Cell-free transcription amplification-based split-type electrochemical sensor using enzyme-linked magnetic microbeads...

Constrained by detecting techniques, patients with acute promyelocytic leukemia (APL) are often confronted with minimal residual disease (MRD) and a high risk of relapse. Thus, a pragmatic and robust method for MRD monitoring is urgently needed. Herein, a novel split-type electrochemical sensor (E-sensor) was developed by integrating nucleic acid sequence-based amplification (NASBA) with enzyme-linked magnetic microbeads (MMBs) for ultra-sensitive detection of the PML/RARα transcript. In this system, NASBA facilitated efficient amplification under isothermal conditions, generating a large amount of RNA amplicons, which mediated the quick binding between horseradish peroxidase (HRP) and MMBs. The separately HRP-linked MMBs were subsequently transferred onto the surface of magnetic glass carbon electrode, producing a remarkably strong electrochemical signal in the presence of the HRP substrate. The proposed split-type E-sensor could detect the PML/RARα transcript with a high sensitivity (a limit detection of 100 aM), a high specificity (single base discrimination) as well as a high stability (a relative standard deviation of 8.3 % for 10 fM target RNA and 6.0 % for 100 fM target RNA). Finally, it could achieve both direct detection of serum cell-free RNA and specific intracellular RNA detection. Owing to its isothermal characteristics, robustness, and suitability for point-of-care testing, this method offers a powerful tool for the early diagnosis of APL and the monitoring of MRD, which holds a great significance for facilitating treatment response assessment and making treatment decisions.