Nanoparticle detection of respiratory infection

Abstract

Respiratory viruses are a constant concern for all demographics. Examples include established viruses such as respiratory syncytial virus (RSV), the leading cause of respiratory infection in infants and young children, and emerging viruses such as severe acute respiratory syndrome (SARS), which reached near pandemic levels in 2003, or H1N1 (swine) influenza. Despite this prevalence, traditional methods of virus detection are typically labor intensive and require several days to successfully confirm infection. Recently, however, nanoparticle-based detection strategies have been employed in an effort to develop detection assays that are both sensitive and expedient. Each of these platforms capitalizes on the unique properties of nanoparticles for the detection of respiratory viruses. In this article, several nanoparticle-based scaffolds are discussed. Gold nanoparticles (AuNPs) have been functionalized with virus specific antibodies or oligonucleotides. In each of these constructs, AuNPs act as both an easily conjugated scaffolding system for biological molecules and a powerful fluorescence quencher. AuNPs have also been immobilized and used as electrochemical transducers. They efficiently serve as a conducting interface of electrocatalyic activity making them a powerful tool in this application. Quantum dots (QDs) posses unique fluorescence properties that have also been explored for their application to virus detection when combined with direct antibody conjugation or streptavidin-biotin binding systems. QDs have an advantage over many traditional fluorophores because their fluorescence properties can be finely tuned and they are resistant to photobleaching. The development of these nanoparticle-based detection strategies holds the potential to be a powerful method to quickly and easily confirm respiratory virus infection.

Figure 1

(a) Schematic depicting gold nanoparticles (AuNP)‐based biosensor. Fluorescence labeling is followed by assembly of the biosensor complex via binding of protein A to capture antibody. Antigen binding causes conformational change in the antibody, leading to a detectable Förster resonance energy transfer response. (b) AuNP biosensor specificity is demonstrated by comparing response of porcine reproductive and respiratory syndrome virus sample to a control well and the nonspecific antigen bovine serum albumin (BSA). Negligible response to the BSA by AuNP biosensor indicates minimal nonspecific binding.18.

Figure 2

(a) Schematic of Au/Ag nanowire detection system. (b) Representative results from a 5‐plexed virus detection experiment. Each specific nanowire can be identified based on its reflectivity under illumination with 400 nm light (top). The fluorescence of a wire whose complement antigen is present will be high compared to the other wires (bottom).23.

Figure 3

A 5‐plex assay using oligonucleotide targets. The experiment was performed in triplicate and the signals from the noncomplementary target negative control was used to perform a background subtraction. FMI refers to fluorescence mean intensity (arbitrary units). HIV, human immunodeficiency virus (white bars); SARS, severe acute respiratory syndrome (black bars); HAV, hepatitis A virus (gray bars); WNV, West Nile virus (horizontal lines); HCV, hepatitis C virus (diagonal lines).23.

Figure 4

(a) Schematic representation of the genosensor device. (b) Analytical signals obtained for 50 pM of biotinylated target with 25% formamide (to apply stringency conditions) using the genosensor to discriminate between target DNA, 1‐base mismatch, 2‐base mismatch, and 3‐base mismatch. The decrease in signal as a function of degree of mismatch indicates high specificity for the genosensor device.25.

Figure 5

Streptavidin‐based antibody labeling scheme for respiratory syncytial virus F protein and G protein.31.

Figure 6

(a) Confocal images showing the sucessful labeling of the F protein32 and G protein18 in respiratory syncytial virus (RSV)‐infected Hep‐2 cells using quantum dots (QDs). The dual labeled sample showed areas of colocalization33 between the two proteins, and analysis of the orthogonal slices XZ2 and YZ23 suggest the proteins are predominately located at the surface of the cells. (b) Fluorescence images of the F protein labeled with QDs at 1 h (1:1), 24 h (2:1), 48 h (3:1), 72 h (4:1), and 96 h (5:1) after infection. The G protein fluorescence can also be seen after subsequent labeling of the same infected monolayer at each time point (1:2–5:2).31.

Figure 7

The viral titer of RSV‐infected Vero cells was determined at 3 days postinfection (a) and 5 days postinfection (b) using both conventional immunostaining techniques and the anti‐F QDs. The anti‐F QD labels samples were able to detect infection at 3 days while traditional staining was not. By 5 days however, both methods were able to detect similar degrees of infection. Circles represent the respiratory syncytial virus‐nanoparticle (RSV‐NP) label and triangles represent the conventional label.34.

Figure 8

Anti‐F quantum dots (QDs) detection of infected lung tissue from respiratory syncytial virus (RSV)‐infected BALB/c mice (a) compared to uninfected control mice (b). When compared to conventional immunohistochemistry (IHC) staining, the RSV‐infected lungs gave similar regions of positive signal (c) and the background was less than what was seen of the IHC of uninfected control samples (d).34.

Figure 9

Fluorescence spectra for the quantum dot (QD) biosensor, where Förster resonance energy transfer (FRET) response is indicated by increasing acceptor fluorescence, with porcine reproductive and respiratory syndrome virus (PRRSV) antigen concentrations ranging from 0 to 60 particles/µl.18.

Figure 10

(a) Schematic illustration for severe acute respiratory syndrome (SARS) gene detection by the combination of functionalized nanoparticles and polymerase chain reaction (PCR)‐based assay. (b) The agarose gel electrophoresis for PCR products. Lane M, marker; Lane A, PCR products before treatment with superparamagnetic nanoparticles (SMNPs); Lane B, PCR products after treatment with SMNPs.38.