Circulating microRNAs carried by exosomes have emerged as promising diagnostic biomarkers for cancer because of their abundant amount and remarkable stability in body fluids. Exosomal microRNAs in blood are typically quantified using the RNA isolation-qRT-PCR workflow, which cannot distinguish circulating microRNAs secreted by cancer cells from those released by non-tumor cells, making it potentially less sensitive in detecting cancer-specific microRNA biomarkers.
Researchers at SUNY Buffalo have developed a sensitive and simple tethered cationic lipoplex nanoparticles (tCLN) biochip to detect exosomal microRNAs in human sera. The tCLN biochip allows the discrimination of tumor-derived exosomes from their non-tumor counterparts, and thus achieves higher detection sensitivity and specificity than qRT-PCR. The researchers have demonstrated the clinical utility of the tCLN biochip in lung cancer diagnosis using sera from normal controls, therapy-naive early stage and late stage non-small cell lung cancer (NSCLC) patients. Total five microRNAs (miR-21, miR-25, miR-155, miR-210, and miR-486) were selected as the biomarkers. Each microRNA biomarker measured by tCLN assay showed higher sensitivity and specificity in lung cancer detection than that measured by qRT-PCR. When all five microRNAs were combined, the tCLN assay distinguished normal controls from all NSCLC patients with sensitivity of 0.969, specificity of 0.933 and AUC of 0.970, and provided much better diagnostic accuracy than qRT-PCR (sensitivity = 0.469, specificity = 1.000, AUC = 0.791). Remarkably, the tCLN assay achieved absolute sensitivity and specificity in discriminating early stage NSCLC patients from normal controls, demonstrating its great potential as a liquid biopsy assay for lung cancer early detection.
Overview of tCLN biochip and its detection mechanism
In the tCLN biochip, cationic lipoplex nanoparticles containing molecular beacons (CLN-MB) are tethered on the surface of a gold-coated cover glass. Exosomes are fused with CLN-MB through electrostatic interaction to form CLN-exosome complexes. Inside the complexes, MB hybridize to exosomal microRNAs and generate fluorescence signals, which are captured by total internal reflection fluorescence (TIRF) microscopy. The fluorescence intensity of each CLN-exosome complex is individually analyzed to measure the expression of exosomal microRNAs. The tCLN assay uses 60 uL serum and total assay time is about 2.5 h.