Label-free imaging, detection, and mass measurement of single viruses by surface plasmon resonance

Abstract

We report on label-free imaging, detection, and mass/size measurement of single viral particles in solution by high-resolution surface plasmon resonance microscopy. Diffraction of propagating plasmon waves along a metal surface by the viral particles creates images of the individual particles, which allow us to detect the binding of the viral particles to surfaces functionalized with and without antibodies. We show that the intensity of the particle image is related to the mass of the particle, from which we determine the mass and mass distribution of influenza viral particles with a mass detection limit of approximately 1 ag (or 0.2 fg/mm(2)). This work demonstrates a multiplexed method to measure the masses of individual viral particles and to study the binding activity of the viral particles.

Fig. 2.

(A) SPRM images of H1N1 influenza A virus and three different sized silica nanoparticles in PBS buffer. (Insets) Nanoparticle images generated by numerical simulation. (B and C) The SPR intensity profiles of selected particles along X and Y directions (indicated by dashed lines in A, respectively. (Insets) Corresponding profiles from simulated images.

Fig. 1.

Schematic of the SPRM experiment setup (drawing not to scale). A detailed description of the setup can be found in Materials and Methods.

Fig. 3.

SPRM images of influenza A virus on bare gold. (A) SPRM image sequence of Influenza Virus. The color map shows the relative SPR image intensity in mDeg. (B) SPR intensity shifts over time at regions (indicated by rectangles) where individual viral particle adsorb onto the gold surface.

Fig. 4.

(A) SPR intensity vs. time profiles for influenza A viral particles on PEG and antiinfluenza A antibody-functionalized surfaces. (B) Histogram showing relative binding probabilities of influenza A on PEG and antiinfluenza A antibody-functionalized surfaces. The analysis of control experiment, HCMV on antiinfluenza A antibody-functionalized surfaces, is also shown for comparison. Note the vertical axis of the histogram is the probability of a particle stays on the surface (determined from the time profiles similar to A).

Fig. 5.

(A) Histograms of relative SPR intensity distributions of individual silica nanoparticles and influenza A viral particles. The solid red lines are Gaussian fittings of the distributions. Arrows indicate peaks that are likely due to the formation of dimmers. (B) Calibration curve of SPR intensity plotted vs. particle volumes. The average volume of an influenza particle is obtained from the calibration curve and the average SPR intensity in the histogram plotted in A. The vertical error bars are standard deviations of the Gaussian fittings of the histograms of the SPR intensities. The horizontal error bars for the silica nanoparticles are standard deviations calculated from the coefficient of variation of particle diameter given by the manufacturers, and the horizontal error bars for the influenza are estimated from the standard deviation of the volume extracted from the SPR intensity.