(A) Linear response of sensors 27B, 31B and 32B to corresponding RNA trigger at 0 nM, 3 nM, 30 nM and 300 nM. Each point represents the mean of triplicate data taken at 60 min.

(B) Orthogonality of sensors 27B, 7A and 32B to treatments of 3000 nM of trigger RNA from each of the three sensors. The absorbance output (570 nm) of the sensors at each time point was converted to a ratio of the maximum absorbance of respective sensor at the 90 min time point and plotted as a heat map. Yellow indicates no sensor activation and purple indicates maximum sensor activation.

(C) Reproducibility of NASBA reactions. Samples of Zika RNA in water or 7% human serum were amplified in three independent 2 hr NASBA reactions. Each NASBA reaction was diluted 1:7 in water and used to rehydrate three freeze-dried, paper-based reactions containing sensor 27B for a total of nine replicates. Fold change was calculated from absorbance (570 nm) after 30 min at 37°C. Error bars represent SD from nine replicates for the 3 pM sample and three replicates for the 0 pM sample.

(D) Effect of NASBA reaction time on sensitivity. Samples of Zika RNA in 7% human serum were amplified in NASBA reactions for 30, 60, and 90 min. Diluted NASBA reactions (1:7) were tested with sensor 32B. Fold change was calculated as above. Error bars represent SD of three replicates.

(E) NASBA with freeze-dried reagents. Samples of Zika RNA in 7% human serum were amplified by NASBA reagents in the standard formulation and by reagents freeze-dried in-house. Fold change and error bars were calculated as above after 60 min.

(F) Removing the 65°C step from NASBA protocol. Samples of Zika RNA in 7% human serum incubated at 95°C for two minutes, mimicking viral lysis, and then amplified by NASBA according to the standard procedure without the 65°C step. Fold change and error bars were calculated as above after 60 min.