Facile preparation of bifunctional monolayers through diazonium grafting and “click” postfunctionalization: A first step towards efficient aptasensing interfaces

Herein, we present an efficient approach for developing electrochemical aptasensing interfaces, by "click" postfunctionalization of phenylethynyl-grafted glassy carbon substrates with mixed monolayers containing biorecognition elements and phosphorylcholine zwitterionic groups. Typically, controlling the composition of multicomponent surface layers by grafting from a mixture of aryldiazonium salts is challenging due to differences in their chemical reactivity. Our approach circumvents this issue by employing the electrochemical reduction of a single aryldiazonium salt containing a silyl-protected alkyne group followed by deprotection, to create phenylethynyl monolayers which can subsequently accommodate the concurrent immobilization of bioreceptors and zwitterionic groups through "click" postfunctionalization. We show that the surface ratio of the components in the bifunctional monolayers, estimated through XPS and electrochemical methods, can be accurately controlled by adjusting the mole ratio of the corresponding azide reagents in the "click" coupling solution. Moreover, electrochemical impedance spectroscopy and fluorescence microscopy investigations on bifunctional monolayers containing ssDNA and phosphorylcholine groups reveal that they effectively prevent nonspecific protein adsorption, while maintaining sufficiently low impedance to facilitate electrochemical detection. Finally, we demonstrate that proof of concept aptasensing interfaces based on binary layers containing a ferrocene-tagged cocaine/quinine aptamer and phosphorylcholine groups exhibit a trade-off between an improved analytical response and antifouling efficiency.