Multiplexed electrochemical detection of trypsin and chymotrypsin based on distinguishable signal nanoprobes

In this work, we developed a novel multisignal output for simultaneous detection of multiple proteases by using nanoprobes labeled with distinguishable electrochemical probes. First, biotinylated peptide1 (S1) and biotinylated peptide2 (S2) were associated with biotinylated DNA1 and DNA2 via biotin-streptavidin interaction, forming DNA1-S1 and DNA2-S2, respectively. Two distinguishable signal nanoprobes (DNA1'-Au NPs-Thi and DNA2'-Au NPs-Fc) were prepared by initial assembling DNA1' and DNA2' on the Au NPs surface, respectively, and then carrying corresponding thionine (Thi) and 6-(Ferrocenyl)hexanethiol (Fc). Then, the peptide substrates (DNA1-S1 and DNA2-S2) were immobilized on gold electrode surface through Au-S bonds, and the DNA1'-Au NPs-Thi and DNA2'-Au NPs-Fc were assembled to the peptide-DNA-modified electrode surface via DNA hybridization. The targets of trypsin and chymotrypsin can specifically recognize and cleave peptides with different sequences, releasing DNA1'-Au NPs-Thi and DNA2'-Au NPs-Fc from the electrode surface into solution, thus decreasing the current of Thi and Fc. The decrease in the electrochemical currents of the two signal nanoprobes enables us to simultaneously and quantitatively determine the targets trypsin and chymotrypsin. More importantly, this strategy can be extended easily by designing various proteases-specific peptide substrates and utilizing corresponding electrochemical detectable elements for simultaneous multiplex protease assay in various biosystems.