Use of Dimethyl Pimelimidate with Microfluidic System for Nucleic

Jun 21, 2017 - The design of the microfluidic chip was first drawn using AutoCAD (Autodesk, Inc., San Rafael, CA) and then printed with a laser-cuttin...
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Use of Dimethyl Pimelimidate with Microfluidic System for Nucleic Acids Extraction without Electricity Choong Eun Jin,†,+ Tae Yoon Lee,‡,+ Bonhan Koo,† Kyung-Chul Choi,§ Suhwan Chang,∥ Se Yoon Park,⊥,# Ji Yeun Kim,⊥ Sung-Han Kim,⊥ and Yong Shin*,† †

Department of Convergence Medicine, Asan Medical Center, University of Ulsan College of Medicine Biomedical Engineering Research Center, Asan Institute of Life Sciences, Asan Medical Center, 88 Olympicro-43gil, Songpa-gu, Seoul 05505, Republic of Korea ‡ Department of Technology Education and Department of Biomedical Engineering, Chungnam National University, Daejeon 34134, Republic of Korea § Department of Biomedical Sciences and Department of Pharmacology, University of Ulsan College of Medicine, Seoul 05505, Republic of Korea ∥ Department of Biomedical Sciences, University of Ulsan College of Medicine, Seoul 05505, Republic of Korea ⊥ Department of Infectious Diseases, Asan Medical Center, University of Ulsan College of Medicine, Seoul 05505, Republic of Korea # Division of Infectious Diseases, Department of Internal Medicine, Soonchunhyang University Seoul Hospital, Soonchunhyang University College of Medicine, Seoul 140-743, Republic of Korea S Supporting Information *

ABSTRACT: The isolation of nucleic acids in the lab on a chip is crucial to achieve the maximal effectiveness of point-of-care testing for detection in clinical applications. Here, we report on the use of a simple and versatile single-channel microfluidic platform that combines dimethyl pimelimidate (DMP) for nucleic acids (both RNA and DNA) extraction without electricity using a thin-film system. The system is based on the adaption of DMP into nonchaotropic-based nucleic acids and the capture of reagents into a low-cost thin-film platform for use as a microfluidic total analysis system, which can be utilized for sample processing in clinical diagnostics. Moreover, we assessed the use of the DMP system for the extraction of nucleic acids from various samples, including mammalian cells, bacterial cells, and viruses from human disease, and we also confirmed that the quality and quantity of the nucleic acids extracted were sufficient to allow for the robust detection of biomarkers and/or pathogens in downstream analysis. Furthermore, this DMP system does not require any instruments and electricity, and has improved time efficiency, portability, and affordability. Thus, we believe that the DMP system may change the paradigm of sample processing in clinical diagnostics.

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diagnosis with the existing technologies due to constraints such as low sensitivity and specificity, labor-intensive nature, high cost, need for highly skilled technicians, and need for automation and portability of the system that requires the integration of sample preparation and detection techniques.4−7

ecent advances in innovative technologies for clinical diagnostics have enabled the rapid and accurate diagnosis of several human diseases. Nevertheless, emerging diseases caused by novel pathogens (i.e., bacteria, viruses) remain a major threat to human life, due to the lack of appropriate diagnostic tools for clinical use at the early disease stage. Hence, various technologies have been evaluated for the diagnosis of diseases such as cancer, as well as neurological and infectious diseases.1−3 However, there are several unmet needs in terms of © 2017 American Chemical Society

Received: March 31, 2017 Accepted: June 20, 2017 Published: June 21, 2017 7502

DOI: 10.1021/acs.analchem.7b01193 Anal. Chem. 2017, 89, 7502−7510

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Figure 1. Schematic representation of the principle of the DMP system for nucleic acid extraction. (A) Chemical structure of DMP (B) A plastic type microfluidic cartridge with a 3D disposable chip that was fabricated using a laser cutting machine for nucleic acid extraction. Schematic drawing for assembling of the 3D disposable chip. (C,D) Schematic work flows of the DMP system for RNA (C) and DNA (D) extraction. A mixture solution including lysis buffer, samples, and DMP is added to the 3D disposable chip to extract nucleic acids. The solution is incubated at either RT for 10−20 min (for RNA) or 56°C for 20 min (for DNA) to capture nucleic acids through DMP reagent on the amine-modified surface of the thin film, for the isolation and purification of the complex. Finally, the nucleic acids (RNA or DNA) are quickly washed and eluted. (C,D) A photograph of the working prototype of the DMP system for RNA (RNA-AMC [Asan Medical Center]) and DNA (DNA-AMC) extraction.

