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  • A novel peptide-based electrochemical biosensor for the determination of a metastasis-linked protease in pancreatic cancer cells.

A novel peptide-based electrochemical biosensor for the determination of a metastasis-linked protease in pancreatic cancer cells.

Analytical and bioanalytical chemistry (2020-01-29)
Cristina Muñoz-San Martín, María Pedrero, Maria Gamella, Ana Montero-Calle, Rodrigo Barderas, Susana Campuzano, José M Pingarrón
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. Graphical abstract.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
p53 human, recombinant, expressed in E. coli, buffered aqueous glycerol solution
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Proteinase K from Tritirachium album, lyophilized powder, BioUltra, ≥30 units/mg protein, for molecular biology
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IgG from human serum, reagent grade, ≥95% (HPLC), buffered aqueous solution
Sigma-Aldrich
α-Chymotrypsin from bovine pancreas, (TLCK treated to inactivate residual tryspin activity), Type VII, essentially salt-free, lyophilized powder, ≥40 units/mg protein
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Trypsin from porcine pancreas, Type IX-S, lyophilized powder, 13,000-20,000 BAEE units/mg protein
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Hemoglobin human, lyophilized powder
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Pepsin from porcine gastric mucosa, lyophilized powder, ≥3,200 units/mg protein
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Lysozyme human, Lysobac, recombinant, expressed in rice, ≥100,000 units/mg protein, lyophilized powder