Rozprawy doktorskie na temat „Biomolecular Sensors”

Electrochemistry is a powerful technique that offers multiple possibilities and which is in constant evolution. Simple modifications of the electrode surface can result in an improvement of the selectivity and sensitivity of the method. However some situations require more complex modifications such as the incorporation of an external agent to the electrode surface, or within the actual electrode. This thesis describes the development and characterization of a range of novel electrochemical sensors for multiple applications covering agri-food, biomedical and environmental contexts. The foundations of the approach rest upon the development of carbon-loaded polycarbonate composite films. Their fabrication is described and the ease with which they can be modified and physically adapted is highlighted and critically evaluated. The response of the resulting sensors have been validated against conventional techniques. An overview of the technologies employing carbon electrodes is presented in Chapter 1 and serves to set the context of the subsequent research. The various methodologies employed are outlined in Chapter 2. Preliminary modifications of the analytical process has evolved from the ex situ functionalisation of the conventional carbon electrodes with copper (Chapter 3) through to the examination of the versatility and complexities of sample pre-treatment (Chapter 4). The pre-treatment of the sample using naphthoquinones as labeling agents has been developed and this work was extended to examine a wholly new derivatisation agent which could have analytical and clinical/veterinary diagnostic merit. A new direction was sought to overcome the limitations of the conventional analytical approach and composite systems were envisaged as providing an accessible yet flexible method of developing electrochemical sensors for discrete probe and flow systems. The basic procedure has been characterization and optimized for a range of analytes such as neurotransmitters (Chapter 5), anti-oxidants (Chapter 6), purine metabolites (Chapter 8) and phosphate (Chapter 9). Each chapter highlights a different aspect and applicability of the composite and go from simple physical surface modification (Chapter 5) to the incorporation of chemical agents (Chapter 6) and more complex systems such as enzymes (Chapters 8 and 9).

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 2003.
Includes bibliographical references.
System-level understanding of biological processes requires the development of novel biosensors capable of quantitative, real-time readout of molecular interactions. Label-free detection methods can minimize costs in time and resources by obviating preparatory steps necessary with label-based methods. They may further be valuable for monitoring biomolecular systems which are difficult or impossible to tag, or for which reporter molecules interfere with biological function. Field-effect sensing is a method of directly sensing intrinsic electrical charge associated with biomolecules without the need for reporter molecules. Microfabrication of field-effect biosensors enables their integration in compact microanalytical systems, as well as the potential to be scaled down in size and up in number. Applying field-effect sensing to the detection and real-time monitoring of specific molecular interactions has long been of interest for protein and nucleic acids analysis. However, these applications are inhibited by serious practical limitations imposed by charge screening in solution. The development of effective measurement techniques requires inquiry into aspects of device engineering, surface chemistry, and buffer conditions. This thesis describes a body of experimental work that investigates the feasibility of label-free analysis of biomolecular interactions by field-effect. This work begins with the microfabrication of field-effect sensors with extremely thin gate oxide, which enables improved surface potential resolution over previously reported sensors.
(cont.) The performance of these sensors has been characterized in terms of drift, noise, and leakage. To better understand the applicability of these sensors, we have characterized the sensors' response to pH, adsorption of polyelectrolyte multilayers, and high-affinity molecular recognition over a range of buffer conditions. Direct, label-free detection of DNA hybridization was accomplished by combining the high-resolution sensors, with enabling surface chemistry, and a differential readout technique. Finally, we explore the lateral scaling limits of potentiometry by applying a novel nanolithographic technique to the fabrication of a single electron transistor that demonstrates Coulomb oscillations at room temperature.
by Emily Barbara Cooper.
Ph.D.

Thesis (M. Eng.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 2004.
Includes bibliographical references (p. 52-53).
Microfabricated silicon field-effect sensors with integrated poly(dimethylsiloxane) microfluidic channels have been demonstrated. These devices are designed for the label-free detection and recognition of specific biomolecules such as DNA. Label-free methods eliminate the time-consuming and costly step of tagging molecules with radioactive or fluorescent markers prior to detection. The devices presented here are sensitive to the intrinsic charge of the target molecules, which modulates the width of the carrier-depleted region of a lightly-doped silicon sensor. The variable depletion capacitance is precisely measured, indicating changes in sensor surface potential of less than 30[micro]V. The integrated microfluidic channels enable the delivery of small (nanoliter-scale) amounts of fluid directly to the sensors. Capacitance-voltage curves were recorded using phosphate buffered saline (PBS) as the test electrolyte; a maximum slope of 44pF/V was measured in depletion. pH sensitivity was also demonstrated using modified PBS solutions. A device with dual 80x80Om sensors yielded a response of 40mV/decade, referenced to the fluid electrode. A device with dual 50x50[micro]m sensors yielded a response of 12mV/decade, referenced to the sensors.
by Peter R. Russo.
M.Eng.

The highly specific recognition processes between biomolecules mediate various crucial biological processes. Uncovering the molecular basis of these interactions is of great fundamental and applied importance. This research work focuses on understanding the interactions of several biomolecular recognition systems and processes that can provide fundamental information to aid in the rational design of sensing and molecular recognition tools. Initially, a reliable and versatile platform was developed to investigate biomolecular interactions at a molecular level. This involved several techniques, including biomolecule functionalization to enable attachment to self-assembled monolayers as well as atomic force microscopy (AFM) based force spectroscopy to uncover the binding or rupture forces between the receptor and ligand pairs. It was shown that this platform allowed determination of molecular binding between single molecules with a high specificity. The platform was further adapted to a general sensing formulation utilizing a group of flexible and adaptive nucleic acid recognition elements (RNA and DNA aptamers) to detect specific target proteins. Investigation of interactions at the molecular level allowed characterization of the dynamics, specificity and the conformational properties of these functional nucleic acids in a manner inaccessible via traditional interaction studies. These interactions were then adapted to aptamer-based detecting methods that at the ensemble or bulk scale, specifically taking advantage of mechanisms uncovered in the biophysical study of this system. A quartz crystal microbalance (QCM) was used to detect protein targets at the bulk level and the affinities and binding kinetics of these systems were analyzed. Along with AFM-based force spectroscopy, ensemble-averaging properties and molecular properties of these interactions could be correlated to contribute to bridging the gap across length scales. Finally, more broadly applicable sensing platform was developed to take advantage of the unique properties of aptamers. DNA was employed both as a carrier and as a molecular recognition agent. DNA was used as a template for nanoconstruction and fabricating unique shapes that could enhance the aptamer-based molecular recognition strategies. With aptamers tagged to distinct nanoconstructed DNA, a novel shape-based detecting method was enabled at the molecular level. The results demonstrated that this is a flexible strategy, which can be further developed as ultrasensitive single molecule sensing strategy in complex environments.

