Microfluidic assay based on G-quadruplex aptamer for nucleolin detection in prostate cancer

Abstract

Prostate cancer (PCa) diagnosis methods lack specificity and sensitivity or are extremely invasive. Thus, the fast, simple, reliable and accurate detection of low-grade PCa biomarkers could have a great clinical impact, saving lives and reducing patient uncertainty. Studies reported the overexpression of the protein nucleolin (NCL) on the cell surface of PCa cells and can be considered as a cancer-selective target. NCL binds specifically G-rich sequences, namely DNA G-quadruplex (G4) aptamers like AS1411, which can be modified and act as a molecular beacon (MB) for detection. In the present work, we have designed a microfluidic platform for the detection of NCL using a modified AS1411 aptamer beacon (AS1411-N5). When the MB recognizes NCL, it is prompted to undergo a conformational change, separating the fluorophore from the quencher and restoring fluorescence. Firstly, we started to characterize structurally the MB. Circular dichroism and Nuclear Magnetic Resonance spectroscopies indicated that the MB forms a parallel G4 structure. The affinity with NCL was determined fluorometric titration. Localization of MB was assessed in the PCa cell line (PC-3), further demonstrating the ability of the MB to recognize cell-surface NCL. To assess the clinically relevant NCL concentration of human PCa, ELISA assay was performed using peripheral blood mononuclear cells of PCa patients. Finally, the microfluidic assays showed the capacity of the microfluidic device in recognizing NCL in complexed samples like spiked human plasma. Concluding, it is here demonstrated that the microfluidic platform based on the AS1411-N5 could be used as a specific and selective approach to detect NCL in biological samples and contribute to the early-stage diagnosis of PCa.