62 research outputs found
Design, Synthesis and Characterization of Oriented Glyco-Affinity Macroligands for Glyco-Capturing, Glycomics and Glycoproteomics Applications
Cell surface carbohydrates existing as parts of glycoproteins, glycolipids, and other conjugates present the first information about cell to the outside world and are intimately involved in various biological events such as intercellular communication, and molecular and cellular targeting. However, mechanisms of most processes at the molecular level are still unclear. Therefore, it is very important to develop carbohydrate-specific binding molecules for rapid, efficient, sensitive purification and accurate analysis of complex carbohydrate structures as well as their functions. Furthermore, carbohydrate-specific binding molecules can be expected to be used in medical diagnostic applications for carbohydrate biomarkers. In this thesis study, oriented and multivalent carbohydrate-binding macromolecules were designed and developed based on a chain-end functionalized boronic acid-containing polymer (boropolymer) for glyco-capturing, glycomics and glycoproteomics applications. Namely, a biotin chain end and O-cyanate chain-end functionalized boropolymers were synthesized via aryalamine initiated cyanoxyl-mediated free radical polymerization in a one-pot fashion. The resultant boropolymers were characterized by 1H-NMR and 13C NMR spectroscopy. In our first study we demonstrated the efficient glyco-capturing followed by direct MALDI mass spectrometry identification of the captured carbohydrate by using magnetic beads functionalized with the biotin boropolymer via streptavidin/biotin interaction. In our second study we demonstrated oriented and covalent immobilization of O-Cyanate chain-end functionalized boropolymer on to amine-modified solid surfaces and its specific glyco-capturing capacity by QCM and AFM techniques. We further studied the multivalent interactions of the immobilized O-Cyanate chain end functionalized boropolymer with five different carbohydrate conjugated AuNPs. Our studies showed that different carbohydrates have different binding constants. Furthermore, the multivalent binding between carbohydrate
Design, Synthesis and Characterization of Oriented Glyco-Affinity Macroligands for Glyco-Capturing, Glycomics and Glycoproteomics Applications
Cell surface carbohydrates existing as parts of glycoproteins, glycolipids, and other conjugates present the first information about cell to the outside world and are intimately involved in various biological events such as intercellular communication, and molecular and cellular targeting. However, mechanisms of most processes at the molecular level are still unclear. Therefore, it is very important to develop carbohydrate-specific binding molecules for rapid, efficient, sensitive purification and accurate analysis of complex carbohydrate structures as well as their functions. Furthermore, carbohydrate-specific binding molecules can be expected to be used in medical diagnostic applications for carbohydrate biomarkers. In this thesis study, oriented and multivalent carbohydrate-binding macromolecules were designed and developed based on a chain-end functionalized boronic acid-containing polymer (boropolymer) for glyco-capturing, glycomics and glycoproteomics applications. Namely, a biotin chain end and O-cyanate chain-end functionalized boropolymers were synthesized via aryalamine initiated cyanoxyl-mediated free radical polymerization in a one-pot fashion. The resultant boropolymers were characterized by 1H-NMR and 13C NMR spectroscopy. In our first study we demonstrated the efficient glyco-capturing followed by direct MALDI mass spectrometry identification of the captured carbohydrate by using magnetic beads functionalized with the biotin boropolymer via streptavidin/biotin interaction. In our second study we demonstrated oriented and covalent immobilization of O-Cyanate chain-end functionalized boropolymer on to amine-modified solid surfaces and its specific glyco-capturing capacity by QCM and AFM techniques. We further studied the multivalent interactions of the immobilized O-Cyanate chain end functionalized boropolymer with five different carbohydrate conjugated AuNPs. Our studies showed that different carbohydrates have different binding constants. Furthermore, the multivalent binding between carbohydrate
Peptoid and Antibody-based GFP Sensors
In this work, we have made and characterized a pair of immunobiosensors for detecting the green fluorescent protein (GFP) in an aqueous matrix. An anti-GFP antibody-based biosensor was assembled to detect GFP, while a novel peptoid (N-substituted oligomers of glycine designated as IOS-1) biosensor was also assembled for GFP detection. A quartz crystal microbalance (QCM) gold sensor was used as the supporting substrate for self-assembly of the immunobiosensors. Gravimetric measurements of the QCM gold sensor during immunobiosensor construction and operation were available in real-time using a QCM instrument. X-ray photoelectron spectroscopy, Fourier transform infrared spectroscopy, and Fluorescence microscopy were used to characterize the immunobiosensors. Dose-dependent calibration curves were developed to contrast the performance of the peptoid immunobiosensor and the antibody-based immunobiosensor. The sensitivity of the biosensors shows that the peptoid could detect GFP at 8 nM, unlike the antibody immunobiosensor, which starts to measurably detect GFP at 40 nM. IOS-1 peptoid immunobiosensor had more adsorption capacity for GFP than the antibody-based immunobiosensor and could be reused through multiple adsorption/ desorption cycles. The peptoid immunobiosensor had a binding constant of 2.197 x 10(7) M(-1) with GFP