Los procesos de reconocimiento biomolecular entre receptores y ligandos son muy importantes en biología. Estas biomoléculas pueden desarrollar complejos muy específicos y tener una variedad de funciones como replicación y transcripción genómica, actividad enzimática, respuesta inmune, señalamiento celular, etc. La complementariedad inequívoca mostrada por estos componentes biológicos es ampliamente utilizada para desarrollar biosensores. Dependiendo de la naturaleza de las señales que se convierten, los biosensores pueden ser clasificados en ópticos, eléctricos o mecánicos. Entre los sensores mecánicos, los microcantilevers son los más comunes. Han sido utilizados como sensores de estrés superficial o como sensores de masa en detección de biomoléculas, desde hace más de 10 años. El enlace de las moléculas a sus superficies funcionalizadas se puede detectar midiendo la deflexión en modo estático o la variación de la frecuencia de resonancia en modo dinámico. Para lograr la máxima resolución, la deflexión es medida por un láser y un fotodetector. Este método limita las medidas en fluidos transparentes, la portabilidad del instrumento, e incrementa la complejidad de medición multiplexada. El desarrollo de cantilevers sensibles a la deflexión mediante la integración de piezoresistores o transistores de efecto de campo (MOSFET) implementados en el mismo voladizo, resuelve este problema. Sin embargo, simultáneamente se disminuye la resolución del sensor debido al incremento del ruido electrónico. Por otro lado, se puede detectar moléculas midiendo la fuerza de enlace entre una molécula y su receptor, estirando el complejo molecular, mediante espectroscopia de fuerza atómica (AFS), técnica basada en el microscopio de fuerza atómica (AFM). A pesar de la elevada resolución en fuerza, el AFM no ha logrado aún convertirse en instrumento analítico debido principalmente a la complejidad del mismo y de su uso. Un biosensor basado en cantilevers que puedan detectar su propia deflexión y que emplee la AFS, tendría resolución de una molécula, podría ser utilizado en fluidos opacos, tendría potencial de multiplexado y su integración a una celda microfluídica sería viable. Considerando esto, se desarrollaron cantilevers dotados de resolución de pN y compatibles con líquidos. Se diseñaron y modelaron cantilevers basados en silicio cristalino y se ha optimizado el proceso de fabricación para aumentar la sensibilidad y el rendimiento. Asímismo, se ha trabajado sobre el modelo, el desarrollo y la fabricación de cantilevers con un MOSFET integrado. Se concluye que el primer sensor ofrece una solución tecnológica más directa, aunque el segundo puede ser una buena alternativa. Simultáneo a la fabricación de sensores, se desarrollaron también nuevas técnicas y montajes para la rápida caracterización eléctrica y electromecánica de los sensores de manera precisa y fiable. Esto fue crucial a la hora de validar el proceso de producción y los dispositivos finales. Después de obtener muy alta resolución (<10 pN en líquido) con elevado rendimiento en la producción, los sensores fueron utilizados para el estudio de procesos de reconocimiento molecular entre avidina y biotina. Para lograr este objetivo, los sensores fueron integrados en un AFM comercial para aprovechar su elevada estabilidad mecánica y el desplazamiento nanométrico del piezoactuador. Se detectaron con éxito las fuerzas de enlace relacionadas a la formación del complejo molecular biotina-avidina, resaltando de esta manera, la posibilidad de detección label-free de biomoléculas en condiciones cuasi fisiológicas con resolución de una molécula. Además de la elevada sensibilidad, estos sensores pueden utilizarse sin restricciones en fluidos opacos, se pueden integrar fácilmente en celdas microfluídicas y demuestran capacidad para el multiplexado. Este resultado abre nuevas perspectivas en detectores de marcadores biológicos con elevada sensibilidad y que puedan trabajar en condiciones fisiológicas.
Biorecognition processes between receptors and their conjugate ligands are very important in biology. These biomolecules can build up very specific complexes displaying a variety of functions such as genome replication and transcription, enzymatic activity, immune response, cellular signaling, etc. The unambiguous one-to-one complementarity exhibited by these biological partners is widely exploited also in biotechnology to develop biosensors. Depending on the nature of the transduction signals, biosensors can be classified in optical, electrical and mechanical. Among mechanical biosensors, the microcantilevers play a prominent role. They have been used as stress or mass transducers in biomolecules detection for already more than a decade. The binding of molecules to their functionalized surface is detected by measuring either the deflection in static mode or the resonant frequency shift in dynamic mode. The deflection of the cantilever is converted optically by a laser and a photodetector in order to have the highest possible resolution. This limits the measurements in transparent liquids, the portability of the instrument and increases the complexity for multiplexing. The development of self-sensing cantilevers by integrating piezoresistors or metal-oxide-semiconductor field effect transistors (MOSFET) into the cantilever solves this issue. However, at the same time, this decreases the bending and frequency shift resolution due to the higher transducer noise. On the other hand, the detection of a single molecule can be attained measuring the unbinding force between two molecules of a complex pulling them apart, using the atomic force spectroscopy (AFS) measuring approach. This technique is based on the atomic force microscope (AFM). Despite the high force resolution, AFM has still not become an analytical instrument and it is mainly due to the complexity of the instrument and of its use. A biosensor based on AFS and on self-sensing cantilever would allow single molecule resolution, working in opaque fluids, easy multiplexing capability, and relatively easy integration in microfluidics cells. In this perspective, we worked to obtain self sensing-probes endowed with pN resolution and compatible with liquid media. Cantilevers based on single crystalline silicon have been modeled and the fabrication process has been optimized to improve the force sensitivity and to obtain high fabrication yield. At the same time we worked also on the modeling, development and fabrication of cantilevers with embedded MOSFET piezoresistive transducers. It turned out that the probes with integrated piezoresistor offer a more straightforward solution, but also the MOSFET cantilever can offer a good alternative. Alongside the force sensors fabrication, new high-throughput set-ups and techniques have been developed and optimized to measure the electrical and electromechanical characteristics of micro-electro-mechanical systems (MEMS) in a precise and reliable way. This was of key importance to correctly validate the new technological processes involved in production as well as characterize the final devices. After achieving very good sensor performances (resolution < 10 pN in liquid environment) with high production yield, we used the force probes to investigate the biorecognition processes in the avidin-biotin complex. For this purpose we integrated the sensor into a commercial AFM to take advantage of the high mechanical stability of this equipment and the highly reliable displacement of the piezo actuator. We detected the forces related to the avidin-biotin complex formation, highlighting the possibility of biomolecule label-free recognition in nearly physiological conditions and at single molecule resolution. Beside the very high sensitivity attained, the sensor can be used with no restrictions in opaque media; it can be easily integrated in microfluidic cells and it displays a high multiplexing potentiality. This result opens new perspectives in highly sensitive label free biomarkers detectors in nearly physiological conditions.

MEMS (Microelectromechanical Systems) acoustic sensors are a promising platform for Point-of-Care biosensing. In particular, piezoelectrically driven acoustic sensors can provide fast results with high sensitivity, can be miniaturized and mass produced, and have the potential to be fully integrated with sample handling and electronics in handheld devices. Furthermore, they can be designed as multiplexed arrays to detect multiple biomarkers of interest in parallel. In order to develop a microfabricated biosensing platform, a specific and high affinity biodetection platform must be optimized, and the microfabricated sensors must be designed to have high sensitivity and maintain good performance in a liquid environment. A biomolecular sensing system that uses high affinity peptide aptamers and a passivation layer has been optimized for the detection of proteins of interest using the quartz crystal microbalance with dissipation monitoring (QCM-D). The resulting system is highly specific to target proteins, differentiating between target IgG molecules and other closely related IgG subclasses, even in complex environments such as serum. Piezoelectrically actuated MEMS resonators are designed to operate in flexural microplate modes, with several modes shown to be ideally suited for fluid based biosensing due to improved performance in the liquid environment. The increase in quality factor of these MEMS microplate devices in liquid, as compared to air, is further investigated through the analytical and finite element modeling of MEMS fluid damping mechanisms, with a focus on acoustic radiation losses for circular microplate devices. It is found that the impedance mismatch at the air-water interface of a droplet is a key contributor to reduced acoustic radiation losses and thus improved device performance in water. Microplate acoustic sensors operating in flexural plate wave and microplate flexural modes are then integrated with a fluidic cell to facilitate protein sensing from fluid samples. Flexural plate wave devices are used to measure protein mass adsorbed to the sensor surface and initial results toward microplate flexural mode protein sensing are presented. Finally, challenges and areas of future research are discussed to outline the path towards finalization of a sensing platform taking advantage of the combination of the sensitive MEMS acoustic sensor capable of operating in a liquid environment and the specific and high affinity biomolecular detection system. Together, these form the potential basis of a novel Point-of-Care platform for simple and rapid monitoring of protein levels in complex samples.

The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file. Title from PDF of title page (University of Missouri--Columbia, viewed on September 21, 2009). Thesis advisor: Liqun Gu Includes bibliographical references.

Nanoparticles (nPs) demonstrate significant advantages over other sensor and marker technologies. The most useful optical nanosensor and label platform for biological samples would be non-toxic, hydrophilic, resistant to non-specific protein interactions and degradation over time or under harsh conditions, highly retentive of entrapped components, and easily functionalized for target specificity. The work described here is part of an investigation into the fabrication and application of polyacrylamide, polyacrylamide/silica hybrid, and polystyrene-core silica-shell nPs. Polyacrylamide (PA) nP nitric oxide (NO) sensors were made by co-entrapping 4, 5-diaminofluorescein (DAF-2) and Texas Red dextran in 60 nm PAnPs. Sensors were used to measure NO produced by a diazeniumdiolate NO donor in solution, and have a response time of 30 seconds or less. Entrapped DAF-2 was protected from non-specific interactions with bovine serum albumin (BSA). Sensor response to NO in FBS solutions was reduced compared to buffer, although improvement over free dyes was observed. The sensors were applied to J477A.1 macrophages as well as a HT1080 cell line (HTRiNOS) in preliminary studies for measuring intracellular NO production. Polyacrylamide/silica hybrid nPs were fabricated and nP architecture was evaluated by transmission electron microscopy. Isopycnic centrifugation of nP samples indicates that the hybrid nPs have a density between 1.70 and 1.76 g/cm³. Silica in the hybrid nPs was covalently labeled with Texas Red, suggesting that the hybrid nPs may be used as ratiometric or possibly multiplexed sensors. Hybrid nPs coated with 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) exhibit reduced adsorption of TRITC-BSA compared to uncoated hybrid nPs. Hybrid nP pH sensors were prepared and responded reproducibly and reversibly to changes in pH, nominally from pH 6.0 to 8.0. Core-shell nPs for scintillation proximity assay (SPA) were fabricated by entrapping the scintillants p-terphenyl and 4-bis(4-methyl-5-phenyl-2oxyzolyl)benzene in polystyrene, onto which silica shells were subsequently added. Core-shell nPs were found to have a scintillation response similar to that of shell-less polystyrene cores, indicating that the presence of the silica shells does not reduce scintillation efficiency. Preliminary studies using core-shell nPS for biotin-streptavidin binding SPA do not indicate an enhancement in scintillation efficiency, although this may be due to high nP:radiolabeled analyte ratios.

