Pyrophosphate Sensor Based on Principal Component Analysis of Conjugated Polyelectrolyte Fluorescence

The pyrophosphate anion (PPi) plays an important role in biochemical processes. Therefore, a simple but reliable analytical technique is essential for selective detection of PPi in biochemical systems. Here, we present a principal component analysis (PCA) method for analytical determination of PPi concentration using a fluorescent conjugated polyelectrolyte (CPE) combined with a polyamine modifier. The CPE has anionic side chains and dissolves molecularly in water, as indicated by its structured fluorescence emission spectrum. However, addition of tris(3-aminoethyl) amine (tetraamine or N4) quenches the CPE fluorescence emission. Tetraamine, which is a polycation at neutral pH, binds multiple anionic CPE chains, leading to aggregate formation, resulting in aggregation-induced fluorescence quenching. Addition of PPi to the polymer-amine aggregate reverses the process, resulting in fluorescence recovery. The relatively higher concentration of PPi compared to that of the polymer allows it to effectively compete to bind the amine, thus releasing molecularly dissolved polymer chains. Fluorescence correlation spectroscopy of the P1/N4 complex and of P1/N4/PPi confirms the change in size of the CPE aggregates that occurs upon reversible aggregation. Application of PCA to the fluorescence emission data set of standard samples yields two principal components, which are used to create a predictive model for PPi analysis. The PCA method is able to directly determine the concentration of PPi with approximately 95% accuracy within the concentration range from 100 mu M to 3 mM, without the need for a reference state as is typically needed for ratiometric fluorescence assays.