancer, pancreatic cancer, endometrial cancer and colon cancer. The platform allows reaction to be carried out in a small sample and with low noise. The results showed that the developed detection platform could be used for protein tyrosine kinases activity assay and for drug screening of these protein tyrosine kinases.

碩士
國立清華大學
動力機械工程學系
98
ii ABSTRACT In this thesis, an electrochemical biosensing platform using carbon nanotubes electrodes, which can be used to evaluate drug release profiles of antibiotic nanocapsules in real time and continuous mode, has been successfully designed, developed, and characterized. First, the electrochemical sensing electrode has been fabricated by MEMS technologies. Then, the biosensing platform is connected to the sensing circuit and LabVIEW program for signal acquire and processing. Finally, the developed carbon nanotubes electrode and electrochemical biosensing platform have been applied for pre-clinical evaluation of drug release profiles of antibiotic nanocapsules. The electrode is one of the key components for electrochemical sensing. The materials and the surface characteristics of electrodes play an important role in electrochemical sensing. Here, we successfully combined the carbon nanotubes electrodes with electrochemical biosensor, using electrophoresis deposition or drop-coating method to deposit carbon nanotubes on the surfaces of the gold electrodes. The measurement results show that the maximum affordable current has been improved 0.0208 mA to o.6680 mA. And, the sensing signals are amplified up to 13.75 times using carbon nanotube-modified electrodes. The linear range of the developed electrochemical biosensing platform using carbon nanotubes electrodes is from 1 g/ml to 10g/ml (R2=0.9837). The sensitivity of the developed system is 0.023 mA‧ml/g. The HPLC and other traditional instrument could not detection the drug release from nanocapsules in real time and continuous mode. According to the measurements using our developed electrochemical biosensing platform, it shows that antibiotic nanocapsules start to increase the drug release on the 4th day and the release rate is 0.0258 μg/ml．hr. The drug release of antibiotic nanocapsules reached 24.98 μg/ml on the 7th day. The antibiotic biosensor platform using carbon nanotube electrodes for preclinical evaluation of drug release profile of nanocapsules presented in this work showed good performance in sensing of antibiotic Teoplanin drug samples. The antibiotic biosensor platform could be further integrated with a micro fluidic platform for controlled synthesis of nanocapsules to feedback the drug release profile for optimization of the synthesis process. In addition, the developed biosensor can be integrated with wireless passive transmission module to be an implantable biomedical microsystem for health monitoring in future.

碩士
國立交通大學
應用化學系分子科學碩博士班
108
The proBrain Natriuretic Peptide is a 108-amino acids prohormone secreted by cardiomyocytes in the heart ventricles in response to volume expansion and pressure overload. The proBrain Natriuretic Peptide can be cleaved by enzyme and produce N-terminal pro hormone BNP (76-amino acid) and BNP (32-amino acid). N-terminal prohormone BNP (NT-proBNP) is a non-active prohormone containing the first 76 amino acid residues of proBNP, Brain natriuretic peptide (BNP) is a active hormone including the remaining 32 amino acids. Both proBNP, BNP and NT-proBNP are produced resulting from the change of pressure inside the heart. Therefore, to measure the concentration of these proteins can be used for evaluation of heart failure. The proBNP was employed for phage display library screening. After several generations of the screening, two single-chain fragment variable (scFv) regions with high binding affinity against proBNP were obtained. The dissociation constant (Kd) of the two selected anti-proBNP scFvs was measured to be 3.4± 0.8 μM and 476 ± 38 nM via the application of microscale thermophoresis. The measurement of proBNP was further performed by electrochemical impedance spectroscopy. The measurement limit of proBNP using the anti-proBNP scFv1 is at μM level, which is not feasible for Kd value estumation, whereas the Kd of the anti-proBNP scFv2 platform towards proBNP is 74±14 nM. The linear range of proBNP was measured from 12.3 to 333 nM. Though the selected scFvs can recognize and specifically bind to proBNP, the binding affinity needs to be improved in order for establishing an effective detection of proBNP.

Urine culture, the current gold standard for urinary tract infection (UTI) diagnosis, does not produce results in an acceptable length of time. An ultra-sensitive, cost-effective electrochemical biosensing platform with nanostructured microelectrodes was designed to address the need for a rapid, point-of-care (PoC) test that could achieve a sample-to-answer time in less than an hour. Printed circuit boards and metallized glass slides were processed using various techniques and then tested for their ability to form nanostructured microelectrodes. Peptide nucleic acid probes for the bacteria and yeast as well as ten probes for antibiotic resistance genes were designed and synthesized for use with the new platform. Validation of the sensor's specificity was performed using high concentrations (100nM) of synthetic DNA oligomers. Furthermore, a clinically relevant sensitivity of 103 cfu/mL was demonstrated by detecting 4 pathogen lysates (Staphylococcus saprophyticus, Pseudomonas aeruginosa, Enterococcus faecalis and Klebsiella pneumoniae) in a buffered solution.

