Measurement of pyrophosphate concentration in cells by simple terpyridine-Zn(II) probe

Abstract

Pyrophosphate (PPi) anion is an integral part of our physiology. It partakes in the crucial energy transactions in cells and proffers energy for the harmonious functioning of the cell machinery. Discrepancies in PPi concentrations have been associated with several pathological aberrations such as chronic pyrophosphate arthropathy, calcium pyrophosphate deposition disease, cancer etc. However, there still exists a strong demand for an efficient and specific cell based assay for PPi in order to tap its importance to full potential. Terpyridine-Zn2+ complex (ZnCl2L probe) binds with PPi in 1:3 stoichiometry. This association engenders an emission band (λem) at ~591 nm when excited at 440 nm with spectrophotometer. It has lowest detection limit (LOD) of 0.8 nM. Water media based, ultra sensitive detection attribute of fluorescent chemosensor ZnCl2L probe was implemented to investigate pyrophosphate concentrations. In addition to that available commercial kits from Life Technologies, Lonza & Abcam manufacturers were also used for comparison. The probe showed remarkable detection ability for PPi. It generated a linear curve from 0-13.32 μM concentration. Higher concentration (>50 μM) of probe solution should be implemented in order to test PPi concentrations higher than 13.32 μM. The measured pyrophosphate content with the probe in cells was found to be in micromolar range. Contribution of interference from other molecules still remains to be tested. The stability of the PPi-probe-complex was tested against several time points, and was found stable until 24 hr time point. Intense reddish-orange fluorescence in cells stained with ZnCl2L probe under confocal microscope setup confirmed the phenomenon. In conclusion, the ZnCl2L probe proved to offer a reliable method to measure PPi. It could hopefully be used to make measurements from various body fluids. Thus, PPi analysis may assist in diagnosis of diseases where increased levels of PPi are present.