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Lab-On-A-Chip Platforms Embedding Self-Powered Electrochemical Sensors: a Walkthrough Approach for Bio-Analytical Applications

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Lab-On-A-Chip Platforms Embedding Self-Powered Electrochemical Sensors: a Walkthrough Approach for Bio-Analytical Applications DOUTORAMENTO EM QUÍMICA SUSTENTÁVEL Lab-on-a-chip platforms embedding self-powered electrochemical sensors: a walkthrough approach for bio-analytical applications Álvaro Miguel Carneiro Torrinha D 2019 Álvaro Miguel Carneiro Torrinha Lab-on-a-chip platforms embedding self-powered electrochemical sensors: a walkthrough approach for bio-analytical applications Tese do 3° Ciclo de Estudos Conducente ao Grau de Doutor em Química Sustentável Trabalho realizado sob a orientação do Professor Doutor Alberto Nova Araújo e co-orientação da Professora Doutora Maria Conceição Branco Montenegro Julho 2019 De acordo com a legislação em vigor, não é permitida a reprodução de qualquer parte desta tese ii Já fiz uma parte de nada. Agora falta o resto de tudo iii iv Acknowledgements The accomplishment of the work present herein was possible due to the expertise of my supervisors, Alberto Araújo and Maria Conceição Branco Montenegro. I want to thank all the help provided, friendship and for the integration in the Sensors and Biosensors lab. I also like to thank Celia, always cheerful and with a smile to give, for her help and encouragement. I want to thank all my colleagues and friends from FFUP for all the good moments. I am truly grateful to my former supervisor and Professor Simone Morais for believing in me and make me believe that was possible obtaining a PhD degree. Thank you Vânia and Eng. Manuela for all the favours done, all the help provided and friendship. I want to thank Fundação para a Ciência e Tecnologia for the funding through the grant PD/BD/109660/2015. A special thanks to my parents and my brother Pedro for the support and patience. For last and not least a very special thought to my wife Isabel Sofia. You have been tireless with all your support and friendship. About 4 years of my life just flew by really quickly and I am sure that the door will remain always open whenever needed. Thank you. v vi Abstract The present dissertation considers the development of enzymatic biofuel cells embedded in lab-on-a-chip platforms, with ability for self-powered biosensing. Since the generated power varies proportionally with the fuel concentration, a biofuel cell can itself be used as a biosensor or instead applied as energy supplier for an external sensor/biosensor. When integrated in a microfluidic platform, other unit operations can be performed and controlled with reduced sample/fuel volumes meeting therefore an ecological and sustainable approach. The coupling of enzymes to the electrodes allied to their substrate specificity makes possible the operation at mild chemical conditions. At the same time, it simplifies the construction of biofuel cells by avoiding the necessity of separation between anolyte and catholyte, therefore enabling their miniaturization. Nevertheless, the immobilization procedure of enzymes is complex and a challenging process. First, because the catalytic centre of enzymes is buried in an insulating glycoprotein shell which needs proper orientation or cross-linking for direct electron transfer (DET) or requiring the use of diffusional mediators for a successful electronic communication with the electrode. And second, because the use of enzymes outside living organisms lead to stability problems reducing the lifetime of biosensors and biofuel cells. Almost all experimental work was performed using pencil graphite electrodes (PGEs) as enzymatic conductive supports (transducers). These type of electrodes are a practicable alternative to other traditionally used electrodes due to their comparable electrochemical performance, availability and reduced cost. PGEs were thoroughly characterized by cyclic voltammetry (CV) and modified with carbon based nanostructures in order to enhance the electrochemical signal. Modification with reduced graphene (rGO) gave the best results and was henceforth applied in the construction of PGE bioelectrodes. An alternative approach as biocatalysts support based on conductive transducers produced through vacuum- filtration of a Vulcan carbon black suspension resulting in flexible, paper-like electrodes, was equally assessed and used in one of the developed lab-on-a-chip-platforms. In biofuel cells, enzymatic oxidation of a fuel occurs at the bioanode with the generated electrons being transferred to the biocathode for the enzymatic reduction of an oxidizer compound, e.g. oxygen. Oxygen reduction bioelectrodes to be used as biocathodes were initially studied. In a first attempt, laccase enzyme from Rhus vernicifera was first immobilized in the PGE-rGO surface alongside single-walled carbon nanotubes (SWCNT) by entrapment in a