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DNAzyme 10-23 - Based Nanomachines for Nucleic Acid Recognition
Published on: February 9, 2024
Exploiting amplification byproducts to enable colorimetric ctDNA detection via nanozyme inhibition
Xiaohua Yuan1, Lianhua Liu2, Xun He3
1Frontiers Science Center for Disease-related Molecular Network, West China Hospital, Sichuan University, Chengdu, 610041, China; Department of Laboratory Medicine, Clinical Laboratory Medicine Research Center, Sichuan Clinical Research Center for Laboratory Medicine, Institution of Medical and Engineering Integration for Molecular Diagnosis, West China Hospital, Sichuan University, Chengdu, 610041, China.
Abstract:
Sensitive circulating tumor DNA (ctDNA) analysis is increasingly important for mutation monitoring and molecular stratification in cancer management, yet most current approaches depend on complex signal transducers, fluorescent labels and sophisticated instrumentation. Here, we present an amplification byproduct-induced nanozyme suppression (ABINS) strategy that uses amplification byproducts as functional signals for colorimetric ctDNA detection. By coupling exponential amplification reaction (EXPAR) with Cu3(PO4)2 microflower nanozymes (Cu3(PO4)2 MFs), pyrophosphate (PPi) generated during amplification selectively inhibits the peroxidase-like activity of the nanozymes, resulting in a straightforward signal-off visual readout. This assay enables highly sensitive detection of KRAS G12D ctDNA at the femtomolar level and effectively discriminates target mutants from wild-type sequences. Furthermore, as a proof-of-concept, the sensing paradigm reliably discriminated KRAS-mutant patients from healthy donors in clinical plasma samples, showing good agreement with PCR-based analysis. The ABINS strategy provides a simple amplification-coupled and label-free colorimetric approach for mutation-associated ctDNA analysis, while broader validation in larger clinical cohorts and more representative ctDNA models will still be needed to further define its practical performance.
Insights
A new colorimetric assay detects circulating tumor DNA (ctDNA) using amplification byproducts. This simple, label-free method shows high sensitivity for KRAS G12D mutations in patient plasma.
Area of Science:
- Biochemistry
- Molecular Biology
- Nanotechnology
Background:
- Sensitive circulating tumor DNA (ctDNA) analysis is crucial for cancer management, but current methods often require complex equipment and labels.
- Existing ctDNA detection strategies face challenges in simplicity and accessibility for routine clinical use.
Purpose of the Study:
- To develop a simple, label-free, and highly sensitive colorimetric assay for detecting specific ctDNA mutations.
- To introduce the amplification byproduct-induced nanozyme suppression (ABINS) strategy for ctDNA analysis.
Main Methods:
- Coupling exponential amplification reaction (EXPAR) with Cu3(PO4)2 microflower nanozymes (Cu3(PO4)2 MFs).
- Utilizing pyrophosphate (PPi) generated during EXPAR to inhibit nanozyme activity, creating a signal-off colorimetric readout.
- Assessing the detection of KRAS G12D ctDNA at femtomolar concentrations and discriminating mutants from wild-type sequences.
Main Results:
- The ABINS assay achieved femtomolar-level detection sensitivity for KRAS G12D ctDNA.
- The method effectively distinguished target mutants from wild-type sequences.
- Proof-of-concept demonstrated reliable discrimination between KRAS-mutant patients and healthy donors in plasma samples, correlating well with PCR analysis.
Conclusions:
- The ABINS strategy offers a straightforward, amplification-coupled, label-free colorimetric approach for mutation-associated ctDNA detection.
- This method simplifies ctDNA analysis by using amplification byproducts as functional signals.
- Further validation in larger clinical cohorts and diverse ctDNA models is necessary to establish its full clinical utility.
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