Single particle-based colorimetric assay of pyrophosphate ions and pyrophosphatase with dark-field microscope

Abstract

We propose a sensitive colorimetric method for the detection of P2O74− and pyrophosphatase (PPase). Without Na4P2O7 and PPase, under the catalysis of gold nanoparticles (AuNPs), Cu(II) can be reduced to Cu by nicotinamide adenine dinucleotide (NADH), leading to the Cu-coating onto the surfaces of AuNPs, accompanied by the color change from green to red under the dark-field microscope observation. While in the presence of Na4P2O7, the formation of Au@Cu NPs is suppressed due to the strong complexation reaction between Cu(II) and Na4P2O7. Thus, the NPs exhibit uniform green color in the dark-field image. By quantitatively counting the integrated optical density (IOD) of red dots in the dark-field images, P2O74− concentration can be accurately determined. A linear logarithmic dependence on P2O74− concentrations from 0.1 nM to 10 mM with a limit of detection (LOD) of 0.0324 nM was obtained. The addition of PPase to the Na4P2O7-triggered dispersed AuNPs restores the formation of Au@Cu NPs owing to the hydrolysis of Na4P2O7 into sodium phosphate under the catalysis of PPase, resulting in reliable quantification of color change of NPs from red to green in the dark-field images. The LOD is as low as 0.0007 U/mL with a linear dynamic range of 0.005–10 U/mL.