inexpensive method with dimethyl pimelimidate (DMP) for the extraction of nucleic acids (RNA and DNA) in combination with a microfluidic-based thin-film platform, to overcome the limitations of nucleic acid extraction from various samples using the existing methods. To the best of our knowledge, this approach is the first to employ dimethyl pimelimidate (DMP) as a nonchaotropic reagent to avoid the use of hazardous chemicals in the chaotropic assays (i.e., conventional nucleic acid extraction assays), and to set up an inexpensive microfluidic platform with thin-film material (created with a laser cutting machine) for nucleic acid extraction. DMPs are known to be cross-linking reagents that covalently bond with proteins and cells, but not with nucleic acids, through a reaction between two imidoester groups and the primary amine groups to form amidine.39 Moreover, in this study, DMP was used as a capturing reagent for nucleic acid (RNA and DNA) extraction from several sources (cancer cells, bacteria, and viruses). Furthermore, to overcome the limitations of conventional assays, such as the high risk of contamination, need for instruments and prepreparation steps, labor-intensive and timeconsuming nature, and difficulty of integration with the detection technique for POC testing,37,38 we coupled the DMP reagent with the microfluidic system using a thin-film platform to create a micro total analysis system (μTAS). The μTAS offers a completely closed system that minimizes the risk of sample contamination, and allows for the portability of the analytical method. Although the μTAS has been found to have these advantages, several complex steps remain unexplored, including the need for valves and pumps, bonding, and microchannel fabrication.40−42 The thin-film-based rapid prototyping method in the present study was used to create a

In order to overcome these constraints, most studies have focused on the improvement of detection technologies,8−13 and accordingly, advancements in detection technologies are being continuously made. In contrast, only few efforts have focused on the development of sample preparation (nucleic acid extraction) technologies,14−17 which are also a major requirement. Extraction of nucleic acids (DNA, RNA) is important not only for clinical diagnostics but also for life sciences research, such as for the identification of DNA biomarkers, assessment of DNA-based therapeutics, and understanding gene-disease relationships.18−22 Hence, the high quality and quantity of nucleic acids extracted from various human samples using innovative technologies would be desired for the improvement of the sensitivity and specificity of the diagnostics tools. Moreover, an ideal nucleic acid extraction technique should be useful for various sample volumes (one drop to microliter scale) and should be easy to integrate with detection techniques for developing point-of-care (POC) diagnostic devices. Based on the present status of the nucleic acid extraction techniques, the current methods, including conventional (liquid phase-based)23−26 and recently developed (solid phasebased)27−29 methods, for nucleic acid extraction are useful but have many limitations, including the degradation of nucleic acids by organic solvents;30−36 high risk of contamination; requirement of instruments including centrifuge, vacuum, and others; requirement of prepreparation steps including fabrication steps for filter/membrane, particles, and reagent coating; labor-intensive and time-consuming nature, including the sample handling steps; and unsuitable platform for POC testing including the difficulty of integration with the detection technique.37,38 Here, we report on the use of a simple and 7503