碩士
國立臺灣大學
化學研究所
100
Silicon nanowire field-effect transistors (SiNW-FETs) have drawn great attention because of their potential as a label-free, real-time, and ultra-sensitive sensor for biomolecular detections. As a biological sensor, the surface of a SiNW-FET device was conventionally all area modified (AAM) with receptors, covering not only the minute SiNW surface area but also the relatively massive surrounding substrate area. However, target molecules could be captured on the upstream substrate area before reaching the SiNW surface in sensing measurements, thus jeopardizing the detection sensitivity. In this study, we have successfully fabricated SiNW-FETs with the selective surface modification (SSM) of receptors only on the SiNW sensing surface via gas-phase premodification and a bottom-up fabrication technique. Our results show that a SSM SiNW-FET, exhibiting desirable electrical characteristics with regard to ohmic contact and high transconductance, has the merits of faster response time, less sample requirements, and much improved detection sensitivity. Besides, we integrated SiNW-FET with a lipid bilayer to mimic the cell membrane for biological research, especially for the membrane protein studies. Our results show that a 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipid bilayer membrane with single or double lipid bilayers could be homogeneously formed on the SiNW-FET surface via a vesicle fusion method. However, because the shielding of the lipid bilayers on the underlying SiNW, signals were reduced in electrical measurement. To improve the signal acquisition from a lipid bilayer membrane covered SiNW-FET, we demonstrated that the electrical signals and the detection limit can be enhanced by utilizing a multiple-parallel-connection (MPC) SiNW-FET system.

碩士
國立中正大學
機械工程系研究所
104
The present study develops a low-cost biochip system employing guided-mode resonance biochip. The advantage of the guided-mode resonance biochip lies in its simple structure and the fact that it is a label-free biochip. By altering the geometric structure of the guided-mode resonance biochip, the sensitivity may be increased over 200 nm/RIU, and thus applicable for high-sensitivity bio-chemical detection. The present study first discussed the impact the geometrical effect has on sensitivity of biochips, and whether it could increase the sensitivity of guided-mode resonance biochip. We perform finite element simulation and transmission experiment to study the impact that depth and thickness of gratings have on sensitivity. The result of finite element analysis shows that, for both thickness and depth, sensitivity peaks at a certain value; increase or decrease from that particular value would cause the sensitivity to drop. Further, it is noticeable that when the geometric size of grating was optimized, the chip’s sensitivity is five times higher than those were not. In other words, optimizing the geometric size of the chip may increase its sensitivity by fivefold. Combining this result to other effects, the performance of guided-mode resonance biochip could be significantly improved. The present study then turned to developing a low-cost biochip detection system. We employed LED as light source, and used the variation in light intensity as detection mechanism to compare the performances of transmissive and reflective structure. The present study found that the reflective light intensity measurement system shows better performance than the transmissive light intensity measurement system, where the resolution of the reflective system, comparing to that of the transmissive system, is increased by approximately 427%. In order to proof the reflective system’s capability in bio-chemical detection, the present study also conducted a DNP/anti-DNP bio-chemical detection experiment. And the result of the calculations shows a detection limit of 5.38×10^(-8) g⁄ml for the reflective guided-mode resonance biochip that we developed, which proves it to possess outstanding bio-signal detection capability.

碩士
國立中正大學
化學暨生物化學研究所
104
The objectives of this work are to develop two novel multiplex chemical and biochemical sensing platforms, namly a reflection-based tubular waveguide particle plasmon resonance (RTW-PPR) biosensing platform, and a reflection-based fiber optic particle plasmon resonance (RFO-PPR) biosensing platform. The principle of inventions are based on measuring the light intensity after consecutive total internal reflections (TIRs) along a noble metal nanoparticles-modified waveguide (tube or optical fiber), wherein the evanescent wave excites the particle plasmon resonance of the nanoparticles at the reflection interface. When a noble metal nanoparticle is influenced by the change of the refractive index on its surrounding environment, its particle plasmon resonance condition will change. This phenomenon can be used as the basis of chemical and biological sensing. In the first part ：we used Poly(methyl methacrylate) PMMA as waveguide material to form a tubular waveguide and utilized 3-mercaptopropylsilatrane (MPS) to reduce the modification time. A variety of experiments were carried out to validate the sensitivity and refractive index resolution of the sensing platform. Using different weight percent of sucrose in pure water as samples, a refractive index resolution of 4.34×10-5 RIU and a sensor sensitivity of 5.39 RIU-1 have been achieved by the platform. In the biochemical detection experiments, OVA was used to functionalize the gold nanoparticle in order to detect anti-OVA. Results show that the calibration curve is linear (R2>0.99) and the limit of detection (LOD) is about 4.64×10-6 g/mL (3.09×10-8 M). In the second part：the RFO-PPR platform has achieved the absorbance sensitivity of 4.83 AU/RIU-1 and the sensor resolution of 4.6×10-5 RIU by using gold nanospheres as the sensing element. By the similar configuration, but using gold nanorods as the sensing element, the absorbance sensitivity of 3.81 AU/RIU-1 and the sensor resolution of 3.7×10-5 RIU have been achieved. In the biochemical detection experiments, DNP was used to functionalize the gold nanorods in order to detect anti-DNP antibody. Results show that the calibration curve is linear (correlation coefficient >0.99) and the detection limit is less than 3.88×10-10 M.

