| The main thrusts in the area of sensors research are toward new and/or improved recognition elements and transduction methods as well as the miniaturization and incorporation of sensing systems into planar platforms with integrated microfluidics (i.e., lab-on-a-chip type devices). This dissertation explores both the development of assays that are target-specific for clinical and pharmaceutical applications as well as the integration of protein-based assays into a microfluidics platform. Further, the utilization of magnetoelastic transduction as a novel means for monitoring biological processes is discussed.; Fluorescent proteins, particularly green fluorescent protein (GFP), are commonly used as labels in the development of homogeneous protein-based assays for quantitative analysis with clinical and pharmaceutical applications. A portion of this research outlines the use of two fusion proteins, which incorporate enhanced green fluorescent protein (EGFP) in the development of assays using the hydrolytic enzyme, β-lactamase, and the binding protein, calmodulin, as sensing elements. The fluorescence signal that is generated by EGFP allows cleavage, to be detected. In the first study, the hydrolysis of a β-lactam substrate leads to the production of protons. The decrease in local pH alters the microenvironment of the attached fluorophore and consequently its fluorescence properties. Assays of this nature serve as a means to study enzyme activity in vitro.; The second assay utilized the calcium-binding protein, calmodulin. Calmodulin is known to bind phenothiazine derivatives, and was therefore used to develop an assay for their detection. The integration of this assay into the centrifugal microfluidics platform was the main focus of this project. The platform consists of a series of channels and reservoirs machined into a disk-shaped polymer substrate. Fluid propulsion is achieved via rotation of the disk, while the incorporation of valves based on surface tension, allow controlled releases of each reaction component. By miniaturizing this assay and putting it on a planar substrate with microfluidics, multiple assays could be performed in parallel on a single platform, which lends itself to high-throughput screening applications.; Magnetoelastic sensors have been used to study the phenomena of blood coagulation and fibrinolysis. These sensors have a characteristic resonance frequency that is dependent on the properties of the strip. The fundamental frequency of the strip will shift in response to changes in physical and chemical parameters, particularly viscosity. The viscosity changes that occur during the hemostatic process, i.e., coagulation and fibrinolysis, were monitored using magnetoelastic transduction, thus allowing for the development of profiles, from which qualitative and quantitative information about these processes can be gathered. The combination of new recognition elements and transduction methods, as well as their integration into planar platforms allow for the development of sensing systems for both clinical and pharmaceutical applications. |