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(ABS) sheets. After mounting the chip on the lower plastic portion, they were assembled with the upper plastic part using four wrench bolts to build the system. Finally, in order to use the DMP (Figure 1A) as a nonchaotropic reagent with the thinfilm platform for nucleic acid extraction, the surface modification protocol was optimized. Briefly, in order to create an amine group at the inner surface of the 3D disposable chip, the surface was first treated with oxygen plasma (Covance Model, Femtoscience) for 3, 5, 7, and 10 min to change the surface properties of the inner film from being hydrophobic to hydrophilic, and immersed in a solution of 2% 3-aminopropyltriethoxysilane (APTES, Sigma-Aldrich) in H2O solution for 10−60 min at 65 °C, followed by thorough rinsing with deionized water. After silanization with APTES on the inner surface for 60 min at 65 °C, the surface hydrophilicity was increased (approximately 30−40 °C), as reported previously.45,46 At this point, the system (RNA-AMC [Asan Medical Center] for RNA and DNA-AMC for DNA) was prepared for the extraction of nucleic acids from various sources. This system can be stored at room temperature until use. To extract nucleic acids using the system, we optimized the assay solution. For an optimized reaction, a lysis buffer (100 mM Tris-HCl (pH 8.0), 10 mM ethylenediaminetetraacetic acid, 1% sodium dodecyl sulfate, and 10% Triton X-100) with either proteinase K for DNA or proteinase K and DNase I for RNA to break down unwanted proteins and DNAs was mixed with various concentrations (50, 100, 150, 200, and 250 mg/ mL) of DMP as the reaction solution. To conduct the reaction, 100−140 μL of sample from mammalian cells, bacteria, or blood plasma was mixed with 200 μL of the assay solution, and then injected into the system by the syringe with hands. For RNA extraction, the system was placed at room temperature for 10 min to extract RNA from the sources (Figure 1C). For DNA extraction, the system was placed in an incubator at 56 °C for 20 min to extract DNA from the sources (Figure 1D). The DMP can capture the nucleic acids through a complex on the surface. After washing with PBS at 50 μL/min of flow rate to remove the debris from the samples using syringe pump (KD Scientific, MA), an elution buffer (10 mM sodium bicarbonate, pH < 10.6, flow rate; 50 μL/min) was used to collect the nucleic acids that were extracted within a few minutes. The quantity and purity of the nucleic acids extracted were measured using the ratio of the optical densities of the samples at 260 and 280 nm, with Nano Drop (Thermo Fisher Scientific, Waltham, MA). The amount of protein in the sample was measured optical density using spectrophotometer at 595 nm after the mixing with Bradford reagent (Sigma-Aldrich, St. Louis, MO). To compare the system with a conventional nucleic acid extraction method, either QIAamp Viral RNA Mini Kit or QIAmp DNA mini kit was used according to the manufacturer’s protocol (Qiagen, Germany). Testing Samples. For testing the utility of the system with various samples, cancer cell lines (AGS [stomach, ATCC_CRL-1739], HCT116 [colorectal, ATCC_CCL-247], and MCF7 [breast, ATCC_HTB-22]) were maintained in plastic culture dishes with high-glucose Dulbecco’s Modified Eagle’s Medium (DMEM, Life Technology), supplemented with 10% fetal bovine serum (FBS), in a 37 °C humid incubator with 5% ambient CO2. After culturing, the cells were used for nucleic acid extraction with the system and Qiagen kit. For DNA extraction, the DNA was extracted from the cells using proteinase K to break down unwanted proteins and QIAmp DNA mini kit (Qiagen, Germany). End-point PCR and

closed single microfluidic channel system to extract nucleic acids from the samples without any contamination. Hence, this system is based on the combination of DMP for capturing nucleic acids and a thin-film-based microfluidic system as a platform for nucleic acid extraction in a single channel microfluidic platform without any instruments. This would lead to a major change in the sample processing techniques used in the clinical settings.

