Integrated bacterial cell lysis and DNA extraction using paper-based isotachophoresis.

Abstract

Bacterial infections remain a global threat, particularly in low-resource settings, where access to accurate and timely diagnosis is limited. Point-of-care nucleic acid amplification tests have shown great promise in addressing this challenge. However, their dependence on complex traditional sample preparation methods remains a major challenge. To address this limitation, we present a paper-based sample preparation device that integrates bacterial cell lysis, DNA purification, and concentration using an electrokinetic technique called isotachophoresis (ITP). This is the first device that (i) integrates electrochemical bacterial lysis with ITP and (ii) demonstrates the focusing of whole bacterial genomic DNA (gDNA) in paper. Characterization with buffers showed that the paper-based ITP sample preparation module (p-ITPrep) concentrated bacterial gDNA with an average concentration factor of 12×, and DNA could be extracted from a sample containing as few as 10² CFU mL^-1<i>Mycobacterium smegmatis</i> (<i>Msm</i>). From complex biological matrices - human saliva, human blood serum, and artificial urine - p-ITPrep extracted DNA from samples containing 10² CFU <i>Msm</i> per mL saliva or artificial urine and 10³ CFU <i>Msm</i> per mL serum within 20 min. The extraction procedure involved only 3 user steps, in contrast to conventional solid phase extraction kits that require more than 10 user steps. p-ITPrep may provide a simple, inexpensive, and versatile alternative to conventional multi-step nucleic acid extraction protocols for point-of-care diagnostics.