Techn. Hochsch., Diss., 2004--Aachen.

Magister Scientiae - MSc / Glucose oxidase (GOx) and horseradish peroxidase (HRP) are important enzymes for the development of amperometric enzyme linked immunosensors. The selectivity of each enzyme towards its analyte deepens its importance in determining the sensitivity of the resultant immunosensor. In designing immunosensors that have customized transducer surfaces, the incorporation with FAD and iron based enzymes ensures that electron kinetics remains optimal for electrochemical measurement. Various different immobilization strategies are used to produce response signals directly proportional to the concentration of analyte with minimal interferences. The combination of self-assembled monolayers and supramolecular chemistry affords stability and simplicity in immunosensor design. In this work, two electrochemical strategies for the detection of human chorionic gonadotropin (hCG) is presented. This involves the modification of a gold surface with a thiolated β- cyclodextrin epichlorohydrin polymer (βCDPSH) to form a supramolecular inclusion complex with ferrocene (Fc)-functionalised carboxymethyl cellulose polymer (CMC). Cyclic voltammetry indicated that ferrocene is in close proximity to the electrode surface due to the supramolecular complex formed with βCDPSH. Furthermore, strategy (a) for the detection of hCG used α-antihCG labelled (HRP) as reporter conjugate. Strategy (b) maintained the CMC bifunctionalised with Fc and recognition antibody for hCG hormone. However, the system was functionalised with a HRP enzyme and detection is done by using GOx reporter conjugates for in situ production of hydrogen peroxide. The reduction of H2O2 was used for the amperometric detection of hCG by applying a potential of 200 mV. The sensitivity and limit of detection of both strategies were calculated from calibration plots. For strategy (a) the LOD was found to be 3.7283 ng/mL corresponding to 33.56 mIU/mL and a sensitivity of 0.0914 nA ng-1 mL-1. The corresponding values for strategy (b) are 700 pg/mL (6.3 mIU/mL) and 0.94 nA ng-1 mL-1.

>Magister Scientiae - MSc / Prostate cancer is a deadly disease that occurs in the male’s prostate gland. A prostate gland is a walnut structure that forms part of the male’s reproductive system. Prostate cancer is caused by high level than normal of PSA (Gleason score > 4 ng ml-1) in human blood. Some symptoms associated with high levels of PSA include blood in urine, pain when urinating, difficulty in getting and keeping an erection, blood in semen and pain in upper thigh. An immunosensor is a type of biosensor that has an antigen or antibody fragment as its biological recognition component. The specificity of the molecular recognition of antigen by antibodies to form a stable complex is the basis of immunosensor technology. In this work, overoxidized polypyrrole (OvoxPpy) was electrosynthesized as a novel sensor platform on glassy carbon electrode (GCE). The OvoxPpy was then doped with osmium telluride quantum dots(OsTe2QDs) by drop-coating method to form OsTe2QDs|OvoxPpy|GCE system. The morphology and the size of OsTe2QDs|OvoxPpy|GCE nanocomposite were determined using scanning electron microscopy. The size of thioglycolic acid capped osmium telluride quantum dots (TGA-OsTe2QDs) used as support material for the biosensor was about 2.289 nm. These quantum dots showed an excellent photo-absorption properties with an ultraviolet- visible (UV-Vis) photo absortion band occurring at 406nm associated with high band energy of 3.05 eV. The electrochemical immunosensor for PSA was prepared by immobilizing anti- PSA-antibody onto the OsTe2QDs|OvoxPpy|GCE by drop-coating and allowing it to dry for 2h. The nanocomposite sensor platform and the immunosensor were electrochemically characterised by voltammetric and impedimetric techniques. The phase shift in Bode diagram at maximum frequency was indicative of kinetic changes. Charge transfer resistance, Rct, was used as the analytical parameter for measuring the interfacial kinetics which occurred as a result of the bio-recognition event between anti-PSA-antibody and PSA. The impedance of the quantum dot electrode (TGA-OsTe2QDs-Nafion|GCE) was lower (1.490 x 104 kΩ) than the impedance of the immunosensor platform (BSA-Anti-PSA-antibody|TGA-OsTe2 QDs|OvoxPpy|GCE), 2.754 x 104. The Rct of the immunosensor was found to increase with increasing concentration of PSA. The linearity of the immunosensor at the very low concentration range (1.266 - 4.207 fg ml-1) tested, confirms its high sensitivity for PSA.

