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 molecularlydissolved polymer chains. Fluorescence correlation spectroscopy ofthe >P1/>N4 complex and of >P1/>N4/PPi confirms the change in size of the CPE aggregatesthat occurs upon reversible aggregation. Application of PCA to thefluorescence emission data set of standard samples yields two principalcomponents, which are used to create a predictive model for PPi analysis.The PCA method is able to directly determine the concentration ofPPi with approximately 95% accuracy within the concentration rangefrom 100 μM to 3 mM, without the need for a reference stateas is typically needed for ratiometric fluorescence assays.
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