Generic phosphatase activity detection using zinc mediated aggregation modulation of polypeptide-modified gold nanoparticles

Abstract

A challenge in the design of plasmonic nanoparticle-based colorimetric assays is that the change in colloidal stability, which generates the colorimetric response, is often directly linked to the biomolecular recognition event. New assay strategies are hence required for every type of substrate and enzyme of interest. Here, a generic strategy for monitoring of phosphatase activity is presented where substrate recognition is completely decoupled from the nanoparticle stability modulation mechanism, which enables detection of a wide range of enzymes using different natural substrates with a single simple detection scheme. Phosphatase activity generates inorganic phosphate that forms an insoluble complex with Zn²⁺. In a sample containing a preset concentration of Zn²⁺, phosphatase activity will markedly reduce the concentration of dissolved Zn²⁺ from the original value, which in turn affects the aggregation of gold nanoparticles functionalized with a designed Zn²⁺ responsive polypeptide. The change in nanoparticle stability thus provides a rapid and sensitive readout of the phosphatase activity. The assay is not limited to a particular enzyme or enzyme substrate, which is demonstrated using three completely different phosphatases and five different substrates, and thus constitutes a highly interesting system for drug screening and diagnostics.