Development of electrochemical biosensors for the detection of biological warfare agents

En aquesta tesi, s’ha desenvolupat un sensitiu bio-sensor electroquímic, amb capacitat de multiplexió, simple, de baix cost i portable, per a la detecció ràpida i fiable d’agents de guerra biològica en diferents situacions com la seguretat nacional, operacions militars i seguretat en instal•lacions dels transports públics. En el desenvolupament de l’immuno-sensor es van explorar diferents superfícies químiques, utilitzant fragments d’anticossos o anticossos sencers per a la detecció de cèl•lules bacterianes. També es va explorar la detecció d’anticossos d’anti-Francisella tularensis en mostres de sèrum animal infectades amb tularemia. Els resultats van mostrar un bon grau de correlació en ésser comparats amb els obtinguts mitjançant mètodes ELISA. En el desenvolupament del bio-sensor d’ADN, es va dur a terme la detecció simultània de vuit (8) espècies virulentes, emprant un conjunt de sensors, amb diferents dissenys de sondes. El conjunt de bio-sensors i l’immuno-sensor van ser integrats amb micro-fluids localitzats en un dispositiu de testeig. Utilitzant un mètode de nano-plantilles (diferents fases de surfactant octaethylene glycol monohexadecyl ether) per a una millor distribució de les sondes, es va aconseguir millorar la sensibilitat i el límit inferior de detecció del bio-sensor d’ADN, millorant l’eficiència d’hibridació. Les superfícies modificades d’elèctrode d’or van ser avaluades mitjançant fluorescència i força atòmica microscòpica i electroquímica. En general, aquest treball constitueix una completa visió del desenvolupament de bio-sensors electroquímics per a la detecció de cèl•lules bacterianes de F. tularensis, anticossos anti-F. Tularensis, així com d’un conjunt de bio-sensors d’ADN multiplexats, altament sensitius i selectius per a la detecció de productes RCP.n esta tesis, se desarrolló un sensitivo bio-sensor electroquímico, con capacidad de multiplexión, simple, de bajo coste y portable, para la detección rápida y fiable de agentes de guerra biológica en diferentes situaciones como la seguridad nacional, operaciones militares y seguridad en instalaciones de los transportes públicos. En el desarrollo del inmuno-sensor, se exploraron diferentes superficies químicas usando fragmentos de anticuerpos o anticuerpos enteros para la detección de células bacterianas. También se exploró la detección de anticuerpos de anti-Francisella tularensis en muestras de suero animal infectadas con tularemia. Los resultados mostraron un buen grado de correlación al ser comparados con los obtenidos mediante métodos ELISA. En el desarrollo del bio-sensor de ADN se llevó a cabo la detección simultánea de ocho (8) especies virulentas utilizando un conjunto de sensores, con diferentes diseños de sondas. El conjunto de bio-sensores i el inmuno-sensor fueron integrados con micro-fluidos localizados en un dispositivo de testeo. Usando un método de nano-plantillas (diferentes fases de surfactante octaethylene glycol monohexadecyl ether) para una mejor distribución de las sondas, se consiguió mejorar la sensibilidad y el límite inferior de detección del bio-sensor de ADN, mejorando la eficiencia de hibridación. Las superficies modificadas de electrodo de oro fueron evaluadas mediante fluorescencia y fuerza atómica microscópica y electroquímica. En general, este trabajo constituye una completa visión del desarrollo de bio-sensores electroquímicos para la detección de células bacterianas de F. tularensis, anticuerpos anti-F. Tularensis, así como de un conjunto de bio-sensores de ADN multiplexados altamente sensitivos y selectivos para la detección de productos RCP.In this thesis, a simple, low cost, portable, multiplexing capable and sensitive electrochemical biosensor was developed for rapid and reliable detection of biowarfare agents for different situations like homeland security, military operations, public transportation securities such as airports, metro and railway stations. In the development of immunosensor, different surface chemistry using antibody fragments or whole antibodies were explored for bacterial cells detection. The detection of anti-Francisella tularensis antibodies in animal serum samples known to be infected with tularemia was also explored. The results obtained were compared to that obtained using ELISA methods with a good degree of correlation. In the development of multiplexed DNA biosensor, simultaneous detection of eight (8) virulent species using a sensor array was developed using different designs of capture probes. The developed multiplexed biosensor array and immunosensor for detecting bacterial cells were integrated with microfluidics housed in a tester set-up device. The search to improve sensitivity and lower limit of detection of a DNA biosensor was achieved using a nanotemplating method for a better probe distribution enhancing hybridisation efficiency. Different phases of the surfactant octaethylene glycol monohexadecyl ether were used as templates. Fluorescence and atomic force microscopy as well as electrochemistry were used to evaluate the modified surfaces of gold electrode. Overall, this work constitutes a complete overview of the development of electrochemical biosensors for the detection of bacterial cells of F. tularensis, anti-F. tularensis antibodies as well a highly sensitive and selective multiplexed DNA biosensor array for the detection of PCR products

