Microelectronics-Based Biosensors Dedicated to the Detection of Neurotransmitters: A Review

Dysregulation of neurotransmitters (NTs) in the human body are related to diseases such as Parkinson's and Alzheimer's. The mechanisms of several neurological disorders, such as epilepsy, have been linked to NTs. Because the number of diagnosed cases is increasing, the diagnosis and treatment of such diseases are important. To detect biomolecules including NTs, microtechnology, micro and nanoelectronics have become popular in the form of the miniaturization of medical and clinical devices. They offer high-performance features in terms of sensitivity, as well as low-background noise. In this paper, we review various devices and circuit techniques used for monitoring NTs in vitro and in vivo and compare various methods described in recent publications

Diamond MEMS Biosensors: Development and applications

This research focuses on the development a dielectrophoresis-enhanced microfluidic impedance biosensor (DEP-e-MIB) to enable fast response, real-time, label-free, and highly sensitive sensor for bacterial detection in clinical sample. The proposed design consists of application of dielectrophoresis (DEP) across a microfluidic channel to one of the impedance spectroscopy electrodes in order to improve the existent bacterial detection limits with impedance spectroscopy. In order to realize such a design, choice of electrode material with a wide electrochemical potential window for water is very important. Conventional electrode material, such as gold, are typically insulated for the application of DEP, and they fail when used open because the DEP voltages avoiding electrolysis do not provide enough force to move the bacteria. First, the use of nanodiamonds (ND) seeding gold surface to widen the electrochemical potential window is examined, since diamond has a wider potential window. ND seed coverage is a function of sonication time, ND concentration, and solvent of ND dispersion. Examining these parameters allowed us to increase the ND surface coverage to ~35%. With the highest ND coverage achievable, such electrodes are still susceptible to damage from electrolysis, however yield a unique leverage for impedance biosensing. When NDs is seeded at a 3x3 interdigitated electrode array, which act as electrically conductive islands between the electrodes and reduce the effective gap between the electrodes, thus allowing to perform impedance spectroscopy in solutions with low electrical conductivity such as ITS. The changes obtained in resistance to charge transfer with bacterial capture is nearly twice than that obtained with plain electrodes. Secondly, the feasibility of using boron-doped ultra nanocrystalline diamond (BD-UNCD) to apply DEP is tested without constructing a 3x3 IDE array. BD-UNCD electrodes can be used for DEP through tagging of the bacteria with immunolatex beads. This allows applying a larger DEP force on the bacteria. Since historically bead based assays are plagued with problems with non-specific binding, the role of different parameters including bead bioconjugation chemistry, bead PEGylation, BD-UNCD surface PEGylation, and DEP on specific and non-specific binding are tested. Most importantly DEP increases the specific binding and PEGylation of beads decreases the specific binding. Finally, a 3x3 IDE array with BD-UNCD was fabricated, and used impedance spectroscopy to test the suitability of BD-UNCD IDEs for impedance biosensing. The huge electrode resistance and the charge transfer resistance at BD-UNCD IDEs poses a problem for impedance biosensing as it will lead to lower sensitivity. BD-UNCD is the material of choice for applying DEP at open electrodes however gold is the choice of material for designing the chip interconnects. So the BD-UNCD layer should be as thin as possible and the interface between gold IDEs and the solution phase during DEP. The findings in this dissertation put us closer to realizing a DEP-eMIB

Conducting polymers and hybrid derivates with specific applications as sensors and bioactive platforms

