Electrokinetic Nucleic Acid Amplification (E-NAAMP) Using Paper-PDMS Microfluidics and High-Frequency Joule Heating

We present a novel paper-based nucleic acid amplification (NAA) technique using electrokinetic nucleic acid amplification (E-NAAMP). In E-NAAMP, a high radio frequency (RF) potential is applied across a conductive aqueous sample to induce an Ohmic current and drive the sample temperature to increase by Joule heating. Using this RF approach, we investigate the ability to induce E-NAAMP in pressurized paper-based microfluidic channels. We use the microfluidic pressure-in-paper (µPiP) method to encapsulate synthetic and natural fiber-based paper channels between thin sheets of polydimethylsiloxane (PDMS) with two strips of conductive PDMS that have been infused with carbon black (PDMS-CB) to act as electrodes in contact with the paper channels. A high-frequency (38 MHz) voltage is applied across a conductive NAA sample via the PDMS-CB electrodes to generate Joule heating within the paper structure. Here, we show that µPiP-based E-NAAMP can amplify NAs using the loop-mediated isothermal amplification (LAMP) reaction. We first investigate the pore-scale temperature profile numerically by solving the relevant energy transport equations within digitized paper fiber domains obtained using micro-computed tomography (micro-CT) scans. We compare these temperature-voltage predictions to those measured experimentally and demonstrate good agreement, suggesting that RF Joule heating is a viable method for electrokinetically heating paper-based microfluidic platforms. We next examine the effect of a porous substrate on NAA and demonstrate that the carrier protein, bovine serum albumin (BSA), is required for paper-based NAA reactions by preventing polymerase adsorption within the paper structure. Finally, we show successful NAA in paper using E-NAAMP with multiple paper fiber types while also further demonstrating BSA's necessity for paper E-NAAMP success. Our results demonstrate that paper-based microfluidic NAA using Joule heating is a viable alternative to traditional microfluidic NAA devices by offering substantial heating element miniaturization and decreased fabrication complexity when compared to both traditional and Joule-heated microfluidic NAA devices.