Click reaction-based single-particle dark-field microscopy: A visual platform for the highly sensitive and selective detection of pyrophosphatase

Inorganic pyrophosphatase (PPase) plays significant roles in diverse pathophysiological processes, therefore, developing a reliable method to measure PPase activity is crucial for the early diagnosis of the related diseases. In this work, a novel platform with single-particle dark-field microscopy (DFM), consisting of core gold nanoparticles (AuNPs), satellite AuNPs, pyrophosphate ions (PPi), copper ions (Cu²⁺), and sodium ascorbate (NaAsc), is designed for the selective and sensitive analysis of PPase according to a click reaction. After the introduction of PPase, the coordination between PPi and Cu²⁺ can be destroyed by catalyzing the hydrolysis of PPi to release free Cu²⁺ which can be reduced by NaAsc to produce cuprous ions (Cu⁺). Then the formed Cu⁺ promotes the click reaction between azide-modified core AuNPs and alkynyl-modified satellite AuNPs to cause the significant redshift of the localized surface plasmon resonance (LSPR) spectra of core AuNPs. Thus, PPase can be ultra-sensitively detected with a low detection limit of 0.003 U/L in a wide linear range from 0.01 to 1 U/L. This click reaction-based DFM technique can also be used to assess PPase inhibition, which suggests that it has a lot of potential for both the screening of PPase inhibitors and the diagnosis of PPase-related disorders.