abstract: Biosensors offer excellent diagnostic methods through precise quantification of bodily fluid biomarkers and could fill an important niche in diagnostic screening. The long term goal of this research is the development of an impedance immunosensor for easy-to-use, rapid, sensitive and selective simultaneously multiplexed quantification of bodily fluid disease biomarkers. To test the hypothesis that various cytokines induce empirically determinable response frequencies when captured by printed circuit board (PCB) impedance immunosensor surface, cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) methods were used to test PCB biosensors versus multiple cytokine biomarkers to determine limits of detection, background interaction and response at all sweep frequencies. Results indicated that sensors for cytokine Interleukin-12 (IL-12) detected their target over three decades of concentration and were tolerant to high levels of background protein. Further, the hypothesis that cytokine analytes may be rapidly detected via constant frequency impedance immunosensing without sacrificing undue sensitivity, CV, EIS, impedance-time (Zt) methods and modeling were used to test CHITM gold electrodes versus IL-12 over different lengths of time to determine limits of detection, detection time, frequency of response and consistent cross-platform sensor performance. Modeling and Zt studies indicate interrogation of the electrode with optimum frequency could be used for detection of different target concentrations within 90 seconds of sensor exposure and that interrogating the immunosensor with fixed, optimum frequency could be used for sensing target antigen. This informs usability of fixed-frequency impedance methods for biosensor research and particularly for clinical biosensor use. Finally, a multiplexing impedance immunosensor prototype for quantification of biomarkers in various body fluids was designed for increased automation of sample handling and testing. This enables variability due to exogenous factors and increased rapidity of assay with eased sensor fabrication. Methods were provided for simultaneous multiplexing through multisine perturbation of a sensor, and subsequent data processing. This demonstrated ways to observe multiple types of antibody-antigen affinity binding events in real time, reducing the number of sensors and target sample used in the detection and quantification of multiple biomarkers. These features would also improve the suitability of the sensor for clinical multiplex detection of disease biomarkers. / Dissertation/Thesis / Ph.D. Bioengineering 2012

Die vorliegende Arbeit befasst sich mit der Entwicklung und Anwendung elektrochemischer Biosensoren zur Erweiterung oder zum Ersatz herkömmlicher Diagnostikverfahren. Als Basis für die Biosensoren wurden Elektrodenarraychips entworfen und im Reinraum gefertigt. Die als 9WPtE bezeichneten Elektrodenarrays waren aus 3 x 3 Elektrodenpaaren im 96-well-Maßstab (ANSI-Standard) aufgebaut. Jedes Elektrodenpaar bestand aus einer kreisrunden Arbeitselektrode mit einem Durchmesser von 1,9 mm und einer Gegenelektrode als offenem Kreisring um die Arbeitselektrode mit einem Durchmesser von 7 mm. Außerhalb des Reinraums wurden separate Messkammern und Ag/AgCl-Referenzelektroden integriert. Sowohl das Referenzsystem als auch die Signalqualität der 9WPtE-Elektrodenarraychips wurden mittels Zyklovoltammetrie, Impedanzspektroskopie und Rasterkraftmikroskopie analysiert und anhand dieser Untersuchungen optimiert. Das Augenmerk lag hierbei auf den Produktionsprozessen zur Herstellung der Elektrodenarraychips, auf den Elektrolytbedingungen für die elektrochemischen Messungen und auf der Recyclebarkeit der Chips. Die Funktionalisierung der Arbeitselektroden der 9WPtE-Chips erfolgte mit sich selbst-organisierenden Schichten aus Thiolen. An die Thiole wurden mittels Chemoligation die biologischen Erkennungskomponenten kovalent gekoppelt. Mit dem 9WPtE-Elektrodenarray wurde auf diese Weise ein funktionsfähiger kompetitiver Immunosensoren gegen den Tumormarker Tenascin C entwickelt. Außerdem wurden der 9WPtE-Chip und ein zusätzlich entwickelter Durchflusssensor, basierend auf dem Prinzip des 9WPtE, genutzt, um die Möglichkeit der Detektion ganzer eukaryotischer Zellen zu untersuchen.