Single molecule biosensing techniques offer unique advantages and opportunities for basic biological studies and medical diagnostic applications. However, their signal levels are intrinsically very weak and can be easily masked by the noise from the sensor itself or the measurement electronics. Thus, the biosensing systems and devices must be carefully characterized and optimized to reduce noise. This thesis first presents optimizations that enable high bandwidth, single channel recordings of the calcium-induced calcium release channel ryanodine receptor 1. By directly integrating a suspended bilayer with a complementary metal oxide semiconductor transimpedance amplifier, the total input capacitance and, therefore, high frequency noise are lowered, enabling gating events to be recorded at an order of magnitude higher bandwidth than the previous state of the art. Next, low frequency noise optimizations for carbon nanotube transistors are explored using hexagonal boron nitride substrates. These devices have improved 1/f noise performance compared to equivalent devices on silicon oxide and demonstrate evidence of contact limited noise. Finally, a basic characterization of 1/f noise in carbon nanotubes is developed using correlated transport and noise measurements in crossed carbon nanotube homojunction devices. These methods of optimizing and characterizing noise can aid in the development of single molecule biosensors with improved temporal resolution and error rates.

碩士
國立陽明大學
生醫光電研究所
102
Nanostructure-based surface plasmon resonance sensors are capable of sensitive and label-free detection for chemical and biological sensing applications. However, low-cost mass-production techniques and development of portable low-cost sensing platforms are the main issues which should be addressed. In this study, double-layer gold nanoslit arrays were fabricated on a cyclic olefin polymer (COP) film using hot embossing nanoimprinting lithography and metal sputtering techniques and then utilized to detect methicillin-resistant staphylococcus aureus (MRSA). In the experiment, penicillin-Binding Protein 2α present in MRSA was detected using the plasmonic biochips and the minimum detectable concentration of penicillin-Binding Protein 2α was 100 ng/mL. In order to improve the sensitivity of the biochips, double-layer gold nanoslit array with a period of 1000 nm was fabricated and tested. The result shows that the wavelength sensitivity of the chip was 926 nm/RIU and the figure of merit value was up to 272. In addition, we combined an inexpensive transmission-type scanner, a 632.8 nm laser line filter and plasmonic biochips to establish a portable low-cost sensing platform capable of high-throughput detection. An antigen-antibody interaction experiment in aqueous environment was conducted using the platform to verify the detection sensitivity in surface binding event. The proposed sensing platform has the advantages of label-free high-throughput detection, simple operation method, quick detection, low price and portable. It can benefit various sensing applications and is suitable for point-of-care detection.

The quick, reliable, and sensitive detection of bacterial contamination is desired in areas such as counter bioterrorism, medicine, and food/water safety as pathogens such as E. coli can cause harmful effects with the presence of just a few cells. However, standard high sensitivity techniques require laboratories and trained technicians, requiring significant time and expense. More desirable would be a sensitive point-of-care device that could detect an array of pathogens without sample pre-treatment, or a continuous monitoring device operating without the need for frequent operator intervention.

Optical microring resonators in silicon photonic platforms are particularly promising as scalable, multiplexed refractive index sensors for an integrated biosensing array. However, no systematic effort has been made to optimize the sensitivity of microrings for the detection of relatively large discrete analytes such as bacteria, which differs from the commonly considered cases of fluid or molecular sensitivity. This work demonstrates the feasibility of using high finesse microrings to detect whole bacterial cells with single cell resolution over a full range of potential analyte-to-sensor binding scenarios. Sensitivity parameters describing the case of discrete analyte detection are derived and used to guide computational optimization of microrings and their constituent waveguides, after considering a range of parameters such as waveguide dimension, material, modal polarization, and ring radius. The sensitivity of the optimized 2.5 µm radius silicon TM O-band ring is experimentally demonstrated with photoresist cellular simulants. A multiplexed optimized ring array is then shown to detect E. Coli cells in an experimental proof of concept.