Real-time detection of virus particles and viral protein expression with two-color nanoparticle probes

Abstract

Respiratory syncytial virus (RSV) mediates serious lower respiratory tract illness in infants and young children and is a significant pathogen of the elderly and immune compromised. Rapid and sensitive RSV diagnosis is important to infection control and efforts to develop antiviral drugs. Current RSV detection methods are limited by sensitivity and/or time required for detection. In this study, we show that antibody-conjugated nanoparticles rapidly and sensitively detect RSV and estimate relative levels of surface protein expression. A major development is use of dual-color quantum dots or fluorescence energy transfer nanobeads that can be simultaneously excited with a single light source.

FIG. 1.

Schematic illustration of single-virus detection and surface protein determination. A virus bound with complementary nanoparticle-antibody probes flows into a confocal volume (confined by blue lines), where fluorescent nanoparticles are excited by a laser. The photons from green and red fluorescent nanoparticles are separated (spectra at the bottom) and analyzed for time correlation (coincidence). (a) The virus that does not bind to both probes (left) will not show coincidence signals (dotted lines), while the virus that binds to both probes (right) will produce time-correlated photons from the red and green detection channels. (b) When a single-color probe is used, the virus that binds to several probes (virus 1) will exhibit greater intensity in the integrated photon count spectrum. The bottom graph shows the expected trend in average intensity calculated from several photon count spectra.

FIG. 2.

Time-correlated coincidence signals for virus detection in solution. Forty-nanometer nanoparticles conjugated to RSV anti-F or anti-G protein monoclonal antibodies were used to produce red or green photon emission, respectively. PIV3, used as a control, produced low red or green photon counts and did not show coincidence signals (a), while coincident peaks were observed for the RSV/A2 (b). The time for detection was 8 s, and signals were acquired and analyzed in real time (without any delay).

FIG. 3.

Time-correlated coincidence signals for virus detection in solution. Forty-nanometer nanoparticles conjugated to RSV anti-F or anti-G protein monoclonal antibodies were used to produce red or green photon emission, respectively. RSVΔG, used as a control, produced low green photon counts, as expected, and did not show coincidence signals (a), while coincident peaks were observed for RSV/A2 (b). The magnitude of the red channel signal for RSVΔG was low compared to RSV/A2, suggestive of lower F protein expression compared to RSV/A2. The time for detection was 8 s, and signals were acquired and analyzed in real time (without any delay). a.u., arbitrary units.

FIG. 4.

Measurement of RSV F protein expression on virus particles. QDs coupled with anti-RSV F protein monoclonal antibody and incubated with different viruses generated different photon counts based on anti-F protein nanoparticle aggregation. No signal above the unconjugated QD control was observed for PIV3. Differences in the signal intensities obtained between RSV/A2 and RSVΔG are suggestive of differences in the level of F protein expression.

FIG. 5.

Virus protein expression determined by average photon peak intensities. Photon counts were determined from 35 individual runs (8 s each) and averaged for each virus. The bars indicate the mean ± standard deviations.