O desenvolvimento de biossensores é um tema de pesquisa bastante promissor, uma vez que permite monitorar diversas classes de substâncias, que muitas vezes apresentam grande interesse nas mais diversas áreas da ciência. Biossensores são pequenos dispositivos que utilizam componentes biológicos como elementos de reconhecimento, ligados a um sistema dedetecção, transdução e amplificação do sinal gerado na reação com o analito-alvo. Podem ser utilizados diversos elementos, sendo os principais, atualmente, aqueles baseados em aptâmeros e nanomateriais, por sua alta especificidade e sensibilidade. Seu potencial de utilização varia desde a detecção e tratamento de doenças ou a medição de componentes nos fluidos biológicos, até o monitoramento ambiental e prevenção de contaminação e bioterrorismo. Neste projeto foi desenvolvido um biossensor eletroquímico cujo objetivo é identificar toxinas inoculadas em pacientes que sofreram acidentes com animais peçonhentos. Foram utilizados os anticorpos/imuniglobulinas comerciais e a peçonha bruta de jararaca (Bothrops) para fazer o estudo/desenvolvimento do biossensor. Neste trabalho foram apresentados os resultados da imobilização dos anticorpos (imunoglobulinas) sobre o eletrodo de trabalho, bem como sua resposta eletroquímica utilizando voltametria cíclica. As soluções utilizadas foram de NaCl 0,9% e tampão fosfato (0,1 mol.L-1) com pH=7,4 por ser bem próximo ao pH fisiológico, pois, posteriormente quer se investigar em plasma sanguíneo. Os voltamogramas cíclicos e a microscopia eletrônica de varredura mostraram a diferença do eletrodo com e sem a imobilização das imunoglobulinas, evidenciando que o biossensor é eficaz para o sistema analisado, sendo promissor aos estudos. Com o biossensor construído, foi investigada a resposta eletroquímica relativa à interação antígeno-anticorpo (veneno-soro) para analisar a interação específica entre eles, sendo o resultado positivamente o esperado. / The development of biosensors is a research topic very promising, since it allows you to monitor various classes of substances, which often exhibit great interest in several areas of science. Biosensors are small devices that use biological recognition elements and components, bound to a selfAdetecting system, transduction and amplification of the signal generated in the reaction with the target analyte it. Various elements, the main currently those based on aptamers and nanomaterials for their high specificity and sensitivity can be used. Their potential use varies from the detection and treatment of diseases or measurement of components in biological fluids to the environmental monitoring and contamination prevention and bioterrorism. In this project we developed an electrochemical biosensor whose objetico is inoculated identify toxins in patients who have suffered accidents with poisonous animals. Antibodies / commercial imuniglobulinas and the crude venom of pit viper (Bothrops) were used to study / development of the biosensor. In this work the results of immobilization of antibodies (immunoglobulins) on the working electrode and its electrochemical response using cyclic voltammetry were presented. The solutions used were 0.9% NaCl and phosphate buffer (0.1 mol L-1) of pH = 7.4 to be close to physiological pH and therefore further investigated whether in blood plasma. Cyclic voltammetry and scanning electron microscopy showed the difference of the electrode with and without immobilization of immunoglobulins, indicating that the biosensor is effective for the system analyzed, and promising to studies. With the biosensor constructed, we investigated the electrochemical response on the antigen-antibody (venom antiserum) interaction to analyze the specific interaction between them, the result being positively expected.

