A biosensor for the nucleic acid-free amplification and label-free quantitative detection of miR-221-3p using liquid crystal

As a representative exosomal miRNA, miR-221-3p has a certain regulatory role in a variety of tumorigenesis, microangiogenesis and lymph node metastasis, and blood miR-221-3p detection is of great significance for early detection, prognosis assessment and individualized treatment of diseases. In this paper, a liquid crystal biosensor based on complementary strand molecular recognition was constructed based on miR-221-3p biomarkers, which was used for nucleic acid-free amplification and label-free rapid detection of miR-221-3p in human blood. The liquid crystal-based biosensor consists of a molecularly oriented and functionalized upper and lower substrates, and the lower substrate surface is sequentially silane and aldehyde functionalized to immobilize the miR-221-3p complementary strand (RNA(C)). The 4-cyano-4 '-pentylbiphenyl (5CB) nematic liquid crystal molecules in the sensor were vertically oriented under the induction of DMOAP and RNA(C), and the double-helix spatial structure formed by the complementarity of RNA(C) and miR-221-3p through the bases disrupted the original vertical alignment of 5CB, which led to the polarized microscopic images from "dark" to "light". The results of polyacrylamide gel electrophoresis, atomic force microscopy and fluorescence microscopy showed that the double helix structure and liquid crystal sensor were successfully constructed by molecular hybridization between RNA(C) and miR-221-3p. In the presence of the target, there was a linear correlation between the bright area coverage (Br) of polarized light microscopy images and the logarithm of miR-221-3p concentration, with a linear range of 100 pM similar to 1 mu M, a detection limit (LOD) of 0.22 pM, and a recovery rate of 97.00 %similar to 101.14 % for blood samples spiked with miR-221-3p. Liquid crystal biosensors without nucleic acid amplification have the advantages of low detection limit and strong specificity, and are expected to be used for the label-free quantitative detection of miR-221-3p in blood samples.