Plasmonic Enhancement of Selective Photonic Virus Inactivation

Abstract

Femtosecond (fs) pulsed laser irradiation techniques have attracted interest as a photonic approach for the selective inactivation of virus contaminations in biological samples. Conventional pulsed laser approaches require, however, relatively long irradiation times to achieve a significant inactivation of virus. In this study, we investigate the enhancement of the photonic inactivation of Murine Leukemia Virus (MLV) via 805 nm femtosecond pulses through gold nanorods whose localized surface plasmon resonance overlaps with the excitation laser. We report a plasmonically enhanced virus inactivation, with greater than 3.7-log reduction measured by virus infectivity assays. Reliable virus inactivation was obtained for 10 s laser exposure with incident laser powers ≥0.3 W. Importantly, the fs-pulse induced inactivation was selective to the virus and did not induce any measurable damage to co-incubated antibodies. The loss in viral infection was associated with reduced viral fusion, linking the loss in infectivity with a perturbation of the viral envelope. Based on the observations that physical contact between nanorods and virus particles was not required for viral inactivation and that reactive oxygen species (ROS) did not participate in the detected viral inactivation, a model of virus inactivation based on plasmon enhanced shockwave generation is proposed.

Figure 1

Schematic of experimental setup for photoacoustic measurements. The following abbreviation are used: fs: femtosecond, QC: quartz cuvette, PA: photoacoustic, and OW: optical window.

Figure 2

(a) UV-Vis spectra of nanorods recorded after 10 s of fs laser irradiation with different average powers. Control refers to rods without irradiation. Inset: TEM image of nanorod. Size bar = 5 nm. (b) Peak wavelengths (gray) and widths (black) of the longitudinal nanorod plasmon mode as function of the average incident laser power.

Figure 3

TEM image of nanorods before (a) and after (b) irradiation with 35 fs laser pulses for 10 s (average power 3 W). (c) Histogram of nanoparticle length before (control) and after laser irradiation. (d) 2% Agarose gels of rod samples collected before (control) and after laser irradiation with specified average powers. The formation of a second band at high laser powers indicates agglomeration.

Figure 4

(a) Virus log-reduction-value (LRV) LRV measured for virus (V) and virus + PEGylated nanorods (NR) both in the presence of IgG antibody (P) as function of average 805 nm laser power. Laser pulse duration was 35 fs. The irradiation time was held constant at t = 10 s. Control = no laser irradiation. (b) Binding curves for the IgG antibody (P) recovered from the experiments in (a) in the presence (red) and absence (black) of nanorods as determined by ELISA. (c) LRV obtained for virus (V) alone, virus + PEGylated nanorods (NR), virus + annexinV functionalized nanorod (NR+A5) and virus + spherical nanoparticles (NP) after irradiation to a pulsed (35 fs) 805 nm laser with an average power of 3 W for 10 s exposure time. Inset: TEM image of laser irradiated virus + PEGylated nanorod. Size bar = 20 nm. (d) Binding curves for antibody (P) irradiated with the pulsed 805 nm laser for 10 s at an average power of 3 W as determined by ELISA. Control = no laser irradiation (e) LRV obtained under similar laser conditions as in (c) but with two-fold increased virus concentration. The increase in LRV with virus concentration indicates that the LRV values are limited by the sensitivity of the infection assay (f) Percentage of viable cells for no-treatment control (mock), cells after incubation with a nanorod solution that was not exposed to the laser, and cells treated with a nanorod solution that was exposed to the laser. Laser irradiation conditions were identical to (e).

Figure 5

(a) LRV obtained after 10 s irradiation with pulsed 805 nm laser as function of average power for virus + ROS scavenger mix (white) and virus + PEGylated nanorod + scavenger mix (gray). All samples contained IgG hybridoma (P). The scavenger does not suppress viral inactivation. (b) Ultrasonic wave emitted from 0.3 W laser irradiated nanorods recorded by photoacoustic transducer. (c) Relative virus fusion (normalized by no treatment control) obtained after 10 s irradiation with pulsed 805 nm laser with 3 W. Laser exposure in the presence of nanorods reduces viral fusion.