Characterization & Application of Immobilized Biomacromolecules using Microcantilever and QCM Sensors
Biswal, Sibani Lisa
Doctor of Philosophy
The structure and function of immobilized biomacromolecules are likely to be altered because of the solid surface. The long-term objective of this thesis is to develop surface-based biosensors for the characterization and application of biomacromolecules at the liquid-solid interface. In this study, two analytical surface-sensitive sensors are utilized: microcantilevers and quartz crystal microbalance with dissipation (QCM-D). Each offers unique information regarding the molecules of interest. In particular, the systems that are covered in this thesis include the detection of target analytes using specific recognition elements and the characterization of supported lipid membranes. This research has led to a better understanding of the effect of solid surfaces on protein structure and function, as well as the ability to engineer biomolecular surfaces with great control. There are two detection systems that were studied: a phage-derived peptide system for the detection of pathogenic bacteria Salmonella and an antibody displacement assay for the detection of an explosive, 2,4,6-trinitrotoluene (TNT). The microcantilever responds to changes in the surface free energy on the sensor surface by monitoring changes in its deflection. The physisorption or chemisorption of molecules to the cantilever surface induces a mismatch in the surface stress, causing the cantilever to bend. The multiplexed measurement is able to quickly determine the binding affinities of various phage-derived peptides, improving the screening efficiency of the peptides derived from phage display libraries for Salmonella detection. The microcantilever-based technique provides a novel biosensor to rapidly and accurately detect pathogens and holds potential to be further developed as a screening method to identify pathogen-specific recognition elements. QCM measures mass changes on the sensor surface by monitoring the frequency change of the crystal. The combination of a competition assay with QCM using an anti-TNT antibody is able to distinguish a TNT molecule among molecules of similar structure at low concentrations, leading a sensitive and selective assay. The reliability of this method was further investigated in more real environments simulated by fertilizer solution and seawater. Furthermore, this method could be also applied in gas phase detection of TNT, as well as the detection of other chemicals, such as environmental pollutants and illegal drugs. In both of these detection assays, a mathematical model was developed to quantify the binding of the target molecules with the molecules of interest. In the second half of the thesis, the microcantilever sensor is applied to characterize supported lipid bilayers (SLBs), an interesting biomacromolecular assembly that holds great importance as a model system for membranes. Through monitoring the cantilever deflection, the formation of the SLB, its temperature induced phase transitions, and its interactions with membrane-active molecules are investigated. With increasing temperature, the lipid acyl chains transition from an ordered state to a disordered state, accompanied by a changes in the surface stress that can be readily detected using microcantilever. The phase transition temperature of SLBs is different from that of a lipid monolayer, indicating that the existence of the solid support aﬀects the monolayer structure. Two amphipathic membrane-active molecules, peptide (PEP1) and a triblock copolymer (Pluoronic), are studied for their associations with SLBs. PEP1’s association with SLBs highly depends on the ratio of peptide over lipid, while the Pluoronic interacts with SLBs as a function of temperature and the length of lipophilic block in the copolymer. Therefore, the microcantilever sensor is capable of measuring the conformational change of surface-bound molecules, as well as characterizing the kinetics of membrane-peptide interactions with great sensitivity.
Macromolecule; Biosensors; Chemical engineering