DNA Nanoflower-Powered CRISPR/Cas12a Biosensing Platform for Ultrasensitive Protein Detection in Clinical Samples

Protein markers secreted by various human cells provide crucial insights for the early diagnosis and prognostic assessment of clinical diseases. However, restricted by efficient protein marker signal amplification in real clinical samples with complex compositions, accurate, sensitive, and rapid detection of protein markers remains largely challenging. Herein, a DNA nanoflower (DNF)-powered CRISPR/Cas12a biosensing platform (DNF-CRISPR) is presented that employs the DNF in upstream to amplify input signals for protein markers, while utilizing the CRISPR system in downstream to amplify output signals by trans cleavage. This upstream and downstream cascade amplification sensing platform exhibits high sensitivity (500 fg mL^-1), rapid (≤2 h), and a broad dynamic range (2.5 pg mL^-1 to 25 ng mL^-1). As a proof of concept, DNF-CRISPR biosensing platform enables the quantitative detection of neutrophil gelatinase-associated lipocalin (NGAL) biomarkers in blood and urine samples from kidney injury patients with 91% accuracy. This study provides a powerful and versatile approach for the accurate diagnosis of protein markers in clinical settings, facilitating the application of CRISPR/Cas12a-based sensing platforms for non-nucleic acid markers.