Fig. 2. Ultrasensitive multiplexed quantification of nucleic acid and protein biomarkers.

a We developed a triplex assay where two DNA signals and one protein signal can be detected in the same droplet digital-PCR reaction. The operating principle of the triplex digital assay is shown in a. Three biomarkers (GN, GP bacteria, and IL-6) can be measured simultaneously by multiplexing the two fluorescence channels and the fluorescence intensity of positive droplets (as is the case for GP and IL-6 in the right panel). b–d Calibration curves for quantification of GN, GP bacterial, and the bacterial antibiotic-resistant gene bla_TEM, respectively. CPN/μl stands for the absolute DNA molecule copy number per microliter. In (b–d), the solid lines are the linear regression of the calibration curve, and the horizontal dashed lines indicate the LOD of the assay. Note the scale of the axes in (b, c): it is linear between 0 and 0.1, and logarithmic from 0.1 and higher. Note the scale of the axes in (d): it is linear between 0 and 1, and logarithmic from 1 and higher. Data in (b–d) are presented as mean ± s.e.m., and each concentration has three technical replicates.