A cascade amplification platform integrating entropy-driven DNA nanomachine with CRISPR/Cas12a for microRNA-21 and Listeria monocytogenes detection

Sep 6, 2025Biosensors & bioelectronics

Enhanced detection system combining DNA nanomachine and CRISPR for microRNA-21 and Listeria bacteria

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Abstract

The platform achieved a low limit of 6.1 pM for microRNA-21 detection.

Entropy-driven catalytic strategies utilize toehold-mediated strand displacement for biological sensing.
A cascade platform combining DNA nanomachines with CRISPR/Cas12a improved reaction velocity by 2-fold compared to traditional methods.
High sensitivity and selectivity were demonstrated through specific recognition by the CRISPR/Cas12a system.
The platform successfully detected both nucleic acid and non-nucleic acid targets.
Detection limits were 6 CFU/mL for Listeria monocytogenes, with good performance in diluted serum samples.
Consistency with traditional culture methods was observed in pork testing.

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As one of enzyme-free amplification strategies, entropy-driven catalytic (EDC) based on toehold-mediated strand displacement reaction could achieve efficient amplification without cumbersome temperature changing and expensive enzymes, which shows great potential in biological sensing. However, the limitations in reaction velocity and sensitivity need to be further improved. Herein, a cascade platform integrating entropy-driven DNA nanomachine with CRISPR/Cas12a was proposed. Benefiting from the increased local concentration of DNA on AuNPs, the reaction velocity was enhanced 2-fold compared to solution-based EDC efficiently and the signal was cascade amplified through specific recognition by the designed CRISPR/Cas12a with high sensitivity and selectivity. Impressively, utilizing the flexible design capabilities of DNA molecules, the proposed method achieved both nucleic acid and non-nucleic acid targets detection. The platform achieved a low limit of 6.1pM for microRNA-21 detection and 6 CFU/mL for Listeria monocytogenes detection. Moreover, it showed good performance in 10-fold diluted serum with 98.6-102.3 % recovery. And it has achieved good consistency with traditional plate culture methods in pork. Here, a rapid and sensitive platform based on entropy-driven DNA nanomachine coupled with CRISPR/Cas12a is proposed with great potential of application for early disease detection and food safety screening.

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A cascade amplification platform integrating entropy-driven DNA nanomachine with CRISPR/Cas12a for microRNA-21 and Listeria monocytogenes detection

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