Protein biomarkers in blood have been widely used in the early diagnosis of disease. However, simultaneous detection of many biomarkers in a single sample remains challenging. Herein, we show that the combination of a sandwich assay and DNA-assisted nanopore sensing could unambiguously identify and quantify several antigens in a mixture. We use five barcode DNAs to label different gold nanoparticles that can selectively bind specific antigens. After the completion of the sandwich assay, barcode DNAs are released and subject to nanopore translocation tests. The distinct current signatures generated by each barcode DNA allow simultaneous quantification of biomarkers at picomolar level in clinical samples. This approach would be very useful for accurate and multiplexed quantification of cancer-associated biomarkers within a very small sample volume, which is critical for non-invasive early diagnosis of cancer.

Both protease overexpression and local pH changes are key signatures of cancer.However,the sensitive detection of protease activities and the accurate measurement of pH in at umor environment remain challenging.H ere,w ed evelop ad ual-response DNAp robe that can simultaneously monitor protease activities and measure the local pH by translocation through a-hemolysin (aHL). The DNAp robe bears as hort peptide containing phenylalanine at ap redesigned position. Enzymatic cleavage of the peptide either exposes or removes the N-terminal phenylalanine that can form ac omplex with cucurbit[7]uril. Translocation of the DNAh ybrid through aHL generates current signatures that can be used to quantify protease activities.Furthermore,the current signatures possess ap H-dependent pattern that reflects the local pH. Our results demonstrate that the versatile DNAp robe mayb ef urther explored for simultaneously measuring multiple parameters of ac omplex system such as single cells in the future.