Fig. 4: Multiplexed DNA-encoded synthetic urine biomarkers for portable monitoring of invasive colorectal cancer.

a, Multiplexed DNA-SUBs are comprised of a polymeric nanocarrier (eight-arm PEG) functionalized with protease-activated peptides barcoded with oligonucleotides. b, Left: Timeline of longitudinal tumour monitoring with DNA-SUB. D, day. Right: histological lung staining of BALB/c mice bearing CRC lung tumours at 11 days (upper) and 21 days (lower) after tumour inoculation. Scale bar, 200 µm. c, Left: Schematic of the fluorogenic assay to identify peptide substrates specifically cleaved by lung tissue homogenates collected 21 days after tumour inoculation. Middle: Peptide cleavage by CRC-bearing and healthy lung tissue homogenates were monitored and cleavage rates normalized to healthy tissue are shown in bar graphs (n = 5 mice per group; mean ± s.e.m.; unpaired two-tailed t-test with Welch’s correction, ****P < 0.0001 for PAP7, **P = 0.005 for PAP9, ****P < 0.0001 for PAP11, **P = 0.008 for PAP13, ****P < 0.0001 for PAP15). Right: ROC analysis of data in the middle panel. d, Pooled DNA-SUBs were administered to CRC lung tumour-bearing BALB/c mice (tumour, T) and saline-injected control animals (sham, S) at day 11 or 21 after tumour initiation. Urine samples were collected at 1 h after sensor administration. Cas12a trans-cleavage assays were performed against each DNA barcode and the initial reaction velocities (V₀) were calculated and normalized to those of saline-injected control animals (day 11, n = 10 mice per tumour group; day 21, n = 8 mice per tumour group; n = 8 mice per control group; mean ± s.e.m.; unpaired two-tailed Mann–Whitney test, day 11 *P = 0.0343 for PAP7; day 21 *P = 0.0379 for PAP7; unpaired two-tailed t-test with Welch’s correction, **P = 0.0054 for PAP9; *P = 0.0171 for PAP15). e, Representative paper readout of Cas12a activation by urine samples collected in d. Band intensities were quantified using ImageJ. The top peak of the curve shows cleaved FAM reporter and the bottom peak shows uncleaved FAM–biotin reporter. f, ROC analysis showing the ability of individual or combinational DNA-SUBs to distinguish diseased mice and healthy controls with fluorescent readout in d. g, With the paper readout in e, the ROC analysis utilized the ratio of quantified cleaved reporter band over its corresponding control band. Dashed line represents an AUC of a random biomarker classifier (0.5); the AUC of a perfect biomarker is 1.0.

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