Nanomaterials for Healthcare Biosensing Applications
In recent years, an increasing number of nanomaterials have been explored for their applications in biomedical diagnostics, making their applications in healthcare biosensing a rapidly evolving field. Nanomaterials introduce versatility to the sensing platforms and may even allow mobility between different detection mechanisms. The prospect of a combination of different nanomaterials allows an exploitation of their synergistic additive and novel properties for sensor development. This paper covers more than 290 research works since 2015, elaborating the diverse roles played by various nanomaterials in the biosensing field. Hence, we provide a comprehensive review of the healthcare sensing applications of nanomaterials, covering carbon allotrope-based, inorganic, and organic nanomaterials. These sensing systems are able to detect a wide variety of clinically relevant molecules, like nucleic acids, viruses, bacteria, cancer antigens, pharmaceuticals and narcotic drugs, toxins, contaminants, as well as entire cells in various sensing media, ranging from buffers to more complex environments such as urine, blood or sputum. Thus, the latest advancements reviewed in this paper hold tremendous potential for the application of nanomaterials in the early screening of diseases and point-of-care testing
Molecularly Imprinted Sensors — New Sensing Technologies
In this chapter we discus molecular imprinting technology (MIT), molecular imprinted polymers (MIPs), and their compatibility on a proper transducer to construct a sensing system. Molecularly imprinted sensors (MISens), in other words, artificial receptor-based sensors synthesized in the presence of the target molecule, are capable of sensing target molecules by using their specific cavities and are compatible with the target molecule. This MIP technology is a viable alternative of artificial receptor technology, and the sensor technology is capable of detecting any kind of molecule without pre-analytic preparations. In this chapter, you can find examples, sensor construction techniques and fundamentals of MIP and sensor combinations to look forward in your studies. For sensor technology, we explained and discussed the new sensing technologies of MIP-based electrochemical, optical (especially surface plasmon resonance, SPR), and piezoelectric techniques. Therefore, this chapter presents a short guideline of MISens
Soft and flexible material-based affinity sensors
Recent advances in biosensors and point-of-care (PoC) devices are poised to change and expand the delivery of diagnostics from conventional lateral-flow assays and test strips that dominate the market currently, to newly emerging wearable and implantable devices that can provide continuous monitoring. Soft and flexible materials are playing a key role in propelling these trends towards real-time and remote health monitoring. Affinity biosensors have the capability to provide for diagnosis and monitoring of cancerous, cardiovascular, infectious and genetic diseases by the detection of biomarkers using affinity interactions. This review tracks the evolution of affinity sensors from conventional lateral-flow test strips to wearable/implantable devices enabled by soft and flexible materials. Initially, we highlight conventional affinity sensors exploiting membrane and paper materials which have been so successfully applied in point-of-care tests, such as lateral-flow immunoassay strips and emerging microfluidic paper-based devices. We then turn our attention to the multifarious polymer designs that provide both the base materials for sensor designs, such as PDMS, and more advanced functionalised materials that are capable of both recognition and transduction, such as conducting and molecularly imprinted polymers. The subsequent content discusses wearable soft and flexible material-based affinity sensors, classified as flexible and skin-mountable, textile materials-based and contact lens-based affinity sensors. In the final sections, we explore the possibilities for implantable/injectable soft and flexible material-based affinity sensors, including hydrogels, microencapsulated sensors and optical fibers. This area is truly a work in progress and we trust that this review will help pull together the many technological streams that are contributing to the field
Antibodies immobilization
Human Cytomegalovirus (HCMV) is a herpes virus that establishes a lifelong latent infection which, in most of the immunocompetent individuals is normally subclinical. Severe infections occur more frequently in immunocompromised ones, or those with immature immune system, for which can be fatal. Glycoprotein B (gB) is the dominant antigen of HCMV envelope being regarded as a promising component in the establishment of new diagnostic tests.