Non-invasive monitoring of pH and oxygen using miniaturized electrochemical sensors in an animal model of acute hypoxia

Background: One of the most prevalent causes of fetal hypoxia leading to stillbirth is placental insufficiency. Hemodynamic changes evaluated with Doppler ultrasound have been used as a surrogate marker of fetal hypoxia. However, Doppler evaluation cannot be performed continuously. As a first step, the present work aimed to evaluate the performance of miniaturized electrochemical sensors in the continuous monitoring of oxygen and pH changes in a model of acute hypoxia-acidosis. Methods: pH and oxygen electrochemical sensors were evaluated in a ventilatory hypoxia rabbit model. The ventilator hypoxia protocol included 3 differential phases: basal (100% FiO2), the hypoxia-acidosis period (10% FiO2) and recovery (100% FiO2). Sensors were tested in blood tissue (ex vivo sensing) and in muscular tissue (in vivo sensing). pH electrochemical and oxygen sensors were evaluated on the day of insertion (short-term evaluation) and pH electrochemical sensors were also tested after 5 days of insertion (long-term evaluation). pH and oxygen sensing were registered throughout the ventilatory hypoxia protocol (basal, hypoxia-acidosis, and recovery) and were compared with blood gas metabolites results from carotid artery catheterization (obtained with the EPOC blood analyzer). Finally, histological assessment was performed on the sensor insertion site. One-way ANOVA was used for the analysis of the evolution of acid-based metabolites and electrochemical sensor signaling results; a t-test was used for pre- and post-calibration analyses; and chi-square analyses for categorical variables. Results: At the short-term evaluation, both the pH and oxygen electrochemical sensors distinguished the basal and hypoxia-acidosis periods in both the in vivo and ex vivo sensing. However, only the ex vivo sensing detected the recovery period. In the long-term evaluation, the pH electrochemical sensor signal seemed to lose sensibility. Finally, histological assessment revealed no signs of alteration on the day of evaluation (short-term), whereas in the long-term evaluation a sub-acute inflammatory reaction adjacent to the implantation site was detected. Conclusions: Miniaturized electrochemical sensors represent a new generation of tools for the continuous monitoring of hypoxia-acidosis, which is especially indicated in high-risk pregnancies. Further studies including more tissue-compatible material would be required in order to improve long-term electrochemical sensing

Miniaturized Electrochemical Sensors to Monitor Fetal Hypoxia and Acidosis in a Pregnant Sheep Model

Perinatal asphyxia is a major cause of severe brain damage and death. For its prenatal identification, Doppler ultrasound has been used as a surrogate marker of fetal hypoxia. However, Doppler evaluation cannot be performed continuously. We have evaluated the performance of a miniaturized multiparametric sensor aiming to evaluate tissular oxygen and pH changes continuously in an umbilical cord occlusion (UCO) sheep model. The electrochemical sensors were inserted in fetal hindlimb skeletal muscle and electrochemical signals were recorded. Fetal hemodynamic changes and metabolic status were also monitored during the experiment. Additionally, histological assessment of the tissue surrounding the sensors was performed. Both electrochemical sensors detected the pO2 and pH changes induced by the UCO and these changes were correlated with hemodynamic parameters as well as with pH and oxygen content in the blood. Finally, histological assessment revealed no signs of alteration on the same day of insertion. This study provides the first evidence showing the application of miniaturized multiparametric electrochemical sensors detecting changes in oxygen and pH in skeletal muscular tissue in a fetal sheep model.This research was funded by CELLEX FOUNDATION. This work was financially supported by The Cellex Foundation and the Agència de Gestió d’Ajuts Universitaris i de Recerca (Grant 2017 SGR 1531). Additionally, E.E. has received support from the Departament de Salut (Grant SLT008/18/00156). The Nanobioengineering group at the Institute of Bioengineering of Catalonia (IBEC) has received support from the Commission for Universities and Research of the Department of Innovation, Universities, and Enterprise of the Generalitat de Catalunya (No. 2017 SGR 1079) and is part of the CERCA Programme / Generalitat de Catalunya and is supported by the Severo Ochoa programme of the Spanish Ministry of Science and Competitiveness (Grant SEV-2014-0425 (2015–2019)). CIBER-BBN is an initiative funded by the VI National R&D&i Plan 2008–2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund. L.R. would also like to acknowledge her support within the BEST Postdoctoral Programme, funded by the European Commission under Horizon 2020’s Marie Skłodowska-Curie Actions COFUND scheme (Grant Agreement No. 712754)

