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Pneumatic Nano-Sieve for CRISPR-based Detection of Drug-resistant Bacteria

Pneumatic Nano-Sieve for CRISPR-based Detection of Drug-resistant Bacteria


Authors: Ruonan Peng, Xinye Chen, Fengjun Xu, Richard Hailstone, Yujie Men, and Ke Dua

Abstract

The increasing prevalence of antibiotic-resistant bacterial infections, particularly methicillin-resistant Staphylococcus aureus (MRSA), presents a significant public health concern. Timely detection of MRSA is crucial to enable prompt medical intervention, limit its spread, and reduce antimicrobial resistance. Here, we introduce a miniaturized nano-sieve device featuring a pneumatically-regulated chamber for highly efficient MRSA purification from human plasma samples. By using packed magnetic beads as a filter and leveraging the deformability of the nano-sieve channel, we achieve an on-chip concentration factor of 15 for MRSA. We integrated this device with recombinase polymerase amplification (RPA) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas detection system, resulting in an on-chip limit of detection (LOD) of approximately 100 CFU/mL. This developed approach provides a rapid, precise, and centrifuge-free solution suitable for point-of-care diagnostics, with the potential to significantly improve patient outcomes in resource-limited medical conditions.



Fig. (A) Beads pattern in the channel without MRSA. (B) Experimental setup of multiplexing separation of target bacteria under the fluorescence microscope. (C) Beads stacking with MRSA under fluorescence microscope. (D) The original bacterial sample and (E) retrieved bacterial sample were compared to indicate the on-chip concentration capability of the nano-sieve device.


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Keywords: Nano-sieve; drug-resistant bacteria; RPA/CRISPR-Cas; microfluidics; low-concentration detection; pressure controller


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