Nowadays there are several diagnosis methods, however, these are expensive, or/and require long time to perform, or/and need skilled operators, or even leads to the possibility of false results. So, in previously work, a disposable immunosensor was developed based on electrochemical silver oxidation as response for gB concentration increasing, using screen-printing carbon electrodes. This method allows a faster and inexpensive way to detect that viral protein. Despite that, a lack on device reproducibility and sensitivity, due to the randomly antibody adsorption was found. It is known that the oriented immobilization has become critical for optimized antigen detection on solid surfaces; therefore, those results lead us to employ other immobilization techniques to improve the immunosensor performance.
In this work the immobilization of antibodies was carried out through glutaraldehyde cross-linking, by covalent immobilization using diazonium salts and finally by using the boronic acid affinity towards the carbohydrate present on the antibody molecules. The results were conclusive only for the cross-linking, which has disadvantages when compared with adsorption.
Additionally, the importance of the gB on diagnosis tests for HCMV, lead us to study its isolated electrochemical behavior. Questionable results were found demonstrating the impossibility of its use in the determination of gB in biological samples.O Citomegalovírus humano (HCMV) é um herpes vírus que pode originar infeções primárias, a partir das quais a reativação poderá ser frequente. Quando os hospedeiros deste agente viral são indivíduos imunocompetentes, é geralmente associada uma latência do HCMV que eventualmente poderá resultar em sintomas subclínicos. No entanto, em indivíduos imunocomprometidos, tais como os portadores do vírus da imunodeficiência humana (HIV) e os sujeitos a terapêuticas imunossupressoras ou ainda em indivíduos cujo sistema imune é imaturo, como é o caso de fetos e recém-nascidos, a infeção adquire proporções graves, podendo resultar na morte dos mesmos.
Este vírus apresenta um invólucro rico em proteínas virais das quais a glicoproteína B (gB) se destaca, pois está presente em aproximadamente 100% dos indivíduos infetados, sendo também reconhecida por desencadear a produção de anticorpos neutralizantes que levam à eliminação das células infetadas. Assim, esta proteína pode ser vista como um componente essencial no diagnóstico da infeção por HCMV.
A definição de um diagnóstico ideal para o HCMV tem sido difícil de implementar devido às desvantagens apresentadas pelos métodos existentes, que se baseiam em informações clinicas e imunológicas. Definido como método convencional, o isolamento do vírus em culturas de fibroblastos, obtido a partir de biópsias ou de fluidos dos hospedeiros, tem associadas as desvantagens de exigir assepsia total e longos períodos de tempo para a sua execução. Para contrapor as dificuldades desta técnica, um método idêntico, o “Shell-vial”, reduz o tempo do ensaio através de um passo de centrifugação que aumenta a penetração do HCMV nos fibroblastos, que por sua vez pode ser avaliada por imunofluorescência. No entanto, o sucesso deste método continua a depender das condições assépticas usadas. Por outro lado, o PCR (“Polimerase Chain Reaction”), analisa amostras clínicas com uma rápida performance e elevada sensibilidade, conseguida na amplificação de ADN viral. Contudo, o elevado custo e a dificuldade de realização contrapõem-se ao seu uso como técnica de diagnóstico corrente. Outra técnica, ELISA (“Enzyme-Linked Immunosorbent Assay”), deteta a presença de anticorpos específicos no sangue, o que pode levar a falsos positivos devido a reações cruzadas com o fator reumatoide, anticorpos antinucleares e outros membros da família herpesviridae. Usado para medir a afinidade e avidez do anticorpo para o antigénio viral, o “Western Blotting” apresenta uma baixa disponibilidade comercial conjugada também com a possibilidade de falsos positivos. Por último, testes citológicos/histológicos permitem observar inclusões virais em biópsias de tecidos do hospedeiro, contudo apresentam igualmente uma baixa sensibilidade tendo apenas 50% de sucesso na identificação de falsos negativos. Todos os métodos têm associadas desvantagens quanto a falsos resultados, ou equipamentos e/ou procedimentos caros, que podem estar ou não relacionados a uma difícil manipulação e elevado tempo de realização. Desta forma, para contrariar estes inconvenientes, num estudo anteriormente realizado neste grupo de investigação, foi desenvolvido um imunossensor eletroquímico descartável para a deteção do HCMV, tendo por base uma imunorreação do tipo sandwich na qual o anticorpo secundário estava marcado com nanopartículas de ouro (AbNPs). Esta marcação permitiu a posterior deposição catalítica de nanopartículas de prata (AgNPs), que geraram um sinal eletroquímico na sua redissolução anódica por voltametria de pulso diferencial. O uso de elétrodos serigrafados (SPEs) como base para o immunosensor, acrescenta vantagens tais como a miniaturização, baixo custo, versatilidade e principalmente a possibilidade de uso como “point of care”. Adicionalmente, a facilidade de produção destes dispositivos por impressão sequencial de camadas de tintas, oferece vantagens na manipulação de padrões e geometrias conforme o pretendido.