The principal focus of this Thesis is the development and design of promising hybrid nanocomposites based on conducting polymers with the main objective of achieving applications in the field of biotechnology and biomedicine. The main lines of research can be summarized as follows;1) Preparation, characterization and evaluation of N-substituted polypyrrole derivatives and poly(3,4-ethylenedioxythiophene) (PEDOT) for electrochemical detection of dopamine, one of the neurotransmitters associated to neurological disorders. In order to examine this purpose, different strategies have been taken into account such as, polymerization method using individual or even combined conducting polymers, the incorporation of gold nanoparticles, the use of soft templates, and other approachs. 2) Design of synthetic amino acids bearing an EDOT group to develop peptide-PEDOT hybrids materials based on chemical similarity concepts. The conjugates have shown that the electrical and electrochemical properties of the conducting polymers are preserved. Therefore, one of their potential applications would be as candidates for the development of platforms with bioactive and bioelectrocompatible properties. 3) Preparation and characterization of organic hybrid materials formed by an all-conjugated polythiophene backbone andwell-defined polyethylene glycol (PEG) grafted chains, which have powerful applicability as active surfaces for the selective adsorption of proteins and as bioactive platforms. Among several factors which influence on the structure and properties of graft copolymers, one of the most important is the molecular weight of the PEG chains which provokes a considerably reduction in the backbone conjugation length. 4) Preparation and characterization of new bionanocomposites formed by PEDOT and CREKA, which is a biologically active linear pentapeptide. The incorporation of CREKA into a PEDOT matrix has been carried out under different experimental conditions and has shown a positive effect on the electrochemical properties of conducting polymer and indicating a favourable cellular proliferation due to the ability to bind fibrin. Some research findings provided in this Thesis have been published or accepted for publication in scientific journals: 1. An electroactive and biologically responsive hybrid conjugate based on chemical similarity. G. Fabregat, G. Ballano, E. Armelin, L. J. del Valle, C. Cativiela and C. Alemán, Polym. Chem., 2013, 4, 1412. 2.Hybrid materials consisting of an all-conjugated polythiophene backbone and grafted hydrophilic poly(ethylene glycol) chains. A.-D. Bendrea, G. Fabregat, L. Cianga, F. Estrany, L. J. del Valle, I. Cianga and C. Alemán, Polym. Chem., 2013,4, 2709. 3.Polythiophene-g-poly(ethylene glycol) graft copolymers for electroactive scaffolds.A.-D. Bendrea, G. Fabregat, J. Torras, S. Maione, L. Cianga, L. J. del Valle, I. Cianga and C. Alemán, J. Mater. Chem. B, 2013,1, 4135. 4.Design of hybrid conjugates based on chemical similarity.G. Fabregat, G. Ballano, J. Casanovas, A. D. Laurent, E. Armelin, Luis J. del Valle, C. Cativiela, D. Jacquemin and C. Alemán, RSC Adv., 2013, 3, 21069. 5.Controlling the morphology of poly(N -cyanoethylpyrrole). G. Fabregat, M. T. Casas, C. Alemán and E. Armelin, J. Phys. Chem. B, 2012, 116, 5064. 