Most electroanalytical detection schemes for DNA markers require considerable time and effort from expert personnel to thoroughly follow the analysis and obtain reliable outcomes. This work aims to present an electrochemical assay performed inside a small card-based platform powered by microfluidic manipulation, requiring minimal human intervention and consumables. The assay couples a sample/signal dual amplification and DNA-modified magnetic particles for the detection of DNA amplification products. Particularly, the sul1 and sul4 genes involved in the resistance against sulfonamide antibiotics were analyzed. As recognized by the World Health Organization, antimicrobial resistance threatens global public health by hampering medication efficacy against infections. Consequently, analytical methods for the determination of such genes in environmental and clinical matrices are imperative. Herein, the resistance genes were extracted from E. coli cells and amplified using an enzyme-assisted isothermal amplification at 37 °C. The amplification products were analyzed in an easily-produced, low-cost, card-based set-up implementing a microfluidic system, demanding limited manual work and small sample volumes. The target amplicon was thus captured and isolated using versatile DNA-modified magnetic beads injected into the microchannel and exposed to the various reagents in a continuously controlled microfluidic flow. After the optimization of the efficiency of each phase of the assay, the platform achieved limits of detections of 44.2 pmol L-1 for sul1 and 48.5 pmol L-1 for sul4, and was able to detect down to ≥500-fold diluted amplification products of sul1 extracted from E. coli living cells in around 1 h, thus enabling numerous end-point analyses with a single amplification reaction.
Keywords: Antimicrobial resistance; Genosensor; Isothermal amplification; Magnetic particles; Microfluidics; Recombinase polymerase amplification.
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