O maior desafio na construção deste tipo de biossensores, passa pela imobilização dos anticorpos na superfície dos elétrodos. Está descrito na literatura que a imobilização de anticorpos de forma orientada resulta numa melhor exposição dos locais de ligação aos antigénios, exibindo melhores capacidades de ligação e posterior deteção dos mesmos. De facto, foram detetadas algumas limitações relacionadas com este passo de construção do imunossensor, uma vez que os anticorpos anti-HCMV se encontravam adsorvidos de uma forma aleatória na superfície do elétrodo de trabalho. Desta forma, as moléculas de anticorpo apresentam uma orientação nem sempre ideal afetando a reprodutibilidade e a sensibilidade do dispositivo na resposta a concentrações de gB do HCMV.
Assim, com o objetivo de melhorar as características do immunosensor descartável, neste trabalho foram aplicadas várias técnicas para a imobilização dos anticorpos anti-HCMV, tais como a reticulação, imobilização covalente através de sais de diazónio e por último usando a afinidade do ácido borónico para os açúcares presentes nas moléculas de anticorpo. O glutaraldeído, usado como agente reticulante, permitiu a imobilização das moléculas de anticorpo anti-HCMV na superfície dos SPEs. O sistema mostrou responder às concentrações incubadas de gB, levando a respostas dependentes das mesmas. Comparativamente aos resultados obtidos para a adsorção, a reticulação demonstrou ter associadas algumas desvantagens em termos de reprodutibilidade e de sensibilidade, devido à imposição da ligação dos anticorpos pelos domínios de ligação ao antigénio. Por outro lado, em condições favoráveis, a adsorção pode resultar numa orientação favorável dos anticorpos, uma vez que as moléculas têm a liberdade para se adaptarem à superfície pela conjugação de diversos fatores, nomeadamente a sua reorientação favorável na zona de saturação. Quanto à imobilização covalente foram encontradas interferências na ativação da superfície dos elétrodos. As vias condutoras de prata presentes nos elétrodos serigrafados geram uma espécie desconhecida de prata que impossibilita o estudo da eficiência de imobilização, levando a uma incompatibilidade com o método de deteção usado no imunossensor. Por fim, uma primeira abordagem foi realizada para a imobilização dos anti-HCMV através da afinidade do ácido borónico para os resíduos de açúcar presentes na estrutura dos anticorpos. Os resultados preliminares demonstraram ser promissores para uma futura aplicação neste imunossensor.
Finalmente, a reconhecida importância da gB no diagnóstico do HCMV remeteu-nos para o estudo do seu comportamento eletroquímico. Este estudo permitiu ainda inferir sobre a possibilidade de esta proteína viral interferir no sinal obtido durante a redissolução anódica das AgNPs no immunosensor. A análise foi primeiramente conduzida em SPE, os quais não possibilitaram a procura de um sinal associado à gB, permitindo no entanto concluir que não há interferência desta molécula no imunoensaio. Quando aplicado num sistema eletroquímico convencional, a gB gerou um sinal em meio tamponado, o que não se confirmou quando aplicado a amostras reais (urina)
Applications of Nanoporous Gold to Drug Release and Glycoscience
Nanoporous gold (NPG) is a versatile material because of its three-dimensional nanoscale network, facile surface functionalization, biocompatibility, and potential usage in biotechnology applications. The field of glycoscience is growing in significance as the importance of glycans in human health and disease becomes more fully understood at the molecular level. NPG can be applied to several needs in the field of glycoscience. Our lab has applied NPG to applications in glycoscience including the capture and release of glycoproteins, and the detection of glycoprotein interactions by using either electrochemical methods or localized surface plasmon spectroscopy (LSPR). The capture of glycoproteins onto high surface area NPG is demonstrated using both lectin-glycan interactions and interaction between glycoproteins and NPG modified with boronic acid functional groups. Thermogravimetric analysis and use of a UV-visible HPLC detector in a flow cell containing monoliths of NPG were applied to monitor the capture of glycoprotein and its elution by the flow of free ligand. The modification of NPG by self-assembled monolayers (SAMs) with terminal boronic acid groups has been used together with LSPR to monitor the capture of glycoprotein by the induced shift in the LSPR peak wavelength. Square-wave voltammetry methods can also be applied to monitor the binding of glycoproteins to NPG modified either by SAMs with terminal boronic acid groups or by conjugated lectins.
