The adaptation of standard integrated circuit (IC) technology for biosensors in drug discovery pharmacology, neural interface systems, environmental sensors and electrophysiology requires electrodes to be electrochemically stable, biocompatible and affordable. Unfortunately, the ubiquitous IC technology, complementary metal oxide semiconductor (CMOS), does not meet the first of these requirements. For devices intended only for research, modification of CMOS by post-processing using cleanroom facilities has been achieved by others. However, to enable adoption of CMOS as a basis for commercial biosensors, the economies of scale of CMOS fabrication must be maintained by using only low-cost post-processing techniques. The scope of this work was to develop post-processing methods that meet the electrochemical and biocompatibility requirements but within the low-cost constraint. Several approaches were appraised with the two most promising designs taken forward for further investigation. Firstly, a process was developed whereby the corrodible aluminium is anodised to form nanoporous alumina and further processed to optimise its impedance. A second design included a noble metal in the alumina pores to enhance further the electrical characteristics of the electrode. Experiments demonstrated for the first time the ability to anodise CMOS metallisation to form the desired electrodes. Tests showed the electrode addressed the problems of corrosion and presented a surface that was biocompatible with the NG108-15 neuronal cell line. Difficulties in assessing the influence of alumina porosity led to the development of a novel cell adhesion assay that showed for the first time neuronal cells adhere preferentially to large pores rather than small pores or planar aluminium. It was also demonstrated that porosity can be manipulated at room temperature by modifying the anodising electrolyte with polyethylene glycol. CMOS ICs were designed as multiple electrode arrays and optimised for neuronal recordings. This utilised the design incorporating a noble metal deposited into the porous alumina. Deposition of platinum was only partially successful, with better results using gold. This provided an electrode surface suitable for electric cell-substrate impedance sensors (ECIS) and many other sensor applications. Further processing deposited platinum black to improve signal-to-noise ratio for neuronal recordings. The developed processes require no specialised semiconductor fabrication equipment and can process CMOS ICs on laboratory or factory bench tops in less than one hour. During the course of electrode development, new methods for biosensor packaging were assessed: firstly, a biocompatible polyethylene glycol mould process was developed for improved prototype assembly. Secondly, a commercial ‘partial encapsulation’ process (Quik-Pak, U.S.) was assessed for biocompatibility. Cell vitality tests showed both methods were biocompatible and therefore suitable for use in cell-based biosensors. The post-processed CMOS electrode arrays were demonstrated by successfully recording neuronal cell electrical activity (action potentials) and by ECIS with a human epithelial cell line (Caco2). It is evident that these developments may provide a missing link that can enable commercialisation of CMOS biosensors. Further work is being planned to demonstrate the technology in context for specific markets.

The main technology currently used in diabetic care, monitors blood glucose and involves an invasive “fingerstick” step. However, low patient compliance and non-continuous glucose monitoring imply poor management of diabetes through this technology, which could lead to adverse and potentially life threatening conditions. In this context, non-invasive glucose sensing appears as an alternative that can bring a change in the prevention and management of the diabetic condition, promising to eliminate patient resistance towards more frequent monitoring and, hence, considerably improving diabetic’s control over glycaemia. However, no non-invasive technology has yet succeeded on the market over the long term. The research field is therefore open to innovative and performant non-invasive technologies. This thesis presents the development of a non-invasive biosensor which as a core principle accesses individual, privileged glucose pathways in the skin (such as hair follicles), allowing the extraction of glucose directly from the interstitial fluid, via reverse iontophoresis (RI). The transdermally extracted glucose is then electrochemically detected in a small size sensor with very high sensitivity. A fully developed technology based on this principle will not require fingerpricking and would thus eliminate users’ main barrier to glucose monitoring. The developed sensor is enzymatic (using glucose oxidase), which electrochemically detects the produced H2O2; while the electrode material is graphene produced by Chemical Vapour Deposition, a promising carbon nanomaterial platform for biofunctionalisation and biosensing. The sensor is a miniature one (typically of 9 mm2 area, containing 24 μL of gel encasing the enzyme), with demonstrated performance parameters that are highly competitive (sensitivity of 2.89 μA.mM-1.cm-2 and limit of detection down to 1 μM), with high specificity towards glucose. The combination of this sensor with glucose extraction by reverse iontophoresis was then validated (with proportionality between subdermal and extracted glucose concentrations demonstrated); as well as enhanced extraction through targeting of hair follicles with the miniature device. The electrochemical determination of glucose concentration was further confirmed by 1H quantitative-NMR detection of glucose. Finally, several such sensors were integrated in a multiplex configuration, and independent sensing, with no cross-talk was demonstrated. The steps demonstrated and implemented so far are proof-of-concept of a highly promising non-invasive, transdermal, future technology for diabetic care.

