Fig 1.
a) Reader instrument b) cross-selection illustration of chip and antibody assay c) picture of the SP-IRIS chip d) micrograph of label-free quality control assay array image e) example SP-IRIS image illustrating that each bright dot is a single captured nanoparticle.
Fig 2.
SP-IRIS nanoparticle response.
Contrast of detected nanoparticle standards for 420 nm and 535 nm wavelengths are plotted. A cubic polynomial fit of the data is used to size other nanoparticles. The dashed line is used for sizing viral particles with refractive index adjusted to n = 1.42. The dotted redline indicates the noise floor of the system measured at 0.5%. The plotted error bars are +/- one standard deviation.
Fig 3.
SP-IRIS virus detection validation with SEM.
SP-IRIS sensors incubated with ZIKV, VSV, and VACV were imaged with SP-IRIS and SEM. To validate accuracy of virus detection, the same area was imaged with SP-IRIS and SEM. The overlay panel aids in visualizing the correlation between the SP-IRIS and SEM images. Red dots in the overlay image represent particles detected by SEM. Contrasts of the SP-IRIS images were differently enhanced for clarity of presentation.
Fig 4.
Measured particle distribution of virus preparation measured by SP-IRIS.
The contrast of the individual detected particle is converted to particle diameter using the response curve created with polystyrene bead standard and refractive index of the virus.
Fig 5.
SP-IRIS detection and characterization of filamentous Ebola VLPs.
a) SP-IRIS of captured Ebola VLP with identified particles and filamentous color-coded. Scale bar 10 micron. b) SEM of Ebola VLP box, outlined in 5a. c) Single filament outlined in 5b shown for both SEM (c) and SP-IRIS (d). Scale bar 1 micron e) Histogram of Ebola VLP with small (green), medium (blue) and large/long (red) particle frequencies.
Fig 6.
Characterization of sucrose gradient fractionated EBOV VLPs.
a) Schematic depiction of Ebola VLPs fractionation on a 20–60% sucrose gradient. Fractions from this gradient were run on an SDS-PAGE gel and VLP was detected with anti-Ebola GP antibody. Fractions 5–8 showed positive signal for Ebola-GP (blot shown at a 90 degree angle to normal viewing) b) Plot of the quantity of Ebola VLP captured on the SP-IRIS sensor by anti-Ebola GP(13F6) vs the size of the detected particles. Fractions shown include 5 (blue circle), 6 (green square), 7 (red star), 8 (green diamond) and a sample with no VLP (black star). c) Graph representing the % of filamentous particles to non-filamentous particles illustrating enrichment of filamentous particles within the gradient. d) individual images illustrating low (top) and high (bottom) filament fractions.
Fig 7.
SP-IRIS detection and characterization of Ebola virus.
a) SP-IRIS image of captured Ebola virus with identified particles and filaments color-coded. b) Histogram of Ebola VLP with small (green), medium (blue) and large/long (red) particle frequencies. Inset in 5b highlights small, medium and large filaments with size distribution of the diffraction limited small particles. Scale bar is 1 micron.
Fig 8.
SP-IRIS real-time detection and characterization of Ebola virus in-liquid.
a) SP-IRIS image of captured Ebola virus with identified particles and filaments color-coded at eighteen minutes. Scale bar 5 micron b) A zoomed portion of Ebola antibody spot highlighted by square box in 7a. Scale bar 1 micron. c) Plot of the number of small particle and medium (blue) and large (red) filamentous Ebola virions as a function of time.