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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 module 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 12X, and DNA could be extracted from a sample containing as few as 10^2 CFU/mL Mycobacterium smegmatis (Msm). From complex biological matrices – human saliva, human blood serum, and artificial urine, p-ITPrep extracted DNA from samples containing 10^2 CFU Msm/mL saliva or artificial urine and 10^3 CFU Msm/mL serum within 20 minutes. 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 diagnostic.
Supplementary materials
Title
Additional results and methods
Description
Fig. S1: Detailed design of p-ITPrep.
Fig. S2 Concentration of 100 copies/µL purified Mtb genomic DNA
Fig. S3 Use of Standard 17 as the ITP membrane
Fig. S4. Qualitative analysis of genomic DNA concentrated using p-ITPrep (using PCR and gel electrophoresis).
Fig. S5. Use of paper discs directly in PCR
Fig. S6. Comparison of isotachophoresis (ITP) and electrophoresis in our paper-based sample preparation.
Fig. S7 Cell lysis of bacteria spiked in trailing electrolyte (TE) on applying a voltage bias of 18 V (for 20 min.) in p-ITPrep.
Fig. S8 Effect of proteinase K (PK) and Triton X-100 treatment on DNA isolation from complex human blood serum samples spiked with Msm.
Fig. S9 Cell lysis of bacteria spiked in different crude biological samples on applying a voltage bias of 18 V (for 20 min.) in p-ITPrep.
Fig. S10 Current vs time plot of ITP experiments in p-ITPrep.
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