Development of Nanomechanical Sensors for Environmental Contaminate Screening Using Protein Functionalized Microcantilevers

The development of real time, label-free biosensors based on ligand-induced nanomechanical responses of microcantilevers (MCs) allows for sensitive and selective detection. High sensitivity is afforded by the MCs small dimensions. Immobilizing biomolecular recognition phases imparts selectivity from bioaffinity interactions. Biological sensors on a MC platform utilize various proteins, such as antibodies and nuclear receptors, which can be used to detect and screen for potential environmental contaminants. The interaction between contaminants and immobilized receptors induces an apparent surface stress that leads to static bending of the MC, which is monitored by an optical beam bending technique. Biofunctionalized MCs can provide high sensitivity and selectivity on a relatively inexpensive platform that requires small amounts of analyte. The goal of this research is to develop and optimize MCs as biosensors to detect low concentrations of contaminants. Initially, the research utilized specific receptors and antibodies to detect and screen for contaminants that are deemed endocrine disrupting chemicals (EDCs). Immobilizing estrogen receptors and specific antibodies on the MC surface may provide information on the ever expanding list of EDCs, along with fundamental endocrine studies. Then, the MC surface was morphologically and chemically optimized. This optimization included the thickness and metal ratio of the dealloyed surface. The concentration, reaction time, and pH of chemical immobilization reagents, which include aminoethanethiol and glutaraldehyde, were optimized by using an anti-body test system. Antibody and protein functionalization conditions, which are incubation time and concentration, were optimized using the anti-immunoglobulin G (anti-IgG) receptor: IgG and an anti-biotin:biotin test systems. The optimized immobilization conditions were applied to the detection of thyroid disrupting chemicals (TDCs) using MCs functionalized with the transport protein thyroxine-binding globulin. The final project involved developing a nanomechanical transducer to study xenobiotic and EDC interactions with the bioreceptor PXR’s ligand binding domain (LBD). The combination of immobilized LBD PXR with a nanostructured microcantilever (MC) platform allows for the study of ligand interaction with the receptor’s binding domain. PXR shows real-time, reversible responses when exposed to specific pharmaceutical, EDC, and xenobiotic ligands. Three binding interactions that involve EDCs are tested, which include phthalic acid, nonylphenol, and bisphenol A, with PXR