Surface plasmon resonance (SPR) biosensors have attracted great attention in scientific research in the past three decades. Extensive studies on the immobilisation of biorecognition elements have been conducted in pursuit of higher sensitivity, but trialled formats have focussed on a thin layer modification next to the plasmon film, which usually requires in situ derivatization. This thesis investigates an 'off-chip' immobilisation strategy for SPR biosensing using silica particles and considers the implications of a particle-modified evanescent field on the signal amplitude and kinetics, for an exemplar affinity binding between immobilised IgG and its anti-IgG complement. Submicron silica particles were synthesized as carriers for the bio-recognition elements. They were then immobilised to form a sub-monolayer on the gold film of an SPR biosensor using two methods: thiolsilane coupling and physical adsorption aided by mechanical pressure. The bio-sensitivity towards an antigen/antibody interaction was lower than an SPR biosensor with an alkanethiolate SAM due to the difference in ligand capacity and position in the evanescent field. The binding kinetics of antigen/antibody pair was found to follow the Langmuir model closely in a continuous flow configuration but was heavily limited by the mass transport from the bulk to the sensor surface in a stop-flow configuration. A packed channel configuration was designed with larger gel particles as ligand carriers, packed on top of a gold film to create a column-modified SPR biosensor. This sensor has comparable bio-sensitivity to the previous sub-monolayer particle-modified systems, but the binding and dissociation of the analyte was heavily dependent on mass transport and binding equilibria across the column. A bi-directional diffusion mechanism was proposed based on a two-compartment mass transport model and the expanded model fitted well with the experimental data. The column-modified sensor was also studied by SPR imaging and analyte band formation was observed and analysed. Using the lateral resolution, a multiplexing particle column configuration was explored, and its potential in distinguishing a multicomponent analyte.

