An electrophoretic DNA extraction device using a nanofilter for molecular diagnosis of pathogens

Jae-Hyun Kang a, Yong Tae Kim bc, Kidan Lee a, Hyun-Mi Kim a, Kyoung G. Lee b, Junhyoung Ahn d, JaeJong Lee d, Seok Jae Lee *b and Ki-Bum Kim *a
aDepartment of Materials Science and Engineering, Seoul National University, Seoul 08826, Republic of Korea. E-mail: kibum@snu.ac.kr
bNano-bio Application Team, National Nanofab Center, 291 Daehak-ro, Yuseong-gu, Daejeon 34141, Republic of Korea. E-mail: sjlee@nnfc.re.kr
cDepartment of Chemical Engineering & Biotechnology, Korea Polytechnic University, 237 Sangidaehak-ro, Siheung-si, Gyeonggi-do 15073, Korea
dKorea Institute of Machinery and Materials, 156 Gajeongbuk-ro, Yuseong-gu, Daejeon 305-343, Republic of Korea

Received 18th December 2019 , Accepted 7th February 2020

First published on 10th February 2020


Rapid and efficient nucleic acid (NA) extraction and concentration are required for point-of-care analysis in order to prevent an epidemic/pandemic disease outbreak. Typical silica-based NA extraction methods have limitations such as being time-consuming, requiring human intervention, and resulting in a low recovery yield. In this study, we have developed a pathogenic DNA extraction device based on electrokinetic separation incorporated with a silicon nitride (SiNx) nanofilter, which expedites the DNA extraction procedure with advantages of being convenient, efficient, and inexpensive. This DNA extraction device consists of a computer numerical control (CNC) milled-Teflon gadget with a cis-chamber as a cell lysate reservoir and a trans-chamber as a elution solution reservoir, with the SiNx nanofilter being inserted between the two chambers. The SiNx nanofilter was fabricated using a photolithographic method in conjunction with nanoimprinting. Approximately 7.2 million nanopores of 220 nm diameter were located at the center of the nanofilter. When a DC electric field is applied through the nanopores, DNA is transferred from the cis-chamber to the trans-chamber to isolate the DNA from the cell debris. To demonstrate the DNA extraction performance, we measured the absorbances at 260 and 280 nm and performed a real-time polymerase chain reaction (real-time PCR) using the recovered DNA to verify its feasibility for downstream genetic analysis. Moreover, the DNA extraction device was successfully operated using a 1.5 V alkaline battery, which verifies the portability of the device for point-of-care testing. Such an advanced DNA extraction system can be utilized in various fields including clinical analysis, pathogen detection, forensic analysis, and on-site detection.


Introduction

Waterborne organisms such as Escherichia coli, Salmonella, and dengue virus have been linked to the most common and leading serious illnesses worldwide1 and brought huge economic burden for disease diagnosis and treatment. To identify such pathogens, molecular diagnostics has attracted a lot of attention due to its high sensitivity, accuracy and specificity, which are crucial disease monitoring factors for appropriate treatment.2,3 The current molecular diagnostics typically involves nucleic acid (NA) extraction from cells, amplification of the isolated NAs and amplicon detection with optical, mechanical, and electrochemical analysis.4–6 Although each step is significant, the key step for a successful molecular diagnostics is to obtain highly pure NAs for downstream analysis.

Since the first report of a chaotropic effect on DNA separation, silica-based solid phase DNA extraction using glass powder was invented, which involves a cell decomposition process with enzymes, chemical lytic agents, or mechanical forces (bead milling, sonication, etc.) and a debris removal step through centrifugation after the absorption of the DNA on a silica column or silica-coated magnetic beads in the presence of chaotropic salts.7–10 DNA was then recovered using an elution buffer with an anti-chaotropic agent to desorb the DNA from the hydrophilic silica matrix. While solid phase extraction has become a gold standard method over the past few decades, limitations such as complex protocol, long running time, and need for skilled professionals can significantly affect the purity or quantity of the recovered DNA. Moreover, the requirement of bulky instruments including centrifuges and heating blocks makes it difficult to create portable set-ups for on-site pathogen detection aiming to minimize disease transmission.11,12