Thiolated β-cyclodextrin modified NPG wire was used for the pH-sensitive release of doxorubicin (DOX) in a controlled manner, with an ultra-high DOX payload. Thiolated β-cyclodextrins are attractive macrocycles as they can form supramolecular inclusion complexes with doxorubicin affording the possibility of altering the controlled release behavior. Doxorubicin is one of the most potent anti-tumor drugs used in the treatment of different cancers. The binding of thiolated β-cyclodextrin with the anti-cancer drug doxorubicin has been examined with the use of spectroscopy and electrochemistry. Moreover, the prepared structure exhibited excellent properties for controlled drug release outlining the potential of a pH-sensitive drug implant or carrier for biomedical application. This delivery system could improve localized targeting of the drug as well as alter the rate of release of the doxorubicin near a tumor
Electrochemically controlled patterning for biosensor arrays.
Existe una demanda creciente de dispositivos de análisis multianalito, con aplicaciones
potenciales en los campos de la biomedicina y biotecnología, así como en el ámbito
industrial y ambiental. Para el desarrollo de estos dispositivos resulta esencial un buen
control espacial durante la etapa de inmovilización de las biomoléculas de interés; cada
una de ellas debe ser depositada de forma precisa sobre la superficie del sensor (por
ejemplo, un transductor amperométrico), evitando solapamientos que puedan
comprometer la especificidad del sistema.
El objetivo de esta tesis es desarrollar diferentes métodos de patterning para la
inmovilización selectiva de biomoléculas. El primer método consiste en la
electrodeposición selectiva de nanopartículas de oro biofuncionalizadas para el
desarrollo de biochips. Se trata de un método de patterning controlado
electroquímicamente, en el que las nanopartículas de oro se modifican en primer lugar
recubriéndolas con diversos enzimas y a continuación se electrodepositan
selectivamente sobre la superficie de un electrodo. Como parte de esta metodología, se
prepararon nanopartículas de oro biofuncionalizadas utilizando tres estrategias
diferentes: a través del enlace dativo oro-tiol, por adsorción directa o mediante
interacción electrostática siguiendo la técnica layer-by-layer (capa por capa). Para la
funcionalización de las nanopartículas de oro se emplearon distintas biomoléculas,
como los enzimas peroxidasa de rábano (HRP), glucosa oxidasa (GOX) y albúmina de
suero bovino (BSA), y finalmente oligonucleótidos modificados con moléculas
fluorescentes y grupos tiol. Las nanopartículas biofuncionalizadas fueron caracterizadas
mediante técnicas de espectroscopía UV-visible, microscopía electrónica de transmisión
(TEM) y medida del potencial zeta. Mediante espectroscopía UV-visible se observó un
pico de resonancia de plasmón característico de las nanopartículas modificadas,
relacionado con la estabilidad de la preparación. La medida del potencial zeta permitió
la caracterización de las nanopartículas de oro modificadas capa por capa con polímero
redox y enzimas. También se estudiaron los cambios en el potencial zeta de
nanopartículas modificadas con BSA a distintos valores de pH. Tras la preparación de
las partículas biofuncionalizadas, se llevaron a cabo estudios fundamentales de
electrodeposición de nanopartículas de oro modificadas con BSA y un polímero redox,
con el fin de analizar el efecto de varios parámetros: potencial aplicado, tiempo de deposición, distancia entre los electrodos, superficie del electrodo auxiliar y pH del
medio. Para estudiar el comportamiento electrocatalítico de las nanopartículas
modificadas una vez electrodepositadas, se llevaron a cabo experimentos utilizando
coloides de oro modificados con HRP y GOX. A continuación se empleó esta
metodología para el desarrollo de biochips, utilizando dos configuraciones diferentes.
En la primera, se electrodepositaron nanopartículas de oro funcionalizadas con GOX y
HRP y modificadas con un polímero redox sobre la superficie de un chip de electrodos
interdigitados (IDE), consiguiendo eliminar por completo las repuestas no específicas.