Aquest treball discuteix difrerents aspects relacionats amb el disseny de sensors i sistemes de biodetecció. Descriu la fabricació i caracterització de transductors electrics particulars, així com el desenvolupament de nous sistemes de transduccio i el descobriment de noves methodologies per la fabricacio de nanomatrius de proteines.
En primer lloc, es presenta un nou tipus de transductor impedimetric (I). Es va escollir un disseny basat en dos electrodes interdigitats per dos motius principals. Primer, aquesta geometria permet monitoritzar tant la resistència como la constant dieléctrica d'una solució, la qual cosa fa dels electrodes interdigitats eines més versatils que altres tipus transductors. Segon, els electrodes presenten una curta penetració del camp electric, la qual cosa els fa mes sensibles als canvis que tenen lloc a prop de la seva superfície. Aquest fet permet monitoritzar canvis locals en les magnituds d'interés. Finalment, són apropiats no nomes per construir sensors sinó també actuadors. Aquesta geometria sembla ser útil en experiments de dielectroforesi. Una innovació introduïda en aquesta tesi es el material escollit per fabricar els electrodes: silici policristal-lí o polisilici. El polisilici pot ser facilment modificat per donar lloc a superficies amb particulars propietats químiques i físiques, fent d'aquest material un excel-lent candidat per a la manufactura de biosensors, comparable a altres aproximacions com la quemisorció de alcanotiols sobre electrodes d'or.
Els esmentats electrodes interdigitats es van fer servir per probar dos nous sistemes de transducció. Ambdues aproximacions comparteixen un tret comu: aprofiten la capacitat dels electrodes interdigitats per mesurar canvis local en les propietats elèctriques del medi on es troben submergits. En II, aquest fet és utilitzat per monitoritzar una reacció enzimàtica, i es mostra com la característica de mesura local en electrodes interdigitats dóna lloc a una detecció més sensible. A més, es demostra que aquesta aproximació es adequada per la detecció de proteïnes fent servir l'enzim com a marca en un immunoassaig. En III, els electrodes interdigitats actuen com a sensor i actuador. Com a actuador, els electrodes son capaços de concentrar esferes de làtex a la seva superficie. Com a transductors, la presencia de les micropartícules aïllants a la seva superficie dóna lloc a un canvi en la geometria de la cel-la, que pot ser detectat monitoritzant tant la resistència com la capacitat de la solucio. Aquest mode de funcionament es paral-lel al dels sensors magnetoresistius, i el principi de transduccio proposat es presenta com a una alternativa a ells.
Finalment, un quart treball es presenta en aquesta tesi (anex). Comparteix dues característiques en comú amb els treballs previs: el sustrat (silici) i una metodologia per la inmoblització de biomolecules (silanització). Les seves aplicacions son, però, diferents i cobreixen un rang més ampli d'aplicacions. En concret, una nova metodologia pel nanoestructurat de superfícies, de baix cost i fàcil disponibilitat és presentada. Es van aconseguir motius fets amb molècules de silà amb dimensions inferiors als 10 nm. En el marc de la biodetecció, aquesta nova tècnica per nanoestructurat superficial es propossa com a alternativa a la nanolitografia dip-pen per la manufactura de nanomatrius de biomolècules. Les petites dimensions dels motius obtinguts obren el cami per la consecució de nanomatrius d'una única molècula.
This work discusses different aspects related to the design of biosensors and biodetection systems. It describes the fabrication and characterization of particular electric transducers together with the development of new transduction systems and the finding of new methodologies for biomolecule nanoarray fabrication.
Firstly, a new type of impedimetric transducer is presented (I). A two-electrode interdigitated design was chosen, mainly for three reasons. First, this geometry allows the monitoring of both the resistivity and the dielectric constant of a solution, thus making interdigitated electrodes more versatile tools than other kind of transducers. Second, they present short electric field penetration depths, which make them more sensitive to changes occurring close to their surface. This fact enables the monitoring of local changes in the magnitudes of interest. Finally, they are suitable for constructing not only sensors but also actuators. This geometry appears to be useful in dielectrophoresis experiments. One innovation introduced in this thesis is the material chosen to fabricate the electrodes: polycrystalline silicon, also known as polysilicon. Polysilicon can be easily modified to render surfaces with distinct physical and chemical properties, thus making this material an excellent approach for biosensors manufacture, comparable to other approaches like alkanethiol chemisorption on gold electrodes.
The aforementioned interdigitated electrodes were used to test two new transduction principles. The two approaches share a common feature: they rely on the ability of interdigitated electrodes to measure local changes in the electrical properties of the medium where they are immersed. In II, this is used to monitor an enzymatic reaction, and it is shown that the characteristics of measuring local changes at interdigitated electrodes result in a more sensitive detection. Furthermore, the feasibility of this approach for protein detection is demonstrated by using the enzyme as a label for performing an immunoassay. In III, the interdigitated electrodes act both as a transducer and as an actuator. As an actuator, the electrodes are able to concentrate latex beads at their surface. As a transducer, the presence of the insulating microparticles at their surface results in a change in the geometry of the cell, that can be detected by monitoring either the resitance or the capacitance of the solution. Such device performance is parallel to that of magnetoresistive biosensors, and the proposed transduction principle is envisaged as a suitable alternative to them.
Finally, a fourth work is presented in this thesis (Annex). It shares two features in common with the previous works: the substrate (silicon) and a method for biomolecule immobilization (silanization). However, the applications are somehow different, and cover a wider range. Precisely, a new methodology for low cost, easily available nanopatterning is shown. Features made of silane molecules, with dimensions less than 10 nm are successfully patterned. In the frame of biodetection, this new nanopatterning technique is proposed as an alternative to dip-pen nanolithography in nanoarray manufacture. Moreover, the small dimensions of the obtained patterns pave the way for the achievement of single-molecule nanoarrays.

The development of a novel protocol for the covalent immobilisation of biomolecules containing primary amines using either polythiol compounds or novel, inexpensive and simple polymers is presented in this thesis. When developing biosensors, the method used for the immobilisation of the sensing elements is very important. The immobilisation needs to be fast, cheap and most importantly should not affect the biorecognition activity of the immobilised receptor. The chemistry used for the immobilisation is based on the well known reaction between primary amines and thioacetal groups, formed upon reaction of o-phthaldialdehyde (OPA) and thiol compounds. Initially the possibility to use this chemistry to immobilise receptors and develop biosensors was proved using commercially available polythiol compounds. Such compounds can be irreversibly adsorbed, creating self-assembling monolayers (SAMs), on noble metal transducer surfaces. These SAMs were immobilised on Biacore surface plasmon resonance (SPR) gold chips and then used to study kinetic of biomolecules interactions and to detect cells. A general protocol suitable for the immobilisation of enzymes and antibodies such as anti-prostate specific antigen (anti-PSA) and anti-Salmonella typhimurium antibody was optimised. Kinetic data were obtained for PSA binding to anti-PSA antibody and they were compared to the results obtained using commercially available Biacore chips, CM1. For Salmonella typhimurium cells, a detection limit of 5 × 106 cells ml-1 with minimal non-specific binding of other biomolecules was obtained. An interesting capability shown by these SAMs, in contrast with commercially available chips, was the opportunity to immobilise any proteins, even those with very low or high isoelectric points, pI. In addition protein immobilisation was achieved with a simple step, without requirement of any activation. These findings make this immobilisation technique a very promising alternative to peptide bond formation for amine coupling. Even though, the developed SAMs showed to be useful for certain type of applications (kinetic study and detection of very large analyte), it was clear that due to a combination of factors (e.g. limited and steric hindrance), they were not suitable for the development of biosensors good enough for practical applications. Therefore to overcome the drawbacks shown by polythiol SAMs, a novel 3-D polymer was developed. The main advantage of this polymer is the tridimensional (3D) network, which, after immobilisation, ensures the availability of a high percentage of receptor binding sites. As the polythiol SAMs, also the 3-D polymer contains thioacetal groups, which do not need any activation to react with primary amines in proteins. The novel 3-D polymer also contains thiol derivative groups (disulphide groups or thioethers) that promote self-assembling on metal surfaces. As before, the polymer was immobilised on SPR gold chips and the resulting layer was characterised using contact angle meter, atomic force microscopy (AFM) and ellipsometry. Contact angle demonstrated that the immobilisation of polymer on sensor surface produced a relatively hydrophobic surface. The thickness of polymer layer was determined by applying ellipsometry, whereas AFM showed the change of surface roughness after polymer attachment. A general protocol suitable for the immobilisation of BSA, enzymes and antibodies such as polyclonal anti-microcystin-LR and monoclonal anti-prostate specific antigen (anti-PSA) antibody was then optimised. The affinity characteristics of developed immunosensors were investigated in reaction with microcystin-LR, and PSA. The calculated detection limit for analytes depended on the properties of the antibodies. The detection limit for microcystin-LR was 10 ng ml and for PSA 0.05 ng ml. The 3-D polymer chips were stored for up to 2 months without any noticeable deterioration in their ability to react with proteins. The performance of 3-D polymer chips were also compared with commercially available Biacore chips, as CM5. The main advantages were found to be the low cost, the possibility to immobilise biomolecules at physiological pH (pH 7.4), the lack of any activation step for biomolecules immobilisation and the opportunity to immobilise proteins with very different pI (also very low pI). Despite the successful detection of PSA achieved in buffer (detection limit 0.05 ng ml-1) using 3-D polymer chips, the detection of proteins in serum resulted to be very challenging due to the complex nature of the matrix, which contains a high content of many different compounds. Different techniques were applied in order to reduce the non specific adsorption of serum on 3-D polymer sensors with antibodies immobilised on the surface. Satisfactory results were finally obtained by including the surfactant P20 into the measuring system. The detection of PSA in serum using 3-D polymer sensors, however, became possible only by switching from a direct detection to a ‘sandwich detection’. In this sandwich format, after injecting samples of PSA (prepared both in buffer or 20% serum) onto a specific antibody (capture-Ab, C-Ab) immobilised on the 3-D polymer surface, the analytical signal is recorded by injecting a second specific Ab (detection-Ab, prepared in PBS), which recognises a different epitope of the antigen. With this format, the analytical signal is recorded in absence of any complex matrix, avoiding interference from non specific adsorption. The detection limit for PSA, obtained using the sandwich immunosensor (developed on 3-D polymer chips) was 0.1 ng ml-1 in buffer and 5 ng ml-1 in 20% serum, which is very close to the sensitivity necessary for detection of the prostate biomarker in real samples. Therefore this study has demonstrated the opportunity to apply the novel 3-D polymer for development of biosensors suitable for applications in real samples.

