Detection of multiple pathogens in serum using magnetic capture of silica-encapsulated SERS nanotags

Authors: NING, JENG - University Of Wyoming; LI, YENA - University Of Wyoming; ASHLEY, DRISCOLL - University Of Wyoming; Wilson, William; JOHNSON, PATRICK - University Of Wyoming

Submitted to: Biosensors and Bioelectronics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/9/2018
Publication Date: 5/7/2018
Citation: Ning, J., Li, Y., Ashley, D., Wilson, W.C., Johnson, P. 2018. Detection of multiple pathogens in serum using magnetic capture of silica-encapsulated SERS nanotags. Biosensors and Bioelectronics. vol 41 pages 316-321. https://dx.doi.org/10.1016/j.bios.2012.08.048.
DOI: https://doi.org/10.1016/j.bios.2012.08.048

Interpretive Summary: In this work, we report a robust and highly sensitive SERS-based immunoassay using magnetic capture of silica-encapsulated SERS nanotags to simultaneously detect three antigens, West Nile virus (WNV) envelope protein, Rift Valley fever virus nucleocapsid protein and capsular antigen fraction 1 from Yersinia pestis in simulated serum samples. In this assay all three antigens were incubated with the mixture of silica-coated SiNTGs and magnetic nanoparticles, and both SiNTGs and MNPs were conjugated with polyclonal antibodies specific for each antigen. Detection was provided by magnetic pull-down of the SiNTG/antigen/MNP immuno-recognition complexes followed by laser excitation of the complexes. To date, only a few studies have demonstrated trace level detection of pathogens in serum or blood. To our knowledge, this is the first time to quantitatively and simultaneously detect three antigens in simulated serum samples in a single SERS-based assay. This design includes several distinguishing features: 1) high level of multiplexing: three antigens were detected; 2) high assay sensitivity: the LOD for three antigens in 20% fetal bovine serum, 0.01 ng/ml, was 100-fold lower than the LOD previously reported for single antigen capture assay in 10% sera; 3) high specificity and robustness: the protective silica shell not only further immobilized the Raman labels on the SERS nanotag surface preventing cross-talk, but also blocked the unfavorable competitive adsorption of the heterogeneous proteins in serum or blood samples; 4) point-of-care testing capability: in addition to the sample size of tens of microliters, the multiplex assay demanded no complicating process especially like purification for crude samples. All of these factors make it possible to conduct the pathogen detection in the field or in a point-of-care facility with a portable Raman device.

Technical Abstract: A robust and highly sensitive surface enhanced Raman scattering (SERS)-based immunoassay has been developed for simultaneous detection of three antigens: West Nile virus (WNV) envelope (E) protein, Rift Valley Fever virus (RVFV) nucleocapsid (N) protein and capsular antigen fraction 1 (F1) spiked in serum samples using magnetic capture of three silica-encapsulated SERS nanotags respectively labeled with corresponding Raman dyes. The specific capture of all three antigens in a single assay was performed by incubating the antigens with the mixture of silica-coated nanotags (SiNTGs) and magnetic nanoparticles (MNPs), both conjugated with polyclonal antibodies specific for each antigen. Detection was provided by magnetic pull-down of the SiNTG/antigen/MNP immune-recognition complexes followed by laser excitation of the complexes. The SiNTGs provided distinct spectra that correspond to each antigen capture and were impervious to external interference. The magnetic pull-down resulted in aggregation of the SERS substrates and concentration of the Raman labels leading to significant enhancement of the detection sensitivity. The limit of the detection (LOD) was ~ 0.01 ng/ml in 20% fetal bovine serum (FBS) for all three antigens, a significant improvement over previous studies in terms of sensitivity, level of multiplexing and medium complexity. This high level multiplex immunoassay platform provides a promise to detect various antigens directly in crude serum samples without the tedious process of sample preparation, which is desirable for onsite diagnostic testing and real time disease monitoring.