To overcome these shortcomings, micropatterning techniques have been used to miniaturize the DNA extraction platform and simplify the operation process.13–15 Kamat et al.16 introduced a polydimethylsiloxane (PDMS) microchip with chitosan-coated magnetic particles to extract DNA in a pH-dependent manner. Gan et al.17 integrated chitosan-modified filter paper in a poly(methyl methacrylate) microfluidic device to develop a low-cost and rapid DNA extraction method. Zhang et al.18 proposed sol–gel coating on a polycarbonate (PC) surface with bis[3-(trimethoxysilyl)propyl]aminosilane for purifying DNA from a crude sample as well as one-step bonding of two PC substrates to fabricate a monolithic microchip. Although the microchip-based DNA separation has been improved in terms of portability and simplicity, DNA recovery yield is still a limitation due to the use of the solid phase DNA extraction mechanism.

Electrokinetic biomolecule delivery technology has been used to extract DNA because of its advantages such as no additional step required for DNA purification and concentration with rapid processing time. Physical dielectrophoresis was introduced to extract cell-free DNA in blood by the Heller group.19–21 They separated the desired particles using the polarity difference between media and particles by generating a non-uniform electric field with a specially designed electrode. Kim et al.22 proposed an electrokinetic biomolecule separation platform incorporated with a nanoporous anodized aluminum oxide (AAO) membrane. The electrokinetic movement of single-stranded DNA (ssDNA) across the nanoporous membrane was affected by the pore size, DNA concentration, and applied voltage. However, the nanoporous AAO membrane device has a significant problem of durability owing to the brittleness of the AAO membrane.

Here, we developed an electrophoretic DNA extraction device using a silicon nitride (SiNx) nanofilter, which can transport negatively charged DNA by applying a direct current (DC) electric field through nanopores. SiNx nanopore arrays having a uniform diameter of 220 nm were fabricated with conventional semiconductor manufacturing processes and used as DNA separation filters. The DC electric field was applied to the DNA extraction device that consists of a cis-chamber filled with cell lysates, a trans-chamber loaded with an elution buffer, and a nanofilter between the chambers. The purity and concentration of the recovered DNA on the microdevice were evaluated using an ultraviolet (UV) spectrophotometer, and a real-time polymerase chain reaction (real-time PCR) was used to verify the adaptability of the extracted DNA for downstream analysis. To demonstrate the portability of the DNA extraction device for point-of-care testing, a 1.5 V alkaline battery was successfully used as a power source to obtain pure DNAs.

Results and discussion

Basic principle of the electrophoretic DNA extraction chip

In bacterial cell lysates, there are cell debris, proteins, nucleic acids, and so on. Negatively charged DNAs in the lysates are migrated across the nanofilter chip by the electric field. In terms of size and charge, components larger than the pore size like cell debris cannot pass through the nanofilter. On the other hand, nucleic acids, proteins and ions in the solution that are smaller than the pore size and have a negative charge will pass through the pores. Although unwanted impurities (in particular, proteins) with a negative charge migrate through the pores due to the electric field, DNA migrates faster than proteins because at physiological pH, proteins have only 5 positive or negative charges of the 20 amino acids that make them up, but DNAs are highly negatively charged because one negative charge is formed on each phosphate backbone.23

The electrophoretic DNA extraction device

The strength of the electrophoretic DNA extraction chip lies in the simple extraction procedure where centrifuges and toxic chemical reagents such as phenol, chloroform, and chaotropic salts need not be used as well as device miniaturization for point-of-care analysis. The nanofilter-based electrophoretic DNA extraction concept enables us to accomplish DNA concentration and enrichment by applying electric current to the lysate solution. Our electrophoretic DNA extraction system can overcome the intrinsic hurdles of conventional DNA extraction systems.

