Synergistic CRISPR/Cas12a-Nanozyme System for Iontronic Sensing of Site-Specific Septin9 Methylation

Jan 18, 2026Analytical chemistry

Combined CRISPR and Nanozyme System for Ion-Based Detection of Specific DNA Methylation in Septin9

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Abstract

The detection limit of the proposed method for septin9-mC is 34.0 aM, with a linear range from 100 aM to 10 nM.

Methylated septin9 (septin9-mC) is a validated biomarker for colorectal cancer screening.
Conventional detection methods are limited by pretreatment requirements, DNA degradation, and low performance in scarce samples.
The proposed iontronic sensing platform utilizes a combination of methylation-sensitive restriction enzymes, CRISPR/Cas12a, and nanozymes for improved specificity and sensitivity.
The method distinguishes colorectal cancer cells from human colonic epithelial cells effectively.
It also differentiates colorectal cancer patients from healthy individuals in real sample analysis.

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Methylated septin9 (septin9-mC) is a well-validated biomarker for colorectal cancer screening, and accurate detection of such site-specific methylated DNA holds significant clinical value for early disease diagnosis. However, conventional methods suffer from cumbersome pretreatment, DNA degradation risks, and poor performance in low-abundance samples. Herein, we report a synergistic iontronic sensing platform integrating methylation-sensitive restriction enzyme (AciI), CRISPR/Cas12a, Ag-DNAzyme, and Au/Pt heterometallic nanozyme for highly sensitive and specific detection of septin9-mC. AciI selectively cleaves unmethylated septin9 (septin9-C) while sparing septin9-mC, and intact septin9-mC activates Cas12a trans-cleavage activity to trigger catalytic hairpin assembly (CHA), generating Ag-DNAzyme. Activated Ag-DNAzyme induces detachment of Au/Pt nanoparticles from anodic aluminum oxide membranes, reducing the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) to positively charged oxTMB and altering ion transport fluxes in nanochannels, which is read out via current-voltage characteristics. The linear range is 100 aM to 10 nM with a detection limit of 34.0 aM. This method effectively distinguishing colorectal cancer cells from human colonic epithelial cells and colorectal cancer patients from healthy individuals, showing excellent performance in real sample analysis. The proposed method provides a dependable tool for site-specific methylation detection with promising applications in biological research and clinical diagnosis.

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Synergistic CRISPR/Cas12a-Nanozyme System for Iontronic Sensing of Site-Specific Septin9 Methylation

Abstract

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