Micro-needle implantable electrochemical oxygen sensor: ex-vivo and in-vivo studies

Oxygen is vital for energy metabolism in mammals and the variability of the concentration is considered a clinical alert for a wide range of metabolic malfunctions in medicine. In this article, we describe the development and application of a micro-needle implantable platinum-based electrochemical sensor for measuring partial pressure of oxygen in intramuscular tissue (in-vivo) and vascular blood (ex-vivo). The Pt-Nafion® sensor was characterized morphological and electrochemically showing a higher sensitivity of -2.496 nA/mmHg (-1.495 nA/μM) when comparing with its bare counterpart. Our sensor was able to discriminate states with different oxygen partial pressures (pO2) for ex-vivo (blood) following the same trend of the commercial gas analyzer used as standard. For in-vivo (intramuscular) experiments, since there is not a gold standard for measuring pO2 in tissue, it was not possible to correlate the obtained currents with the pO2 in tissue. However, our sensor was able to detect clear statistical differences of O2 between hyperoxia and hypoxia states in tissue

Fetal ischemia monitoring with in vivo implanted electrochemical multiparametric microsensors

Under intrauterine growth restriction (IUGR), abnormal attainment of the nutrients and oxygen by the fetus restricts the normal evolution of the prenatal causing in many cases high morbidity being one of the top-ten causes of neonatal death. The current gold standards in hospitals to detect this relevant problem is the clinical observation by echography, cardiotocography and Doppler. These qualitative techniques are not conclusive and requires risky invasive fetal scalp blood testing and/or amniocentesis. We developed micro-implantable multiparametric electrochemical sensors for measuring ischemia in real time in fetal tissue and vascular. This implantable technology is designed to continuous monitoring for an early detection of ischemia to avoid potential fetal injury. Two miniaturized electrochemical sensors were developed based on oxygen and pH detection. The sensors were optimized in vitro under controlled concentration, to assess the selectivity and sensitivity required. The sensors were then validated in vivo in the ewe fetus model, by means of their insertion in the muscle leg and inside the iliac artery of the fetus. Ischemia was achieved by gradually obstructing the umbilical cord to regulate the amount of blood reaching the fetus. An important challenge in fetal monitoring is the detection of low levels of oxygen and pH changes under ischemic conditions, requiring high sensitivity sensors. Significant differences were observed in both; pH and pO2 sensors under changes from normoxia to hypoxia states in the fetus tissue and vascular with both sensors. Herein, we demonstrate the feasibility of the developed sensors for future fetal monitoring in medical applications

Electrochemiluminescence DNA sensor array for multiplex detection of biowarfare agents

Development of a fully automated electrochemiluminescence (ECL) DNA assay for multiplex detection of six biowarfare agents is described. Aminated-DNA capture probes were covalently immobilised on activated-carbon electrodes and subsequently hybridised to target strands. Detection was achieved via a sandwich-type assay after Ru(bpy)<inf>3</inf>2+-labelled reporter probes were hybridised to the formed probetarget complexes. The assay was performed in an automated microsystem in a custom-designed ECL detection box with integrated fluidics, electronics, and movable photomultiplier detector. The obtained limits of detection were 0.61.2 nmol L1 for six targets ranging from 50 to 122 base pairs in size, with linear range 115 nmol L1. Non-specific adsorption and cross-reactivity were very low. Detection of six targets on a single chip was achieved with subnanomolar detection limits