Electrochemical aptamer-based determination of protein tyrosine kinase-7 using toehold-mediated strand displacement amplification on gold nanoparticles and graphene oxide.

Abstract

An electrochemical method is described for ultrasensitive determination of protein tyrosine kinase-7 (PTK7). It is based on (a) the use of positively charged gold nanoparticles (AuNPs) and negatively charged graphene oxide (GO), and (b) of toehold-mediated strand displacement amplification. A hairpin probe 2 (HP2) containing the sgc8 aptamer was used to modify a glassy carbon electrode (GCE). Its hairpin structure is opened in the presence of PTK7 to form the PTK7-HP2 complex. The exposed part of HP2 partly hybridizes with hairpin probe 1 (HP1) that was immobilizing on the AuNPs and GO modified GCE. On addition of the hairpin probe 3 that was labeled with the redox probe Methylene Blue (MB-HP3), toehold-mediated strand displacement occurs due to complementary hybridization of HP1 with MB-HP3. This causes the release of PTK7-HP2 into the solution and makes it available for the next reaction. Under optimal conditions, PTK7 can be quantified by voltammetry (typically performed at -0.18V) with a detection limit of 1.8 fM. The assay possesses high selectivity for PTK7 due to the employment of the aptamer. It was successfully applied to the determination of PTK7 in the debris of malignant melanoma A375 cells. Graphical abstract Schematic representation of the enzyme-free electrochemical sensor for ultrasensitive determination of protein tyrosine kinase-7 (PTK7) based on the toehold-mediated strand displacement reaction amplification on gold nanoparticles and graphene oxide.