This thesis summarises work towards a Lab-on-Chip (LOC). The request for faster and more efficient chemical and biological analysis is the motivation behind the development of the LOC-concept. Microfluidic devices tend to become increasingly complex in order to include, e.g. sample delivery, manipulation, and detection, in one chip. The urge for smart and simple design of robust and low-cost microdevices is addressed and discussed. Design, fabrication and characterization of such microdevices have been demonstrated using low-cost polymer and glass microfabrication methods. The manufacturing is feasible, to a large extent, to perform outside the clean-room, and has subsequently been the chosen technique for most of the work. Issues of bonding reliability are solved by using polymer adhesive tapes. A planar electrocapture device with LOC-compatibility is demonstrated where beads are immobilised and released in a flowing stream. Retention of nanoparticles by means of electric field-flow fractionation using transparent indium tin oxide electrodes is presented. Moreover, a cast PDMS 4-way crossing is enabling a combination of liquid chromatography and capillary electrophoresis to enhance separation efficiency. Sample transport issues and a new flow-cell design in a quartz crystal microbalance bioanalyzer are also investigated. Fast bacteria counting by impedance measurements, much requested by the pharmaceutical industry for biomass monitoring, is carried out successfully. In conclusion, knowledge in micro system technology to build microdevices have been utilised to manipulate and characterise beads and cells, taking one step further towards viable Lab-on-Chip instruments.

Our understanding of the folding of membrane proteins lags behind that of soluble proteins due to the challenges posed by the exposure of hydrophobic regions during in vitro chemical denaturation and refolding experiments. While different folding models are accepted for soluble proteins, only the two-stage model and the long-range interactions model have been proposed so far for helical membrane proteins. To address our knowledge gap on how different membrane proteins traverse their folding landscapes, Chapter 2 investigates the structural features of SDS-denatured states and the kinetics for reversible unfolding of sensory rhodopsin II (pSRII), a retinal-binding photophobic receptor from Natronomonas pharaonis. pSRII is difficult to denature, and only SDS can dislodge the retinal chromophore without rapid aggregation. Even in 30% SDS (0.998 $\mathit{\Chi}_{SDS}$), pSRII retains the equivalent of six out of seven transmembrane helices, while the retinal binding pocket is disrupted, with transmembrane residues becoming more solvent-exposed. Folding of pSRII from an SDS-denatured state harbouring a covalently-bound retinal chromophore shows deviations from an apparent two-state behaviour. SDS denaturation to form the sensory opsin apo-protein is reversible. This chapter establishes pSRII as a new model protein which is suitable for membrane protein folding studies and has a unique folding mechanism that differs from those of bacteriorhodopsin and bovine rhodopsin. In Chapter 3, SDS-denatured pSRII, acid-denatured pSRII and sensory opsin obtained by hydroxylamine-mediated bleaching of pSRII were characterised by solution state NMR. 1D $^1$H and $^{19}$F NMR were first used to characterise global changes in backbone amide protons and tryptophan side-chains. Residue-specific changes in backbone amide chemical shifts and peak intensities in 2D [$^1$H,$^{15}$N]-correlation spectra were analysed. While only small changes in the chemical environment of backbone amides were detected, changes in backbone amide dynamics were identified as an important feature of SDS- and acid-denatured pSRII and sensory opsin. $^{15}$N relaxation experiments were performed to study the backbone amide dynamics of SDS-denatured pSRII, reflecting motions on different timescales, including fast fluctuations of NH bond vectors on the ps-ns timescale and the lack of exchange contributions on the µs timescale. These studies shed insight on differences in the unfolding pathways under different denaturing conditions and the crucial role of the retinal chromophore in governing the structural integrity and dynamics of the pSRII helical bundle. Hydrogen bonds play fundamental roles in stabilising protein secondary and tertiary structure, and regulating protein function. Successful detection of hydrogen bonds in denatured states and during protein folding would contribute towards our understanding on the unfolding and folding pathways of the protein. Previous studies have demonstrated residue-specific detection of stable and transient hydrogen bonds in small globular proteins by measuring $^1{\it J}_{NH}$ scalar coupling constants using NMR. In Chapter 4, different methods for measuring $^1{\it J}_{NH}$ scalar coupling were explored using RalA, a small GTPase with a mixed alpha/beta fold, as proof-of-concept. Detection of hydrogen bonds was then attempted with OmpX, a beta-barrel membrane protein, both in its folded state in DPC micelles and in the urea-denatured state. While $^1{\it J}_{NH}$ measurement holds promise for studying hydrogen bond formation, further optimisation of NMR experiments and utilisation of perdeuterated samples are required to improve the precision of such measurements in large detergent-membrane protein complexes. Naturally occurring split inteins can mediate spontaneous trans-splicing both in vivo and in vitro. Previous studies have demonstrated successful assembly of proteorhodopsin from two separate fragments consisting of helices A-B and helices C-G via a splicing site in the BC loop. To complement the in vitro unfolding/folding studies, pSRII assembly in vivo was attempted by introducing a splicing site in the loop region of the beta-hairpin constituting the BC loop of pSRII. The expression conditions for the N- and C-terminal pSRII-intein segments were optimised, and the two segments co-expressed. However, the native chromophore was not observed. Further optimisation is required for successful in vivo trans-splicing of pSRII and application of this approach towards understanding the roles of helices and loops in the folding of pSRII.

L’émollience est un terme définissant la capacité d’une matière première à adoucir, amollir, ou lubrifier la peau. Dans le domaine de la cosmétique, les émollients sont utilisés pour modifier la consistance, la viscosité ou la polarité d’une formulation. Il existe un nombre non négligeable d’émollients pouvant être utilisés en cosmétique. Cependant, les données aussi bien physico-chimiques que sensorielles disponibles dans la littérature sont encore très rares, rendant le choix des émollients complexe. De plus, les analyses sensorielles habituellement réalisées par les fournisseurs constituent une méthode de caractérisation particulièrement chronophage et coûteuse.Parmi les différents types d’émollients, les dérivés siliconés se démarquent par des propriétés bien spécifiques. Il s’agit notamment d’un très bon étalement, un toucher doux, non huileux et non collant, ou encore d’un effet sec sans effet de fraicheur. Cependant, malgré ces propriétés sensorielles exceptionnelles, de récentes études soulèvent la question de la toxicité d’un dérivé cyclique particulièrement utilisé dans les produits cosmétiques : le décaméthylcyclopentasiloxane (D5). Ainsi, deux problématiques font le sujet de ces travaux : une portant sur la recherche d’un substituant biosourcé au D5 et pour laquelle des molécules commerciales et synthétisées ont été caractérisés et comparés par des mesures physico-chimiques et sensorielles. La seconde problématique repose sur la recherche de corrélations entre les données physico-chimiques et sensorielles dans le but de faciliter le travail des formulateurs lors du screening des émollients par la prédiction de certaines de leurs propriétés sensorielles
Emolliency is a word used to define the ability of a compound to soften or lubricate the skin. ln the cosmetic field, emollients are used to modify the consistency, the viscosity or the polarity of a formulation. Many emollients can be used in cosmetic products. However, in the literature both physicochemical and sensory data ar still lacking, making it difficult to choose an emollient. Furthermore, the sensory analysis usually performed to characterize emollients are particularly time-consuming and thus, expensive. Among the different chemical families of emollients, silicone derivatives stand out thanks to their specific properties. Indeed, they are characterized by an excellent spreading on skin and hair, a smooth skin feel, non-greasy and non-sticky, or by a dry skin feel without a fresh effect. However, even though these sensory properties are exceptional, recent studies wonder about the toxicity of a cyclic silicone particularly used in cosmetic products: the decamethylcyclopentasiloxane (D5). Thus, this work deals With two main objectives. The first one consists in the research of a bio-based alternative to the D5 For this purpose, a number of commercial and synthesized molecules were characterized and compared With physicochemical measurements and sensory analysis, allowing the observations of trends between structures and properties. The second objective relies on the study of correlations between physico-chemical and sensory data in order to predict the emollient properties of cosmetic ingredients. This would ease the work of formulators during the screening of ingredients