En la segunda configuración, las partículas se modificaron con una capa adicional de
polímero redox, comprobando de nuevo la ausencia total de respuestas no específicas
después de la electrodeposición. Esta método de patterning es genérico y puede
utilizarse para la producción de diversos biochips.
El segundo método de patterning también está basado en el control electroquímico, y
consiste en la modificación de los electrodos con monocapas autoensambladas
electroactivas cuya funcionalidad es modulable en función del potencial aplicado. En
esta metodología, la monocapa electroactiva contiene grupos acetal que pueden ser
desprotegidos selectivamente mediante la aplicación de un potencial en zonas
específicas de la superficie del electrodo. De esta manera quedan expuestos en la
superficie grupos aldehído activos, que pueden ser fácilmente conjugados con aminas
primarias presentes en las biomoléculas de interés. Los enzimas GOX y HRP se usaron
como proteínas modelo para comprobar la versatilidad de esta técnica. Su aplicabilidad
para la fabricación de biochips se demostró con medidas amperométricas y medidas en
tiempo real mediante resonancia de plasmón de superficie combinado con
electroquímica (eSPR).
La tercera metodología es también un sistema de patterning controlado
electroquímicamente, pero en este caso se utiliza la inmovilización del 4,4-bipiridil
como base para la creación de biochips. Se sintetizaron moléculas de 4,4-bipiridil
funcionalizadas con grupos carboxílicos, que fueron caracterizadas electroquímicamente
y a continuación conjugadas con las biomoléculas de interés para la creación de
biochips. La selectividad de estos sistemas se demostró colorimétricamente,
obteniéndose niveles mínimos de respuesta inespecífica.
Por último, el cuarto de los métodos de patterning desarrollados está basado en la
técnica de fotolitografía. Los enzimas glucosa oxidasa y sarcosina oxidasa se
depositaron selectivamente junto con un polímero redox sobre la superficie de
electrodos interdigitados utilizando un proceso de lift off, consiguiendo eliminar por
completo las señales cruzadas o cross-talk. Como parte de esta metodología se
optimizaron varios procedimientos de inmovilización de las biomoléculas, con el fin de
seleccionar la estrategia más adecuada. También se llevaron a cabo ensayos con
diferentes reactivos para eliminar la adsorción inespecífica. Finalmente, el sistema
optimizado fue aplicado sobre IDEs fabricados mediante fotolitografía. Los sensores de
glucosa y sarcosina respondieron de forma selectiva a sus respectivos sustratos, con
ausencia total de cross-talk.
La presente tesis está estructurada en 7 capítulos. En el Capítulo I se exponen las bases
del desarrollo de biochips, métodos de patterning con control electroquímico, otros
métodos de patterning selectivo y las técnicas de fotolitografía, así como un resumen de
la tesis. El Capítulo 2 y 3 describe la síntesis de coloides de oro, la modificación con
biomoléculas, los estudios de estabilidad y los estudios fundamentales de
electrodeposición de las nanopartículas de oro modificadas sobre la superficie de los
electrodos. En el Capítulo 4 se muestra la aplicación de la electrodeposición de
nanopartículas de oro biofuncionalizadas para la creación de biochips. El Capítulo 5
describe la inmovilización selectiva de biomoléculas mediante la desprotección
electroquímica de monocapas autoensambladas electroactivas. En el Capítulo 6 se
muestra la síntesis, caracterización e inmovilización selectiva de derivados de 4,4-
bipiridil funcionalizados con HRP. El Capítulo 7 describe el patterning selectivo en la
escala micrométrica de dos oxidasas sobre un chip de electrodos interdigitados mediante
fotolitografía. Finalmente, el Capítulo 8 resume las conclusiones y el trabajo futuro.There is an increasing demand of multianalyte sensing devices having potential
applications in biomedical, biotechnological, industrial and environmental fields. A
good spatial control during biomolecule deposition step is strictly necessary; each
biomolecule has to be precisely deposited on the surface of the relevant sensor (eg., an
amperometric transducer), avoiding mixing that can compromise the biosensor
specificity.