sol-gel matrix. However, a second approach tested with the bilirubin oxidase (BOx) enzyme would reveal a simpler immobilization procedure with much higher vii performance regarding biocatalytic reduction of oxygen. This procedure consisted in further modification of PGE-rGO with multi-walled carbon nanotubes (MWCNT) followed by the immobilization of enzyme BOx through a pyrene-based succinimidyl ester compound (PBSE). CV was performed to evaluate the immobilization efficiency whereas amperometric analysis revealed a high sensitivity of 648 µA mM-1 cm-2 and a low limit of detection value of 1.8 µM for the PGE-rGO-MWCNT-BOx. When employing the bioelectrode as a biocathode, the polarization curves resulted in an open circuit potential (EOCP) of 0.48 V vs Ag/AgCl and generated a maximum current density of about 500 µA cm-2 at 0.10 V vs Ag/AgCl. The glucose oxidase (GOx) bioanode was assembled through an enzyme precipitate coating method. This was accomplished by cross-linking of GOx to MWCNT after a precipitation step with ammonium sulphate and further solubilisation in nafion and deposition in the PGE-rGO surface. The approach enabled high enzyme activity, improved stability of the biofilm coating over PGE surface and its use under flow regimen inside microfluidic platforms. In this last condition, a sensitivity to glucose of 35 μA mM-1 cm-2 and a LOD of 14.9 μM were achieved in a high analytical range up to 39 mM. The bioelectrode was also successful tested in the detection of cadmium through an inhibitory effect on GOx. As a final work, GOx and BOx paper-like bioelectrodes were conjugated in a biofuel cell, respectively as bioanode and biocathode and finally integrated in a finger pressure-driven microfluidic platform made of poly(methyl methacrylate) - polydimethylsiloxane (PMMA- PDMS). The autonomous, self-powered biosensor device showed a sensitivity of 2.1 µW mM-1 cm-2 up to 20 mM of glucose at physiological conditions. The maximum power density achieved in 50 mM glucose solution was about 70 µW mM-1 cm-2 at 0.19 V vs Ag/AgCl. The work developed in this thesis enabled the fabrication of an autonomous and self- powered biosensors with potential use for in situ measurements. This was possible due to the implementation of efficient immobilization procedures of enzymes in miniaturized electrodes and their integration in a platform featuring human propelled fluidics. Meanwhile, the practical use of PGEs in electrochemistry was extensively demonstrated throughout the work. Keywords: Bioelectrocatalysis, Biofuel cells, Self-powered biosensors, Microfluidics, Enzymes viii Resumo Na presente tese foi equacionado o desenvolvimento de plataformas “lab-on-a-chip” contendo células de biocombustível enzimáticas para serem aplicadas como biossensores autoalimentados. Uma vez que a potência gerada varia proporcionalmente com a concentração de combustível, as células de biocombustível podem ser usadas como biossensores ou serem aplicadas como alimentadores de energia para sensores/biossensores externos. Quando integrados numa plataforma microfluídica, outras operações unitárias podem ser adicionadas e executadas sobre volumes reduzidos de amostra/combustível indo assim ao encontro de uma abordagem ecológica e sustentável. O acoplamento otimizado de enzimas aos elétrodos, aliado à especificidade daquelas pelos seus substratos torna possível o funcionamento das células de combustível em condições amenas. Por outro lado, simplifica-se a sua construção pois evita-se a necessidade de separar o anólito do católito, tornando assim possível a sua miniaturização. Contudo, o procedimento de imobilização enzimática é um processo complexo e desafiante. Primeiro, porque o centro catalítico das enzimas encontra-se geralmente sob um invólucro glicoproteico isolante. Necessita assim de uma imobilização orientada, ou de uma ligação química que propicie o tunelamento eletrónico direto, ou requer o uso de mediadores difusionais que assegurem a ligação eletrónica com o elétrodo. Em segundo lugar, o uso de enzimas no exterior dos organismos conduz a problemas de estabilidade reduzindo o tempo de operação de biossensores e células de biocombustível. Quase todo o trabalho experimental foi realizado usando minas de grafite de lapiseira (PGEs) como suporte condutor enzimático (transdutor). Este tipo de elétrodos constitui uma alternativa viável a outros elétrodos tradicionalmente usados pois que, para além de um desempenho eletroquímico comparável, são facilmente disponíveis e apresentam custo muito reduzido. Os PGEs foram completamente caracterizados por voltametria cíclica (CV) e modificados com nanoestruturas à base de carbono, a fim de potencializar o sinal eletroquímico. A modificação com grafeno reduzido (rGO) proporcionou melhores resultados e passou a ser aplicada por rotina na construção dos bioeletrodos. Como abordagem alternativa de suporte dos biocatalisadores, desenvolveram-se transdutores
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