Activation Methods of Carboxyl Functions for Enhanced Aptamer Immobilization on Glassy Carbon for Application to Electrochemical Biosensing

Electrochemical aptasensors are an attractive class of biosensors for target detection in several complex matrices. The immobilization procedure of the aptamers is currently one of the technological bottlenecks affecting biosensors' performance. It must ensure both the preservation of its affinity toward the target and its stability. Herein, we evaluate carboxyl function activation methods for further aptamer immobilization in the design of glassy carbon-based aptasensors in a three steps strategy. Aptamer immobilization at the glassy carbon surface was conducted in three steps: (i) electrografting of diazonium salts for the functionalization of the electrode with carboxyl groups, (ii) activation of the carboxylic groups, and (iii) immobilization of a DNA aptamer sequence. We focused on the activation step of carboxylic groups by evaluating three coupling agents: the widely reported EDC/NHS carbodiimide agent, bromotripyrrolidinophosphonium hexafluorophosphate (PyBroP), and 2-(1<i>H</i>-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate (TBTU). Cyclic voltammetry, electrochemical impedance spectroscopy, X-ray photoelectron spectroscopy, and water contact angle were used to characterize and confirm each surface modification step, specifically the activation step, which, to our knowledge, has not been investigated before using these activation agents. Aminoferrocene was first used as an electroactive molecule to electrochemically evaluate its coupling with activated carboxylic groups using the different agents. The developed approach for designing this electrochemical aptasensor was subsequently applied to the immobilization of an aptamer sequence for the detection of diclofenac. This work is part of a proof-of-concept study that could be further developed for the design of an electrochemical aptasensor.