First, solid phase DNA extraction leads to operational complexity with repeated DNA seizure, debris washing, and the DNA recovery procedure. In addition, chaotropic salts, which are known PCR inhibitors, should be used for capturing DNA on the silica substrate surface. In contrast, our proposed device does not require such complex steps and simply applies an electric current through the nanofilter that delivers DNAs from the cis-chamber to the trans-chamber without any toxic chemicals. Second, the conventional system necessitates bulky instrumentation such as a centrifugal system to drive the solutions (lysates, washing buffer, and elution buffer) to the recovery column, which would not be adequate for the construction of a portable DNA extraction system for point-of-care genetic analysis application. However, our device requires only an electric power supplier (even possible with an alkaline battery) that can miniaturize the DNA extraction chip without using bulky external operating hardware. Finally, DNA concentration per unit volume is one of the main factors to determine the detection sensitivity of PCR analysis. However, conventional DNA extraction kits with a silica membrane have a limitation to DNA enrichment due to the necessity of a high volume of an elution buffer for sufficiently wetting the whole silica-based membrane to harvest the absorbed DNA from the silica membrane. Moreover, a part of the elution buffer would be adsorbed by the silica membrane that causes a low DNA recovery yield. In contrast, our DNA extraction chip requires only 30 μL of the elution solution for transporting DNA to the trans-chamber, which means DNA could be delivered from the cis- to the trans-chamber with high concentrations. In addition, the nanofilter does not absorb any solution, indicating that most DNAs passing through the nanofilter reach the trans-chamber without any interruption.

In terms of economy, the nanofilter can be fabricated from at least 200 chips on an 8-inch single wafer. In this case, the fabrication cost of a single nanofilter was calculated to be 1 USD. Considering that a column-based commercial DNA extraction kit is priced at about 3 USD, the electrophoretic DNA extraction device is price competitive.

Characterization of the SiNx nanofilter membrane

The morphology of the SiNx nanofilter was observed using a scanning electron microscope (SEM), as shown in Fig. 1. The circular shape of nanopores can be clearly observed in Fig. 1a, which is an SEM image at 100[thin space (1/6-em)]000× magnification with a hexagonal array and 400 nm pitch. The SEM image of the SiNx nanofilter with a software-analyzed nanopore size distribution histogram is shown in Fig. 1b. A uniform array with an average diameter of 220 ± 5 nm was observed. The pore density was 7.2 pores per μm2 and the total number of nanopores was about 7.2 million, which were patterned on the 1 mm × 1 mm nanopore area. The porosity of the nanopore array was 22.7% and the pores were uniformly fabricated over the entire SiNx membrane.
image file: c9nr10675h-f1.tif
Fig. 1 (a) SEM image of the SiNx nanofilter with a hexagonal nanopore array at 100[thin space (1/6-em)]000× magnification (scale bar: 500 nm). (b) SEM image of the SiNx nanofilter at 20[thin space (1/6-em)]000× magnification (scale bar: 2.5 μm) with a histogram of pore size distribution overlaid at 220 ± 5 nm.

Optimization of voltage applied to the device

To determine the optimum DC electric potential for DNA transportation on the electrophoretic DNA extraction chip, 1 to 4 V was applied to the 107E. coli O157:H7 cell lysate and the nanofiltered elution solution for 5 to 30 min. The recovered DNA was amplified using the real-time PCR and the results are shown in Fig. 2. The y-axis represents the cycle threshold (Ct) value of the real-time PCR and the x-axis represents the voltage application time to the microchip. As shown in Fig. 2a and b, the average Ct value constantly decreased over time until it reached 21.7, which was 3.5 cycles lower than that of Ct value using DNA extracted by the commercial kit (the yellow area in Fig. 2 indicates Ct value of amplification of the DNA extracted from 107E. coli O157:H7 cell using the commercial kit). Fig. 2c and d show the Ct values of the real-time PCR at 3 and 4 V, respectively. The Ct value obtained from both experiments had an optimum value at around 15 to 20 min and after that point, the Ct values increased. This result implies that the DNA could be damaged during electroseparation by reactive oxidative ions generated by the electrochemical reaction. In particular, electrochemical products including hypochlorous acid (HOCl; the cell lysate contains chlorine ions) and hydrogen peroxide (H2O2) possibly damage NAs, which were generated during electrophoretic DNA extraction.24 The production mechanism of HOCl and H2O2 at the anode is described as follows:
H2O + Cl → HOCl + H+ + 2e