A dengue é uma doença negligenciada que carece de métodos diagnósticos rápidos nos primeiros dias de infecção. São quatro sorotipos diferentes, cuja monitoração é essencial para o controle da ocorrência de casos graves como a dengue hemorrágica. É urgente o desenvolvimento e disponibilização de um dispositivo capaz de suprir essa demanda, de modo que propomos a utilização de imunossensores potenciométricos, devido a facilidade de miniaturização e produção dos dispositivos e seu baixo custo, além da possibilidade de detecção direta (sem marcadores) e simplicidade de manuseio. Dispositivos sensores de pH, como o transistor de efeito de campo de porta estendida e separada (SEGFET) e amplificadores de instrumentação (AI) podem ser utilizados como transdutores de sinal para a reação antígeno-anticorpo, a partir da utilização de materiais não nernstianos, como o ouro, como plataforma sensível. A proteína NS1 do vírus da dengue é um excelente marcador da infecção, pois é secretada em altas concentrações pelo vírus no sangue de pessoas infectadas logo nos primeiros dias, de modo que o sistema preza pelo diagnóstico precoce da doença. Sua detecção é realizada através da imobilização de anticorpos anti-proteína NS1 na plataforma sensível, permitindo sua quantificação através da detecção da alteração local de carga. O eletrodo foi caracterizado por diversas técnicas de microscopia, entre elas de varredura, confocal e de força atômica, além da utilização de espectroscopia de impedância eletroquímica, permitindo um amplo conhecimento da superfície da membrana sensível. Os imunossensores desenvolvidos apresentaram alta sensibilidade, com capacidade de detecção da ordem de ng.mL-1. Na região linear da curva analítica, foram obtidos sensibilidade correspondente a (15.7 ± 4.4) .10-4 μA.μg.mL-1 para o SEGFET e (3.2 ± 0.3) mV.μg.mL-1 para o AI, sendo que este último apresenta uma maior estabilidade de sinal e dispensa a utilização de uma fonte variável de tensão, reduzindo o custo no desenvolvimento de um dispositivo diagnóstico comercial. Estes resultados levaram a um pedido de patente e o prosseguimento do projeto através da miniaturização do sistema e detecção em amostras reais. / Dengue is a neglected disease that lacks fast diagnosis methods in the first days of infection. There are four different serotypes, which monitoring is essential to the occurrence control of severe cases as dengue hemorrhagic fever. The development of a device capable of fulfilling this demand is urgent, so we propose the use of potentiometric immunosensors, since its ease of miniaturization, mass production, low cost and the possibility of direct detection (label-free). pH sensor devices, as the separated extended gate field effect transistors (SEGFET) and instrumentation amplifiers (AI) can be applied as transducers to the antibody-antigen reaction by using non-nernstian materials such as gold as sensitive membrane. The non-structural 1 (NS1) protein is an excellent marker of infection, since its secreted in high concentration in the blood of infected people by the dengue virus in the first days, prioritizing early diagnosis. Its detection is made by immobilization of anti-NS1 protein antibodies, allowing its quantification by local charge changes. The electrode was characterized by many microscopy methods, including scanning electron, confocal and atomic force, besides electrochemistry impedance spectroscopy, providing a wide knowledge of the membrane surface. The developed immunosensors showed high sensitivity with detection capacity in the order of ng.mL-1. In the linear range of the analytic curve, were obtained sensitivities of (15.7 ± 4.4) .10-4 μA.μg.mL-1 for the SEGFET and (3.2 ± 0.3) mV.μg.mL-1 for the AI, whereas the latter has high signal stability sparring the use of a variable voltage source, minimizing the costs in the development of a commercial diagnostic device. These results led to a patent and the project continues by working in miniaturizing and real samples detection.

