Enzyme‐Free Fluorescent Detection of MicroRNA in Clinical Samples by Catalytic Hairpin Assembly Coupled with Magnetic Bead‐Confined 3D DNA Walking

Abstract

Catalytic hairpin assembly (CHA), as an enzyme‐free isotheral nucleic acid amplification method, can easily cooperate with other amplification procedures to improve the sensitivity and accuracy of detection. Herein, we constructed a cascaded CHA sensing platform for breast cancer biomarker detection. Introducing a short double nucleic acid stand avoids the product of CHA1 to directly trigger the CHA2 reaction, which simplifies the design of the CHA hairpins. Compared with the single CHA2 reaction, the cascaded CHA biosensor activated by microRNA‐155 holds nearly 10 times the amplification efficiency with detection limit down to 47.4 pM and quantifies the target in the range from 50 pM to 200 nM. Besides, the magnetic bead‐confined CHA2 taking 3D DNA walking as the display form contributes to decreasing the environmental interference. As expected, the strategy sensitively distinguishes expression levels of microRNA‐155 in different cell lines and cancer patients, which are consistent with the results of traditional qRT‐PCR method. More importantly, simply adjusting the microRNA recognition sequence of CHA1 can extend the cascaded CHA platform to a wider detection range. Therefore, the robustness and efficiency of the approach enable the potential applications for detection of microRNA and early clinical disease diagnosis.

Bead-enriched catalyzed hairpin assembly for the flow cytometric detection of microRNA via FRET signal readout

“…Second, different from the homogeneous CHA, 22 the target-initiated CHA reactions in this study are all enriched in the tiny space around micron-sized magnetic beads (MBs). Particularly, unlike the traditional bulk measurement by recording the fluorescence signal of the whole solution, 23,24 in this study, the minuscule sized MBs bearing enriched fluorophores are investigated as separate reaction units one-by-one through a flow cytometer (FCM), thus even very weak fluorescence signals can be detected. Third, an on-bead FRET readout mode is introduced to be monitored by versatile FCM.…”