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

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.

Keywords: Isotachophoresis; Nucleic acids; Paper-microfluidics; Protein digestion; Sample preparation.

Figure 1:

Device for extracting DNA from whole blood. (A) Device materials and construction. The device is constructed from various layers of acrylic, PCR tape, and several different membranes. The device is designed for an input sample volume of 33 μL of whole blood. (B) Schematic of assembled device, showing the glass fiber membrane reservoirs for ITP buffers. (C) Image of an assembled device.

Figure 2.

Device protocol to extract DNA from whole blood. (A) Initial device configuration. (B) We add 33 μL whole blood spiked with target DNA and fluorescently labeled tracking DNA onto the Vivid membrane. Blood filtration occurs for 4 minutes, during which plasma and nucleic acids filter through to the Fusion 5 membrane. (C) After filtration, we remove the blood filtration fixturing and Vivid and place a piece of PCR tape over the sample port. Proteolysis then occurs for 15 minutes. (D) We add ITP buffers to their respective reservoirs. Color is added to the image for clarity: red represents trailing electrolyte buffer while blue represents leading electrolyte buffer. (E) Constant current is applied across the device, beginning ITP and focusing the DNA into a narrow plug which then migrates towards the leading electrolyte reservoir. We monitor this process via fluorescent microscopy. (F) Once the ITP plug reaches the eluate port, we stop ITP, remove the eluate port tape, and cut out the exposed portion of Fusion 5 membrane containing the ITP plug. Buffer colors are removed for clarity. (G) We add the strip directly to an RPA reaction, or first dewater the strip using a centrifuge and then add the resulting liquid sample to an RPA reaction. (H) RPA is then performed with the real-time fluorescence recorded.

Figure 3.

SDS-PAGE results of human serum protein digests via proteinase K. All digests were performed for 15 minutes to limit the overall protocol timespan. Lane 1 is a protein ladder (ranging from 250 kDa to 10 kDa) and lane 2 is undigested human serum. Lane 3 represents a commonly used digestion buffer for DNA extraction protocols, with 50 μg/mL proteinase K with 0.5% (w/v) SDS, digested in a microcentrifuge tube in a water bath at 55 °C. Lanes 4–7 show the effect of various proteinase K concentrations (50–1500 μg/mL) without SDS present, in a water bath at 55°C. Without SDS, the digestion is significantly diminished, though there is a clear relationship between digestion completion and proteinase K concentration. Lanes 8–11 show the same proteinase K concentration range using the on-paper digestion protocol at 55 °C. We see a lower digestion completion when compared to the in-tube protocol, though the same concentration dependent relationship is present. Lanes 12–15 and lanes 16–19 show the same proteinase K concentration range, using the on-paper digestion protocol at 37 °C and 22 °C, respectively. No significant difference is observed between the various temperature conditions.

Figure 4.

Spatiotemporal maps of representative ITP progression with varying concentrations of proteinase K, visualized via fluorescently-labeled tracking DNA with the on-paper paper digestion protocol. (A) When no proteinase K is used, the tracking DNA focuses into a diffuse region and does not migrate down the length of the Fusion 5 membrane. (B) and (C) When moderate concentrations of proteinase K are used, the fluorescently labeled DNA focuses into a concentrated plug, although the plug still does not migrate well downstream. (D) When 800 μg/mL proteinase K is used, the tracking DNA is focused into a concentrated plug and migrates down the length of the strip successfully.

Figure 5.

Recombinase polymerase amplification curves using DNA extracted from whole human blood. Input copy numbers of 10,000 cps/trial, 1,000 cps/trial, and 100 cps/trial correspond to input copy concentrations of 3×10⁵ cps/mL, 3×10⁴ cps/mL, and 3×10³ copies DNA per mL of input blood, respectively. NTC is a no template control. (A) Amplification curves using spun-out eluate (n=3 for all trials). (B) Amplification curves using the direct strip addition (n=4 for all trials). All trials amplify down to 100 cps/trial (3×10³ cps input DNA per mL of whole blood).