In this research, a proton exchange membrane fuel cell (PEMFC) sensor was investigated for specific detection of volatile organic compounds (VOCs) for point-of-care (POC) diagnosis of the physiological conditions of humans. A PEMFC is an electrochemical transducer that converts chemical energy into electrical energy. A Redox reaction takes place at its electrodes whereas the volatile biomolecules (e.g. ethanol) are oxidized at the anode and ambient oxygen is reduced at the cathode. The compounds which were the focus of this investigation were ethanol (C2H5OH) and isoflurane (C3H2ClF5O), but theoretically, the sensor is not limited to only those VOCs given proper calibration. Detection in biosensing, which needs to be carried out in a controlled system, becomes complex in a multivariate environment. Major limitations of all types of biosensors would include poor selectivity, drifting, overlapping, and degradation of signals. Specific detection of VOCs in multi-dimensional environments is also a challenge in fuel cell sensing. Humidity, temperature, and the presence of other analytes interfere with the functionality of the fuel cell and provide false readings. Hence, accurate and precise quantification of VOC(s) and calibration are the major challenges when using PEMFC biosensor. To resolve this problem, a statistical model was derived for the calibration of PEMFC employing multivariate analysis, such as the “Principal Component Regression (PCR)” method for the sensing of VOC(s). PCR can correlate larger data sets and provides an accurate fitting between a known and an unknown data set. PCR improves calibration for multivariate conditions as compared to the overlapping signals obtained when using linear (univariate) regression models. Results show that this biosensor investigated has a 75% accuracy improvement over the commercial alcohol breathalyzer used in this study when detecting ethanol. When detecting isoflurane, this sensor has an average deviation in the steady-state response of ~14.29% from the gold-standard infrared spectroscopy system used in hospital operating theaters. The significance of this research lies in its versatility in dealing with the existing challenge of the accuracy and precision of the calibration of the PEMFC sensor. Also, this research may improve the diagnosis of several diseases through the detection of concerned biomarkers.

博士
國立成功大學
醫學工程研究所碩博士班
95
With the suitable immobilizations of biomolecules on the sensing surface of surface plasmon resonance (SPR) sensors, they can be extended as useful tools in the studies of biomolecular interactions. In this dissertation, the study of β-amyloid (Aβ) peptide aggregation, the immunodetection of C-reactive protein (CRP), and the fabrication of a multispot DNA chip were evaluated by the SPR sensing or imaging techniques which constructed the three specific applications. The aim of first application is to observe the process of Aβ aggregation with or without the existence of metal ions. The immunodetection of CRP is to employ SPR biosensor in the measurement of pentamer and modified CRPs with less false signals. Finally, a practical method to fabricate a multispot DNA chip is proposed and evaluated by a SPR imager. Soluble Aβ(1-40) peptide is immobilized on the surface of SPR chip and the aggregating reaction occurs spontaneously or under the induction of metal ions. A first-order kinetics model is applied to analysis the data for getting the kinetic parameters. A metal chelator, EDTA, is used in the experiments for testing its effect on the disruption of Aβ aggregates induced by metal ions. Results revealed the metal ions promoted Aβ aggregation with various propensities. Cu(II) could induce a rapidest initial Aβ aggregation, but it did not promote the formation of large Aβ aggregates. The Aβ aggregates induced by metal ions could be disrupted by the chelator, EDTA. For constructing surface with a well-order immobilization of antibodies, the three monoclonal antibodies (Mabs), C8, 8D8, and 9C9 are immobilized on a protein G layer in the immunodetection of CRP. In all experiments of detecting CRP, no false results were observed in the recognition of modified and pentamer CRPs. In order to prepare a multispot DNA chip, thiolated single-stranded DNAs (ssDNAs) (1 μM) with short sequences and oligo (ethylene glycol) (OEG) alkanethiol (50 μM) mixed in 1 M KH2PO4 is used to spot on the SPR sensing chip. The commercial program, Oligo, is applied to calculate of possibility on formation of DNA secondary structure. The experiments of DNA hybridization are performed at two different temperatures for interpreting the effect of temperature on DNA hybridization. According to the results from the DNA hybridizing experiments evaluated on the SPR imaging system, the fabrication method for making a multispot DNA chip was proved its feasibility. The effect of DNA secondary structure on hybridization was verified that could be minimized by raising experimental temperature. The experimental resluts obtained from the SPR techniques are easily affected by chemical surface modifications and biomolecular immobilizations on the sensing surface. The several methods used in this dissertation are successfully applied in the experiments, and they also can be used in other biomolecular detections. The experimental results can provide the useful examples of applying SPR techniques in the biomedical examinations to other related researchers.

Title: Designing and testing of new metal nanosubstrates for biomolecular sensors based on surface-enhanced Raman scattering (SERS) spectroscopy Author: Vlastimil Peksa Department: Institute of Physics of Charles University Supervisor: doc. RNDr. Marek Procházka, Ph.D., Institute of Physics of Charles University Abstract: This experimental methodical work was aimed at the optimization of selected gold and silver substrates and their use in construction of SERS-based biosensors, including following practical application. Several types of substrates, fabricated via a combination of bottom-up techniques on solid surfaces, were tested. The properties of these substrates were examined with probe molecules, namely methylene blue, porphyrins and tryptophan, on a confocal Raman microspectrometer. Obtained findings about the influence of analyte application, objective focusing and internal intensity standard were exploited for optimization of measurement procedures with regard to sensitivity, accuracy and reproducibility. A method for quantitative detection of food dye azorubine (E 122) in commercially available drinks was developed, based on these findings. Its results have shown its potential as a pre-scan method for field application and preliminary testing. Keywords: Metal nanosubstrates, biomolecules,...

Measurement of molecular interactions is essential for fundamental biological studies as well as for the development of health diagnostics devices. Several label-free and labeled techniques to characterize and quantify molecular interactions have been developed. A major disadvantage of the labeled techniques is the requirement of an additional process step of labeling that increases the system complexity for completely automated systems. There are many label free molecular detections techniques such as electrical (nano wires and nano tubes based), mechanical (cantilever and quartz crystal micro balance based), electrochemical and optical (interferometry, waveguides, optical cavities and surface plasmon resonance (SPR) based). The requirements of an ideal bio-molecular sensing technique are a) cost effective, portable and simple to operate, b) high sensitivity and specificity, c) high reproducibility, d) real time measurement capability and e) ability to do parallel measurements in a Lab-on-Chip format. The goal of the work done in this thesis is to design an optical real time bio molecular sensing technique that should be cost effective and highly sensitive. In this work we describe the use of self-referenced measurement techniques to eliminate common mode signal drifts due to thermal and concentration gradients in the fluid flow cell and demonstrate a nearly 40-fold decrease in the system noise. It started with a reflectance modulation point by point scanning system. Polyelectrolytes (poly allylamine hydrochloride (PAH) and poly acrylic acid (PAA), negatively and positively charged polymers) microarray was created on a SiO2 grown silicon substrate. Reflectance modulation was analyzed from the SiO2 surface and polyelectrolyte microarray on the SiO2. But the limit of detection (LoD) was poor due to laser intensity fluctuations. Therefore, a self-referencing diffractive reflectance modulation system (DRMS) was proposed. There, diffractive microstructures were fabricated in the SiO2 layer on top of silicon substrate and created a flow cell on the diffractive microstructures. When these diffractive microstructures are illuminated by a laser beam, they produce a diffraction pattern in reflected and transmitted light. In the diffraction pattern the zeroth contains average background information whereas higher orders contain signal information of the liquid sample present on the microstructures. By using Si/SiO2 devices we demonstrated bulk (refractive index sensing for water ethanol mixtures) and surface adsorption (real time binding of polymer layers on micro structured Si/SiO2 devices) molecular detection measurements. Instead of doing bio molecular detection measurements on Si/SiO2 devices we started doing measurements on glass devices with the advantages, flexibility to do measurement in transmission and lower substrate cost. In the next part of the thesis, bulk and surface adsorption measurements were demonstrated for glass substrates. For this, a 2D periodic array of wells of 10-micron diameter was created in the glass cover slide with various well depths. A flow cell was constructed on the diffractive micro structured cover slide to inject liquid samples on the microstructures, and this micro patterned glass cover slide was used as our sensor device for bulk and surface detection measurements. With the glass sensor devices bulk and surface adsorption bio molecular detection measurements were demonstrated both in transmission and reflection. To demonstrate the bulk detection, we measured the refractive index of different concentrations of NaCl in water and for bulk measurements a limit of detection (LoD) of 3x10-6 RIU was achieved. For surface adsorption measurements, layer by layer (LBL) deposition of positively (PAH) and negatively (PAA) charged polymers was demonstrated at the glass micro array sensor surface. The experimental data were validated with the model developed using transfer matrix method in chapter 4. Bio-molecular detection measurements were not demonstrated with this technique because this technique is in very preliminary stage of development. This technique needs more work to be done for standardization and optimization of appropriate conditions of detection protocols for biological samples. Large number of trials are necessary to standardize this technique under various control conditions because detection of proteins and DNA samples will add a whole new dimension of complexities depending upon size, coverage, and orientation of biological samples. The same diffractive self-referencing concept can be applied for SPR also. For this, grating structures available on commercial DVDs were used for the excitation of surface plasmons. On these DVD samples, metal dielectric multi layers were coated and demonstrated to couple surface plasmons and give multiple SPR peaks in transmission as a function of incidence angle. By using diffractive self-referencing concept an intensity referenced SPR method was demonstrated for bio molecular detection measurements. With the intensity referenced SPR as a proof of concept for bulk measurements refractive index detection of different concentration of NaCl in water was demonstrated. Similarly, for surface adsorption measurements, polyelectrolytes PAH and PAA were used for the functionalization of surface and detection of Bovine Serum Albumin (BSA) was demonstrated. Only very preliminary experiments were done to demonstrate that the ratio metric self-referencing concept can be exploited in grating coupled SPR also for bulk and surface bio-molecular detection measurements. This technique also needs many trials and repetitions for the standardization and optimization for use with biological samples. A self-referencing method was introduced and demonstrated, and this method was applied to two different optical label-free sensing techniques, namely, interferometric reflectance modulation and SPR. As a proof of concept for bulk detection, real time refractive index measurements were done for different concentrations of ethanol and NaCl in water and for surface adsorption detection, real time layer-by-layer (LBL) assembly of polyelectrolytes PAH and PAA was demonstrated on Si/SiO2 and glass micro array sensor surfaces. These real time self-referencing techniques namely DRMS and intensity referenced SPR need lot more work to be done to realize their true potential for bio-molecular detection measurements. The performance of these techniques was studied from the point of view of simplicity, cost effectiveness, sensitivity, and robustness.