7.Ultrathin Films of Polypyrrole Derivatives for Dopamine Detection. G. Fabregat, E. Córdova-Mateo, E. Armelin, O. Bertran and C. Alemán. J. Phys. Chem. C, 2011, 115,14933.8.Nanostructured conducting polymer for dopamine detection.M. Martí, G. Fabregat, F. Estrany, C. Alemán and E. Armelin, J. Mater. Chem., 2010, 20, 10652.El propósito de la presente tesis es el desarrollo y diseño de nanocompuestos híbridos basados ??en polímeros conductores para su posterior aplicación en el campo de la biotecnología y la biomedicina. Las principales líneas de investigación se resumen de la siguiente manera; 1) Preparación, caracterización y evaluación de derivados N-sustituidos de polipirrol y poli(3,4-etilendioxitiofeno ) (PEDOT) para la detección electroquímica de la dopamina, uno de los neurotransmisores asociados a trastornos neurológicos. Para examinar este propósito, diferentes estrategias han sido consideradas, tales como; el método de polimerización empleando polímeros individuales o combinados, la incorporación de nanopartículas de oro, la utilización de templates, etc. 2) Diseño de aminoácidos sintéticos unidos covalentemente con un grupo EDOT y posterior desarrollo de materiales híbridos (péptido - PEDOT). Los materiales híbridos han demostrado conservar las propiedades eléctricas y electroquímicas del polímero base, siendo posibles candidatos para el desarrollo de plataformas bioactivas y bioelectrocompatible. 3) Preparación y caracterización de materiales híbridos orgánicos formados por una cadena principal de politiofeno y cadenas injertadas de polietilenglicol (PEG), los cuales tienen una elevada aplicabilidad como superficies activas para la adsorción selectiva de proteínas y como plataformas bioactivas. 4) Preparación y caracterización de nuevos bionanocomposites formados por PEDOT y CREKA, el cual es un pentapéptido lineal biológicamente activo. La incorporación de CREKA en una matriz de PEDOT se ha llevado a cabo en diferentes condiciones experimentales, y ha demostrado tener un efecto positivo sobre las propiedades electroquímicas del polímero conductor como también proporcionar una mejora en la proliferación celular debido a la capacidad de éste para unirse a la fibrina. Algunos resultados obtenidos en la presente Tesis han sido publicados o aceptados para su publicación en revistas científicas: 1. An electroactive and biologically responsive hybrid conjugate based on chemical similarity. G. Fabregat, G. Ballano, E. Armelin, L. J. del Valle, C. Cativiela and C. Alemán, Polym. Chem., 2013, 4, 1412. 2. Hybrid materials consisting of an all-conjugated polythiophene backbone and grafted hydrophilic poly(ethylene glycol) chains. A.-D. Bendrea, G. Fabregat, L. Cianga, F. Estrany, L. J. del Valle, I. Cianga and C. Alemán, Polym. Chem., 2013,4, 2709. 3. Polythiophene-g-poly(ethylene glycol) graft copolymers for electroactive scaffolds.A.-D. Bendrea, G. Fabregat, J. Torras, S. Maione, L. Cianga, L. J. del Valle, I. Cianga and C. Alemán, J. Mater. Chem. B, 2013,1, 4135. 4.Design of hybrid conjugates based on chemical similarity.G. Fabregat, G. Ballano, J. Casanovas, A. D. Laurent, E. Armelin, Luis J. del Valle, C. Cativiela, D. Jacquemin and C. Alemán, RSC Adv., 2013, 3, 21069. 5. Controlling the morphology of poly(N -cyanoethylpyrrole).G. Fabregat, M. T. Casas, C. Alemán and E. Armelin, J. Phys. Chem. B, 2012, 116, 5064. 7. Ultrathin Films of Polypyrrole Derivatives for Dopamine Detection. G. Fabregat, E. Córdova-Mateo, E. Armelin, O. Bertran and C. Alemán. J. Phys. Chem. C, 2011, 115,14933.8.Nanostructured conducting polymer for dopamine detection.M. Martí, G. Fabregat, F. Estrany, C. Alemán and E. Armelin, J. Mater. Chem., 2010, 20

