DNA triangular prism-engineered multicolor chemiluminescence capsules for multiplexed visual detection of nucleic...

Chemiluminescence (CL) capsules, featuring long-lasting emission and facile functionalization, hold great potential for multiplexed analysis of nucleic acid biomarkers. Herein, we report a strategy that utilizes DNA triangular prism (TP) scaffolds to spatially engineer trivalent catalytic hairpin assembly (CHA) amplification on multicolor CL capsules for the multiplexed visual detection of avian influenza virus (AIV) DNA biomarkers. Three multicolor CL capsules are fabricated by co-encapsulating luminol alone, luminol with coumarin 6 (C6), and luminol with both C6 and Nile red (NR), denoted as LUM@TP-3H2, C6/LUM@TP-3H4, and NR/C6/LUM@TP-3H6. TP-3DNAzyme exhibits approximately 1.6-fold higher peroxidase-like activity than free DNAzyme in solution, and TP-functionalized capsules show stronger CL signals and higher CL amplification efficiency than capsules with directly anchored CHA hairpins. Upon target-triggered CHA reaction and H₂O₂ addition, these TP-functionalized capsules generate blue, green, and red light, respectively, through chemiluminescence resonance energy transfer (CRET), along with an intense and long-lasting CL emission over 1200 s. The capsule-based CL imaging assay is recorded with a common smartphone, exhibiting a linear range of 10-1000 pM and detection limits of 0.57 pM, 0.82 pM, and 1.97 pM for H1N1, H5N1, and H7N9, respectively, with recoveries of 97.41-104.87% in 100-fold diluted human serum. Altogether, this work provides a versatile CRET platform by using TP-engineered multicolor capsules with trivalent CHA amplification for smartphone-based multiplexed visual detection.