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EXPERIMENTAL SECTION Development and Operation of the System. A low-cost thin-film device for use as a microfluidic system (closed system) was fabricated with a CO2 laser-cutting machine (VLS3.50 (610 × 305 mm); Universal Laser Systems, Scottsdale, AZ) and comprised microfluidic chambers in a single microchannel in combination with DMP for nucleic acid extraction (Figure 1). In contrast to the Qiagen kit (open system), the microfluidic chamber of this system is based on a closed platform to prevent the contamination caused by an open platform. During the washing and elution steps, the reaction samples remain in the microfluidic chamber with the closed platform, which reduces contamination. The shape of the microfluidic chamber was designed to facilitate cell lysis and sample dispersion via inducing the passive micromixing during the nucleic acid extraction. Repeated sudden expansion and contraction in flow cross-sectional area can create microvortices at liquid sample injection.43,44 Thirty-five slot-type microwells were connected to each other with expansion and contraction ratios of 1:5.6 and 5.6:1, respectively, in the chamber to extract the nucleic acids (DNA or RNA) from the sources. The design of the microfluidic chip was first drawn using AutoCAD (Autodesk, Inc., San Rafael, CA) and then printed with a laser-cutting machine, which can be used for the fabrication of rapid prototyping devices with low cost, simplicity, and rapidity. In order to fabricate a 3D disposable chip with three layers (Figure 1B), the laser-cutting machine (10.6 μ CO2 laser sources ranging in power from 10 to 50 W) created the three layers comprising 300-μm-thick double-sided tape (Adhesive 300LSE9495LE, 3M, U.S.A.) as an inner layer and two 100-μm-thick thin films (Kemafoil hydrophilic film, HNW-100, COVEME, Italy) as outer layers (Figure 1B). The outer layers were attached to the permanent adhesive surfaces of the top and bottom of the inner layer (double-side tape) to generate a 3D disposable chip for the DMP reaction (Figure 1B, right). Accordingly, the chamber height was set to approximately 300 μm, and the total volume was 300 μL (Figure 1B). Moreover, to control the sample flow in the microchannel, a cast acrylic sheet (MARGA CIPTA, Indonesia) with 3 mm-thickness, attached to the double-sided tape on one side, was cut and drilled by the laser cutting machine. The fabricated adapters were attached to the inlet and outlet of the 3D disposable chip. Thereafter, precut Tygon tubing (AAC02548; Cole-Parmer, Vernon Hills, IL) was placed in the hole of the adapter and sealed using epoxy (EP-05, AXIA EPOXY GLUE, Japan), thermally stable at 120 °C (Figure 1C,D). Second, for easy handling of the thin-film platform for nucleic acid extraction, a plastic cartridge was fabricated using the laser cutting machine (Figure 1B). The plastic cartridge (upper and lower parts) held the 3D disposable chip during the assay; the dimensions were 105 mm long, 60 mm wide, and 10 mm high. The layout of each plastic component was first designed using AutoCAD (Figure 1B). A milling machine patterned the designed structures on transparent acrylonitrile butadiene styrene 7504

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Figure 2. Fundamental characterization of the DMP system for RNA and DNA extraction. (A) Recovery amounts of the input DNA with DMP. (B) Downstream analysis for DNA genetic testing with the Actin gene with the input DNA (I), without DMP group (N), with DMA (1) and with DMP (2), was performed using PCR. (C,D) The quantity (C) and purity (D) of the DNA extracted from the cells (HCT116, colorectal cancer cell line) using different concentration of DMP (100, 50, 20, and 10 mg/mL). (E) Downstream analysis for RNA genetic testing with the 18S gene, using RNAs extracted from the cancer cells, with the DMP system was performed using one-step qRT-PCR. (F) Downstream analysis for DNA genetic testing with the Actin gene, using DNAs extracted from cancer cells (1 × 106 cells) with the DMP system, was performed using real-time PCR. All error bars indicate standard deviation of the mean based on at least three independent experiments.

Instrument protocol (Roche Diagnostics). Five microliters of DNA was amplified in a total volume of 20 μL containing 10 μL of LightCycler 480 SYBR Green I master, 25 pmol of each primer, and DI water. Five microliters of RNA was amplified in a total volume of 20 μL containing 10 μL of QuantiTect SYBR Green RT-PCR master mix (Qiagen), 25pmol of each primer, QuantiTect RT mix (Qiagen), and RNase-Free water. An initial preincubation cycle of 50 °C for 30 min (only for RNA) and 95 °C for 10 min was followed by 50 cycles of 95 °C for 10 s, 55, 58, or 60 °C (for KRAS and 18s or Actin and E. coli or ST and SFTS) for 20 s, and 72 °C for 20 s, and by cooling step of 40 °C for 30 s. The amplified products with SYBR Green signals were carried out on a LightCycler 480 (Roche Diagnostics). Clinical Samples. To validate the ability of the system in human clinical samples, 140 μL of blood plasma samples from human disease patients were mixed with 200 μL of the assay solution, and then injected into the system for viral RNA and bacterial DNA extraction. The samples from the patients who had either severe fever with thrombocytopenia syndrome (SFTS, Huaiyangshan virus) for RNA or scrub typhus (ST, Orientia tsutsugamushi) for DNA were obtained using protocols approved by the institutional review board of AMC, Republic of Korea. Institutional approval and written informed consent from the patients were obtained. All experiments were performed in accordance with the relevant guidelines and regulations. End-point PCR and real-time PCR were performed to assess the quantity and purity of viral RNA and bacterial DNA for downstream analysis. The forward and reverse primers of several genes were synthesized at the usual length of approximately 24 bp (Table S1).