Targeting human aquaporin function : physiological and chemical approaches

Tese de doutoramento, Farmácia (Bioquímica), Universidade de Lisboa, Faculdade de Farmácia, 2018Aquaporins (AQPs) are a group of small membrane proteins belonging to a highly conserved family of membrane proteins called MIPs (Major Intrinsic Proteins) that are responsible for the bidirectional transport of wate (orthodox aquaporins) and also small uncharged solutes (aquaglyceroporins) across cell membranes, in response to osmotic or solute gradients. Rapid water flux across membranes is crucial to maintain the water homeostasis in many epithelia and endothelia involved in fluid transport. In addition, due to the unique ability of aquaglyceroporins to transport glycerol in addition to water, they have important roles in glycerol metabolism and skin hydration in non-fluid transporting tissues such as skin, fat and liver. The thesis introduction (Chapter 1) presents an overview of aquaporins structure, their main biological functions and related pathologies, with special emphasis on the so far described mechanisms of regulation. In the first part of this thesis (Chapter 2), we report the discovery of a new role for Aquaporin-5 (AQP5, an orthodox aquaporin) in adipocyte biology, where Aquaporin-7 (AQP7, an aquaglyceroporin) has been the mainly characterized protein in adipose tissue responsible for glycerol efflux. A better understanding of aquaporin regulation and gating would allow manipulation of their activity facilitating the identification of new putative modulators. A cellular model optimized to assess the function of aquaporins and discriminate individually each isoform, instead of mammalian cells where more than one isoform is usually expressed, is a useful tool to study aquaporin regulation. The second part of this thesis (Chapter 3) is dedicated to the functional characterization of different mammalian aquaporin isoforms (AQP3, AQP5, AQP7 and AQP10), using a yeast heterologous expression system devoided of endogenous aquaporins, a background where analysis is unlikely to be compromised by the co-expression of other aquaporin isoforms. Using the stopped-flow technique to evaluate the channel permeability for water and for glycerol, we were able to disclose gating mechanisms of aquaporin isoforms, being given special emphasis to the regulation by pH and phosphorylation. In the third part of this thesis (Chapter 4), a screening of several small gold compounds as inhibitors for Aquaporin-3 (AQP3, an aquaglyceroporin) was performed aiming at identifying new modulators with potential therapeutic use.A água possui um papel crucial para a vida devido às suas propriedades únicas. Todos os processos bioquímicos e fisiológicos de um organismo dependem da presença de água, sendo esta o componente fundamental na manutenção da homeostase celular. Nas células eucarióticas, a água encontra-se distribuída pelos vários compartimentos intracelulares separados entre si por membranas intracelulares e do meio extracelular pela membrana plasmática. Estas membranas de composição bilipídica são normalmente impermeáveis à maioria dos solutos polares e/ou carregados, cuja passagem é facilitada através de canais membranares específicos. No entanto estas membranas são bastante permeáveis à água, sendo então propostas três vias de transporte: por difusão simples, por transporte passivo associado ao transporte de iões e solutos e por canais específicos para a água. Atualmente sabe-se que a maioria das células de um organismo possui proteínas específicas – as aquaporinas – que conferem à membrana uma permeabilidade à água de cerca de 5 a 10 vezes superior às membranas que não possuem estas proteínas. Devido às suas características estruturais, as aquaporinas permitem um rápido transporte bidirecional de água, seletivo e regulado, em resposta a gradientes osmóticos, ao mesmo tempo que previnem a passagem de protões e iões através da membrana plasmática. Em mamíferos, são conhecidas até à data treze isoformas (AQP0-AQP12) que são classificadas em três grupos de acordo com a sua sequência primária, localização celular e seletividade em 1) aquaporinas ortodoxas, primariamente seletivas à água; 2) aquagliceroporinas, para além de água também transportam pequenos solutos neutros, como glicerol e ureia; e 3) super-aquaporinas, que são encontradas em membranas intracelulares e possuem menor homologia. No entanto, a lista de substâncias que são capazes de permear as diferentes aquaporinas tem aumentado ao longo do tempo. Recentemente, para além de água e glicerol, foi também descrito o transporte facilitado através de algumas isoformas de arsenito, amoníaco e peróxido de hidrogénio. Devido à grande diversidade de tecidos onde são encontradas as aquaporinas, o seu papel de facilitar o transporte de água e/ou solutos através das membranas plasmáticas é importante em vários processos fisiológicos, tais como: secreção de fluido glandular, mecanismo