Trace Detection of Multiple Macromolecular Biomarkers in Saliva by Enzymatic Responsive Serial-Nanofluids Strategy

Saliva assay is a promising potential strategy for widespread screening and prompt surveillance of oral cancer to improve the prognosis and reduce the financial burden. But current saliva detection methods are hampered from clinical application by their restricted target diversity, complex procedures, and costly equipment. In this work, we introduce an enzymatic responsive serial-nanofluids strategy, enabling simultaneous trace-level detection of multiple humoral markers in saliva. Enzymatic-responsive nanochannels were engineered respectively by modifying the outer surfaces of AAO arrays with polypeptides featuring target-cleavage sites. Thus, the macromolecular markers in saliva could cleave corresponding polypeptide, opening the ion pathway and enhancing the ion flux of nanochannels. Upon the open of a set of nanochannel-arrays tandemly connected, serial-nanofluids are generated and ionic currents are coupled to enable collaborative detection of multiple targets. Employing oral cancer markers of MMP-1 and MMP-3 as models, we developed a mobile nanosensor that can concurrently monitor their trace variation low to 2.14 × 10^{- 1 3} g/mL. Furthermore, the clinical trial indicated that our nanosensor could noninvasively, conveniently, and effectively distinguish healthy individuals from oral cancer patients, and those with lymph node metastasis. This strategy offers a robust framework for multi-marker detection in clinical diagnostics, facilitating high-frequency and large-scale cancer screening through saliva test.