Reliable naked-eye detection of Mycobacterium tuberculosis antigen 85B using gold and copper nanoshell-enhanced immunoblotting techniques

Abstract

A straightforward and naked-eye–sensitive nanoshell-enhanced immunoblotting technique was successfully developed to detect Mycobacterium tuberculosis antigen 85B (Ag85B). The immunoblotting assay included the intensification of the signal of the gold or copper nanoshell on the surface of gold-binding polypeptide fused to 50B14 (GBP-50B14) antibody-conjugated gold nanoparticles (AuNPs). The recombinant GBP-50B14 fusion antibody, which has gold-binding and antigen-binding abilities, shows high affinity toward the AuNP surface as well as Ag85B. The formation of a gold nanoshell or copper nanoshell on the surface of AuNPs leads to the generation of a purple or red spot that can be observed with the naked eye. The gold- and copper-enhanced intensity corresponds to the concentration of Ag85B and provides a detection limit of 0.93 and 0.21 ng/mL by a phone camera and 1.56 and 0.75 ng/mL by the naked eye, respectively. Owing to the higher analytical performance of copper nanoshell enrichment, this immunoblotting technique was successfully applied to determine Ag85B in clinical urine samples. Using this nanoplatform to determine the complexity of Ag85B and CFP10 tuberculosis antigens, the strong reactivity of urine samples from active tuberculosis patients showed 71.4% sensitivity and 86.7% specificity, suggesting excellent application potential in nanomedicine to reliably detect tuberculosis biomarkers for the accurate diagnosis of active Tuberculosis.