Phospholipase Assays Using Conjugated Polyelectrolytes

Water-soluble conjugated polyelectrolytes interact with oppositely charged surfactants forming polymer-surfactant complexes. The polymer urfactant interaction induces changes in the conformation [22[ and optical properties [22-24] of the pol3nner including narrowed absorption and enhanced fluorescence quantum efficiency. In addition, the fluorescence quenching efficiency of 

After optimizing the assay conditions, including ionic strength, pH, temperature, activator (Ca ) concentration, and polymer concentration, a calibration curve was developed, which allows the lipid substrate concentration to be determined from the fluorescence intensity. The calibration curve allows the enzyme catalysis kinetics parameters (e.g.. Km and Vmax) to be measured. This PLC turn-off assay is effectively inhibited by known inhibitors (F and EDTA), which demonstrates that the sensor relies on the specific catalysis reaction by PLC. It has been demonstrated to be a sensitive (detection limit 0.5nM enzyme concentration), fast ( 5 min), and selective (good specificity over phospholipase A and D, and other nonspecific proteins) PLC assay, which can be carried out at very low initial substrate concentration (in the range of micromolar to nanomolar). 

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