Dual-Gated G-Quadruplex DNAzyme-LAMP for Sequence-Specific and Positive Colorimetric Nucleic Acid Detection

Point-of-care nucleic acid diagnostics demand rapid, instrument-free detection with high sensitivity and specificity. While loop-mediated isothermal amplification (LAMP) enables rapid amplification, conventional colorimetric indicators generate false positives by responding to any DNA synthesis, not just target-specific products. We developed a dual-gated G-quadruplex DNAzyme-LAMP that integrates G-quadruplex DNAzyme formation into the loop primer architecture while blocking non-specific activation through locked nucleic acidstabilized probe design. This approach gates colorimetric signal generation to occur only when target amplicons displace a 3'-blocking strand, enabling sequence-specific positive signaling without sacrificing amplification kinetics. When tested with Hepatitis A virus, the assay detected as few as 12 copies per reaction, matching RT-qPCR sensitivity while providing unambiguous positive colorimetric readouts. Specificity was maintained even in the presence of a 10⁹-fold excess non-target DNA. Importantly, the platform requires only inexpensive hemin and chromogenic substrates, avoiding the protein reagents, custom oligonucleotides, and cold-chain logistics that constrain existing sequence-specific platforms. By exploiting the universally adopted loop primer element, this platform offers a generalizable framework for reliable colorimetric detection suitable for resource-limited outbreak settings.