Direct Electrochemistry of Horseradish Peroxidase‐Gold Nanoparticles Conjugate

We have studied the direct electrochemistry of horseradish peroxidase (HRP) coupled to gold nanoparticles (AuNP) using electrochemical techniques, which provide some insight in the application of biosensors as tools for diagnostics because HRP is widely used in clinical diagnostics kits. AuNP capped with (i) glutathione and (ii) lipoic acid was covalently linked to HRP. The immobilized HRP/AuNP conjugate showed characteristic redox peaks at a gold electrode. It displayed good electrocatalytic response to the reduction of H2O2, with good sensitivity and without any electron mediator. The covalent linking of HRP and AuNP did not affect the activity of the enzyme significantly. The response of the electrode towards the different concentrations of H2O2 showed the characteristics of Michaelis Menten enzyme kinetics with an optimum pH between 7.0 to 8.0. The preparation of the sensor involves single layer of enzyme, which can be carried out efficiently and is also highly reproducible when compared to other systems involving the layer-by-layer assembly, adsorption or encapsulation of the enzyme. The immobilized AuNP-HRP can be used for immunosensor applications

Detección electroquímica de peróxido de hidrógeno usando peroxidasa de pasto Guinea (Panicum maximum) inmovilizada sobre electrodos serigrafiados de puntos cuánticos

Electrochemical biosensors are analytical tools of fast and reliable response that have acquired interest in the last years due to the possibility of integrating biomolecules and electrodes made of nanometric materials. In this study, an electrochemical biosensor to detect hydrogen peroxide (H2O2) based on Guinea Grass peroxidase (GGP) immobilized on screen-printed quantum dots electrodes (SPQDE) was developed. GGP was partially purified from Guinea grass leaves having a specific activity of 602 U mg-1.Then, GGP was immobilized by physical adsorption on the surface on SPQDE and the electrochemical behavior was carried out through cyclic voltammetry and chronoamperometry techniques. GGP revealed a well-defined pair of redox signals at 17mV/-141mV corresponding to the redox process of the heme group (Fe2+/Fe3+) of peroxidases. The bioelectrocatalytic reduction of H2O2 has a redox potential of -645 mV vs Ag. This process was controlled by the diffusion of the species on the electrode surface using a scan rate range of 50-500 mV s. Chronoamperometry studies allow us the construction of calibration curves of reduction current vs H2O2 concentration for the determination of analytical parameters such as sensitivity, linear range and minimum detection level. The development of this amperometric biosensor becomes a preliminary step for the construction of a portable and rapid response device for the analysis of H2O2 in samples of environmental and biomedical interest.Os biossensores eletroquímicos são ferramentas analíticas de resposta rápida e confiável que adquiriram interesse especial nos últimos anos, graças à possibilidade de integrar biomoléculas com eletrodos feitos de materiais nanométricos. Neste trabalho, um biossensor eletroquímico foi desenvolvido para a detecção de peróxido de hidrogênio (H2O2) usando peroxidase do capim-da-Guiné (PPG) imobilizada em eletrodos serigráficos de pontos quânticos (ESPC) serigrafados. O PPG foi isolado e parcialmente purificado a partir de folhas de capim-da-Guiné com atividade específica de 602 U mg-1. Posteriormente, o PPG foi imobilizado na superfície do ESPC por adsorção física e estudo do comportamento eletroquímico foi realizado por voltametria cíclica e cronoamperometria. O PPG revelou um par bem definido de sinais redox em 17mV/-141mV correspondente ao processo redox do grupo heme (Fe2+/Fe3+) de peroxidases. A redução bioeletrocatalítica do H2O2 foi observada com um potencial redox de -645 mV vs. Ag. Esse processo foi controlado pela difusão das espécies na superfície do eletrodo em uma faixa de velocidade de varredura linear de 50-500 mV/s. A cronoamperometria permitiu a construção de curvas de calibração entre a corrente de redução e a concentração de H2O2 para a determinação de parâmetros analíticos como sensibilidade, faixa linear e nível mínimo de detecção. O desenvolvimento deste biossensor amperométrico torna-se uma etapa preliminar para a construção de um dispositivo portátil e de resposta rápida para a análise de H2O2 em amostras de interesse ambiental e biomédico.Los biosensores electroquímicos son herramientas analíticas de rápida y confiable respuesta que han adquirido especial interés en los últimos años gracias a la posibilidad de integrar biomoléculas con electrodos hechos a base de materiales nanométricos. En este trabajo se desarrolló un biosensor electroquímico para detección de peróxido de hidrógeno (H2O2) usando peroxidasa de pasto Guinea (PPG) inmovilizada sobre electrodos serigrafiados de puntos cuánticos (ESPC). La PPG fue aislada y parcialmente purificada a partir de hojas de pasto Guinea con una actividad específica de 602 Umg-1. Posteriormente, la PPG fue inmovilizada sobre la superficie del ESPC mediante adsorción física y el estudio del comportamiento electroquímico fue llevado a cabo mediante voltamperometría cíclica y cronoamperometría. La PPG reveló una pareja bien definida de señales redox a 17mV/-141mV correspondientes al proceso redox del grupo hemo (Fe2+/Fe3+) de las peroxidasas. La reducción bioelectrocatalítica del peróxido de hidrógeno se observó a un potencial redox de -645 mV vs Ag. Este proceso fue controlado por difusión de las especies en la superficie del electrodo en un rango de velocidad de barrido lineal de 50-500 mV/s. La cronoamperometría permitió la construcción de curvas de calibración entre la corriente de reducción y la concentración del H2O2 para la determinación de parámetros analíticos como sensibilidad, rango lineal y nivel mínimo de detección. El desarrollo de este biosensor amperométrico se convierte en un paso preliminar para la construcción de un dispositivo portátil y de respuesta rápida para el análisis de H2O2 en muestras de interés ambiental y biomédico

