Fig. 5: Specificity of the multiplexed synthetic urine biomarkers enabling clinical translation via DNA-barcoding strategy.

a, Top: scheme of an immunocompetent, autochthonous KP mouse lung cancer model; bottom: histological display of disease progression showing lung tumour nodules at 7.5 and 12 weeks of lentiviral inoculation of Cre recombinase. Wk, week. Scale bar, 200 µm. b, Pooled DNA-SUBs were administered to KP (tumour, T) and healthy control B6 mice (control, C) at 7.5 and 12 weeks after tumour initiation. Urine samples were collected at 1 h after sensor administration and Cas12a trans-cleavage assays were performed against each DNA barcode with the fluorophore-quencher paired reporter (at 7.5 weeks, n = 12 mice per tumour group; n = 9 mice per control group; mean ± s.e.m. unpaired two-tailed t-test with Welch’s correction, *P = 0.044 for PAP9; at 12 weeks, n = 9 mice per tumour group; n = 8 mice per control group; mean ± s.e.m., unpaired two-tailed t-test with Welch’s correction, **P = 0.004 for PAP9, *P = 0.045 for PAP15). The initial reaction velocity (V₀) refers to the slope of the curve at the beginning of a reaction. c, Representative LFA paper strips of Cas12a activation by mouse urine samples collected in b. Band intensities were quantified using ImageJ. The top peak of the curve shows the freed FAM molecule in cleaved reporter and the bottom peak shows the presence of the uncleaved FAM–biotin reporter. d, ROC curves showing the ability of DNA-SUBs to distinguish KP lung tumour-bearing mice and healthy controls. Black line, fluorescent readout; dark red line, paper readout. Dashed line, AUC of a random biomarker classifier (0.5); the AUC of a perfect biomarker is 1.0. e, PCA to reveal the divergence between lung tumour nodules derived from invasive CRC or primary KP mutants. met., metastasis. f, The relative fold change between disease states was calculated using mean-scaled reporter concentrations normalized to the corresponding healthy controls. Each dot represents one reporter, and size of dots represents the relative fold change (healthy control = 1). g, Left: schematic of massively in-parallel CRISPR-Cas-mediated DNA detection on the Fluidigm microfluidics platform; right: heatmap of trans-cleavage rates of different modified ssDNA activator-crRNA pairs. Assays were performed with ssDNA-spiked human urine samples on a Fluidigm microfluidic platform.

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