(664a) A Low-Cost Paper-Based Sample Preparation Module to Lyse Bacterial Cells and Extract Genomic DNA Using Isotachophoresis

In recent years, much work and effort have been put into developing affordable and simple POC-nucleic acid amplification tests (NAATs) for diagnosing infectious diseases like tuberculosis. While the primary focus has been on nucleic acid (NA) amplification and detection strategies, many POC-NAAT assays still rely on complex sample preparation methods. To overcome this limitation, we demonstrate a POC-compatible paper-based device capable of purifying and concentrating Mycobacterium tuberculosis genomic DNA (gDNA) from viscous mock-sputum using isotachophoresis. We also demonstrate the device's capability to electrochemically lysis M. Smegmatis bacteria, along with purification and concentration of the Mycobacterium smegmatis gDNA. This is the first demonstration of sputum DNA preparation using paper-based ITP.

KEYWORDS: Paperfluidics, sample preparation, NAAT, isotachophoresis (ITP), point-of-care (POC) diagnostics, bacterial lysis

INTRODUCTION

Dependence on off-line sample preparation methods involving complex protocols, instrumentation, and trained technicians has proved to be one of the major roadblocks in the progression of POC-NAATs. The presence of several amplification-inhibitory molecules within complex biological samples such as sputum makes sample preparation a critical pre-analytical step to obtain more clinically relevant results for TB diagnosis. Sample preparation generally involves sample collection, storage, NA isolation (cell lysis), purification, and concentration.

ITP is an electrokinetic separation and concentration technique that utilizes an electric field and a discontinuous buffer system consisting of a fast-moving leading electrolyte (LE) and slow-moving trailing electrolyte (TE). It is used to concentrate the charged species (DNA) at the LE/TE interface. The electrophoretic mobility of the DNA must lie between LE and TE. Paper-based ITP formats have been shown to work well in focusing dyes, short-ssDNA, dsDNA ladder, and extract/amplify DNA (200 bp) spiked in serum and blood samples.^1,2 Moving towards the goal of an integrated sample-to-answer POC-NAAT device for TB, we present an inexpensive paper-based ITP platform capable of lysing bacterial cells and purifying and concentrating gDNA from viscous mock-sputum within 15 min.

EXPERIMENTAL

The paper-based ITP (Fig. 1) device consists of an 11-layered foldable structure with circular disks of Whatman filter paper sandwiched between the two reservoirs (containing TE and LE buffer) that forms a paper channel for the liquid to flow between the two reservoirs. gDNA (10²-10⁰ copies/µL) mixed with 900 µL of TE buffer (2 mM tris-taurine, TTD) was added to the TE reservoir along with simultaneous addition of 900 µL of LE buffer (1 M tris-HCl, TH) to the LE reservoir. A voltage bias of 18 V was applied between the platinum electrodes in the reservoirs. Post-ITP, DNA gets concentrated in some of the initial paper disks. Each paper disk can be used as a PCR/real-time PCR template, or the concentrated DNA can be eluted out from the paper disk for DNA amplification. The device's capability to purify gDNA in the presence of inhibitory substances was also tested.

In another set of initial experiments, we also demonstrate the device's capability to electrochemically lysis M. Smegmatis bacteria, spiked in mock sputum (TE reservoir), and simultaneous purification and concentration of the Msm gDNA into the paper disks.

RESULTS AND DISCUSSION

Gel electrophoresis images of the gDNA concentrated within the paper layers (L1-L11) after ITP, along with some positive (P: 230 bp target; red arrow) and negative controls (N, NP, TH, TT), are shown in Fig. 2. The concentration of 10² copies gDNA/µL and 1-copy (10⁰) gDNA/µL is depicted in Fig. 2A and 2B, respectively. gDNA mixed with TE buffer (TTD) before undergoing ITP concentration is represented by the green arrow, whereas the bands highlighted within the yellow rectangle represent the gDNA concentrated within the paper layers. The higher intensity of the bands within some of the paper layers compared to the intensity of the band corresponding to their respective TTD signifies the concentration and focusing of gDNA within the paper layers. The number of paper layers with the gDNA after ITP depended on the initial copy number of the sample. The device was able to concentrate and purify the DNA even in the presence of inhibitory molecules present in the mock-sputum (egg yolk emulsion + methylcellulose) (Fig. 2C). qPCR results showed approx. 13x concentration of purified DNA, post-ITP (Fig. 3). Fig. 3 A-C shows the normalized DNA concentration (w.r.t. the liquid sample before performing ITP, TTD) for different gDNA copy numbers ranging from 10³-10¹ copies/µL. PTTD (in Fig. 3) refers to the control in which a blank paper disk was dipped in the DNA sample (TTD) before performing ITP. The qPCR was performed on the concentrated gDNA eluted out of the paper disk (using 1X TE buffer) after completing the ITP experiment. Fig. 3D. depicts the normalized DNA concentration for 3X-diluted mock sputum spiked with 10² gDNA/µL, suggesting that the paper-based ITP device can purify and concentrate the gDNA even in the presence of inhibitory molecules. ITP was performed for 20 minutes at 18 V for mock sputum samples.

Using this paper-based ITP device, we were also able to electrochemically lyse (via electrochemical generation of hydroxide) M. Smegmatis bacteria cells, spiked in mock sputum (TE reservoir), and simultaneously purify and concentrate Msm gDNA into the paper disks. (results not shown here)

CONCLUSION

This low-voltage paper-based ITP platform is the first demonstration of sputum DNA preparation using paper-based ITP. One of the advantages of this device is that it can accommodate large sample volumes (900 µL). It offers a critical pre-analytical step involving bacterial lysis along with purification and preconcentration of gDNA from complex sputum samples and complements paper-based isothermal nucleic acid amplification TB tests.³ Ultimately, we aim that these tools would enable molecular diagnostics of TB in low-resource settings.

REFERENCES

1 A. T. Bender, M. D. Borysiak, A. M. Levenson, L. Lillis, D. S. Boyle and J. D. Posner, Anal. Chem., 2018, 90, 7221–7229.

2 X. Li, L. Luo and R. M. Crooks, Lab Chip, 2015, 15, 4090–4098.

3 N. Kaur, J. S. Michael and B. J. Toley, Sci. Rep., 2019, 9, 15367.