Development of electrochemical immunosensors for HER-1 and HER-2 analysis in serum for breast cancer patients

In this work, two human epidermal growth factor receptors, HER-1 and HER-2, were selected as biomarkers to enable the detection of breast cancer. Therefore, two biosensors were developed using gold sensor chips coupled with amperometric detection of the enzyme label horse radish peroxidase (HRP). The biosensors/immunosensors relied on indirect sandwich enzyme-linked immunosorbent assays with monoclonal antibodies (Ab) against HER-1 and HER-2 attached to the sensors to capture the biomarkers. Detection polyclonal antibodies followed by secondary anti-rabbit (for HER-1) and anti-goat (for HER-2) IgG antibody-HRP were then applied for signal generation. In buffer, the developed sensors showed limits of detections (LOD) of 1.06 ng mL−1 and 0.95 ng mL−1 and limits of quantification (LOQ) of 2.1 ng mL−1 and 1.5 ng mL−1 for HER-1 and HER-2, respectively. In 100% (undiluted) serum, LODs of 1.2 ng mL−1 and 1.47 ng mL−1 and LOQs of 1.5 ng mL−1 and 2.1 ng mL−1 were obtained for HER-1 and HER-2, respectively. Such limits of detections are within the serum clinical range for the two biomarkers. Furthermore, gold nanoparticles (AuNP) labelled with secondary anti-rabbit and anti-goat IgG antibody-HRP were then used to enhance the assay signal and increase the sensitivity. In buffers, LODs of 30 pg mL−1 were seen for both sensors and LOQs of 98 pg mL−1 and 35 pg mL−1 were recorded for HER-1 and HER-2, respectively. For HER-2 the AuNPs biosensor was also tested in 100% serum obtaining a LOD of 50 pg mL−1 and a LOQ of 80 pg mL−1. The HER-2 AuNP electrochemical immunosensor showed high specificity with very low cross-reactivity to HER-1. These findings demonstrate that the two developed sensors can enable early detection as well as monitoring of disease progression with a beneficial impact on patient survival and clinical outcomes

