Immunoassay of Goat Antihuman Immunoglobulin G Antibody Based on Luminescence Resonance Energy Transfer between Near-Infrared Responsive NaYF4:Yb, Er Upconversion Fluorescent Nanoparticles and Gold Nanoparticles

Near-infrared (NIR) light can penetrate biological samples and even tissues without causing sample damage and avoid autofluorescence from biological samples in fluorescence detection. Thus, a luminescence resonance energy transfer (LRET)-based immunoassay that can be excited by NIR irradiation is a promising approach to the analysis of biological samples. Here we demonstrate the use of NIR-to-visible upconversion nanoparticles (UCNPs) as an energy donor, which can emit a visible light upon the NIR irradiation, and gold nanoparticles (Au NPs) as an energy acceptor, which can absorb the visible light emitted from the donor, to develop a sandwich-type LRET-based immunoassay for the detection of goat antihuman immunoglobulin G (IgG). Amino-functionalized NaYF4:Yb, Er UCNPs and Au NPs were first prepared and then conjugated with the human IgG and rabbit antigoat IgG, respectively. The NIR-excited fluorescence emission band of human IgG-conjugated NaYF4:Yb, Er UCNPs (λmax = 542 nm) partially overlaps with the visible absorption band of the rabbit antigoat IgG-conjugated colloidal Au NPs (λmax = 530 nm), satisfying the requirement of spectral overlap between donors and acceptors for LRET. A LRET system was then formed when goat antihuman IgG was added to a mixture of human IgG-modified NaYF4:Yb, Er UCNPs (donor) and rabbit antigoat IgG-modified Au NPs (acceptor). The sandwich-type immunoreactions between the added goat antihuman IgG (primary antibody) and the two different proteins (antigen and secondary antibody on the surface of the donors and acceptors, respectively) cross-bridge the donors and acceptors and thus shorten their spacing, leading to the occurrence of LRET from UCNPs to Au NPs upon NIR irradiation. As a result, the quenching of the NIR-excited fluorescence of the UCNPs is linearly correlated to the concentration of the goat antihuman IgG (in the range of 3−67 μg·mL−1) present in the system, enabling the detection and quantification of the antibody. Such sandwich-type LRET-based approach can reach a very low detection limit of goat antihuman IgG (0.88 μg·mL−1), indicating that this method is applicable for the trace protein detection. This approach is expected to be extended to the detection of other biological molecules once the donor and acceptor nanoparticles are modified by proper molecules that can recognize the target biomolecules.