AuNPs/methylene blue dual-signal nanoimmunoconjugates and electrode activation for electrochemical biosensors

The use of nanoimmunoconjugates displaying multiple electroactive labels for enhanced analyte quantification is scarce in the literature. In this work, new nanoimmunoconjugates, based on gold nanoparticles (AuNPs) and either methylene blue (MB) or an NHS ester MB derivative (MB_NHS), have been developed by two chemical paths. They have then been used as dual-signal nanolabels in electrochemical biosensors by means of the MB oxidation and AuNPs reduction peaks. The optimization on bare screen-printed carbon electrodes (SPCE) revealed that the use of Fe(CN)₆^3−/4- in the measurement solution enhanced the currents displayed by the MB tag. Additionally, electrochemical SPCE activation in 70% ethanol improved significantly the electrode electrochemical properties compared with the untreated ones. Using 1 mM Fe(CN)₆^3−/4-_, we found that this activation increased 6–10 times the peaks registered by CV and the electrode active area, and reduced significantly the charge-transfer resistance (Rct) obtained from the impedance curve (from 11.4 to 0.3 kΩ). This SPCE activation process allowed detecting the nanoimmunoconjugate in a model immunosensor using the MB tags nearly as sensitively as when measuring the AuNPs counterpart tags (limits of detection of 0.77 and 0.55 nM, respectively). However, while MB detection was faster and simpler, Au detection was less prone to sample matrix interference. This provided sensor efficient performance in a wider range of experimental conditions than when measuring each label independently.