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Miniaturized polymerase chain reaction device for rapid identification of genetically modified organisms
Food Control 57 (2015) 238e245
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Miniaturized polymerase chain reaction device for rapid identification of genetically modified organisms Minh Luan Ha a, Nae Yoon Lee a, b, * a b
Department of BioNano Technology, Gachon University, 1342 Seongnam-daero, Sujeong-gu, Seongnam-si, Gyeonggi-do 461-701, Republic of Korea Gachon Medical Research Institute, Gil Medical Center, Inchon 405-760, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 January 2015 Received in revised form 6 April 2015 Accepted 8 April 2015 Available online 1 May 2015
In this study, a polycarbonate (PC) microdevice functioning as a reactor for the polymerase chain reaction (PCR) was fabricated for rapid identification of genetically modified organisms (GMOs). The PC microdevice was fabricated by first modifying its surface with an amine-functionalized alkoxysilane, namely bis[3-(trimethoxysilyl)propyl]amine (bis-TPA), to obtain a hydrophilic surface. Coating of bis-TPA on PC was realized by forming a urethane linkage between the amine terminals of the bis-TPA with the carbonate backbone of PC by aminolysis. This surface enabled the thermal bonding of two PC substrates at a relatively low temperature and atmospheric pressure, thereby maintaining the structure of the microchannel in high resolution. Next, the surface of the microchannel was further treated with a fluorosilane, namely tridecafluoro-(1,1,2,2-tetrahydrooctyl)-triethoxysilane (FTES), to obtain a hydrophobic surface inside the microchannel. This modification was realized by the hydrolysis and subsequent condensation of the alkoxy terminals of both bis-TPA and FTES to form a robust siloxane (SieOeSi) bond. The hydrophobic microchannel improved the PCR performance by stabilizing the fluid flow, particularly under heated conditions, when the flow-through PCR was conducted on a microdevice. Using the microdevice, the 35S promoter sequences and bar gene, which are commonly used for the identification of GMOs, were successfully amplified, resulting in the detection of 234 and 504 bp gene fragments for the 35S promoter sequences and 261 bp gene fragment for the bar gene from the genomic DNA extracted from the leaves of GM soybean. This process took approximately 30e35 min, which was approximately 3-fold faster than when using a conventional thermal cycler. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Genetically modified organism (GMO) Soybean Polycarbonate (PC) microdevice Surface hydrophilic/hydrophobic modification Flow-through polymerase chain reaction (PCR) 35S promoter bar gene
1. Introduction Genetically modified organisms (GMOs) are defined as organisms whose genetic materials have been altered by genetic engineering. The emergence of GMO products has brought numerous benefits such as the enhancement of crop productivity and the increase in nutritional content (Gachet, Martin, Vigneau, & Meyer, 1999). However, besides the above-mentioned advantages, GMOs also pose potential risks to human health (Dona & Arvanitoyannis, 2009). Currently, several methods are available for the identification of GMOs, such as protein-based and nucleotide-based amplification techniques, among which are enzyme-linked immunosorbent assay (ELISA), lateral flow strip, polymerase chain reaction (PCR), western blot analysis, as well as the detection of
* Corresponding author. Tel.: þ82 31 750 8556; fax: þ82 31 750 8774. E-mail addresses: [email protected] (M.L. Ha), [email protected] (N.Y. Lee). http://dx.doi.org/10.1016/j.foodcont.2015.04.014 0956-7135/© 2015 Elsevier Ltd. All rights reserved.
specific promoter and terminator sequences (Brett, Chambers, Huang, & Morgan, 1999; Dong et al., 2008; Liu et al., 2004; Miraglia et al., 2004; Permingeat, Reggiardo, & Vallejos, 2002; Rudi, Rud, & Holck, 2003; Ujhelyi et al., 2008; Xiao et al., 2012; Zhang & Guo, 2011). Among the aforementioned methods, nucleotide-based amplification by polymerase chain reaction (PCR) is the most commonly adopted method worldwide (Meyer, 1999; Pauli et al., 2001). Although PCR is one of the most timeconsuming and labor-intensive processes for genetic analyses, however, this method is indispensable for enhancing the sensitivity of target detection. It has been reported that a large number of GMOs share the same promoter of the 35S subunit of ribosomal RNA of cauliflower mosaic virus (P35S) (Lin, Chiang, & Shih, 2001; Liu, Xing, Shen, & Zhu, 2005), which can be used as a common marker for the detection of GMOs. Besides the 35S promoter sequences, bar gene, which is originally derived from the soil bacterium Streptomyces hygroscopicus and used to engineer herbicideresistant plants, was used to identify GMOs.
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With the development of microfluidic technology, many researchers have endeavored to miniaturize the PCR process (Chow, 2002; Felton, 2003; Stone & Kim, 2001). Among the many biological reactions with miniaturization potential, flow-through PCR on a miniaturized platform significantly facilitated the speed of DNA amplification owing to the high surface-to-volume ratio, smaller thermal capacity, and larger heat transfer rate realized inside the microchannel. Moreover, this technology can take advantage of a reduced use of sample and reagents, potential for parallel reactions, and enhanced portability. For this reason, many researchers have attempted to develop microfluidic PCR devices (Belgrader et al., 2003; Burns et al., 1996; Chen, Wang, Young, Chang, & Chen, 1999; Lagally, Emrich, & Mathies, 2001; Lagally, Medintz, & Mathies, 2001; Matsubara et al., 2005; Nakano et al., 1994; Yang et al., 2002), which have been applied in the fields of biology, chemistry, medicine, forensic science, food technology, and environmental science. Polycarbonate (PC) has been widely utilized as the material of choice for fabricating microdevices because of its high impact resistance, low moisture absorption, relatively high glass transition temperature (Tg), and optical transparency (Chen et al., 2005; Liu et al., 2001; Yang et al., 2002; Zhang, Xu, Ma, & Zheng, 2006). Among these advantages, the high Tg (~145 C) of PC, in particular, makes it suitable for withstanding the reaction temperatures as high as 95 C that are required for the denaturation of nucleic acids. In this study, we fabricated a PC microdevice to perform flowthrough PCR for rapid identification of GM soybeans. To facilitate the thermal bonding of two PC substrates without deformation or collapse of the microchannel, the PC surface was first hydrophilically modified. The hydrophilic modification of PC was easily realized by forming a urethane linkage employing amine-terminated chemicals because the amine functionality can react with the carbonate backbone of PC by aminolysis (Wu & Lee, 2014; Zhang, Trinh, Yoo, & Lee, 2014). Oxidation of the alkoxy terminals of the amine-terminated chemicals on both PC surfaces ensures bonding through the formation of robust siloxane bonds (SieOeSi) under relatively mild conditions such as atmospheric pressure and a temperature lower than the Tg of PC. After assembling two PC substrates, the hydrophilic PC surface inside the microchannel was further modified with a hydrophobic fluorosilane (Wildes et al., 1999). Hydrolysis and condensation between the alkoxy terminals of the fluorosilane and the hydrophilically modified PC enabled the hydrophobic modification of the microchannel. This hydrophobic coating of PC ensured stable fluid flow inside the microchannel under heated conditions (Jankowski, Ogonczyk, Kosinski, Lisowski, & Garstecki, 2011). This device was adopted to perform flow-through PCR on a microdevice for the detection of GM soybeans, paving the way for the development of a portable platform for the rapid identification of GM products. 2. