Fluorescent microspheres lateral flow assay integrated with Smartphone-based reader for multiple microRNAs detection

[Display omitted] •Ligation, rolling circle amplification, and digestion were optimized into one step.•Two target miRNAs were amplified simultaneously in one step and one tube.•Fluorescence strip realized two amplification products detection simultaneously.•Smartphone-based strip reader was successfully designed and fabricated.•The fluorescence intensity of strips was analyzed by smartphone-based strip reader. More and more attention has been paid to microRNAs (miRNAs) detection, as they have been recognized as a kind of important disease-related biomarkers. However, due to the costly and bulky instruments as well as highly skilled and complex operations, the existing detection methods have some limitations in miRNA detection, especially in low-resource areas. The present study developed a method integrated rolling circle amplification (RCA) with fluorescent microspheres (FM) based lateral flow assay (LFA) (RCA-FM-LFA) for portable and multiple miRNAs detection. Particularly, a smartphone-based strip reader with WeChat mini program was designed to analyze the fluorescence intensity of test lines (T lines) and control lines (C lines) on strips for meeting the requirements of point-of-care testing (POCT). The proposed method was sensitive, specific, accurate, relatively fast and portable. Under optimal conditions, the concentrations of miRNA 21 and miRNA let-7a as low as 230.60 pM and 27.89 pM could be simultaneously detected with only 2 μL of sample needed within 3 h, and no interference can be caused by non-target miRNAs or serum matrix. Satisfactory recoveries ranging from 85.00% to 106.83% for miRNA 21 and from 94.40% to 107.33% for miRNA let-7a were obtained in spiked artificial serum. With further development, the proposed method herein can be chosen as a potential analytical tool for miRNAs detection, which might play an important role in early screening, diagnosis, and prognosis of human serious diseases.