COC Chip-Integrated Zinc Finger Protein Array for PCR-Free Detection of RASSF1A Promoter Methylation

The detection of RASSF1A (Ras-associated domain family 1 isoform A) promoter methylation in body fluids can offer a powerful tool for the early diagnosis of bladder cancer. Zinc finger proteins (ZFPs) serve as sequence-specific recognition elements for targeting double-stranded DNA sequences. Here, we report a cyclic olefin copolymer (COC) chip-integrated ZFP array-based molecular sensor that bypasses the need for bisulfite conversion and PCR amplification to recognize the specific site of DNA methylation in the RASSF1A promoter. Building upon the SEER-LAC (SEquence-Enabled Reassembly of β-Lactamase) framework, we engineered a dual-recognition split-enzyme system in which a COC chip-immobilized ZFP array confers sequence specificity while a co-recruited methyl-binding domain (MBD) enforces methylation-dependent gating, together driving the proximity-induced reconstitution of functional β-lactamase at methylated target loci. Accordingly, this sensor specifically reassembles and restores enzymatic activity only in the presence of specific methylated DNA in the RASSF1A promoter region. We demonstrate that this dual-component array effectively differentiates methylation status with high specificity. Given its rapid turnaround and non-PCR-based mechanism, this system can be well-suited for developing diagnostic assays for bladder cancer, offering a potential alternative to conventional epigenetic screening methods.