The aim of this thesis is to develop different patterning methods for the selective
immobilization of biomolecules. The first method is selective electrodeposition of
biofunctionalized Au nanoparticles for biosensor arrays. This is an electrochemically
controlled patterning method where the Au nanoparticles modified by the enzymes
initially and later the enzyme modified Au nanoparticles were electrodeposited
selectively on the electrode surface. As a part of this methodology, initially
biofunctionalized Au nanoparticles were prepared using three different approcahes. One
is Au-thiol dative bonding, the second is direct adsorption and finally electrostatic layerby-
layer approach. Different biomolecules like horse radish peroxidase(HRP), glucose
oxidase (GOX), bovine serum albumin(BSA), and finally fluorescence labelled
oilgonucleotide thiols were used to attch to the Au nanoparticles. Biofunctionalized Au
nanoparticles were characterized by different techniques like zeta sizer, UV-Vis
spectroscopy, transmission electron microscopy (TEM). UV-Vis spectroscopy showed
the successfull modification of Au nanoparticles with a characterstic surface plasmon
peak related to the stability. By using zeta sizer, layer-by-layer modification of the Au
nanoparticles with redox polymer and enzymes were characterized successfully.
Changes of the Au nanoparticles modified with BSA was characterised at different pH s
by using the zeta sizer. After the preparation of biofunctionalized particles, some
fundamental studies were done with electrodeposition of Au nanoparticles modified
with medically important BSA, redox polymer to see how different parameters like
potential, time of deposition, interelectrode distance, counter electrode sized, pH, effect
the electrodeposition. As a part of these fundamental studies Au colloids modified with
HRP and GOX were deposited for studying the electrocalaytic behaviour of the
enzymes on the Au nanoparticles after electrodeposition. Later this methodology was
applied for creating biosensor arrays by using two different approaches. In the first
approach, GOX and HRP functionalized redox polymer modified Au nanoparticles were electrodeposited successfully on an interdigitated electrode (IDE) array with complete
absence of non-specific response. In the second approach the particles were modified
with an extra redox polymer layer and proved that there is complete absence of nonspecific
response after electrodeposition. Moreover, this patterning methodology is
generic and can be used for production of different biochips.
The second method is another electrochemically controlled patterning method where
the electrodes were immobilized with self assembled monolayers with electroactive
functionalities which can be tunable with potentials. In this methodology, electroactive
self-assembled monolayer contains an active ligand aldehyde which can be readily
conjugated to the primary amine group of the biomolecule is protected in the form of
acetal. Later when a active potential was applied to the underlying electrode surface, the
acetal functionality is deprotected to reveal the aldehyde functionality which was further
conjugated to the biomolecule. Two enzymes GOX, HRP were used as model proteins
to prove the versatility of this technique. Amperometric as well as real time
measurements proved the selective applicability of this technique for creation of
biosensor arrays.
The third methodology is also an electrochemically controlled patterning methodology
where the special advantage of the electrochemically-controlled immobilization of the
4,4-bipyridyl was taken as base for the creation of biosensor arrays. In this
methodology, carboxylic acid functionalised 4,4, bipyridyl molecules were synthesized
and characterized by electrochemistry. Later the biomolecules were conjugated to these
special molecules for the creation of sensor arrays. Proof of selectivity was shown using
colourimetrically with minimal non-specific response.
Finally in the fourth method which is based on the photolithography technique, two
different oxidases GOX & SOX were patterned along with redox polymer selectively on
an IDE array using the lift off process with complete absence of cross-talk. As a part of
this methodology, different immobilization methods were optimized initially for
checking the best optimisation strategy. Later different reagents were tried to optimise
the best reagent that prevents the non-specific adsorption. Later this optimised system
was applied on the pholithographically created IDE array. Sarcosine and glucose
sensors responded selectively to their substrates with complete absence of cross talk. This thesis is structured in 7 chapters. Chapter 1 establishes to basics of the biosensor
arrays, electrochemically controlled patterning methods, other selectively patterned
methods, photolithography and summary of this thesis. Chapter 2 describes about the
gold colloid synthesis, modification with the biomolecules, stability studies. Chapter 3
decribes fundamental studies of the electrodeposition of the functionalised Au
nanoparticles on the electrode surface. Chapter 4 describes the application of the
electrodeposition of the protein functionalised Au nanoparticles for the creation of
biosensor arrays. Chapter 5 describes the selective immobilization of biomolecules
through electrochemical deprotection of electroactive self-assembled monolayers.