A pesquisa na área de biossensores de glicose tem crescido muito nos últimos anos, devido a sua grande importância no monitoramento contínuo da glicemia em pessoas com diabetes. O estudo da utilização de novos materiais nestes dispositivos, como os polímeros condutores e nanopartículas de ouro, tem sido alvo de extensas pesquisas. Neste trabalho, a polianilina (PAni), um dos polímeros condutores mais estudados, foi sintetizada quimicamente na presença de poli(óxido de etileno) (PEO) e também na presença de PEO e de ácido cloroáurico (HAuCl4), para a formação de nanopartículas de ouro (NPAu). Estes nanocompósitos foram utilizados na fabricação de um biossensor eficiente para glicose, servindo de suporte para a imobilização da enzima glicose oxidase (GOx) e de facilitadores do transporte de elétrons. Os polímeros foram caracterizados por infravermelho com transformada de Fourier (FT-IR), microscopia eletrônica de transmissão (MET), microscopia eletrônica de varredura (MEV), espectroscopia UV-visível, voltametria cíclica e pelo método padrão de 4 pontas. Para a produção dos biossensores, parâmetros como a quantidade de polímero a ser aplicada sobre os eletrodos, a concentração da GOx, o pH do eletrólito de realização dos ensaios eletroquímicos e a quantidade de mediador no eletrólito, foram avaliadas previamente por voltametria cíclica, a fim de encontrar a máxima resposta eletroquímica do biossensor. Além dos ensaios de voltametria cíclica, os biossensores foram caracterizados por espectroscopia de impedância eletroquímica e por cronoamperometria. Através dos ensaios de cronoamperometria foi verificado que o biossensor de PAni-PEO detecta glicose em uma faixa de concentrações de 1 a 10 mM, com sensibilidade de 16,04 μA mM-1 cm-2, e o de PAni-PEO-NPAu, na faixa de 0,1 a 5,5 mM, com sensibilidade de 5,5 e 0,76 μA mM-1 cm-2, nas faixas de concentração de 0,1 a 0,5 e de 1,5 a 5,5 mM, respectivamente. Além disso, ambos os biossensores apresentaram seletividade a interferentes como ácido ascórbico e ácido úrico, confirmando que o sinal gerado nos ensaios eletroquímicos refere-se efetivamente à detecção da glicose. / Research in the area of glucose biosensors has grown tremendously in recent years due to their great importance in continuous glucose monitoring in patients with diabetes. The study of the use of new materials in these devices, such as conductive polymers and gold nanoparticles, has been the subject of extensive research. In this work, polyaniline (PAni), one of the most studied conductive polymers, was chemically synthesized in the presence of polyethylene oxide (PEO) and also in the presence of PEO and chloroauric acid (HAuCl4) for the formation of gold nanoparticles (AuNP). These nanocomposites were used in the manufacture of an efficient glucose biosensor, serving as support for the immobilization of the enzyme glucose oxidase (GOx) and as electron transport facilitators. The polymers were characterized by Fourier transform infrared (FT-IR), transmission electron microscopy (TEM), scanning electron microscopy (SEM), UV-visible spectroscopy, cyclic voltammetry and standard 4-point pobe method. For the production of the biosensors, parameters such as the amount of polymer to be applied on the electrodes, the concentration of GOx, the electrolyte’s pH of the electrochemical tests and the amount of mediator in the electrolyte were previously evaluated by cyclic voltammetry to find the maximum electrochemical response of the biosensor. In addition to the cyclic voltammetry tests, the biosensors were characterized by electrochemical impedance spectroscopy and chronoamperometry. Through the chronoamperometry assays, it was verified that PAni-PEO biosensor detected glucose in a range of 1 to 10 mM, with a sensitivity of 16,04 μA mM-1 cm-2 and PAni-PEO-NPAu biosensor, in the range of 0,1 to 5,5 mM, with sensitivity of 5,5 and 0,76 μA mM-1 cm-2 in the 0,1 to 0,5 and 1,5 to 5,5 mM ranges, respectively. In addition, both biosensors presented selectivity to interferents such as ascorbic acid and uric acid, confirming that the signal generated in the electrochemical tests effectively refers to the detection of glucose.

This dissertation describes a new type of molecular assay for nucleic acids and proteins. We call this technique a digital microarray since it is conceptually similar to conventional fluorescence microarrays, yet it performs enumerative (‘digital’) counting of the number captured molecules. Digital microarrays are approximately 10,000-fold more sensitive than fluorescence microarrays, yet maintain all of the strengths of the platform including low cost and high multiplexing (i.e., many different tests on the same sample simultaneously). Digital microarrays use gold nanorods to label the captured target molecules. Each gold nanorod on the array is individually detected based on its light scattering, with an interferometric microscopy technique called SP-IRIS. Our optimized high-throughput version of SP-IRIS is able to scan a typical array of 500 spots in less than 10 minutes. Digital DNA microarrays may have utility in applications where sequencing is prohibitively expensive or slow. As an example, we describe a digital microarray assay for gene expression markers of bacterial drug resistance.

