Morphological and chemical optimization of microcantilever surfaces for thyroid system biosensing and beyond

The development of biosensors is vital in many areas of biotechnology and biomedical research. A prominent new class of label-free biosensors are those based on ligand-induced nanomechanical responses of microcantilevers (MCs). The interaction between biologically significant ligands with bioreceptors (e.g., antibodies or nuclear receptor proteins) immobilized on one side of the MC surface causes an apparent surface stress, resulting in static bending of the MC, which can be detected by an optical beam bending technique. The three key performance metrics of sensitivity, selectivity, and reversibility are foci of the work reported herein. The nature of the MC surface and the method by which the bioreceptor is immobilized influence these performance metrics and, hence, optimization studies involving these were conducted. In our work, the gold surface on one side of the MC is first activated via self-assembled monolayer formation with amino ethane thiol (AET) then reacted with glutaraldehyde (GA) as a crosslinker before finally functionalizing with the protein receptor. We report the effect of concentration, reaction time, and pH for these reagents on the magnitude of the nanomechanical responses using an anti-immunoglobulin G (anti-IgG) receptor: IgG ligand test system. By vapor depositing an alloy of silver and gold and then etching away the former, a nanostructured “dealloyed” MC surface is created that outperforms a smooth gold MC in terms of nanomechanical responses. Optimization of the dealloying parameters (thickness, metal ratio) is also reported herein using the aforementioned anti-IgG-IgG system. Maximum response was obtained with these conditions: 150 nm dealloyed surface, 1 mM aqueous solution of AET-incubation time 1 h, 1% GA solution in 10 mM pH 8 phosphate buffered saline (PBS)-incubation time 3 h, and 0.5 mg mL⁻¹ of receptor protein solution in 10 mM pH 7 PBS-incubation time 1 h. Additionally, surprising results are reported when Protein A is immobilized first to properly orient the bioreceptor IgG molecules. We also report the application of optimum and non-optimum conditions to detect thyroid disrupting chemicals (TDCs) using MCs functionalized with the transport protein thyroxine-binding globulin. Selectivity patterns are reported for several TDCs and sensitive detection of thyroxin at sub-nM levels is demonstrated.