2H2O → H2O2 + 2H+ + 2e

image file: c9nr10675h-f2.tif
Fig. 2 C t values versus the voltage application time at (a) 1, (b) 2, (c) 3, and (d) 4 V. These results indicate that DNA is damaged during electrochemical separation by HOCl or H2O2. This causes the Ct value inflection point to be greater than 3 V. The yellow highlight indicates the Ct value of the DNA extracted from the 107E. coli O157:H7 cells using the commercial kit.

The generation of HOCl and H2O2 is proportional to the electric current quantity, as shown by Faraday's laws of electrolysis.25,26

image file: c9nr10675h-t1.tif
where n, I, t, F, and z represent the amount of substance in moles, ionic current in ampere (A), constant current applying time in seconds, Faraday's constant, and the valency number of ions, respectively. To understand the electric current in our system, the average current for 250 s was measured at 1, 2, 3, and 4 V, and the results are shown in Fig. S1. The average electric current was detected to be 1.10 and 1.18 μA at 1 and 2 V, respectively. Surprisingly, the average electric current at 3 and 4 V was significantly increased to 9.9 and 31 μA, respectively. This result indicates that the production rate of reactive oxidative agents increased by several ten times at 3 and 4 V compared to that at 1 and 2 V, further implying that the generation of reactive oxidative ions exponentially increases with an increase in the electric current in the system.27 To test the concentration of reactive chlorine ions (HOCl), a strip-based detection was carried out, and the results are shown in Fig. S2. The concentration of HOCl ions was higher than 25 mg L−1 at 3 and 4 V, and is rarely detected at 1 and 2 V. Therefore, 2 V is the optimum voltage for the application of our proposed DNA extraction chip for minimizing DNA damage.

DNA extraction performance of the electrophoretic DNA extraction device

To demonstrate the performance of the DNA extraction chip, the real-time PCR was carried out with the extracted DNA from different E. coli O157:H7 cell numbers. Following the 10-fold dilution of the bacterial cell solution from 107 to 103, cell lysis was performed. The cell lysate solution was loaded to the cis-chamber while the elution buffer was added to the trans-chamber. By applying a 2 V electric potential through the Pt electrodes, DNA extraction was accomplished and the real-time PCR was performed on the recovered DNA to verify its relative extraction efficiency compared with that of the DNA obtained using the commercial kit. Prior to performing the real-time PCR, the absorbance ratio (A260/A280) of the extracted DNAs was found to be 1.75 ± 0.06 using a NanoDrop™ (Thermo Fisher Scientific, USA) spectrophotometer. As shown in Fig. 3, the Ct value of the DNA extracted from the 107E. coli O157:H7 lysate using the proposed device (Ct value: 23.3) was similar to that of the DNA extracted using the commercial kit (Ct value: 24.0), with the values being within the error range. However, at the 104 cell population, PCR threshold of the DNA extracted by the electrophoretic DNA extraction device (Ct value: 32.5) was almost four cycles lower than that of the DNA extracted by the commercial kit (Ct value: 36.7). No PCR threshold was detected by using an unfiltered lysate (used as a negative control) in all cell populations. In addition, we were able to successfully detect the DNA extracted from the 103E. coli O157:H7 lysate with our device (Ct value: 33.8), while the DNA extracted using the commercial kit was below the detection limit.
image file: c9nr10675h-f3.tif
Fig. 3 Limit of detection test of the electrophoretic DNA extraction device using the real-time PCR with the DNA extracted from E. coli O157:H7. Ct values of the DNA extracted using both our device and the commercial kit were gradually increased with a decrease in the number of cells used for DNA extraction. However, the Ct value of the DNA extracted using the electrophoretic DNA extraction device under 105 cells was lower than that of the DNA extracted using the commercial kit. Moreover, the electrophoretic DNA extraction device can successfully isolate the DNA even from 103 cells, but the commercial kit cannot.

