Fig. 4: Simultaneous detection of multiple model nucleic acid sequences in membrane-less protocell arrays.

Reactions were incubated at 37 °C for 3 h. a Schematic of toehold switch mechanism. Without trigger RNA, the switch mRNA forms an inhibitory hairpin blocking the translation of a reporter (GFP). Addition of trigger RNA unwinds the switch hairpin, allowing GFP translation. b Schematic of protocell array setup for simultaneous detection of multiple nucleic acid sequences. Red and blue circles indicate micro-basins containing toehold switches B and H, respectively. Gray circles indicate CFE protocells without plasmids. c Representative fluorescence image of RNA detection from 10 nM to 1 µM. Images in the same column have the same RNA trigger(s) added. Images in the same row have the same concentration of trigger(s) added. Circle colors are as shown in b. d Quantification of fluorescence images in c and their replicates. Addition of both RNA triggers mutually represses their outputs, but this effect is specific to these triggers. e Representative fluorescence image of linear DNA detection from 20 pM to 2 nM. Images in the same column have the same DNA trigger(s) added. Images in the same row have the same concentration of DNA trigger(s) added. Circle colors are as shown in b. Addition of both DNA triggers also mutually represses their outputs. f Quantification of fluorescence images in e and their replicates. For experiments where the bulk phase had no triggers, data are presented as mean values ± SD of 27 replicates for protocells containing toehold switch sensors (9 biological replicates × 3 technical replicates) and 54 replicates for reactions with no DNA (9 biological replicates × 6 technical replicates). For experiments with triggers (RNA or DNA), data are presented as mean values ± SD of 9 replicates for protocells containing toehold switch sensors (3 biological replicates × 3 technical replicates) and 18 replicates for reactions with no DNA (3 biological replicates × 6 technical replicates). Hollow circles represent the mean of each biological replicate. Scale bar is 1 mm. Data for each biological replicate are provided in Supplementary Figs. 4 and 5. Details on CFE lysate used, plasmid concentrations, and reaction additives are provided in Supplementary Table 1.