A novel peptide-based electrochemical biosensor for the determination of a metastasis-linked protease in pancreatic canc
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A novel peptide-based electrochemical biosensor for the determination of a metastasis-linked protease in pancreatic cancer cells Cristina Muñoz-San Martín 1 & María Pedrero 1 & Maria Gamella 1 & Ana Montero-Calle 2 & Rodrigo Barderas 2 & Susana Campuzano 1 & José M. Pingarrón 1 Received: 10 December 2019 / Revised: 7 January 2020 / Accepted: 13 January 2020 # Springer-Verlag GmbH Germany, part of Springer Nature 2020
Abstract Proteases are involved in cancer‚ taking part in immune (dis)regulation, malignant progression and tumour growth. Recently, it has been found that expression levels of one of the members of the serine protease family, trypsin, is upregulated in human cancer cells of several organs, being considered as a specific cancer biomarker. Considering the great attention that electrochemical peptide sensors have nowadays, in this work, we propose a novel electroanalytical strategy for the determination of this important biomolecule. It implies the immobilization of a short synthetic peptide sequence, dually labelled with fluorescein isothiocyanate (FITC) and biotin, onto neutravidin-modified magnetic beads (MBs), followed by the peptide digestion with trypsin. Upon peptide disruption, the modified MBs were incubated with a specific fluorescein Fab fragment antibody labelled with horseradish peroxidase (HRP-antiFITC) and magnetically captured on the surface of a screen-printed carbon electrode (SPCE), where amperometric detection was performed using the hydroquinone (HQ)/HRP/H2O2 system. The biosensor exhibited a good reproducibility of the measurements (RSD 3.4%, n = 10), and specificity against other proteins and proteases commonly found in biological samples. This work reports the first quantitative data so far on trypsin expression in human cell lysates. The developed bioplatform was used for the direct determination of this protease in lysates from pancreatic cancer, cervix carcinoma and kidney cells in only 3 h and 30 min using low amounts (~ 0.1 μg) of raw extracts. Keywords Cancer . Peptide-magnetic bioconjugate . Screen-printed carbon electrodes . Trypsin . Cell lysates
Introduction Proteases are enzymes whose activity is expressed in proteolysis processes hydrolyzing peptide bonds by attacking the carbonyl groups of peptides [1, 2]. Although they are essential in fundamental biological processes in normal cells regulating cellular processes as gene expression, differentiation, and cell Published in the topical collection featuring Female Role Models in Analytical Chemistry. * Susana Campuzano [email protected] * José M. Pingarrón [email protected] 1
Departamento de Química Analítica, Facultad de CC. Químicas, Universidad Complutense de Madrid, 28040 Madrid, Spain
2
Chronic Disease Programme, UFIEC, Instituto de Salud Carlos III, 28220 Majadahonda, Madrid, Spain
death, proteases are also involved in tumour growth and progression, both at primary and metastatic sites. Around onethird of all proteases are classified as serine (Ser) proteases containing a nucleophili
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