Made available in DSpace on 2015-04-11T13:38:27Z (GMT). No. of bitstreams: 1 Michella Bezerra Lima.pdf: 2830330 bytes, checksum: fabc8826135a63e76710a30d853375fa (MD5) Previous issue date: 2008-08-21 / The development of analytical and low cost methods for fast safe diagnosis and control of illnesses in humans has been one of researchers great interests in the last two decades. This attention is also due to simplicity of use and the great sensitivity in the results. Biosensors based on the surface plasmon resonance (SPR) technique combines the inherent specificity of the recognition elements with high sensitivity of physical transducers, enabling selective and sensitive detection of the analytes of interest without labeling and preliminary purification steps. We proposed the development of a biosensor with immobilized monoclonal antibodies on gold surface, for specific recognition of TNF-α using SPR technique. Design. Anti-TNF-α antibody was covalently immobilized on a gold substrate by silane coupling chemistry. After this, TNF-α antigens aliquots were added on the biosensor surface and the signal was measured during three minutes for TNF-α mass quantification recognized by immobilized antibodies. Results.This biosensor presented 24.0 pg.mm-2 (198,3  28,4) of antibodies immobilized on gold surface. TNF-α human was determined from successive injections, with a linear range from 4.3 up to 34.7 pg/mm-2 (R=0.98, SD=2.18, p<0.0001). Conclusion. We performed a preliminary biosensor to quantify TNF-α for planning of diseases treatment, such as auto immune and periodontal diseases, in which TNF-α inhibitor therapy is usually applied. In this cases, it would be necessary the quantification of this cytokine for therapeutic dose adjustment, improving treatment effectiveness and costs reduction. In the next step we intend to optimize the biosensors conditions preparation as well as to test it in human body fluids samples / Os imunossensores, ou biossensores de imunoafinidade são sistemas que exploram a seletividade e especificidade das interações antígeno-anticorpo, para detecção do analito alvo. O objetivo deste trabalho foi o desenvolvimento de um imunossensor, em que anticorpos monoclonais (anti-TNF-α humano) foram imobilizados em superfícies modificadas de ouro, para reconhecimento específico do Fator de Necrose Tumoral Alfa humano (TNF-α). Para a imobilização dessas imunoglobulinas foi necessária a formação de uma monocamada auto-organizada (SAM) sobre a superfície do sensor, composta por 3-aminopropiltrietóxisilano (APTS), sendo testadas quatro concentrações diferentes (1%, 2,5%, 5% e 10%), em dois tipos de solventes, água deionizada e tolueno, para comparação de metodologias para obtenção de melhores respostas. Os métodos de caracterização adotados foram a espectroscopia de Ressonância de Plásmon de Superfície (SPR), para análise da imobilização de anticorpos, na superfície modificada do sensor e a resposta dos sistemas desenvolvidos para detecção do TNF-α; e, Microscopia de Força Atômica (AFM), para verificação topográfica da SAM da superfície sensora. Os resultados mostraram que as melhores condições de desenvolvimento do biossensor desenvolvido neste trabalho ocorreram com a utilização de APTS 2,5% em tolueno, bloqueado com BSA 1%. O biossensor apresentou 24 pg.mm-2 (198,3  28,4 mgrau) de TNF-α humano imobilizados na superfície de ouro, e após injeções sucessivas de 25 pg.mL-1 de antígeno (n=8), em triplicata experimental, obtivemos uma região linear de resposta de 4,3 a 34.7 pg.mm-2 (R=0.98, SD=2.18, p<0.0001). Em análise topográfica das superfícies sensoras, realizada por AFM, foi observado que as amostras preparadas em tolueno, com melhores respostas de reconhecimento do antígeno, apresentaram menor uniformidade na sua estrutura, em razão da maior rugosidade detectada. Foi desenvolvido um imunossensor experimental, que servirá como modelo para o desenvolvimento de biossensor para aplicação clínica futura para quantificação de TNF- humano em fluidos corporais de pacientes portadores de doenças inflamatórias

