Virus-directed synthesis of emitting copper nanoclusters as an approach to simple tracer preparation for the detection of Citrus Tristeza Virus through the fluorescence anisotropy immunoassay

Citrus Tristeza Virus (CTV) was detected by targeting the virus capsid protein, CP25, through a competitive fluorescence anisotropic immunoassay. In this assay, instead of using chromophore labeled tracers, a simple approach in which the target antigen serves both as the main target and tracer was developed. For this, CP25 was expressed in E.coli and the purified recombinant protein was manipulated for copper nanocluster synthesis. The resultant CP25/CuNCs optically showed quantum dot-like behavior and emitted light in a range of 535–715 nm. The CP25/CuNCs could also interact with the anti-CTV antibody and showed polarized emission. In this assay, the CP25 protein competes with the fluorescent CP25/CuNCs used as a tracer for immune-complex formation with the anti-CTV antibody. The results showed that the FA values decreased linearly from 400 pg/mL to 25 ng/mL with an increase in CP25 protein levels and the limit of detection (LOD) was 220 pg/mL. The procedure of this assay can be useful for the detection of low molecular weight proteins or peptides in general, due to the availability of various kinds of metal nanoclusters and synthesis protocols.