Electrocatalysis by heme enzymes—applications in biosensing

Funding Information: The APC was funded by TIMB3 project, European Union's Horizon 2020 Research and Innovation Program grant agreement No 810856. Funding Information: Acknowledgments: We acknowledge the support from Project LISBOA-01-0145-FEDER-007660 (Microbiologia Molecular, Estrutural e Celular) funded by FEDER funds through COMPETE 2020-Programa Operacional Competitividade e Internacionalização (POCI); from FCT—Fundação para a Ciência e a Tecnologia (PTDC/BIA-BFS/31026/2017 and 2020.05017.BD) and from the European Union's Horizon 2020 Research and Innovation Program, through TIMB3 and B-LigZymes projects (grant agreements No 810856 and 824017, respectively). We thank Edilson Galdino for critical reading of the manuscript and helpful discussions. Funding Information: Funding: The APC was funded by TIMB3 project, European Union's Horizon 2020 Research and Innovation Program grant agreement No 810856. Publisher Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. Copyright: Copyright 2021 Elsevier B.V., All rights reserved.Heme proteins take part in a number of fundamental biological processes, including oxygen transport and storage, electron transfer, catalysis and signal transduction. The redox chemistry of the heme iron and the biochemical diversity of heme proteins have led to the development of a plethora of biotechnological applications. This work focuses on biosensing devices based on heme proteins, in which they are electronically coupled to an electrode and their activity is determined through the measurement of catalytic currents in the presence of substrate, i.e., the target analyte of the biosensor. After an overview of the main concepts of amperometric biosensors, we address transduction schemes, protein immobilization strategies, and the performance of devices that explore reactions of heme biocatalysts, including peroxidase, cytochrome P450, catalase, nitrite reductase, cytochrome c oxidase, cytochrome c and derived microperoxidases, hemoglobin, and myoglobin. We further discuss how structural information about immobilized heme proteins can lead to rational design of biosensing devices, ensuring insights into their efficiency and long-term stability.publishersversionpublishe

Development of electrochemical ZnSe Quantam dots biosensors for low-level detection of 17β-Estradiol estrogenic endocrine disrupting compound

Magister Scientiae - MScThe main thesis hub was on development of two electrochemical biosensors for the determination of 17β-estradiol-estradiol: an estrogenic endocrine disrupting compound. Endocronology have significantly shown that the endocrine disruptors contribute tremendously to health problems encountered by living species today, problems such as breast cancer, reproductive abnormalities, a decline in male population most significant to aquatic vertebrates, reduced fertility and other infinite abnormalities recurring in the reproductive system of mostly male species. The first biosensor developed for the detection of 17β-estradiol-estradiol endocrine disrupting compound; consisted of an electro-active polymeric 3-mercaptoprorionic acid capped zinc selenide quantum dots cross linked to horseradish peroxidase (HRP) enzyme as a bio-recognition element. The second biosensor developed was comprised of cysteamine self assembled to gold electrode, with 3-mercaptopropionic acid capped zinc selenide quantum dots cross linked to cytochrome P450-3A4 (CYP3A4) enzyme in the presence of 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide hydrochloride and succinimide.South Afric

Imaging horseradish peroxidase under electrochemical conditions

Ph. D. Thesis.In this work, potential dependent electrochemical characterisation has been performed on horseradish peroxidase (HRP) molecules immobilised on annealed highly oriented pyrolytic graphite (HOPG) surface immersed in phosphate buffer solution (PBS). Electrochemical impedance spectroscopy (EIS) was applied to investigate the HRP molecular capacitance. The observed HRP capacitance shows significant potential dependency, which is analogous to that of a typical metal oxide semiconductor (MOS) capacitor fabricated with p-type semiconductor materials. Accumulation region and depletion region has been observed from the capacitance (C)-potential (U) curve of the HRP molecules, and the “flat band potential”, at which there is no potential drop within the semiconductor layer, has been determined as the potential where dC/dU-U curve peaks. An MOS capacitor approximation is proposed to describe the potential dependent capacitance behaviour of HRP molecules. Scanning electrochemical potential microscopy (SECPM) and electrochemical scanning tunnelling microscopy (EC-STM) has been applied to monitor the potential drop in HRP molecules at single molecular level. SECPM maps the potential distribution within the electrochemical double layer (EDL) and can resolve HRP molecules with Angstrom resolution. Potential drop within HRP molecules is reflected as potential dependent morphological variation in SECPM images. The observed HRP apparent size peaks at the flat band potential. Similar potential dependency of HRP apparent size is observed using EC-STM, as the potential drop being an influence on the bias voltage between tip and substrate. SECPM was also used to directly measure the EDL profile with alternating electrode potential and electrolyte concentration. An exponential potential decay with reference to the tip-substrate separation has been observed as predicted by Gouy-ChapmanStern model. Delayed response of potential decay, with significant dependence on the local electrical field and ionic strength, was also observed at close tip-substrate distance. A quantitative approach based on a novel mechanism which suggests high resolution SECPM imaging could be attributed to direct electron exchange between tip and substrate is proposed to interpret the delayed response of potential decay