real-time PCR were performed to assess the quantity and purity of DNA for downstream analysis. For RNA extraction, RNA was extracted from cells using proteinase K, DNase I to break down unwanted proteins and DNAs, and the QIAmp Viral RNA mini kit (Qiagen, Germany). One-step end-point reverse transcript (RT)-PCR and one-step real-time reverse transcript (qRT)-PCR were performed to assess the quantity and purity of RNA for downstream analysis. The forward and reverse primers of several genes were synthesized at the usual length of approximately 24 bp (Table S1). Thereafter, we performed PCR-based DNA amplification by using extracted Escherichia coli DNA with the system. All the primers used for conventional PCR assays of E. coli are described in Table S1. E. coli (ATCC 25922) was inoculated in nutrient broth (NB) medium and incubated overnight at 37 °C under shaking conditions. We extracted genomic E. coli DNA from 100 μL of medium broth containing 103 to 108 colony formation units (CFUs)/mL. The PCR process consisted of an initial denaturation step at 50 °C for 30 min (only for RNA) and 95 °C for 15 min; 45 cycles of 95 °C for 30 s, 55, 58, or 60 °C (for KRAS and 18s or Actin and E. coli or ST and SFTS) for 30 s, and 72 °C for 30 s; and a final elongation step at 72 °C for 7 min. Five μL of DNA was amplified in a total volume of 25 μL containing 10× PCR buffer (Qiagen), 2.5 mM MgCl 2 , 0.25 mM deoxynucleotide triphosphate, 25 pmol of each primer, and 1 unit of Taq DNA polymerase (Qiagen). Five microliters of RNA was amplified in a total volume of 25 μL containing 5× OneStep RT-PCR buffer (Qiagen), 0.25 mM deoxynucleotide triphosphate, 25 pmol of each primer, and 1 unit of OneStep RT-PCR Enzyme mix (Qiagen). Gel electrophoresis was used to separate PCR products on a 2% agarose gel containing ethidium bromide (EtBr). The gel was visualized using a GelDoc System (Clinx Science Instruments). For the real-time PCR process, the following procedure is modified from LightCycler 480 7505

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Figure 3. Application of the DMP system for RNA extraction with cancer cell lines. (A) Capacity of the DMP system with HCT116 (colorectal cancer cell line) in concentrations ranging from 1 × 101 to 1 × 105 cells. The quantity of the RNA extracted from the cells was measured using Nano Drop. All error bars indicate the standard deviation of the mean based on at least three independent experiments. (B) Downstream analysis for RNA genetic testing with the 18S gene using the RNAs extracted from the cancer cells with the DMP system was performed using one-step end-point RTPCR. (L: DNA size marker; 1: RNA from 1 × 101 cells; 2: RNA from 1 × 102 cells; 3: RNA from 1 × 103 cells; 4: RNA from 1 × 104 cells; 5: RNA from 1 × 105 cells) (C) Downstream analysis for RNA genetic testing with the 18S gene, using RNAs extracted from HCT116 cancer cells in concentrations ranging from 1 × 101 to 1 × 106 cells either with DMP (black) or Qiagen (gray), was performed using one-step qRT-PCR. The experiment was repeated three times for each concentration, and only one curve for each concentration is shown.