<p>A proof-of-concept for a novel microarray used to study protein-ligand interaction in real-time using label-free detection is presented. Many of todays commercially available instruments lack the ability to immobilize membrane proteins. At the same time, the pharmaceutical industry develops drugs directed towards membrane-bound receptors. The need to study drug-target kinetics and to be able to screen for new medical substances is high. To study the biomolecular interactions in real-time, imaging surface plasmon resonance (iSPR) is used. A patterned sensor surface with hydrophobic barriers assisting in the piezodispensing of NeutrAvidin with complex-bound biotin-ssDNA is created. Histidine-tagged proteins are immobilized at the vesicle surface using divalent nitrilotriacetic acid. The concept of the vesicle immobilization, the protein-binding to vesicles and the protein-ligand interaction is initially studied using a Biacore instrument. The dissociation of the ligand IFNα2 from its receptor ifnar-2 (wt) are in accordance with the literature. In the imaging SPR experiments, it is found that the dissociation of IFNα2 from the ifnar-2 (wt) receptor is slower than expected, probably due to rebinding of the ligand. It is also found that imidazole is needed to avoid vesicle-vesicle interaction. The immobilization of proteins had to be done on-line i.e. when the vesicles were bound to the surface. Depending on the mixture of receptors at the vesicle surface the affinity for the ligand was changed. The results achieved were reproducible.</p>

In recent years, application of fluorescent conjugated polymers to sense chemical and biological analytes has received much attention owing to its technological significance. Water soluble conjugated polymers are interesting towards the developing sensors for biomolecules. In this present contribution, we describe the syntheses and characterization of a series of water soluble conjugated polymers with sulfonic acid groups in the side chain. Such anionic conjugated polymers are designed to interact with biomolecules such as cytochrome-C. All polymers are water soluble and showed strong blue emission. Significant quenching of the fluorescence from our functionalized PPP was observed upon addition of viologen derivatives or cytochrome -C. / Singapore-MIT Alliance (SMA)

Point-of-care (POC) diagnostics presents a giant market opportunity with profound societal impact. In particular, specific detection of DNA and protein markers can be essential for early diagnosis of e.g. cancer, cardiovascular disease, infections or allergies. Today, identification of these markers often requires extensive laboratory work and hence is expensive and time consuming. Current methods for recognition and detection of specific biomolecules are mostly optics based and thus impose severe limitations as to convenience, specificity, sensitivity, parallel processing and cost reduction. Electronic sensors based on silicon nanowire field-effect transistors have been reported to be able to detect biomolecules with concentrations down to femtomolar (fM) level with high specificity. Although the reported capability needs further confirmation, the CMOS-compatible fabrication process of such sensors allows for low cost production and high density integration, which are favorable for POC applications. This thesis mainly focuses on the development of a multiplex detection platform based on silicon nanowire field-effect sensors integrated with a microfluidic system for liquid sample delivery. Extensive work was dedicated to developing a top-down fabrication process of the sensors as well as an effective passivation scheme. The operation mechanism and coupling efficiencies of different gate configurations were studied experimentally with the assistance of numerical simulation and equivalent circuits. Using pH sensing as a model system, large effort was devoted to identifying sources for false responses resulting from the instability of the inert-metal gate electrode. In addition, the drift mechanism of the sensor operating in electrolyte was addressed and a calibration model was proposed. Furthermore, protein detection experiments were performed using small-sized Affibody molecules as receptors on the gate insulator to tackle the Debye screening issue. Preliminary results showed that the directionality of the current changes in the sensors was in good agreement with the charge polarities of the proteins. Finally, a graphene-based capacitor was examined as an alternative to the nanowire device for field-effect ion sensing. Our initial attempts showed some attractive features of the capacitor sensor.

Preliminary work towards the development of novel biosensing interfaces for EMPAS (ElectroMagnetic Piezoelectric Acoustic Sensor) is presented in this manuscript. This method involves the use of unprecedented thiosulfonate-based linkers to construct robust and durable SAMs (Self-Assembling Monolayer) onto piezoelectric quartz crystals, which can chemoselectively immobilize thiol-containing biomolecules under aqueous conditions in a single, straightforward, reliable and coupling-free manner. Initial efforts are devoted to the construction of SAMs and the subsequent immobilization of thiol-containing biomolecules, and then characterization by CAMs (Contact Angle Measurement) and ARXPS (Angle-Resolved X-ray Photoelectron Spectroscopy). This method is then implemented into the construction of biosensing interfaces dedicated to the detection of avidin. With the incorporation of OEG (Oligo(Ethylene Glycol)) backbone and diluent in the method, 14-fold difference in signal response of EMPAS was observed between biotinylated and unfunctionalized SAMs.