碩士
國立臺南大學
通訊工程研究所碩士班
98
In this thesis, two portable multi-channel potentiostats are proposed for the signal processing of electrochemical biosensors. One is designed by modular approach to construct a portable multi-channel potentiostat and the other is directly designed to implement a portable multi-channel auto-range potentiostat. The proposed potentiostats can perform three operation modes which are commonly used in electrochemical experiments: potentiometry, voltammetry, and cyclic voltammetry. In system verification, taking advantage of the function of cyclic voltammetry, the proposed potentiostats are used to integrate an EGCG sensor and a caffeine sensor to measure EGCG and caffeine concentrations in green tea. The experimental results show that the biosensor currents measured by the proposed potentiostats have the same trend as those measured by the commercial potentiostats when the EGCG concentration and the caffeine concentration change. According to the calibration curve, the measured caffeine in green tea can reach 101.3%、93.25% and 97.27% accuracy respectively for three different green tea samples respectively. As for the EGCG measured, the results are 111.19%、100.53% and 122.66% accurate in each sample. In conclusion, the proposed multi-channel potentiostats have the merits of small size, light weight, high portability, low cost, and being able to measure multi-biosensors at the same time. In the future, we will integrate the proposed potentiostats with biosensors to develop the home-care system in daily life.

Optical sensors play vital roles in many applications in today’s world. Photonic technologies used to design and engineer optical sensing platforms can provide distinctive advantages over conventional detection techniques. For instance, when compared to electronic and magnetic sensing systems, optical sensors require physically smaller equipment and have the capability for delivering more analytical information (e.g. spectroscopic signatures). In addition, demand for low-cost and portable bio-analyte detections is a growing area for applications in healthcare and environmental fields. Among other factors to achieve reliable results in terms of selectivity and sensitivity is key for the detection of bio-analytes with analytical relevance. Commonly used bio-analytical techniques (e. g. high performance liquid chromatography) have been appropriately designed based on qualitative and quantitative analysis. However, the requirement of expensive equipment, and complexity of procedures (e.g. biomolecule labelling, calibrations, etc.) restrict the board applicability and growth of these techniques in the field of biosensing. Optical sensors tackle these problems because they enable selective and sensitive detection of analytes of interest with label-free, real-time, and cost-effective processes. Among them, optical interferometry is increasingly popular due label-free detection, simple optical platforms and low-cost design. An ideal substrate with high surface area as well as biological/chemical stability against degradation can enable the development of advanced analytical tools with broad applicability. Nanoporous anodic alumina has been recently envisaged as a powerful platform to develop label-free optical sensors in combination with different optical techniques. This thesis presents a high sensitive label-free biosensor design combining nanoporous anodic alumina (NAA) photonic structures and reflectometric interference spectroscopy (RIfS) for biomedical, food and agricultural applications. NAA is a suitable optical sensing platform due to its optical properties; a high surface area; its straightforward, scalable, and cost-competitive fabrication process, and its chemical and mechanical stability towards biological environments. Our biosensor enables real-time screening of any absorption and desorption event occurring inside the NAA pores. A proper selection of bio-analytes were able to be detected using this platform which offers unique feature in terms of simplicity and accuracy. The most relevant components of this thesis are categorised as below: 1. Self-ordered NAA fabrication and detection of an enzymatic analyte as a biomarker for cancer diagnosis: Fabrication of NAA photonic films using two step electrochemical anodization and chemical functionalisation. Detection of trace levels of analyte enzyme and its quantification by selective digestion. The NAA photonic film with the enzyme acts as a promising combination for a real-time point-of-care monitoring system for early stages of disease. 2. NAA rugate filters used to establish the binding affinity between blood proteins and drugs: Design, fabrication, and optimisation of NAA anodization parameters using sinusoidal pulse anodization approach (i.e. anodization offset and anodization period) to produce rugate filter photonic crystals that provide two comparative sensing parameters. Establishment of highly sensitive and selective device capable for drug binding assessments linked to treating a wide range of medical conditions. 3. NAA bilayers and food bioactive compound detection: Design, fabrication, and optimisation of NAA anodization parameters (i.e. anodization time and number of anodization steps) to obtain NAA bilayered photonic structures that display the effective response of NAA geometry with different types of nano-pore engineering. The photonic properties of the NAA bilayer were studied at each layer of nano-structure under specific binding of human serum albumin and quercetin as target agent. 4. Single nucleotide polymorphism (SNP) detection: The design and implementation of a Ligation-Rolling Circle Amplification assay to detect a single nucleotide polymorphism associated with insecticide resistance in a pest beetle species, Tribolium castaneum. This proof-of-concept SNP detection assay has the potential to provide a method compatible with a biosensor platform such as NAA. This demonstrates the first step towards the potential development of a genotyping biosensor, and a real-world application of insect insecticide resistance monitoring. The results presented in this thesis are expected to enable innovative developments on NAA sensing technology that could result in highly sensitive and selective detection systems for a broad range of bio-analytes detections.
Thesis (Ph.D.) (Research by Publication) -- University of Adelaide, School of Chemical Engineering, 2018

Biological/biochemistry analyses traditionally require bulky instruments and a great amount of volume of biological/chemical agents, and many procedures have to be performed in certain locations such as medical centers or research institutions. These limitations usually include time delay in testing. The delays may be critical for some aspects such as disease prevention or patient treatment. One solution to this issue is the realization of point-of-care (POC) testings for patients, a domain in public health, meaning that health cares are provided near the sites of patients using well-designed and portable medical devices. Transportation of samples between local and central institutions can therefore be reduced, facilitating early and fast diagnosis. A closely related topic in engineering, lab-on-a-chip (LOC), has been discussed and practiced in recent years. LOC emphasizes integrating several functions of laboratory processes in a small portable device and performing analysis using only a very small amount of sample volume, to achieve low-cost and rapid analysis. From an engineer's point of view, LOC is the strategy to practice the idea of POC testing. This dissertation aimed at exploring the POC potentials of porous membrane-base LOC devices, which can be used to simplify traditional and standard laboratory procedures. In this study, three LOC prototypes are shown and discussed. First the protein sensor incorporating with silica nanofiber membrane, which has shown 32 times more improvement of sensitivity than a conventional technique and a much shorter detection time; secondly the bacteria filter chip that uses a sandwiched aluminum oxide membrane to stabilize the bacteria and monitor the efficacy of antibiotics, which has reduced the test time from 1 day of the traditional methods to 1 hour; the third is the sensor combining microfluidics and silica nanofiber membrane to realize Surface Enhanced Raman Spectroscopy on bio-molecules, which has enhancement factor 10^9 and detection limit down to nanomolar, but simple manufacturing procedures and reduced fabrication cost. These results show the porous-base membrane LOC devices may have potentials in improving and replacing traditional detection methods and eventually be used in POC applications.

碩士
國立臺灣大學
電子工程學研究所
100
In recent years, the use of nanowire transistors as a new bio-molecular measurements device has became a good tool in the detection of the disease. This new biosensor could measure bio-molecular signals in a short time, also provides the advantages of high sensitivity, high selectivity, small size and probability to mass production. However, there are still some problems in this new technology need to be overcome, especially the stability and sensitivity characteristics which are the two most widely discussed. The improved methods can be broadly divided into three types, one is bio–abio interface, one is the device structure improvements, and the last one is fabrication technique. There are two parts of discussion in this paper, the first discussion is to discuss the different structures consists of single-nanowire, multiple-nanowires and ribbon- nanowire. The second discussion is to discuss the difference between heavy ion implantation in S/D device and normal ion implantation device. Both discussions are focused on the comparison of the different bio-molecular sensing properties in these two devices. To verify the purpose of this thesis wants to achieve, the various devices are fabricated and experimentally verified. The silicon nanowire thin-film transistor is bio-functionalized to have the specific-binding to testing DNA strands, so the DNA concentration of the test. Then we use the change of the threshold voltage to analyze the sensitivity and stability of bio-molecular sensor of silicon nanowire transistors. By the two analytical results, this paper can clearly demonstrate the effect of the comparison of different structure and different ion concentration in biomolecular sensing.

