[Homogeneous immunoassay technology based on near infrared upconversion fluorescence resonance energy transfer]

A FRET based assay utilizing NaYF4 : Yb3+, Tm3+ UCNPs as an energy donor, which can emit intense near infrared (NIR) upconversion emission around 800 nm ranges under illumination with a 980 nm laser, and GNPs as an energy acceptor, which has an surface plasmon absorption maximum at 784 nm, was demonstrated. Their optical properties satisfy the requirement of spectral overlap between donors and acceptors for FRET. A model assay for human IgG was then constructed, in which amino-functionalized NaYF4 : Yb3+, Tm3+ UCNPs and GNPs were first prepared and then conjugated with the antibody (goat antihuman IgG) and antigen (human IgG), respectively. The mutual affinity of the antigen and antibody brought the nanocrystals close enough together to allow the FRET to occur, resulting in a significant quenching of UCNPs upconversion emission at 800 nm. When free human IgG was added to the immunocomplex, it competitively binds to UCNPs-goat antihuman IgG, thereby replacing human IgG-GNPs from the immunocomplex and inhibits the FRET process. As a result, the gradually increasing the NIR emission was observed. The authors associate the fluorescence enhancement effect with the concentration of human IgG. Under our experimental conditions, the detection limit is 5 microg x mL(-1). This approach is expected to be extended to the detection of other biological fields, enabling measurements without background fluorescence interference.