Integrated dual-biomarker detection: Transforming proteins and nucleic acids into ssDNA for enhanced disease diagnosis

Integrating information from protein and nucleic acid biomarkers enhances detection sensitivity and specificity, offering valuable insights for disease management compared to single biomarkers alone. Thus, there is an increasing demand for a unified diagnostic approach by introducing an innovative assay that simultaneously detects protein and nucleic acid biomarkers within a single test. Here we developed a method that uniquely transforms both protein and nucleic acid markers into single-stranded DNAs (ssDNAs), utilizing a novel combination of aptamers and the CRISPR/Cas9 system. Subsequent amplification of these ssDNAs through both linear and exponential rolling circle amplification enables simultaneous and efficient detection. This approach not only simplifies the diagnostic process but also conserves valuable sample volume, reduces the time required for detection, and minimizes the consumption of reagents and the need for extensive instrumentation. In a critical proof-of-concept application, we employed this assay to detect programmed cell death-ligand 1 (PD-L1) protein and epidermal growth factor receptor (EGFR) L858R circulating tumor DNA (ctDNA) in plasma of patients with non-small cell lung cancer (NSCLC), which helps to guide the selection of the most appropriate treatment for NSCLC patients, whether immunotherapy (based on PD-L1 expression) or targeted therapy (based on EGFR mutation). The remarkable results demonstrated high sensitivity (83.3% for EGFR L858R ctDNA and 88.2% for PD-L1) and specificity (100% for EGFR L858R ctDNA and 87.5% for PD-L1). These findings not only validate the effectiveness of our method but also underscore its potential as a transformative tool in the simultaneous monitoring of multiple types of biomarkers, paving the way for more nuanced and effective disease management strategies.