Electrophoretic DNA extraction using a battery

One of the limitations of the conventional DNA extraction system is the requirement of large operational equipment such as a centrifuge that makes the miniaturization of the DNA extraction system difficult for on-site genetic analysis for rapid pathogen identification. Since our system can be operated using a DC power supply with low power consumption, we demonstrated how our devices work with a simple household battery, as shown in Fig. 4a. To construct a portable DNA extraction system, a 1.5 V AA alkaline battery was used instead of the DC power supply and electrophoretic DNA extraction was carried out with the E. coli O157:H7 cell lysate. The results of the real-time PCR using the extracted DNA are shown in Fig. 4b. The Ct value of the DNA extracted from the 107E. coli O157:H7 lysate was 23.6, and it gradually increased to 32.3 with a decrease in the bacterial concentration, showing DNA extraction performance similar to that observed using a DC power supply, especially at a low cell population. These results show that our simple, physical, and electrical DNA extraction system could successfully isolate and concentrate the pathogenic E. coli O157:H7 DNA from a crude cell lysate sample with low power consumption. In addition, alkaline battery-powered DNA concentration is promising for developing a portable DNA extraction system for on-site pathogen identification.
image file: c9nr10675h-f4.tif
Fig. 4 Portability tests using the electrophoretic DNA extraction device driven by a 1.5 V AA battery. (a) A digital photograph of configuration for device portability testing. (b) The DNA obtained from E. coli O157:H7 using the portable system was used in the real-time PCR to ascertain its potential use for on-site DNA analysis.

Experimental

Fabrication of an electrophoretic DNA extraction device

The structure of the electrophoretic DNA extraction device is shown in Fig. 5. The DNA extraction device is composed of a cis-chamber as a cell lysate container, a trans-chamber as an elution solution receptacle, and a SiNx nanofilter as the DNA extraction filter and a barrier to prevent the mixing of two solutions in the containers, as shown in Fig. 5a. The cis- and trans-chambers were fabricated with polytetrafluoroethylene (PTFE, Teflon) using CNC milling and the nanofilter (1 cm × 1 cm) was sandwiched between the Teflon gadgets in the presence of two punched PDMS blocks on both sides. Next to the chambers, two screw threads were carved to fix the two Teflon gadgets and the nanofilter by tightening with screws to avoid reagent leakage. The assembled DNA extraction device (30 mm × 18 mm × 8 mm) is shown in Fig. 5b, and the digital image of the fully assembled DNA extraction device with platinum (Pt) electrodes is shown in Fig. 5c.
image file: c9nr10675h-f5.tif
Fig. 5 Schematic image of an electrophoretic DNA extraction device. (a) An enlarged view of the electrophoretic DNA extraction device with two CNC milled-Teflon gadgets, two bolts and nuts, and a SiNx nanofilter with two PDMS gaskets. (b) Assembled schematics of the electrophoretic DNA extraction device with a cis-chamber as a cell lysate container and a trans-chamber as an elution buffer reservoir. (c) Digital image of the fully assembled electrophoretic DNA extraction device with Pt electrodes.