Chapter 6 describes the synthesis, characterization and selective immobilization of HRP
functionalized 4,4-bipyridyl derivatives. Chapter 7 describes the selective microscale
protein patterning of two oxidases on an IDE array through photolithography. Finally
chapter 8 summarizes the conclusions and the future work
Plastic Antibodies for the detection of Bacterial Proteins and Microorganisms
El diagnosi de moltes malalties és de vital importància per proporcionar el tractament adequat i per tant per controlar les necessitats de salut públiques. Els mètodes estàndard que es fan servir per confirmar la presència de microorganismes consisteixen típicament en l’ús de mètodes de cultiu específics per multiplicar, separar, identificar i comptar les bactèries. La durada d’aquests processos depèn del microorganisme en concret, però en molts casos un resultat confirmatori pot tardar entre uns pocs dies o inclús vàries setmanes. Un dels principals objectius en aquesta àrea és la detecció ràpida de microorganismes, d’una forma acurada i barata. Els polímers d’impremta molecular (PIMs) ofereixen una alternativa robusta i econòmica als anticossos naturals, però encara es requereix el seu desenvolupament pel reconeixement de molècules de gran mida. En aquesta tesi presentem diferents polímers d’impremta molecular amb l’objectiu de desenvolupar una nova aproximació per detectar proteïnes de la superfície de bactèries i microorganismes, aproximació basada en anticossos artificials utilitzats en la construcció de dispositius portàtils i econòmics. Aquests objectius generals s’assoleixen implementant una sèrie d’objectius específics: i. desenvolupament d’un camí simple per la construcció d’anticossos artificials utilitzant processos d’impremta molecular, ii. aplicació d’impedimetria, voltametria d’ona quadrada i potenciometria com a tècniques de detecció conjuntament amb una capa sensora formada per polímers d’impremta molecular, iii. ús d’elèctrodes comercials i de fabricació casolana per la detecció electroquímica en la cerca de dispositius portables i d’un sol ús, iv. impressió molecular i detecció de proteïnes de superfície de bactèries i/o microorganismes.La diagnosis de muchas enfermedades es de vital importancia para proporcionar el tratamiento adecuado y por lo tanto para el control de las necesidades de salud públicas. Los métodos estándar utilizados en la confirmación de la presencia de microorganismos consisten típicamente en el uso de métodos de cultivo específicos para multiplicar, separar, identificar y contar las bacterias. La durada de estos procesos depende del microorganismo en concreto, pero en muchos casos se necesitan entre pocos días o incluso varias semanas para tener una confirmación del resultado. Uno de los principales objetivos en esta área es la detección rápida de microorganismos, de una forma fiable y barata. Los polímeros de impronta molecular (PIMs) ofrecen una alternativa robusta y económica a los anticuerpos naturales, pero aún se requiere su desarrollo para el reconocimiento de moléculas de elevado tamaño. En esta tesis presentamos diferentes polímeros de impronta molecular con el objetivo de desarrollar una nueva aplicación para detectar proteínas de la superficie de bacterias y microorganismos, aproximación basada en anticuerpos artificiales utilizados en la construcción de dispositivos portátiles y económicos. Estos objetivos generales se consiguen implementando una serie de objetivos específicos: i. desarrollo de un camino simple para la construcción de anticuerpos artificiales utilizando procesos de impronta molecular, ii. aplicación de impedimetría, voltamperometría de onda cuadrada y potenciometría como técnicas de detección conjuntamente con una capa sensora formada por polímeros de impronta molecular, iii. uso de electrodos comerciales y de fabricación casera para la detección electroquímica en la búsqueda de dispositivos portátiles y de un solo uso, iv. impresión molecular y detección de proteínas de superficie de bacterias y/o microorganismos.The diagnosis of most illnesses is of vital importance for providing the appropriate cure and hence controlling public health concerns. The standard methods that are used to confirm the presence of microorganisms typically consist of specific enrichment media to multiply, separate, identify and count bacterial cells. The duration of the process depends on the microorganism, but in most cases a confirmatory result can take from a few days to even weeks. One of the major objectives in this area is to detect microorganisms quickly, accurately and cheaply. Molecularly imprinted polymers (MIPs) offer in principle a robust, cost-efficient alternative to natural antibodies, but it is still a challenge to develop such materials for large molecule recognition. In this thesis we present a variety of molecular imprinting approaches with an aim to develop a new approach for detecting bacterial surface proteins and microorganisms based on artificial antibodies for the construction of label-free and cost-effective portable devices. These general objectives are achieved by implementing a series of specific objectives: i. development of an easy pathway to make artificial antibodies by molecular imprinting process, ii. application of impedimetry, square wave voltammetry and potentiometry as detection techniques using molecularly imprinting polymers as the sensing layer, iii. use of homemade and commercially available screen-printed electrodes for the electrochemical detection of targets in the search for disposable and portable devices iv. electrochemical imprinting and detection of bacterial surface proteins and/or microorganisms
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