Bioconjugated nanoparticle detection of respiratory syncytial virus infection

Abstract

The integration of nanotechnology with biology has produced major advances in molecular diagnostics, therapeutics, and bioengineering. Recent advances have led to the development of functionalized nanoparticles (NPs) that are covalently linked to biological molecules such as antibodies, peptides, proteins, and nucleic acids. These functionalized NPs allow for development of novel diagnostic tools and methods, particularly for pathogens, as rapid and sensitive diagnostics are essential for defining the emergence of infection, determining the period that preventive measures should be applied, for evaluating drug and vaccine efficacy, and for controlling epidemics. In this study, we show that functionalized NPs conjugated to monoclonal antibodies can be used to rapidly and specifically detect respiratory syncytial virus in vitro and in vivo. These results suggest that functionalized NPs can provide direct, rapid, and sensitive detection of viruses and thereby bridge the gap between current cumbersome virus detection assays and the burgeoning need for more rapid and sensitive detection of viral agents.

Figure 1

HRTEM images of green and orange CdTe nanoparticles showing an average size of 3 nm (A) and 5 nm (B), respectively. Abbreviations: CdTe, cadmium telluride; HRTEM, high resolution transmission electron microscopy.

Figure 2

RSV-NP detection of RSV-infected Vero cells. Vero cells were mock-infected (A, C) or RSV-infected at a MOI = 1 (B, D) and fixed with acetone: methanol (60:40). Cells were immunostained with by conventional methods (A, B) or by RSV-NPs (C, D), and analyzed using an immunofluorescence microscopy (40X; A, C or 100X; B, D). Abbreviations: MOI, multiplicity of infection; RSV-NP, respiratory syncytial virus-nanoparticles.

Figure 3

RSV-NP virus plaque assay. RSV was serially diluted ten-fold and the dilutions used to infect Vero cells for determination of virus titers. The viral titer determined from a conventional immunostaining plaque assay (A). were compared with RSV-NPs detection (B). Both 540 nm and 585 nm QDs were evaluated as RSV-NPs (B). RSV plaques were enumerated using an Amersham Biosciences Typhoon 9210 scanner. Abbreviations: RSV-NP, respiratory syncytial virus-nanoparticles; QDs, quantum dots.

Figure 4

Dynamic range and sensitivity of detection of RSV-NP plaque assay compared with a conventional plaque assay. The viral titer of RSV-infected Vero cells was determined at day 3 pi (A) and day 5 pi (B) using conventional immunostaining plaque assay or single-step detection using 1:250 dilution of RSV-NPs derived from 585 nm QDs. RSV plaques were enumerated using an Amersham Biosciences Typhoon 9210 scanner. Abbreviations: RSV-NP, respiratory syncytial virus-nanoparticles; QDs, quantum dots.

Figure 5

RSV-NPs detection of RSV-infected lung tissue. Lung tissue sections from RSV-infected (A, C) or naïve, mock-treated BALB/c mice (B, D) were stained by IHC using RSV-NPs (A, B), or by conventional IHC (C, D), and analyzed using an immunofluorescence microscopy. Abbreviations: IHC, immunohistochemistry; RSV-NP, respiratory syncytial virus-nanoparticles.