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RESULTS AND DISCUSSION DMP System for Extraction of Nucleic Acids. The DMP in this study was first assessed as nonchaotropic reagents for the extraction of both RNA and DNA using a thin-film-based microfluidic platform (Figure 1). The chemical structure of DMP consists of methylene groups and bifunctional imidoester groups (Figure 1A). The use of the thin-film-based microfluidic platform involves sample lysis, washing, and elution in a single closed microchannel with a syringe pump only. To extract RNA and DNA from samples with the DMP system (Figure 1C,D), a mixed solution of sample, the lysis buffer, and DMP was added via pipetting into the system, which was previously activated with amine reactive groups on the surface to capture the complex of nucleic acids and DMP. Amino groups of DMP capture the nucleic acids by electrostatic adsorption due to the large numbers of negative charges on the nucleic acids. In addition, DMP reacts with amine groups of ends of the fragmented DNA, which contains a few base pairs of singlestranded DNA at each end of the fragment during lysis reaction, through covalent bonding. Therefore, DMP can capture the nucleic acid by both electrostatic interaction and covalent bonding. The system mixture was then incubated at room temperature (RT) for 10−20 min to extract RNA (Figure 1C) or at 56 °C for 20 min to extract DNA (Figure 1D) according to the Qiagen protocol; subsequently, the amino groups of the nucleic acids interact with the bifunctional groups of the DMP to form cross-links (i.e, covalent bonds) due to use of proteinase K and DNase I in the mixing solution for breaking down the unwanted proteins and DNAs. After the incubation, the purification step was completed by twice PBS washes to remove the debris and unbound molecules. Finally, the elution buffer was added to collect the extracted nucleic acids (RNA or DNA) by breaking the interaction between the complex and inner surface of the system that can be used for the downstream analysis of biomolecules. Characterization of DMP System. First, in order to investigate whether the DMP system can be useful for the extraction of nucleic acids (RNA and DNA), the fundamental characterization of the system was performed in cancer cells and bacterial cells. In Figure 2A, the recovery rate of the input DNA (1 μg of human genomic DNA) was measured with and without DMP. More than 95% of the DNA was recovered with DMP (black), and pH 10), which can be used as a means to capture and release by changing pH. We showed that the efficiency of the elution buffer (sodium bicarbonate (pH > 10)) is better than that of the buffer (pH < 10) (Figure S1C). In contrast to DNA extraction, RNA extraction is more difficult due to the easy degradation of RNA in all-environmental conditions. Nevertheless, the DMP system can be used for the extraction of RNA at the concentrations of cancer cells (1 × 103 and 1 × 106 cells/mL). For PCR compatible testing, we used the RNA extracted from the system with the concentrations for the amplification of the 18S gene, which serves as a good internal control due to the reduced variance in expression,47,48 with both one-step end-point reverse transcript (RT)-PCR and onestep real-time reverse-transcript (qRT)-PCR. We observed that the 18S gene was strongly amplified in both the 106 (CT: 17.86 ± 0.32) and 103 (CT: 31.60 ± 0.2) concentrations of the cells (Figure 2D). For the extraction of DNA using the DMP system, we also used the cancer cells (1 × 103 and 1 × 106 cells/mL) at different concentrations with DMP (range, 50− 250 mg/mL). For PCR compatible testing, we used the DNA extracted from the system at two concentrations for the amplification of the Actin gene with real-time PCR (Figure 2E, Figure S1D). We observed that the Actin gene was amplified at all concentrations of DMP with 1 × 106 cells (CT: 22.11 ± 0.31; Figure 2E) and 1 × 103 cells (CT: 31.73 ± 0.01; Figure 7506

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Figure 4. Application of the DMP system for DNA extraction with cancer cell lines. (A,B) Capacity of the DMP system with MCF7 (breast cancer cell line) cells for DNA extraction. The quantity (A) and purity (B) of the DNA extracted were measured using Nano Drop. (C) Capacity of the DMP system with HCT116 cells in concentrations ranging from 1 × 101 to 1 × 106 cells using real-time PCR. All error bars indicate the standard deviation of the mean based on at least three independent experiments. (D) Capacity of the DMP system with E. coli in concentrations ranging from 1 × 103 to 1 × 108 colony forming units by using real-time PCR. (Qiagen: white, DMP: black). All error bars indicate the standard deviation of the mean based on at least three independent experiments.

