Light-controlled CRISPR–Cas12a one-pot platform for ultrasensitive cell-free DNA detection in systemic lupus erythematosus diagnosis

Systemic lupus erythematosus (SLE), as a complex autoimmune disease with heterogeneous clinical manifestations, presents significant challenges for early diagnosis. Circulating cell-free DNA (cfDNA) has emerged as a promising disease monitoring biomarker due to its correlation with SLE pathological progression in terms of concentration and fragmentation patterns. However, existing detection methods lack sufficient sensitivity and practicality for clinical application. To address this, we developed a spatiotemporally resolved light-controlled biosensor by integrating a photoactivatable CRISPR-Cas12a system with TdT-mediated poly-A tail extension, achieving three major innovations: (1) implementation of NPOM-dt modified crRNA for precise regulation; (2) optimization of 365 nm UV activation protocol to eliminate interference in one-pot reactions; and (3) establishment of a three-phase "Extension-Activation-Detection" workflow. The platform demonstrates outstanding performance with a detection limit of 0.42 pM, excellent linearity (<i>R</i>² = 0.9956) in the 0-0.1 nM range, and the novel DNA Integrity Index (DII) as a diagnostic indicator - showing significantly higher values in SLE patients (9.82 × 10³ nmol g^-1) <i>versus</i> healthy controls (4.2 × 10³ nmol g^-1, <i>P</i> < 0.0001) with an AUC of 0.8947. This study provides an innovative detection platform combining high sensitivity with clinical utility for early SLE diagnosis.