Fig. 2: Validations of the microfluidic pipeline.

a Alternating injection of Alexa-488 or Cascade-Blue from a 96-well plate using the autosampler. Samples being injected in the time window of decreasing and increasing fluorescence signals (grey box) were discarded by ensuring no combinations were generated. Since no drug-barcode plugs from the Braille display were injected during time windows with unstable concentrations (grey box), droplets only contained non-functional barcodes (BC-PCR). Time windows with stable fluorescence signals (blue box) were used to generate drug combinations by the co-injection of 20 plugs generated on the Braille display chip resulting in functional barcodes. b Fluorescence intensities of a plug-sequence from the Braille valves supplemented with either Alexa-488 or Cascade-Blue measured at position M1 (=before combinatorial mixing). The blue overlay connecting (a) and (b) illustrates a time series during which one cycle of 20 plugs (second plug without reference dye) is combined with one sample from the autosampler. Alternating sequences of drugs supplemented with Cascade-Blue or Alexa-488 were used to quantify cross-contamination between specific (e.g., green) fluorescent-positive plugs into the subsequent negative (e.g., blue) sample for both injection modes, separately. c Cross-contamination from green positive plugs into green negative plugs from the Braille display (11 cycles as shown in b). d Cross contaminations of plugs for three different chips: The ratio between fluorescence intensities of a blue or green negative plug and the previous blue or green positive plug was analyzed to quantify the level of cross-contamination between sequential samples (n = 99, n = 80, and n = 171, respectively). e Fluorescence signals of droplets containing combinatorial mixtures. Scatterplot representing the fluorescence intensities measured for droplets generated from only Cascade-Blue labeled plugs and only Alexa-488 injected from the autosampler, measured at the droplet outlet (n = 91,899). f Fluorescence signals of droplets from (e) shown for 180 individual combinations. Colors represent cycles of 20 drug plugs combined with one drug from the autosampler. The boxplots show the median and first and third quartiles as a box, and the whiskers indicate the most extreme data points within 1.5 lengths of the box. M1–M3 indicate positions as shown in Fig. 1c. Source data are provided as a Source Data file.