Split-crRNA Transcription Based Dual-Recognition Cascade Amplification Assay for Sensitive and Accurate Postoperative...

Area of Science:

• Biochemistry
• Molecular Biology
• Biotechnology

Background:

• Postoperative delirium (POD) is a significant complication requiring sensitive biomarker detection.
• Circulating microRNAs (miRNAs) are potential biomarkers for POD.
• Existing amplification methods struggle with both sensitivity and specificity.

Purpose of the Study:

• To develop an ultrasensitive and specific miRNA detection assay for POD biomarkers.
• To integrate multiple amplification techniques for enhanced detection fidelity.
• To establish a versatile platform for nucleic acid detection in diagnostics.

Main Methods:

• A cascade amplification strategy combining catalytic hairpin assembly (CHA), split DNAzyme cleavage, T7 RNA polymerase transcription, and CRISPR-Cas12a trans-cleavage.
• A dual-recognition mechanism for enhanced specificity.
• Validation in spiked human serum and comparison with RT-qPCR.

Main Results:

• Achieved ultrasensitive detection limit of 0.52 fM for miRNA-193 with a wide linear range (1 fM to 100 pM).
• Demonstrated high specificity, discriminating single-base mismatches.
• Showed robust performance in human serum with high recovery rates (97.3-107.2%) and close correlation with RT-qPCR.

Conclusions:

• The developed biosensing platform offers a promising tool for POD-related miRNA analysis.
• The modular framework is adaptable for detecting diverse nucleic acid targets.
• This technology supports advancements in point-of-care diagnostics.