Functionalized gold nanoparticles with zinc finger-fused proteins as a colorimetric immunoassay platform

The quest for highly sensitive and specific detection of disease biomarkers is high, despite many advances in analysis system. Here, we present a sensitive immunoassay platform using DNA-tethered gold nanoparticles and DNA-binding zinc fingers (ZFs). Monomeric alkaline phosphatase (mAP) and human TNF-α were employed as a signal generator and a disease biomarker, respectively. Gold nanoparticles (AuNPs) were first grafted with double-stranded DNAs having specific sequences for two different types of ZFs (QNK and zif268). The alkaline phosphatase and TNF-α-specific protein binder were genetically fused to each of two different types of ZFs, respectively, followed by conjugation with the DNA-tethered AuNPs in a sequence-specific manner. The use of the functionalized AuNPs as a signal generator in a colorimetric immunoassay of TNF-α led to LOD of 120 pg/ml, showing about 161-fold higher sensitivity than a protein binder-fused mAP. The present immunoassay platform could be applied to other analytes by simply replacing a targeting moiety, allowing a versatile and reproducible colorimetric immunoassay. [Display omitted] •AuNPs were functionalized with template DNA and zinc finger proteins (ZFs) in a sequence specific manner.•Functionalized AuNPs were used as a signal generator in colorimetric immunoassay.•The immunoassay platform showed a 161-fold lower limit of detection (LOD) for TNF-α than the previous system.•Developed immunoassay platform can be generally applied to other analytes by simply replacing a targeting moiety.•The immunoassay platform using functionalized AuNPs led to high sensitivity and reproducibility.