Nucleic acid sample preparation from whole blood in a paper microfluidic device using isotachophoresis

Highlights

• A paper-based device is presented for the purification of nucleic acids using isotachophoresis.

• Large sample volumes of whole blood are used, with integrated on-device cell fractionation.

• Paper-based, on-device protein digestion is incorporated and paper-based electrolyte reservoirs simplify buffer loading.

• Recombinase polymerase amplification used to verify extraction and purification.

• Successful isotachophoretic extraction and amplification of as few as 3x10³ cps DNA per mL is shown.

Abstract

Nucleic acid amplification tests (NAATs) are a crucial diagnostic and monitoring tool for infectious diseases. A key procedural step for NAATs is sample preparation: separating and purifying target nucleic acids from crude biological samples prior to nucleic acid amplification and detection. Traditionally, sample preparation has been performed with liquid- or solid-phase extraction, both of which require multiple trained user steps and significant laboratory equipment. The challenges associated with sample preparation have limited the dissemination of NAAT point-of-care diagnostics in low resource environments, including low- and middle-income countries. We report on a paper-based device for purification of nucleic acids from whole blood using isotachophoresis (ITP) for point-of-care NAATs. We show successful extraction and purification of target nucleic acids from large volumes (33 µL) of whole human blood samples with no moving parts and few user steps. Our device utilizes paper-based buffer reservoirs to fully contain the liquid ITP buffers and does not require complex filling procedures, instead relying on the natural wicking of integrated paper membranes. We perform on-device blood fractionation via filtration to remove leukocytes and erythrocytes from our sample, followed by integrated on-paper proteolytic digestion of endogenous plasma proteins to allow for successful isotachophoretic extraction. Paper-based isotachophoresis purifies and concentrates target nucleic acids that are added directly to recombinase polymerase amplification (RPA) reactions. We show consistent amplification of input copy concentrations of as low as 3 × 10³ copies nucleic acid per mL input blood with extraction and purification taking only 30 min. By employing a paper architecture, we are able to incorporate these processes in a single, robust, low-cost design, enabling the direct processing of large volumes of blood, with the only intermediate user steps being the removal and addition of tape. Our device represents a step towards a simple, fully integrated sample preparation system for nucleic acid amplification tests at the point-of-care.

Introduction

There is a need to develop point-of care (POC) infectious disease diagnostic technologies to enable faster, more economic care closer to the patient [1], [2], [3]. This includes nucleic acid amplification tests (NAATs), a crucial diagnostic and monitoring tool for bacterial and viral infections [4], [5], [6], [7]. NAATs have been developed for a variety of diseases, including HIV, hepatitis B, hepatitis C, and tuberculosis [8], [9], [10]. NAATs may be used to determine the presence of a pathogen or as a way to surveil disease progression and inform patient treatment plans. In many cases, NAATs have replaced more traditional techniques (e.g. cell culture or immunoassays) as the gold standard [11], [12], [13], [14]. The majority of currently available NAATs require central laboratory infrastructure, including highly trained personnel, sensitive reagents, and significant logistics related to sample collection and transport [4], [15]. In low- and middle-income countries (LMIC), where disease prevalence remains high, these requirements, as well as high capital costs and other required resources (e.g. stable electricity and climate-controlled facilities) may significantly complicate NAAT implementation [16], [17], [18].

Nucleic acid amplification tests require three main steps: sample preparation, amplification, and detection [4]. Sample preparation isolates and purifies target nucleic acids from potent amplification inhibitors often present in complex biological samples to allow for successful subsequent amplification and detection, and is often considered to be a primary bottleneck in nucleic acid testing [19], [20]. Traditional sample preparation is performed via liquid phase extraction (LPE), such as phenol–chloroform extraction, or solid phase extraction (SPE) techniques, which preferentially bind and release nucleic acids to silica columns, beads, or membranes. These protocols utilize high-molarity toxic chaotropic salts such as guanidine thiocyanate or guanidine hydrochloride, often included in lysis buffers, which help facilitate nucleic acid adsorption onto silica materials [21], [22]. Both LPE and SPE result in purified nucleic acids that are readily added to amplification reactions. These protocols require numerous manual pipetting and centrifugation steps, can take more than an hour to perform, and can be difficult to implement in a point-of-care environment primarily due to their requirements for training, refrigerated reagent storage, and necessary associated equipment (e.g. centrifuges and pipettes) [15].