Development of displacement El objetivo de este trabajo es explorar y explotar los principios de funcionamiento de un Inmunosensor Electroquímico de Desplazamiento [(Displacement Electrochemical Immunosensor (DEI)] y también del ELISA Indirecto Competitivo [(Indirect Competitive ELISA (ICE)] para la detección de 2,4,6-tricloroanisol (TCA). Para tal fin, se lleva a cabo el desarrollo racional de un ELISA Indirecto Competitivo. El ensayo desarrollado resulta capaz de detectar TCA en concentraciones 1ppt a 1 ppm, con un limite de detección de 4.2 ppt. El ensayo desarrollado puede tener un particular interés comercial en situaciones donde el tiempo experimental requerido es de menos de 80 minutos.Se desarrolla también un modelo matemático (MM) cuyo principal objetivo es permitir el desarrollo racional de un Inmunosensor Electroquímico de Desplazamiento (DEI). A pesar de las bajas constantes de afinidad observadas en los anticuerpos obtenidos para este trabajo, se logra desarrollar un DEI funcional cuyo limite de detección de TCA (0.2 ppm) se corresponde con los valores obtenidos a través del MM.La adsorción inespecífica (NSA) de proteínas es identificada como uno de los problemas críticos que impidieron alcanzar limites de detección más bajos. El uso del electroquímicamente compatible Cu UPD como barrera/control de la NSA, junto con la detección amperométrica del desplazamiento son propuestos en este trabajo como base o punto de partida para el desarrollo de inmunosensores que puedan ser operados sin necesidad de marcaje (labelling) o la adición de otros componentes diferentes de la muestra de interés (reagentless and labelless immunosensors). / The purpose of this work is to explore and exploit the principles of Displacement Electrochemical Immunosensing (DEI) and Indirect Competitive ELISA (ICE) to detect 2,4,6-trichloroanisole (TCA). The rational design of indirect competitive ELISA for TCA detection is attempted. The developed assay detects TCA at concentrations from 1ppt to 1 ppm, with a limit of detection of 4.2 ppt. The assay can be commercially useful in situations where less than 80 minutes total assay time is required. A mathematical model (MM) is developed for the rational design of an electrochemical displacement immunosensor (DEI). Despite the low affinity constants of the antibodies obtained for this work a functional DEI is developed with the predicted by the MM high limit of detection for TCA (0.2 ppm). The non-specific adsorption (NSA) of proteins is identified as a critical problem inhibiting further optimization of the DEI. The use non-insulating Cu UPD as NSA controller or electrochemically compatible blocking, together with amperometric displacement detection are proposed as a platform that could permit further development of reagentless and labelless immunosensors.

Ovarian cancer is a high mortality disease where early stage detection may have significant survival benefits. Promising next-generation non-invasive, biomarker-based screening modalities involve longitudinal monitoring of serum biomarkers and multi-marker panel detection. Here, rapid, sensitive, precise and multiplexable diagnostic platforms can facilitate biomarker validation along with early detection and screening, and this work attempts to exploit the programmable bio-nano-chip (p-BNC) immunosensor to address these specific translational needs in ovarian cancer. First, the p-BNC was adapted for Cancer Antigen 125 (CA125) quantitation, the current FDA standard, with prominent implications in novel early detection and screening modalities. Antibody pairs binding to distinct epitopes on CA125 were identified and the p-BNC operating variables (incubation times, flow rates and reagent concentrations) were attuned to deliver optimal analytical performance (inter- and intra-assay precision of 1.2% and 1.9% and Limit-of-Detection (LOD) 1.0 U/mL), competitive with current gold standards, but with a short analysis time of 43 minutes. Further validation of the system with advanced stage patient sera (n=20) demonstrated good correlation with 'gold standard' ELISA (R² = 0.97). Next, the p-BNC was adapted for concomitant analysis of CA125 and Human Epididymis Protein 4 (HE4), a novel multiplexed biomarker panel for early detection and screening. The HE4 immunoassay was developed to perform optimally with the 'rate determining' CA125 assay. Cross-reactivity analysis demonstrated high specificity multiplexing. The dose-response curves for the multiplexed CA125 and HE4 immunoassays were congruous with their singleplex counterparts with respective LODs of 0.51 U/mL and 4.18 pM and a total analysis time of 44 minutes. A small pilot scale clinical study was conducted to discriminate between surgically confirmed patient sera (n=8) and corresponding age-matched healthy controls (n=8) utilizing the multiplexed p-BNC, interpreted with a risk of ovarian malignancy algorithm. Successful discrimination was achieved between the groups with Receiver Operating Characteristic (ROC) curve AUC (Area Under the Curve) values of 1.00, 0.984 and 1.00 respectively for CA125, HE4 and the composite marker combination. Taken together, the analytical and clinical performance, multiplexing capabilities and the short turn-around times on the p-BNC offer methodological advancements over current gold standard techniques, indicating strong promise for ovarian cancer diagnostics. / text

