Abnormal Phospholipase D (PLD) enzyme levels have been associated with various disease conditions including cancer and Alzheimer’s. Thus, PLD can be a potential biomarker for the early detection of these conditions. To detect low quantities of the enzyme, an analytical method with high sensitivity and high selectivity for PLD is essential. In this work, we investigate the development of lipid coated microspheres (lipobeads) and flow cytometry based bio-analytical technique to determine PLD in bio-fluids and cell extracts. Microspheres provide a high surface area to volume ratio medium, thereby allowing us to create reactive surfaces with high lipid concentrations. Additionally, these microsphere-based biomimetic model systems can be easily analyzed using sensitive and high-throughput bioanalytical technique like flow cytometry. PLD hydrolyzes the phosphodiester bond of the glycerophospholipids and generate phosphatidic acid and their respective head groups, thus the presence of the enzyme can be monitored by the lipid-enzyme interaction. The surface of the silica microspheres modified with specific lipid membrane containing fluorescent tags in the head group provides the substrate for the enzymatic interaction. The fluorescent label will be cleaved in the presence of PLD, thereby allowing us to quantify the PLD enzyme through the extent of loss in fluorescence signal. Preliminary experiments with phospholipid model membranes have successfully demonstrated that the proposed biosensor can detect the enzyme at concentrations as low as 1.5 nM in laboratory prepared samples. We are currently investigating its application as a biosensor in measuring PLD activity in biological samples using non-cancerous human breast epithelial cell line (MCF-10A) and breast cancer cell lines (MCF-7 and MDA-MB-231).

Development of lipobead-based phospholipase D biosensor for diagnostic applications

2019 Spring National Meeting

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.