A NASBi–Cas13a E-Sensor for Accurate Diagnosis of Acute Promyelocytic Leukemia: RNA and DNA Cotargeting LbuCas13a by Cell-Free Transcription Amplification

A highly sensitive but accessible molecular diagnostic method is urgently needed for the early diagnosis and minimal residual disease (MRD) monitoring of cancers, such as acute promyelocytic leukemia. Herein, based on the finding of RNA and DNA cotargeting LbuCas13a, we proposed an efficient amplification system that integrated cell-free nucleic acid sequence-based amplification with the bimodal activation of LbuCas13a (NASBi-Cas13a) and accordingly developed a simple electrochemical biosensor platform using magnetic beads for an ultrasensitive detection of the PML/RARα fusion transcript. The developed NASBi-Cas13a E-sensor achieved a detection limit as low as 10 aM for transcript target and demonstrated a linear dynamic range from 500 aM to 100 fM. The biosensor demonstrated exceptional specificity, capable of detecting positive cells amidst a 1000-fold excess of negative cells. A clinical validation study demonstrated that this method achieved a sensitivity and specificity of 100% across 22 blood samples, showing strong concordance with reverse transcription-quantitative polymerase chain reaction results. Furthermore, in five cases, this method accurately monitored changes in transcript levels. This method, due to its simplified process without reverse transcription and thermal cycling as well as the free of clumsy instrument, holds promise as a point-of-care tool for MRD detection and could potentially prove invaluable in the regular follow-up and efficacy evaluation of cancer treatment.