Fig. 4: Counting result of miR375 bridge assay in human serum.

a In the bridge-activated assay process, QD-tags will be pulled down to the PC surface when target miRNAs bridge the formation of a surface-bound complex. b Illustration of the line-scanning process that counts the number of PC-attached QDs. c Line-scanning Imaging: for PCEF enhanced digital counting in human serum. Target concentration: 10 aM to 1 nM. FOV: 300 µm × 300 µm. Scale bar: 40 µm. Data are averages from more than 9 FOVs, and error bars indicate the standard deviation between three independent replicas. Statistical significance was tested using one-way ANOVA between the negative control group and all testing groups with P < 0.0001; Imaging conditions for the assay on the PC: laser power = 1 mW, EM-gain = 40×; Higher laser power (5 mW) and EM-gain (1200×) are needed in order to detect the intensity change at 1–10 pM on glass while avoiding signal saturation at 1 nM; Same integration time (600 ms) and objectives (×50, NA = 0.5) were applied in both surface assays. d Dose–response curve for various concentrations across a 10⁹-fold concentration range after a 2-h incubation for digital counting results. The error bars represent the mean and the standard deviation of n = 3 independent assays. e Single-base mismatch discrimination test. Line-scanning image panel demonstrates the digital resolution of captured QDs for the target miR375 perfect match (PM) group, versus three different SNVs at 100 pM. f Quantification of the perfect match target sequence and the SNV cases at 100 pM concentration. Statistical significance was tested using independent t tests (two-sided); ****P < 0.0001. The error bars represent the mean and the standard deviation of n = 3 independent assays.