The schematic illustration of the SiNx nanofilter fabrication process is shown in Fig. 6. Conventional semiconductor fabrication processes were used to manufacture the SiNx nanofilter. A 500 nm thick low-stress SiNx was deposited on a double-side polished 4-inch silicon wafer (DASOM RMS, South Korea) by low-pressure chemical vapor deposition (LPCVD) (Fig. 6a). SiNx was chosen as the membrane material due to its mechanical strength (8.5 on the Mohs scale, a scale of mineral hardness), inertness in both acidic and basic solutions, and well-established fabrication processes for manufacturing a free-standing membrane.28 Nanoimprinting patterning was used to develop the nanopore array on the SiNx nanofilter.29,30 After spin-coating a UV curable imprint resin (MINS-511RM, Minuta Technology, South Korea) on one side of the wafer (Fig. 6b), a 200 nm circular patterned polydimethylsiloxane (PDMS) imprint mold, which was fabricated using electron-beam lithography, was stamped on the spin-coated imprint resin, as shown in Fig. 6c. Following the imprint resist curing, the cured resin was etched using an inductively coupled plasma (ICP) etcher (Oxford instrument, UK) with SF6 and CHF3 gas as etchants to expose the surface of SiNx (Fig. 6d). The nanopore pattern was then engraved on the exposed SiNx using the ICP etcher with etchants including SF6, CHF3, and argon gas (Fig. 6e). The etching depth of SiNx was limited to 400 nm to prevent the exfoliation of the SiNx film from the Si-substrate during KOH wet etching. In order to perform backside etching, a photoresist (AZ5214, AZ electronic materials, Luxembourg) was spin-coated on the backside of the wafer (Fig. 6e) and developed. The exposed SiNx layer was removed using the ICP etcher with SF6 and CHF3 gas (Fig. 6g). Following Si etching using KOH (Fig. 6f), the photoresist was removed with Piranha solution (H2SO4[thin space (1/6-em)]:[thin space (1/6-em)]H2O2 = 3[thin space (1/6-em)]:[thin space (1/6-em)]1; Fig. 6g). To manufacture a free-standing SiNx membrane, the Si-exposed wafer was immersed in 6 M KOH at 78 °C for 12 h. In order to perforate the nanopore on the SiNx free-standing membrane, the back surface was dry etched with the ICP etcher and a 100 nm thick SiNx membrane with nanopore arrays was obtained as shown in Fig. 6h.


image file: c9nr10675h-f6.tif
Fig. 6 Schematic of the SiNx nanofilter fabrication process (figures not in scale). (a) A 500 nm thick SiNx deposited by LPCVD on a double-sided polished 4-inch wafer. (b) Spin-coated UV-curable imprint resist. (c) Pattern of the nanopore array of a diameter of 200 nm on the imprint resin. (d) Dry etching of the imprint resin for exposing the SiNx layer. (e) Partial etching of the SiNx thin film using the ICP etcher. (f) Square backside SiNx opening. (g) Fabrication of a free-standing SiNx membrane with the KOH wet etching of Si. (h) Dry etching of the backside of the SiNx membrane to perforate the nanopore array.

Characterization of the SiNx nanofilter

SEM image analysis was performed to characterize the circular shape and pore size of the prepared nanofilter (SUPRA 55VP, Carl Zeiss, Germany). In order to investigate the nanopore uniformity, image analysis was carried out using commercial image analysis software (Image Pro Plus version 4.5, Media Cybernetics, Inc., USA).

Electrophoretic bacterial DNA extraction

E. coli O157:H7 (ATCC 9637) was chosen as a model pathogen to confirm DNA extraction performance by the electrophoretic DNA extraction device. Pathogenic bacteria were cultured in 10 mL of Luria–Bertani broth (LPS solution, Daejeon, Korea) at 37 °C until the optical density at 600 nm reached 1.0, rinsed with 0.1 M phosphate-buffered saline (PBS) and 10-fold serially diluted from 107 to 103 cells for downstream DNA extraction. The bacterial cells were lysed using the G-spin™ genomic DNA extraction kit (iNtRON Biotechnology, Inc., Seongnam, Korea). The cell solution in the 2 mL tube was centrifuged at 12[thin space (1/6-em)]000g for 1 min at room temperature and the supernatant was removed. After adding 300 μL of the G-buffer solution (lysis buffer) to the pellet, it was submerged in a 62 °C water bath for 15 min to obtain a cell lysate.

