Design Experiments From Reported Results

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Paired Droplet-Evaporation and Infectivity Timecourse: Experimental Design Plan

Droplet Generation: Use interfacial emulsification to produce monodisperse 20–30 μm droplets with precise size control (CV < 5%) from virus-laden media with defined organic loads (e.g., 0.5–5 mg/mL mucin or BSA in PBS). This method leverages shearing force at the air-liquid interface and is cost-effective and easy to assemble Interfacial emulsification for monodisperse droplets. Validate size distribution by laser diffraction or high-speed microscopy immediately post-generation.

Evaporation Measurement: Track droplet diameter in real time via shadow photography at 10 s intervals inside a temperature- and RH-controlled chamber (6 mm minichannel configuration with Peltier heating, ±0.2°C precision). Substrate temperature governs evaporation rate; gas flow also affects evaporation with maximal effect at early times Droplet evaporation physics in controlled channel. Record diameter-versus-time curves for each T-RH condition to extract the evaporation constant K from the D² law fit (d² = d₀² − Kt).

Concurrent Infectivity Assay: At timepoints spanning pre-equilibrium, equilibration, and post-evaporation phases, collect droplets onto chilled collection media and perform standard PFU plaque assay. Critically, single-cell analyses show that PFU output has a coefficient of variation of ~0.66 even within a homogeneous cell population, with mean PFU/cell ranging 860–976 and individual values spanning 0–4225 Biological noise in viral PFU measurements. This biological noise mandates ≥5 independent biological replicates per condition-timepoint and the use of geometric mean PFU for analysis.

Design Matrix and Analysis: The full factorial design spans 4 temperatures × 3 RH levels = 12 conditions. For each condition, collect PFU at ≥5 timepoints (t = 0, 0.5×t_evap, 1×t_evap, 2×t_evap, 5×t_evap). Fit infectivity decay as first-order: ln(PFU/PFU₀) = −k·t. Compare k across T and RH to resolve non-monotonicity. Use two-way ANOVA (T × RH interaction term) to formally test for the hypothesized intermediate-RH survival maximum.

Note: Evaporation times above are simulated estimates from the D² diffusion-limited evaporation model assuming a 25 μm pure water droplet in still air. Organic load will extend evaporation time; actual times must be measured experimentally.

Key Controls: Include (1) non-biological surrogate droplets (fluorescent beads) to independently validate evaporation kinetics without biosafety constraints, (2) no-virus organic-load controls to verify that solute crystallization or phase separation does not interfere with plaque assay recovery, and (3) t=0 PFU from bulk suspension to establish baseline infectious titer before aerosolization stress.

Statistical Power: With expected CV ≈ 0.66 for PFU measurements, detecting a 2-fold infectivity difference between adjacent RH levels (e.g., 50% vs. 80%) requires n ≥ 6 replicates per condition-timepoint (power = 0.80, α = 0.05, two-tailed). For the full 12-condition matrix with 5 timepoints, this totals 360 experimental runs.

Critical Blindspots: (1) Organic load may alter both evaporation kinetics (by forming a skin) and virus protection (by physical shielding), confounding the physics-infectivity link. (2) The local minimum in evaporation rate observed in controlled channels suggests non-monotonic evaporation itself, which could complicate timepoint selection. (3) PFU assay recovery from partially evaporated droplets with high local salt concentrations may underestimate true infectivity if virions remain adherent to collection surfaces.