In this dissertation, guided surface acoustic wave sensors were investigated theoretically and experimentally in detail for immunosensing applications. Shear horizontal polarized guided surface acoustic wave propagation for mass loading sensing applications was modeled using analytical modeling and characterized by perturbation analysis. The model verification was performed experimentally and a surface acoustic wave immunosensor case study was presented. The results of the immunosensing were also investigated using the perturbation analysis. Guided surface acoustic wave propagation problem was investigated in detail for gravimetric (or mass loading) guided wave sensors, more specifically for immunosensors. The analytical model was developed for multilayer systems taking viscoelasticity into account. The closed form algebraic solutions were obtained by applying appropriate boundary conditions. A numerical approach was used to solve dispersion equation. Detailed parametric investigation of dispersion curves was conducted using typical substrate materials and guiding layers. Substrate types of ST-cut quartz, 41° YX lithium Niobate and 36° YX lithium tantalate with guiding layers of silicon dioxide, metals (chromium and gold), and polymers (Parylene-C and SU-8) were investigated. The effects of frequency and degree of viscoelasticity were also studied. The results showed that frequency only has effect on thickness with same shaped dispersion curves. Dispersion curves were found to be unaffected by the degree of viscoelasticity. It was also observed that when there was a large shear velocity difference between substrate and guiding layer, a transition region with a gradual decrease in phase velocity was obtained. However, when shear velocities were close, a smooth transition was observed. Furthermore, it was observed that, large density differences between substrate and guiding layer resulted in sharp and with nearly constant slope transition. Smooth transition was observed for the cases of minimal density differences. Experimental verification of the model was done using multi-layer photoresists. It was shown that with modifications, the model was able to represent the cases studied. Perturbation equations were developed with first order approximations by relating the slope of the dispersion curves with sensitivity. The equations were used to investigate the sensitivity for material selection (substrate, guiding layer, and mass perturbing layer) and degree of viscoelasticity. The investigations showed that the sensitivity was increased by using guiding layers with lower shear velocities and densities. Among the guiding layers investigated, Parylene C showed the highest sensitivity followed by gold and chrome. The perturbation investigations were also extended to viscoelasticity and to protein layers for immunosensing applications. It was observed that, viscous behavior resulted in slightly higher sensitivity; and sensitivity to protein layers was very close to sensitivity for polymers. The optimum case is found to be ST-cut quartz with Parylene-C guiding layer for protein layer sensing. Finally, an immunosensing case study was presented for selective capture of protein B-cell lymphoma 2 (Bcl-2), which is elevated in many cancer types including ovarian cancer. The immunosensor was designed, fabricated, and experimentally characterized. An application-specific surface functionalization scheme with monoclonal antibodies, ODMS, Protein A/G and Pluronic F127 was developed and applied. Characterization was done using the oscillation frequency shift of with sensor used as the feedback element of an oscillator circuit. Detection of Bcl-2 with target sensitivity of 0.5 ng/ml from buffer solutions was presented. A linear relation between frequency shift and Bcl-2 concentration was observed. The selectivity was shown with experiments by introducing another protein, in addition to Bcl-2, to the buffer. It was seen that similar detection performance of Bcl-2 was obtained even with presence of control protein in very high concentrations. The results were also analyzed with perturbation equations.