Surface- Enhanced Raman Scattering (SERS) and Tip-Enhanced Raman Scattering (TERS) exploit the enhancement of the local electromagnetic field induced by optical nanoantennas and nano-tips to largely amplify the Raman scattering of molecules located in the near-field of these structures, the so called hot-spots. With SERS or TERS, Raman scattering amplifications up to ten orders of magnitude can be obtained. SERS sensors based on nanoparticles of different materials and shapes have been developed for applications in the detection of pollutants, biomarkers, explosives, food pathogens, etc. Large area, high reproducibility, chemical stability, even flexibility, combined to high enhancement factors are key factors that are driving the research efforts in this field. TERS, in addition to SERS, permits to place and spatially control the position of the hot spot at the tip apex, allowing for imaging with nanoscale resolution or, under proper conditions, atomic scale resolution. Several TERS commercial setups are now available, coupling compact Raman spectrometers with with scanning probe microscopy (SPM) platforms, like atomic force microscopy (AFM), scanning tunneling microscopy (STM) or Shear-Force Microscopy (ShFM). These techniques can offer new original approaches in many fields, such as analytical chemistry, biology and biotechnology, forensic science and cultural heritage. One of the most appealing application field for SERS and TERS is biomolecular spectroscopy. Biomolecular sensors have become fundamental in biomedical research and the human health improvement. One of the crucial applications is in early stage diagnosis of diseases, through the detection, identification and quantification of some specific proteins, generally called biomarkers, that are present in very low quantities in body fluids (sub-nanomolar range). Immunoassays, such as enzyme-linked immunosorbent assay (ELISA), protein arrays, Western blots, immune-histochemistry and immune-fluorescence are well assessed probes The sensitivity is generally in the µM range, although new strategies have been proposed to achieve sub-femtomolar sensitivity. Moreover, these techniques are “indirect” methods, characterized by long operation times (several hours) and, especially at ultralow concentrations, can yield a large number of false positive detection events. Surface- and Tip- Enhanced Raman spectroscopies can tailor molecular sensitivity down to the atto-molar range, and to the single-molecule level in some particular configurations. Different concepts of SERS-based biosensors have been demonstrated so far. Raman dye-labeled sensors exploit SERS-active labels (NPs coated with high Raman cross-section dyes and functionalized with antibodies against the target molecule) to spot proteins, permitting their indirect detection (the signal of the dye is monitored) also in-vivo. Direct, label-free SERS sensors, in which the vibrational fingerprint of the target molecule is used for detection, are, however, desirable. This is due to operational rapidity, simplicity and richness of information embedded in the vibrational spectrum (e.g. on the functional state of a protein). Recently much effort has been addressed to SERS detection of biomolecules in liquid environment, i.e. their natural habitat. New tools for the manipulation of plasmonic nanoparticles in liquid environments like proteins buffered solutions, or in cells cultures are required for this task. The use of optical forces exerted by tightly focused laser beams on micro and nanostructures, is among the most promising emergent strategies for manipulation, control and SERS detection of molecular and biomolecular compounds in liquid. Optical forces can be used to attract or push micro and nano-objects from the focus of a lens (typically a microscope objective) in a contactless way. In particular, the use of the radiation pressure, has no restriction on excitation wavelength and permits to push nanoparticles along the beam direction, permitting to induce and control the formation of SERS-active aggregates in liquid. This latter point is yet largely unexplored among the scientific community. A crucial point for an efficient biosensor is the selective interaction of its active surface with biomolecules. In this framework the employment of bioreceptors increases the affinity of the sensor with the target molecule. Antibodies have been used in many SERS detection schemes for the functionalization of metal nanoparticles, but usually a Raman-active dye molecule is used as label for protein detection, due to the large dimensions of antibodies that prevent biomolecules to feel the plasmonic field enhancement. A very interesting alternative to antibodies is represented by DNA aptamers, whose nucleobases sequence can be properly designed for the specific binding with target biomolecules. Aptamers are more efficient and smaller with respect to antibodies, allowing for a direct characterization of the vibrational fingerprint of the biomolecules selectively linked to DNA strands. In addition chemical manipulation of aptamers is simpler, enabling the possibility to include a free thiol group at the end of DNA sequence for increasing the affinity with gold surfaces of SERS biosensors. Therefore the combination of the high sensibility and specificity within a label free approach, make SERS biosensors very appealing tools that can overcome the limitations of the conventional immunoassays in the way towards early diagnosis of cancer diseases. The work presented in this Thesis was focused, on one side, on the study of the basic electromagnetic processes in SERS, including polarization- issues, targeted at optimizing the excitation geometry of a nanosensor and, on the other side, on the development of new strategies for high sensitivity and specific detection of biomolecules in dry and liquid conditions by means of Surface- and Tip- enhanced Raman spectroscopy. In the initial part the attention has been focused on polarization issues related to the twofold electromagnetic enhancement mechanism in Surface enhanced Raman Scattering (SERS). We have discussed how the re-radiation effect can strongly alter the polarization state of the SERS radiation, influencing the SERS depolarization ratio. We have developed a model to relate the SERS depolarization to the molecular depolarization ratio, an intriguing physical quantity that provides information on the molecular orientation. Furthermore, we have studied, both theoretically and experimentally, the polarization properties of SERS from near-field coupled nanowires excited with circularly polarized light. From a practical point of view, this configuration turns out to be very attractive since the signal intensity becomes insensitive to the exact orientation of the sample, making sensors more robust to optical misalignments. Afterwards, driven by the necessity to develop new SERS sensors featuring large area, low cost, highly reproducibility and field enhancement, we have characterized SERS enhancement of novel nanostructures, namely Au nanocrescents evaporated over monolayers of polymeric nanospheres and asymmetric Au nanoclusters grown on flexible PDMS substrates. In both cases we have found a signal amplification up to four orders of magnitude. We have shown the possibility to employ Au nanocrescents in the detection of haemoglobin, reaching a detection limit of 100 pM. Au/PDMS plasmonic substrates were employed for first measurements on mitochondria, suggesting the possibility to detect via SERS the properties of cytochrome c molecules contained in these organelle. Subsequently we faced the problems related to the in-liquid SERS detection of biomolecules. We have exploited a strategy (LIQUISOR) based on optical forces to push and form SERS-active aggregates of gold nanorods (NR) mixed with proteins. Working on Bovine Serum Albumin (BSA), we have reached a limit of detection of 50nM, we have studied the growth kinetics of the optically induced aggregate, its morphology by SEM images and demonstrated that LIQUISOR can provide quantitative information on the protein concentration. A model has been developed that describes the process of nanorod/protein binding and size increase of the bio-nanorod composite, supported by dynamic light scattering measurements. To assess the efficiency and versatility of the LIQUISOR methodology we have applied it to the detection of Lysozyme (Lys), Hemoglobin (Hgb) and Catalase (Cat). Detection at physiological pH was demonstrated in all cases, reaching sensitivities of few ng/mL (picomolar) and high SERS gains (seven orders of magnitude for Hgb). Finally, we carried out first proof of principle experiments of of high sensitive and selective LIQUISOR detection of MnSOD (4.5 nM) and Ochratoxin A (1 µM), exploiting aptamers to functionalize the gold NRs and add specificity to the LIQUISOR methodology. In the last part of this thesis the attention was moved to Tip enhanced Raman spectroscopy. Firstly we have developed a fast and efficient double-step electrochemical etching for the fabrication of nanotips featuring radius of curvature lower than 35 nm starting from Au wires of 125 µm diameter. Homemade tips were used to obtain TERS spectra of Rhodamine 6G (R6G), Methylene Blue, Crystal violet (CV) and Alizarin. TERS enhancement factors EF higher than four orders of magnitude were obtained. TERS imaging was demonstrated on a molecular film of R6G and CV adsorbed on a flat gold substrate to discriminate aggregates of molecules with a spatial resolution of 3 nm. TERS was finally applied in the biomolecular field to high sensitivity spectroscopy of HypF-N oligomers, analogues of amyloid oligomers, the constitutive elements of amyloid fibrils, responsible for several neurodegenerative diseases. Our first results highlight the potentiality of TERS effect for the detection of small biological systems. Notably, our results evidence the possibility to discriminate among different structural conformation of the oligomers, one of which exhibiting a toxic behavior that leads to the formation of misfolded amyloid fibrils in Parkinson’s disease. Several possible developments are envisaged for both the LIQUISOR and the TERS methodologies. The LIQUISOR is of rapid use (few minutes), experimentally simple (standard micro-spectrometers and commercial nanorods are used), reliable and intrinsically scalable to lab-on-chip devices. Higher specificity can potentially be achieved by centrifuging functionalized NR mixed to the protein in order to separate free molecules in complex fluids from the target proteins interacting with aptamer functionalized NR or employing functionalized surfaces (instead of glass slides) to increase the affinity between BIO-NRCs and substrates and thus to speed up the aggregation process. On the other side higher sensitivity can be potentially achieved adopting silver nanoplatelets, as well as core-shell nanostructures. The use of laser beams in the optical transparency window of biological tissues could enable the application of our scheme in combination with optical injection of nanoparticles into living cells for in-vivo SERS biomolecular detection. TERS, with its high sensitivity and spatial resolution has already demonstrated potentialities in the field of DNA sequencing and can have unique applications in proteomics, allowing for the detection of single amino-acid alterations, e.g. phosphorylation, in complex proteins. The fabrication of new tips, more efficient, less expensive and more reproducible is another future challenge. Besides applications in the nanomedicine field, TERS can have important applications in cultural heritage field, for identification of inks on paper, dyes on statues which can be essential for dating, restoring and conserving the artwork.