RNA extracted with the DMP system depends on the number of cells (Figure 3A). In order to assess the sufficient removal of PCR inhibitors in the RNAs extracted with the system for use in downstream analysis, we performed both conventional PCR assays for the analysis of the 18S gene (Figure 3B−C). The gene was found to be strongly amplified on one-step end-point RT-PCR of RNA extracted from the cell line (Figure 3B). Figure 3C indicates the dependence of the cycle threshold (CT value) in the one-step qRT-PCR on the concentrations of the HCT116 cells. Good linearity (R2 = 0.9907) between CT and the concentrations was observed. In addition, the RNA extracted from the system was comparably amplified with that obtained from the Qiagen kit (Figure 3C). To further evaluate the versatility of the DMP system as a DNA extraction assay, we tested the DNA extracted from the cancer cell lines using conventional PCR assays. To assess the critical parameters of the DMP assay for downstream analysis, the purity, quantity, and PCR compatibility of the DNA extracted from the system were tested. Figure 4A,B showed that the DNA extracted with DMP was sufficient for PCR. We also compared the system with the Qiagen kit using the same concentration of cells (1 × 106 cells/mL). The DNA extracted from the DMP system was comparably amplified with that obtained from the Qiagen kit in AGS and HCT116 cells (Figure S3A-B). Moreover, we evaluated the DMP system with sample solutions containing HCT116 cells in concentrations ranging from 1 × 101 to 1 × 106 cells in a volume of 100 μL. In real-time PCR, we observed that the DNA extracted from the DMP system (Figure 4C) was amplified gradually, depending on the serial dilution of the cells. In addition, to test the flexibility of the system in various sample sources, we extracted DNA from two bacteria (E. coli and Brucella) from 100 μL of medium broth containing 1 × 100 to 1 × 108 CFU/mL. We

S1C). Additionally, we assessed some issues such as reducing assay solution, use of battery, and long storage conditions for portable platform. According to the WHO (World Health Organization) guidelines, sample processing could be done with low-cost, fewer instruments, and no electricity in actual settings. We tested the quantity and amplification efficiency of the DNA extracted from the DMP system with the different amount of the assay solutions (Figure S2A-B). We showed that the quantity and the amplification efficiency of the DNA extracted from the system with 100 μL (ratio 1:1 of lysis buffer and DMP) of the assay solution was similar to that of the system with 200 μL of the assay solution, which reduces the cost of testing. Although the DMP system does not require the use of large instruments including centrifuges, vortexes, and others, the system involves the use of a syringe pump with electricity for optimal results. Therefore, we examined the proof of concept of the system without instruments (no electricity). In end-point PCR, we observed that the DNAs extracted from the DMP system without the syringe pump (no electricity) were also well amplified. Moreover, the efficiency of amplification by PCR in the no syringe pump group was similar to that in the syringe pump group (Figure S2C). Then, we performed storage testing to assess the duration for which the extracted nucleic acid can be stored at refrigerator for downstream analysis. We showed that the extracted nucleic acids from the DMP system could be used within 20 days under the storage conditions (at −20 °C) (Figure S2D). Application of DMP System in the Variety of Sample Sources. Thereafter, to further evaluate the versatility of the DMP system as an RNA extraction assay, we tested the RNA extracted from HCT116 (colorectal cancer cells) with serial dilutions ranging from 1 × 101 to 1 × 105 cells/mL, in a volume of 100 μL (Figure 3A−C). We observed that the quantity of the 7507

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Figure 5. Validation of the DMP system in clinical samples. (A) Bacterial DNA extraction from Orientia tsutsugamushi in the blood plasma of a scrub typhus (ST) patient. All error bars indicate the standard deviation of the mean based on at least three independent experiments. (B) Viral RNA extraction from Huaiyangshan virus in the blood plasma of a severe fever with thrombocytopenia syndrome (SFTS) patient. The colors represent the Qiagen kit (white), and DMP assay (black). (C) Amount of proteins in plasma samples before and after DMP. The proteins can be removed completely (99%) by washing step using DMP system.

