Sensitive Measurement of Waterborne Parasites and Cell Culture Mycoplasma with Cantilever Sensor.

摘要

The objective of the research presented in this dissertation is to develop rapid and sensitive assays for waterborne parasites and mycoplasma contamination detection, as well as assess viability of these targets using piezoelectric excited millimeter-sized cantilever (PEMC) sensor.;Molecular and traditional culture methods in current use are time-consuming and laborious. PEMC sensor is a novel type of mass-sensitive sensor that enables rapid detection in liquid at high sensitivity. Typically it was functionalized with specific antibodies and exposed to the target analytes in a flow apparatus. When analytes bind to the antibody, mass of the sensor increases and causes decrease of sensor's resonant frequency. Real-time monitoring of resonant frequency changes was used for low concentration analytes detection. Limit of detection (LOD) for C. parvum was found to be 5 oocysts/mL, for G. lamblia was found to be 10 cysts/mL in buffers and in complex matrixes (milk, tap water and river water). LOD for mycoplasma (Acholeplasma laidlawii) was found to be 1,000 CFU/mL in cell culture samples. Detection was confirmed with second antibody binding or low pH release or scanning electron microscope. Operating in complex matrixes showed lower sensor response and slower binding kinetics, which are attributed to the hindering of antigen transport and masking of antibody sites by alien material. A novel method was developed for cell viability assessment using a dye that accumulates in viable cells and not in dead cells. PEMC sensor was successfully used for sensing mass change caused by 2',7'-bis(2carboxyethyl)-5,6-carboxyfluorescein (BCECF) accumulation in viable E. coli cells.;At a fundamental level, a novel measurement method based on monitoring sensor impedance magnitude was shown to be feasible. Impedance magnitude of the sensor was monitored not at resonant frequency, but at a fixed frequency near the initial resonant frequency. Results showed that impedance monitoring at a fixed frequency provides measurement equivalent to resonant frequency, with superior signal-to-noise ratio. Minute liquid density changes and antigen-antibody interactions were measured using this approach. This alternate approach lends itself to a simplified implementation for either a single sensor or a bank of multiple sensors. Because of its simplicity, the method would be suitable for high-throughput systems where multiple sensors are used simultaneously.