碩士
大同大學
生物工程學系(所)
95
Fiber-optic surface plasmon resonance (SPR) can be used as a rapid and quantitative assay, and is also an important tool for monitoring biomolecular interactions. Furthermore, the use of fiber-optic SPR provides a number of potential advantages. Because the fiber optic sensors are reproducible and inexpensive to be disposable, binding kinetics and screening analyses can be performed in complex media without fear of irreversibly fouling expensive chipbased SPR platforms. Additionally, the small size of the fibers optic probe and sensing area make the sensors ideal for studying small volumes of samples. The applications are more and more than before. In this study, we used the fiber-optic SPR sensor designed and obtained from Graduate Institute of Electro-Optical Engineering in Tatung University to determine the small biomolecules. The nanogold particle and precipitation of 4-choro-1-naphthol were used to amplify the detection signals of SPR. The amplification method using nanogold particle was insignificant, but one using precipitation by reducing 4-choro-1-naphthol was able to enhance significantly the SPR signals. The signal changes of wavelength shift were consistent to the increasing in the concentrations of biotin-conjugated albumin bovine serum (biotin-BSA) and ochratoxin A-conjugated ovalbumin (OTA-OVA). Therefore, these results indicated that the amplification method of reducing precipitation by enzyme was suitable to the fiber-optic SPR detection system. The detection limiting for biotin-BSA was 0~10 �慊/ml, and 10~20 ng/ml for biotin.

碩士
大同大學
材料工程學系(所)
95
The plasma deposition and surface grafting polymerization have been used to gradually formed the organic layers on inorganic substrates such as interdigital electrodes devices for single strand DNA immobilization. In this study, the semiconductive tin oxide (SnOX) organic-like thin films were deposited by PECVD of TMT and O2 mixtures on the comb-shaped electrodes as the humidity sensor devices. To improve the sensitivity and stability for humidity sensing, the surface of the deposited flims was subsequently grafted with AAm (acrylamide). Hence, GA as cross-linking reagent to chemical bonding NH2-oligonucleotide probes on the surface of interdigital electrodes. As the surface of sensitive electrodes was immobilized the single strand DNA fragment of Vibrio parahaemolyticus or antibody, ionic molecules, etc. The grafted ionic molecules film enhances the impedance variations with humidity. When surface immobilization of antigen, the results appear the best sensitive range for humidity is R.H. 35∼95％, and the impedance variations decreases by over 5 orders of magnitude from 107 to 102 W .

Zinc oxide (ZnO) is a well-known II-VI semiconductor material that has gained renewed interest in the past decade due to the developments of growth technologies and the availability of high-quality ZnO bulk single crystals. Owing to a wide direct band gap (3.37 eV), large exciton binding energy (60 meV), and high electron mobility (440 cm2 V-1 s-1), ZnO has been used for applications including actuators, optoelectronics, and sensors. ZnO nanoparticles can be synthesized in a broad variety of morphologies, such as nanotetrapods, nanotubes, and nanowires. Among these nanostructures, the tetrapods have attracted significant attention due to their unique morphology consisting of four legs connected together in a tetrahedral symmetry. Recently, it has been reported that nano-microstructured ZnO tetrapods (ZnO-Ts) can be synthesized by flame transport synthesis (FTS) in a rapid and up-scalable approach. Compared to conventional ZnO nanoparticles, the nano-microstructured ZnO-Ts can reduce cellular uptake, while still exhibiting specific nanomaterial properties due to the nanoscale tips. Moreover, the anisotropic ZnO-Ts have the advantages of multiple electron transfer paths, chemical stability, and biocompatibility, which make the ZnO-Ts promising candidates for biomolecule sensing applications. This work herein reports a systematical study on the structural, optical and electrochemical properties of the ZnO-Ts, which were synthesized by FTS using precursor Zn microparticles. The morphology of the ZnO-Ts was confirmed by scanning electron microscopy (SEM) as joint structures of four single crystalline legs, of which the diameter of each leg is 0.7-2.2 μm in average from the tip to the stem. The ZnO-Ts were dispersed in glucose solutions to study the photoluminescence as well as photocatalytic activity in a mimicked biological environment. The photoluminescence (PL) intensity in the ultraviolet (UV) region decreased with linear dependence on the glucose concentration up to 4 mM. The ZnO-Ts were also attached with glucose oxidase (GOx) and over coated with Nafion® to form the active media for electrochemical glucose sensing. The active layers were confirmed by Fourier transform infrared spectroscopy (FT-IR). Furthermore, the current response of the active layers to glucose was studied by cyclic voltammetry (CV) in various glucose concentration conditions. Stable current response to glucose was detected with linear dependence on the glucose concentration up to 12 mM, which confirms the potential of ZnO-Ts for biomolecule sensing applications.

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

abstract: My research centers on the design and fabrication of biomolecule-sensing devices that combine top-down and bottom-up fabrication processes and leverage the unique advantages of each approach. This allows for the scalable creation of devices with critical dimensions and surface properties that are tailored to target molecules at the nanoscale. My first project focuses on a new strategy for preparing solid-state nanopore sensors for DNA sequencing. Challenges for existing nanopore approaches include specificity of detection, controllability of translocation, and scalability of fabrication. In a new solid-state pore architecture, top-down fabrication of an initial electrode gap embedded in a sealed nanochannel is followed by feedback-controlled electrochemical deposition of metal to shrink the gap and define the nanopore size. The resulting structure allows for the use of an electric field to control the motion of DNA through the pore and the direct detection of a tunnel current through a DNA molecule. My second project focuses on top-down fabrication strategies for a fixed nanogap device to explore the electronic conductance of proteins. Here, a metal-insulator-metal junction can be fabricated with top-down fabrication techniques, and the subsequent electrode surfaces can be chemically modified with molecules that bind strongly to a target protein. When proteins bind to molecules on either side of the dielectric gap, a molecular junction is formed with observed conductances on the order of nanosiemens. These devices can be used in applications such as DNA sequencing or to gain insight into fundamental questions such as the mechanism of electron transport in proteins.
Dissertation/Thesis
Doctoral Dissertation Physics 2019

This doctoral thesis investigates the application of laser-induced breakdown spectroscopy (LIBS) for detection of labeled biomolecules on nitrocellulose paper. Nitrocellulose paper is a material often used for assays involving the concentration and labeling of a target analyte, followed by label detection. Among paper-based diagnostics are lateral-flow immuno-assays (LFIAs). Research efforts have made LFIAs into accessible, portable,and low-cost tools for detecting targets such as allergens, toxins,and microbes in food and water.Gold (Au) nanoparticles are standard biomolecular labels among LFIAs, typically detected via colorimetric means.Other labels, such as quantum dots, are also often metallic, and research is ongoing to expand the number of portable instrumentations applied to their detection. A wide diversity of lanthanide-complexed polymers (LCPs) are used as immunoassay labels but have been inapt for portable paper-based assays owing to lab-bound detection instrumentation, until now. LIBS is a multi-element characterization technique which has recently developed from a bench-top to a portable/hand-held analytical tool. This is among the first studies to show that LCPs can be considered as options for biomolecule labels in paper-based assays using bench-based and hand-held LIBS as label detection modalities.

博士
國立中央大學
化學工程與材料工程研究所
93
Biological events are initiated by bio-molecular interactions, e.g. protein-protein, protein-nucleic acid, protein-lipid and chemical ligand-receptor interactions. The kinetics, thermodynamics and conformational matching involved in the contact between two or more molecules determine whether the interaction has any biological meaning in the real world. To clearly characterize the process of molecular interactions and further explore their true behaviors in biological systems, highly sensitive, specific, precise and fast biosensors are required to obtain the real-time and quantifiable information of interactions. In this study, we investigated the application of surface plasmon resonance sensor (SPR) in the bio-molecular interactions analysis. The core of molecular recognition lies on the study on immobilized bio-molecule conformation, which is a very challenging research field.　In the regard of uncertainty analysis of SPR, we evaluated the source and composition of signals of angle measurements and established the principle of data analysis. Based on this, we disclosed the effect of interactions between bulk solution, solute and immobilized matrix on measurement. Further more, the direct observation of conformational changes of immobilized proteins in unfolding and refolding derived a lot of important structural information of protein, such as the compactness of conformations, the exposure of hydrophobic region and the effects of intrinsic disulfide bonds on structural stability of immobilized proteins. On the other side, the combination of real-time monitoring capacity of QCM and microscopic surface morphology by AFM was employed to investigate the effects of liposome composition on the structure of supported lipid bilayer and the kinetics mechanism of absorption at the solid-liquid interface. The formula of stable vesicular lipid bilayer was established. We also demonstrated the possibility of application of vesicular lipid bilayer in biophysical and biochemical studies by the transmembrane process of melittin. We believe that the mimetic membrane systems would be ideal platform candidates to study bio-molecules interactions in the application of biosensors.