Several fully integrated sample-to-answer point-of-care NAAT platforms are commercially available, which automate the sample preparation, such as the as the m-PIMA system (Abbott), the GeneXpert (Cepheid), and the Cobas Liat platform (Roche) [23]. These solutions typically rely on solid-phase extraction techniques, using complex hydraulics and electromechanical pathways and robotics to replace manual steps, significantly increasing platform costs [24], [25], [26]. Various microfluidic approaches have also been investigated, though these often require external pressure sources for sample introduction and valving [27], [28]. This additional complexity complicates overall device design and implementation.

Isotachophoresis is an electrokinetic separation technique that has been applied to purification of nucleic acids from complex biological samples [29], [30], [31], [32], [33]. First recognized for its ability to separate and concentrate metal ions from organic solutions [34], isotachophoresis (ITP) is appealing for nucleic acid sample preparation because it uses simple buffers and requires no moving parts or valving. ITP relies on a discontinuous buffer system containing a high electrophoretic mobility leading electrolyte (LE) and a low mobility trailing electrolyte (TE). As an electric field is applied to the system, a charged sample species with an intermediate mobility, such as nucleic acids, focuses at the interface between the two electrolytes. Species with higher or lower mobilities than the respective LE and TE, for example proteins, are excluded from the interface plug region, resulting in the purification of the species of interest. Kondratova et al. were the first to show successful isotachophoretic extraction of nucleic acids from complex biological samples, including human plasma/serum and urine in agarose gel rods, though this required significant sample preparation, including overnight incubation with proteinase K/SDS and dialysis [32], [33]. Since then, isotachophoresis has been demonstrated in microchannel and paper based formats, and has been used to separate and purify nucleic acids from a range of biological samples, including urine, milk, and blood [30], [35], [36], [37], [38].

Blood is a particularly compelling sample for POC NAATs as many of the world’s most widespread diseases, such as HIV, malaria, dengue, and HBV are bloodborne. Persat et al. were the first to extract and purify nucleic acids from diluted whole blood lysate using ITP [30]. They targeted genomic DNA with an estimated extraction efficiency of 30–70% and processed 2.5 nL of whole blood that had been diluted 10-fold with lysis buffer, for a total processed sample volume of 25 nL. Eid et al. extracted nucleic acids from Listeria monocytogenes cells in diluted whole blood via ITP and amplified using off-chip recombinase polymerase amplification, achieving a limit-of-detection (LoD) of 5 × 10³ cell-equivalents per mL when using purified genomic DNA and 2x10⁴ cells per mL when using L. monocytogenes cells [39]. In both experiments, 2.5 µL of whole blood was spiked with target and diluted 10-fold with lysis buffer and modified LE. Marshall et al. purified and detected genomic DNA from P. falciparum parasites using ITP, with a limit of detection reported as 500 parasites per µL blood [40]. In this work, 1 µL of whole blood was added to a 14 µL reservoir of TE and proteinase K, utilizing ITP with a semi-infinite sample injection scheme to separate the nucleic acids after pathogen lysis.

