Sensitive and Specific Y-Shaped Ratio Biosensor for Detecting Serum miR-18a: Potential Early Scanning Tool for Non-Small Cell Lung Cancer

miR-18a has been identified as a significantly expressed microRNA (miRNA) in non-small cell lung cancer (NSCLC) and plays a vital role in cancer cell transformation, metastasis, and carcinogenesis. Herein, a pair of binary probes from numerous probe pairs based on single nucleotide polymorphism analyses of miR-18a and miR-18b was first designed and screened to develop a Y-shaped ratio biosensor for accurate detection of serum miR-18a in NSCLC. The special structure of the binary probes combined with the hairpin showed strong specificity for miR-18a, which was confirmed by polyacrylamide gel electrophoresis assay and square wave voltammetry assay. Furthermore, it is beneficial to immobilize single-stranded DNA (ssDNA) probes due to the large specific surface area of nanoporous gold, thereby improving the sensitivity of the biosensor. The Y-shaped ratio biosensor exhibited a wide detection range and can quantify the concentration of miR-18a in the range of 10 fmol/L–100 pmol/L, with a limit of detection of 0.211 fmol/L (S/N = 3). Moreover, it exhibits excellent detection capabilities in serum samples since the biosensor showed a high selectivity toward the coexistence of miR-18a and miR-18b. Therefore, the prepared Y-shaped ratio biosensor is a highly sensitive and specific miR-18a detection tool, capable of identifying microscopic amounts of miR-18a in serum samples, providing great potential for early screening of NSCLC.