To extract DNAs electrokinetically, 300 μL of the cell lysate and 30 μL of the Tris/borate/EDTA (TBE) buffer were loaded in the cis- and the trans-chambers, respectively. After introducing Pt electrodes at each chamber, DC current was applied using a Keithley 237 high-voltage source-measure unit (Tektronix, USA) to transport DNAs from the cis- to the trans-chamber. To optimize the applied voltage, the voltage was varied from 1 to 4 V and extraction time was also optimized by varying the operation time from 5 to 30 min.

PCR amplification of the stx2 gene

To identify E. coli O157:H7 DNA in the extracted solution, the stx2 gene was targeted for performing the PCR. The recovered DNAs were amplified using SpeedSTAR™ HS DNA polymerase (Takara Bio Inc., Shiga, Japan) in the presence of a forward primer (5′-GGG CAG TTA TTT TGC TGT GGA-3′), a reverse primer (5′-TGT TGC CGT ATT AAC GAA CCC-3′), and a TaqMan probe (5′-FAM-CTA TCA GGC GCG TTT TGA CCA TCT TCG-TAMRA-3′) on the Mic real-time PCR system (Bio Molecular Systems, Australia).31 The total volume of the real-time PCR reagent was 25 μL, which consisted of 2.5 μL of 10× fast buffer I, 2.5 μL of SpeedSTAR DNA polymerase, 3.0 μL of dNTPs, 2 μL of primers (0.15 μM each), the TaqMan probe (0.065 μM) mixture, 1 μL of the extracted DNA, and water. Thermal cycling temperatures under real-time PCR conditions were as follows: an initial denaturation at 95 °C for 5 min followed by 40 cycles at 95 °C for 5 s, 60 °C for 15 s, and 72 °C for 10 s, and a final extension step at 72 °C for 3 min.32 Fluorescence signals released during real-time PCR were measured at every extension step. To demonstrate the performance of the DNA extraction device, the E. coli O157:H7 cell lysate was produced from the cells 10-fold serially diluted from 107 to 103. Following the DNA electrotranslocation in the lysate, the DNA in the trans-chamber was used for the real-time PCR to confirm the ability of the DNA extraction device.

To verify the portability of the system, DNA extraction was conducted using a 1.5 V AA battery (Energizer Holdings, USA). Cell lysates were prepared from 107, 106, 105, and 104 cells for confirming the extraction performance with various bacterial concentrations. The isolated DNA in the trans-chamber was used for the real-time PCR to analyze the extraction efficiency using the battery.

Conclusions

In summary, we successfully developed an electrophoretic bacterial DNA extraction device with a nanofilter for the detection of pathogenic bacteria with on-site DNA recovery capability. The use of the nanofilter membrane enables us to easily transport DNA from the crude E. coli O157:H7 cell lysate to the elution solution by applying a DC electric field, which simplifies the DNA extraction step and reduces the time consumption. The quality of the extracted DNA was verified by measuring the absorbances at 260 and 280 nm. The real-time PCR on the recovered pathogenic DNA was performed to demonstrate its usability for downstream analysis and relative extraction efficiency compared to that of the DNA obtained using the commercial kit. The results of the real-time PCR showed that the proposed DNA extraction device isolated and concentrated DNA from 105 or lower cell populations better than the commercial kit used. DNA extraction was also accomplished using a 1.5 V AA alkaline battery, which makes it possible to construct a portable DNA recovery system. Such an advanced electrophoretic-based portable DNA extraction system can be used for applications in clinical diagnosis, pathogen detection, and forensic analysis.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This research was supported by the BioNano Health-Guard Research Center funded by the Ministry of Science and ICT (MSIT) of Korea as a Global Frontier Project (grant number H-GUARD_2013M3A6B2078943), the Nano Material Technology Development Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (MSIT) of Korea (grant number 2015M3A7B4050454 and 2019R1A2C2005783) and the academic promotion system of Korea Polytechnic University.

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Footnotes

Electronic supplementary information (ESI) available. See DOI: 10.1039/c9nr10675h
These authors contributed equally to this work.

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