Much of the previous work has focused on isotachophoretic nucleic acid extraction and purification has been performed in microchannel devices manufactured via traditional photolithography or injection molding [41], [42]. These devices have a number of advantages, such as precisely defined geometries, minimal channel adhesion sample loss, and readily available mass manufacturing methods. Challenges of microchannel based ITP devices include complex LE/TE buffer loading procedures (often requiring vacuum assistance) and minimally contained free-surface liquid reservoirs [42], complicating potential application at the point-of-care. Additionally, in microchannel based ITP, the sample volumes have been very limited and recovering ITP-focused target analyte can be challenging. Paper-based ITP systems are robust, extremely inexpensive to manufacture, and allow for larger sample volumes. They also fill with electrolyte solutions and samples automatically owing to the natural wicking properties of the hydrophilic porous membranes. Large scale paper-based ITP was originally used for laboratory separations of ions [43], [44]. More recently, microfluidic based paper devices have been developed for point-of-care applications [37], [38], [45], [46]. Our group first demonstrated preconcentration in a paper based microfluidic device and how this can be used to improve the limit of detection in rapid immunoassays [37], [47]. Rosenfeld and Bercovici created a paper-based ITP system by patterning wax onto a nitrocellulose membrane to constrain the LE/TE buffers, and showed 20,000 fold sample focusing and amplification-free detection of target DNA in buffer [46]. Previously, our group extracted target DNA from human serum utilizing paper-based ITP, and amplified on-device via recombinase polymerase amplification with a limit of detection of 10⁴ copies DNA per mL from an initial sample volume of 20 µL [31]. We also showed proof-of-concept data describing extraction and amplification of DNA from whole blood while using a significant concentration of target DNA.

While ITP represents an attractive solution for nucleic acid extraction and purification from complex samples such as whole blood, additional sample preparation steps such as off-chip sample dilution are often still required. Dilution is frequently used because the high salt and protein content of blood and plasma can make isotachophoretic extraction difficult due to effects on viscosity, ionic strength, pH, and conductivity of the system [48]. For applications that are primarily concerned with pathogen presence and binary diagnoses, where the concentration of the target is high, dilution may not be problematic. Dilution can be detrimental, however, for applications where low limits of detection are necessary, such as HIV viral load monitoring [49]. In applications where low detection limits are required there is often only a single target nucleic acid per microliter, or less, so large biological sample volumes are critical to ensure sufficient target is present in the device. In much of the previous work where whole blood was used as a sample in ITP, the total processed sample volume was typically 10–25 µl, while the volume of blood processed was 1–2.5 µl. For this reason, there have been several works that have focused on increasing ITP sample volume or biological sample concentration. For instance, van Kooten et al. focused DNA and E. coli bacteria diluted in 50 µL trailing electrolyte into a volume of 500 pL by carefully designing their microchannel-based system [41]. While this volume is impressive in an isotachophoretic system, complex biological samples were not tested. Bender et al. extracted nucleic acids from 20 µL of human serum, representing one of the largest volumes of undiluted complex sample used in isotachophoretic systems to date [31]. For bloodborne pathogen detection in the point-of-care setting, it is often necessary to process large volumes of blood to reach low limits of detection in downstream nucleic acid amplification assays. This requires an integrated approach that addresses many unique aspects of using whole blood as a sample, including leukocyte/erythrocyte depletion and endogenous protein digestion to successfully extract and purify target nucleic acids in an inexpensive, semi-automated manner with minimal user steps.

In this paper, we present a paper-based, isotachophoretic nucleic acid sample preparation platform that extracts target DNA from human whole blood. Our device directly processes relatively large volumes (33 µL) of whole blood, so no preliminary dilution or pretreatment user steps are required. We verify nucleic acid isotachophoretic extraction and purification by real-time imaging of fluorescently labeled DNA and off-device recombinase polymerase amplification (RPA). We show an optimized integrated plasma separation membrane for cell fractionation, as well as on-paper proteolytic digestion of endogenous plasma proteins via proteinase K to enable isotachophoretic extraction, verified using SDS-PAGE. We discuss design decisions used to develop a platform that decreases the number of user steps and potential sources of error, including the first integration of paper-based ITP buffer reservoirs. The nucleic acid sample preparation platform presented here represents a step towards a simple, robust, low-cost sample preparation technique for point-of-care NAATs for bloodborne diseases.