Applying NV center-based quantum sensing to study intracellular free radical response upon viral infections

Abstract

Although viruses are known to modify the free radical concentration in infected cells, the exact location and concentrations of such changes remain unknown. Although this information is important to understand the virus pathogenesis and design better anti-viral drugs or vaccines, obtaining it with the conventional free radical/ROS detection techniques is impossible. Here, we elucidate the utility of diamond magnetometry for studying the free radical response of baby hamster kidney-21 cells upon Semliki Forest virus infection. Specifically, we optically probe the alterations in free radical concentration near infectious viruses via measuring the spin-lattice relaxation (T₁) of NV defect ensembles embedded in intracellular nanodiamonds. We performed measurements both at random locations as well as close to the virus entry by conjugating viruses to nanodiamond sensors. We observed alterations of T₁, which represent the intracellular free radical concentration during the viral replication process. Moreover, relaxometry is also used to monitor real-time free radical variation during the early infectious process.

Keywords: Diamond magnetometry; Fluorescent nanodiamonds; Free radicals; NV centers; ROS; Viral infections.

Fig. 1

Schematic representation of probing general intracellular free radical response (a), and free radical response near viral particles (b) of BHK-21 upon SFV infection using relaxometry.

Fig. 2

(a) Schematic representation of SFV-FND formation. (b) Represent fluorescent images of SFV-FND conjugates.

Fig. 3

The time required for SFV and SFV-FND to enter the cell. Representative time-lapse image sequences demonstrating the change in intensity of the DiO fluorescence after (a–c) adding the DiO-SFV or (e–g) DiO-SFV-FND to the cells. In (b) and (f) we show the time series when the uptake is inhibited with NH₄Cl. (c) and (g) show the respective particles in an empty Petri dish (control). Change in normalized fluorescence intensity with respect to time. (d) and (h) show the data for DiO-SFV and DiO-SFV-FND respectively. (a complete time series is shown in the supplementary material in Section S2).

Fig. 4

Individual T₁ measurements recorded every 2 h post infection (hpi) till 10 hpi in untreated cells (negative control), incubated with inactivated SFV and challenged with infectious SFV. All the timepoints across all the groups have at least 8 independent measurements. (b) Graphical representation of the FND movement within the cell cytoplasm to evaluate whether more active areas of greater movements were experiencing different T₁ times. We plot T₁ relaxation correlated with the maximum FND displacement for the cells treated with (d) plain cell-culture medium (negative control), (e) infectious SFV and (f) inactivated SFV group. Green and red color represent 0 hpi (time control) and 10 hpi, respectively. The off-green and red colors indicate the intermediate timepoints. Plots in b-d, have ∼40 data points each obtained in three independent experiments. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

(a) Representative fluorescent images of SFV-FND conjugates in BHK-21 cells at different incubation times. 0 hpi represents DiO-SFV-FND conjugates and BHK-21 cells were co-cultured at 4 °C for 1 h; 2, 4, and 6 hpi represent DiO-SFV-FND conjugates and BHK-21 cells co-cultured for 2, 4, and 6 h at 37 °C, 5% CO₂, followed by fixing and staining. (b) T₁ measurements recorded from 2 hpi to 10 hpi during incubation with infectious SFV-FND and inactivated SFV-FND. Cells treated with SA-FND were set as negative control group. All the timepoints across all the groups have at least 8 independent measurements. (c) Representation of the location and events that diamond particles are detecting by using SFV-FND. * indicates statistical significance as determined by the one-way Anova test. A two-way Anova with post-hoc test (Sidak) indicates that time has a significant effect on T₁ but infectious and inactivated viruses cause similar responses (P = 0.0229, *).

Fig. 6

(a) Graphical representation of the ‘moving window’ algorithm used for analyzing the data to deduce pseudo real-time variation in the intracellular free radical concentration near viral particles. Time-dependent early viral infection process was superimposed to ‘moving window’ algorithm. The orange dash square represents the endocytosis process, and green square represents the fusion & uncoating process, respectively. (b) Experimental results obtained for cells treated with infectious SFV-FND, inactivated SFV-FND, and SA-FND (negative control). The data was shown as median of at least 8 independent measurements for each group. (c) Average of independent normalized T₁ curves for each group. Two-way Anova with post-hoc test (Turkey) indicates the significant difference between infectious/inactivated SFV-FND and SA-FND groups (P < 0.001, *). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)