Abstract
An amperometric magnetoimmunosensor for the determination of human p53 protein is described in this work using a sandwich configuration involving the covalent immobilization of a specific capture antibody onto activated carboxylic-modified magnetic beads (HOOC-MBs) and incubation of the modified MBs with a mixture of the target protein and horseradish peroxidase-labeled antibody (HRP-anti-p53). The resulting modified MBs are captured by a magnet placed under the surface of a disposable carbon screen-printed electrode (SPCE) and the amperometric responses are measured at −0.20 V (vs. an Ag pseudo-reference electrode), upon addition of hydroquinone (HQ) as a redox mediator and H2O2 as the enzyme substrate. The magnetoimmunosensing platform was successfully applied for the detection of p53 protein in different cell lysates without any matrix effect after a simple sample dilution. The results correlated accurately with those provided by a commercial ELISA kit, thus confirming the immunosensor as an attractive alternative for rapid and simple determination of this protein using portable and affordable instrumentation.
Highlights
Following heart disease, cancer is one of the main occurring diseases worldwide, the number of new cases is expected to rise by about 70% in the two decades [1,2,3]
After a blocking protocol with ethanolamine of the unreacted activated groups on the MBs and washing with the casein blocker solution, AbC-MBs were incubated with the samples supplemented with the horseradish peroxidase (HRP)-labelled detector antibody (HRP-AbD), the target protein being sandwiched between the AbC immobilized on the MBs and the HRP-AbD
MBs, bearing the sandwich immunocomplexes, were captured magnetically on the working electrode surface by placing the screen-printed electrode (SPCE) on a custom-fabricated magnetic holding block, and the extent of the biorecognition event was monitored by amperometry in stirred solutions of the reduction current generated upon H2 O2 addition in the presence of HQ
Summary
Cancer is one of the main occurring diseases worldwide, the number of new cases is expected to rise by about 70% in the two decades [1,2,3]. To reduce and control cancer, implementation of evidence-based strategies for its prevention, early detection and management of patients is needed. Nowadays quite a number of cancer-related proteins and biomarkers have been identified, to be clinically useful simple and reproducible analysis procedures need to be developed for routine use. The implementation of simple, accurate, and low-cost detection systems for clinical biomarkers ideated to be used at home or in the field for personal healthcare and diagnostic is, one of the main objectives in the clinical research field. P53 is a DNA binding protein known in cancer biology as a critical tumor suppressor and transcription factor, proposed as the master regulator of cell fate and regarded as “the guardian of the genome” [5,6,7]. It is considered to play a crucial role in the regulation of the cell cycle, DNA repair, and programmed cell death, Biosensors 2016, 6, 56; doi:10.3390/bios6040056 www.mdpi.com/journal/biosensors
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