Robust and highly sensitive fluorescence approach for point-of-care virus detection based on immunomagnetic separation.
In this work,  robust approach for a highly sensitive point-of-care   virus detection was established based on immunomagnetic nanobeads and fluorescent quantum dots (QDs). Taking advantage of immunomagnetic nanobeads functionalized with the monoclonal antibody (mAb) to the surface protein hemagglutinin (HA) of avian influenza virus (AIV) H9N2 subtype, H9N2 viruses were efficiently captured through antibody affinity binding, without pretreatment of samples. The capture kinetics could be fitted well with a first-order bimolecular reaction with a high capturing rate constant k(f) of 4.25 × 10(9) (mol/L)(-1) s(-1), which suggested that the viruses could be quickly captured by the well-dispersed and comparable-size immunomagnetic nanobeads. In order to improve the sensitivity, high-luminance QDs conjugated with streptavidin (QDs-SA) were introduced to this assay through the high affinity biotin-streptavidin system by using the biotinylated mAb in an immuno sandwich mode. We ensured the selective binding of QDs-SA to the available biotin-sites on biotinylated mAb and optimized the conditions to reduce the nonspecific adsorption of QDs-SA to get a limit of detection low up to 60 copies of viruses in 200 μL. This approach is robust for application at the point-of-care due to its very good specificity, precision, and reproducibility with an intra-assay variability of 1.35% and an interassay variability of 3.0%, as well as its high selectivity also demonstrated by analysis of synthetic biological samples with mashed tissues and feces. Moreover, this method has been validated through a double-blind trial with 30 throat swab samples with a coincidence of 96.7% with the expected results.
Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, Research Center for Nanobiology and Nanomedicine (MOE 985 Innovative Platform), Wuhan University, Wuhan, 430072, P. R. China.
Wei
W
Zhao
Wan-Po
WP
Zhang
Zhi-Ling
ZL
Zhang
Rui-Li
RL
He
Yi
Y
Lin
Min
M
Xie
Han-Zhong
HZ
Wang
Dai-Wen
DW
Pang
D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000276:immunology; D001710:Biotin / Q000737:chemistry / Q000378:metabolism; D002645:Chickens; D005243:Feces / Q000821:virology; D018189:Immunomagnetic Separation; D053127:Influenza A Virus, H9N2 Subtype / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D005585:Influenza in Birds / Q000821:virology; D007700:Kinetics; D008099:Liver / Q000821:virology; D008168:Lung / Q000821:virology; D018613:Microscopy, Confocal; D019095:Point-of-Care Systems; D045663:Quantum Dots; D019809:Streptavidin / Q000737:chemistry / Q000378:metabolism
D000906:Antibodies; D001710:Biotin; D019809:Streptavidin