Surface-enhanced Raman scattering based immunoassay platforms for viral pathogen detection.

摘要

Surface-enhanced Raman scattering (SERS) based biosensors have been widely applied in biomedical diagnostics for its robustness, high sensitivity, improved levels of multiplexing and potential to perform detection in a point-of-care platform. Recent progress in SERS has been demonstrated for a wide range of biosensor applications, ranging from nucleic acid and immunoassay detection to tumor imaging and cancer diagnosis. SERS-based immunoassays have gained considerable attention for target pathogen detection in food safety and public health assurance. In this work, we have employed different versions of this strategy in the low-level detection of proteins (e.g., immunoglobulins and viral antigens). Therefore, we can detect either the exposure to the pathogen or the existence of pathogen.;First, a novel SERS immunoassay for detection of exposure to viral antigen has been developed which used antigen-coated gold nanoparticles (GNPs) for antibody capture and Raman label-conjugated protein A/G (pA/G) as a bi-functional reporter. GNPs were first coated with a recombinant His+-tagged fusion of the West Nile virus (WNV) envelope (E) protein and then incubated with rabbit antiserum raised against recombinant E protein and finally mixed with malachite green (MG)-labeled protein A/G (p/AG). Laser interrogation of the GNP/WNV E antibody/MG-pA/G immunocomplex in solution provided an intense characteristic Raman spectrum of MG which indicated the presence of WNV E antiserum and provided a limit of detection (LOD) of ∼2 ng/ml total serum protein. Compared to enzyme-linked immunosorbent assay (ELISA) that were performed using the same antigen/antibody reagents, the LOD for the SERS immunoassay was found to be >400-fold more sensitive.;In the next study, we proposed a cost-effective, highly sensitive and reproducible magnetic nanoparticle (MNP) capture-based SERS assay for the detection of two viral antigens. GNPs were conjugated with the capture antibodies and the corresponding Raman reporters, followed by hybridizing with the West Nile virus antigen (WNV) and the Rift Valley fever virus (RVFV) antigen as well as the capture MNPs conjugated with specific antibodies. Using an external magnet source, two different hybridization complexes, Infrared-792 (IR-792) labeled GNP/WNV antigen/MNP and Nile Blue (NB) labeled GNP/RVFV antigen/MNP, were removed from solution followed by laser interrogation of the compacted pellet. Laser excitation of the pelleted material provided a mixed spectrum representing the combination of IR-792 and NB signals obtained from each SERS probes, which is diagnostic for recognition of both antigens. The LOD was ∼5 fg/ml for assays conducted in phosphate buffered saline buffer (PBS) and ∼25 pg/ml for assays containing PBS spiked with fetal bovine serum.;Finally, we demonstrated a robust SERS-based immunoassay for the simultaneous detection of three viral antigens spiked in serum through magnetic capture of the silica-encapsulated SERS nanotags. The specific capture of all the antigens in individual assay reactions was performed by incubation with mixtures of silica-coated nanotags (SiNTGs) and magnetic nanoparticles (MNPs), both conjugated with polyclonal antibodies specific for antigen recognition. The SiNTGs provided distinct spectra that correspond to each antigen capture and were impervious to matrix effects. Detection was provided by magnetic pull-down of the three different SiNTG/antigen/MNP immune-recognition complexes and laser excitation of the three spectrally distinct Raman labels. The LOD was ∼ 0.01 ng/ml in 20% fetal bovine serum (FBS) for three antigens, a significant improvement over previous studies in terms of the sensitivity, level of multiplexing and medium complexity.