de concentração urinária, excitabilidade neuronal, metabolismo dos lípidos, hidratação epidérmica e balanço de água no cérebro. A observação do fenótipo de ratinhos geneticamente modificados com knock-out de determinadas aquaporinas revelou funções fisiológicas muito importantes no aparecimento e desenvolvimento de várias patologias, como epilepsia, edema cerebral, glaucoma, cancro e obesidade. No Capítulo 1 é apresentada uma introdução geral que visa proporcionar um conhecimento abrangente sobre as principais funções das aquaporinas humanas e patologias associadas, dando especial atenção aos diferentes mecanismos de regulação já conhecidos. Na primeira parte dos resultados desta tese (Capítulo 2), através da construção de linhas celulares de pré adipócitos de ratinho 3T3-L1 com diferentes níveis de expressão da Aquaporin-5 (cenário de ganho e perda de função) foi possível estabelecer um novo e determinante papel desta aquaporina na diferenciação dos adipócitos. Na segunda parte dos resultados desta tese (Capítulo 3), pretendeu-se usar um sistema de expressão heteróloga em Saccharomyces cerevisiae (S. cerevisiae) para permitir avaliar de forma individual a função de cada aquaporina. A levedura S. cerevisiae é considerada um valioso sistema de expressão heteróloga para estudar inúmeras proteínas devido à elevada homologia funcional entre esta e os eucariontes superiores, incluindo mamíferos. Pelo facto de existir uma grande biblioteca de estirpes disponíveis, ser de fácil manipulação genética, ser pouco dispendioso em comparação com as culturas de células animais e poderem ser testadas uma variedade de condições externas, este sistema oferece condições experimentais ótimas para estudar a especificidade e regulação das aquaporinas. Após a expressão e confirmação da sua localização celular, procedeu-se à caracterização da função de cada isoforma, utilizando a técnica de interrupção brusca de fluxo, seguindo a variação de volume celular por fluorescência quando se introduz uma perturbação no meio extracelular. Os fluxos de água através da membrana celular causados por gradientes de pressão osmótica (de solutos impermeantes ou permeantes) provocam alterações transitórias de volume, até se atingir um novo volume final de equilíbrio osmótico. A velocidade com que as alterações de volume ocorrem e o tempo que a célula leva a re-estabelecer o seu novo equilíbrio osmótico dependem diretamente das características intrínsecas de transporte da membrana, em particular da quantidade de canais específicos para a água e para o soluto em questão. No Capítulo 3 foram estudadas quatro isoformas diferentes (AQP3, AQP5, AQP7 e AQP10) e os seus mecanismos de regulação por pH e fosforilação foram revelados pela primeira vez. Vários esforços têm vindo a ser feitos com o intuito de desenvolver possíveis fármacos para tratamento das aquaporinopatias, mas até agora nenhum composto se revelou qualificado para estudos in vivo, quer pela sua fraca solubilidade quer pela sua baixa capacidade de inibição. Na terceira parte dos resultados desta tese (Capítulo 4), deu-se especial atenção à descoberta de novos compostos organometálicos, inibidores da função da Aquaporina-3, que poderão ser usados para benefício clínico na prevenção ou tratamento das várias patologias associadas.Fundação para a Ciência e a Tecnologia (FCT), SFRH/BD/52384/2013, Programa de Doutoramento Medical Biochemistry and Biophysics Doctoral Programme (M2B-PhD

Biomimetic Radical Chemistry and Applications

The enormous importance of free radical chemistry for a variety of biological events, including ageing and inflammation, has attracted a strong interest in understanding the related mechanistic steps at the molecular level. Modelling the free radical chemical reactivity of biological systems is an important research area. When studying free-radical-based chemical mechanisms, biomimetic chemistry and the design of established biomimetic models come into play to perform experiments in a controlled environment that is suitably designed to be in strict connection with cellular conditions. This Special Issue gives the reader a wide overview of biomimetic radical chemistry, where molecular mechanisms have been defined and molecular libraries of products are developed to also be used as traces for the discovery of some relevant biological processes. Several subjects are presented, with 12 articles and 6 reviews written by specialists in the fields of DNA, proteins, lipids, biotechnological applications, and bioinspired synthesis, having “free radicals” as a common denominator

43rd Rocky Mountain Conference on Analytical Chemistry

Final program, abstracts, and information about the 43rd annual meeting of the Rocky Mountain Conference on Analytical Chemistry, co-sponsored by the Colorado Section of the American Chemical Society and the Rocky Mountain Section of the Society for Applied Spectroscopy. Held in Denver, Colorado, July 29 - August 2, 2001