Development of an enzymatic flow-injection chemiluminescence system for determining inorganic pyrophosphate ion

Detection techniques of inorganic pyrophosphate ions (PPi) enable monitoring of nucleic acids and metabolic activity. For PPi determination, we used an enzymatic phosphate ion (Pi) measuring system that was developed in our previous study. It is an automatic flow-injection (FI) system combining a luminol chemiluminescence (CL) reaction and a pyruvate oxidase G (POG; from Aerococcus viridans) reaction. Inorganic pyrophosphatase (IP; from bakers yeast, E.C. 3.6.1.1) was used in an enzymatic FI-CL system for PPi determination. IP and POG were co-immobilized onto a N-hydroxysuccinic-acidimido gel and packed into a stainless steel column. The characterization of the enzymatic FI-CL system produced a calibration curve with a linear range between 100 nM and 100 (mu)M PPi (7 points of PPi conc., r^2=0.9997). The obtained detection limit was 100 nM PPi. Under these conditions, obvious differences of the CL responses to four 30 (mu)M deoxyribonucleoside 5ʹ-triphosphates (dNTPs) with PPi were observed.