Extended Data Fig. 4: Simultaneous detection of six biomarkers—CA19-9, CEA, AFP, SCCA, NSE, and CA125—in a single sample.

(a) Working curves for CA19-9: y = -11.86 lgx + 110.37, R² = 0.989. (b) Working curves for CEA: y = -11.15 lgx + 105.76, R² = 0.992. (c) Working curves for AFP: y = 10.58 lgx + 6.22, R² = 0.998. (d) Working curves for SCCA: y = - 15.69 lgx + 137.29, R² = 0.993. (e) Working curves for NSE: y = 11.08 lgx + 24.40, R² = 0.998. (f) Working curves for NSE: y = 17.17 lgx – 2.30, R² = 0.993. All data were acquired in the buffer of 3.6 M KCl, 10.0 mM PBS, pH 5.0 in trans, 1.0 M KCl, 10.0 mM PBS, pH 5.0 in cis, with the transmembrane potential held at +200 mV. Number of individual experiments n = 3. Each data comes from three independently prepared standard solution samples of the same concentration and three independent nanopore translocation experiments. Data are presented as mean ± SD. Experimental procedure: Standard sample solutions of different concentrations were used for the NELISA tests. Sample solutions of gradient concentrations (CA19-9: 50 mU/mL - 500 U/mL; CEA: 50 pg/mL - 500 ng/mL; AFP: 40 pg/mL - 400 ng/mL; SCCA: 100 pg/mL - 1.0 μg/mL; NSE: 1.0 ng/mL - 10.0 μg/mL; CA125: 100 mU/mL - 1.0 kU/mL) were added to 96-well plates pre-coated with specific capture antibodies, forming a sandwich complex with subsequently added enzyme-labeled detection antibodies. Corresponding peptide probes were then added to the wells containing the sandwich complexes and incubated under optimal conditions for 1 h (Supplementary Figs. 9–21). After the enzymatic cutting step, the reaction solutions from the same sample were combined and subjected to nanopore translocation experiments. For these experiments, the ratio of peptide probes is as follows. FGK(Gal)GGD₈: PBAP-FGLD₈: FGpYD₈: PBAP-FGED₈: FGpTD₈: PBAP-FGFD₈ = 125: 30: 25: 25: 25: 30 (nM). Under these experimental conditions, the working curves for various biomarkers were obtained.

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