observed that the E. coli gene was strongly amplified using the DNAs extracted from the serially diluted samples. The efficiency of the amplification was comparable for the extracted DNA from both the DMP system and the Qiagen kit (Figure 4D). The amplification efficiency of the DNA extracted from the DMP platform in the low concentration of Brucella were superior to that obtained from the Qiagen kit (Figure S3C). Moreover, we examined whether the point mutation of KRAS gene, which is a well-known biomarker in cancer,49,50 can be detected from the DNAs extracted through the system (Figure S4A). The DNAs from AGS cells, which carry the G12D substitution mutation with no codon 13 mutation, and HCT116 cells, which carry the G13D substitution mutation with no codon 12 mutation, were used as templates. To demonstrate that the purified DNA allows specific PCR analysis, two PCR amplicons identifying codon specific mutations in the KRAS gene of cancer cells were used and observed to correctly identify their specific targets (Figure S4A). Furthermore, in our previous report of DTS (dimethyl adipimidate/thin-film-based sample processing,45 we used DMA (dimethyl adipimidate) as a nonchaotropic reagent for the extraction of DNA only from the variety of sources. Thus, we compared the performance of the DNA extracted from both DTS and DMP methods to report the best possible method. The amplification efficiency of RT-PCR with the DMP assay was 2−3 cycles earlier as compared to that with the DTS (DMA) kit (Figure S4B). In Figure S4B, the amplification efficiency of the DNA extracted from the DMP system were superior to that obtained from the DTS (DMA) method. Validation of DMP System in Clinical Samples. Finally, we sought to determine whether the DMP system could be used to extract either viral or bacterial nucleic acids (DNA and RNA) from clinical samples. Blood plasma samples from ST and SFTS were used as the clinical samples in this study.51,52 Bacterial DNA was also extracted from Orientia tsutsugamushi in the blood plasma of ST patients using the Qiagen kit, and DMP. The amplification efficiency of one-step qRT-PCR for the extraction of bacterial DNA with the system was comparable with that with the Qiagen kit (Figure 5A). The viral RNA was extracted from Huaiyangshan virus in the blood plasma of SFTS patients using both the Qiagen kit and the DMP system. The amplification efficiency of RT-PCR with the

DMP assay was 2−3 cycles longer as compared to that with the Qiagen kit (Figure 5B). This might be optimized with further clinical study. Nevertheless, the system can completely removed (99%) the proteins (Figure 5C), which can be interfered with the covalent bonding between the amino groups of nucleic acids and DMP, in clinical samples. Therefore, the system can be useful for the extraction of the nucleic acids (RNA and DNA) from the clinical samples.

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CONCLUSION Our current findings indicate that the combination of a single channel microfluidic-based thin-film platform with DMP is a novel sample processing strategy that facilitates rapid and simple RNA and DNA extraction, which can be vital in genetic and epigenetic analysis in clinical applications. In contrast to the previous DTS method (Table 1), the developed system Table 1. Comparison of DMP and DTS system novelty types of nucleic acids types of sample sources

1

DNA

2 3 4 5

RNA cancer cells (culture) bacterial cells (culture) viral RNA from clinical sample (plasma) bacterial DNA from clinical samples (plasma)

6

DMP (current)

DTS (previous)

O O (single point mutations) O O O O

O X

O

X

X O O X

offers cost-effective sample processing option for nucleic acid extraction from clinical samples, eliminating the need for large experimental instrument (i.e., centrifuges, vortexer, and electrical devices) and laborious prepreparation (i.e., coating, bonding). The DMP system also provides a nonchaotropic alternative that can reduce the need for PCR inhibitors and solvents, and can permit simple integration with other detection techniques, which would be helpful for establishing a portable system. Finally, the quality and quantity of the nucleic acids extracted from the system are sufficient for use in downstream analysis of various sample sources (cancer cells, 7508

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bacteria, and viruses from blood plasma). Nevertheless, the system requires the optimization of the assay protocol with a large clinical cohort due to its noncommercialized status. Thus, the DMP system may cause a paradigm shift in the field of sample processing in clinical diagnostics. We believe that the DMP system could be crucial for sample processing in the life sciences research and could also have clinical applications.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.7b01193. Fundamental characterization of the DMP system; assay solution and storage days testing of the DMP system; application of the DMP system with cancer cell lines; mutation testing using the DMP system; and primer sequences (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +82-2-3010-4193. Fax: +82-2-3010-4193. ORCID

Yong Shin: 0000-0002-7426-4717 Author Contributions +

These authors (C.E.J. and T.Y.L.) contributed equally in this study

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (HI16C-0272-010016).

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REFERENCES

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DOI: 10.1021/acs.analchem.7b01193 Anal. Chem. 2017, 89, 7502−7510

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DOI: 10.1021/acs.analchem.7b01193 Anal. Chem. 2017, 89, 7502−7510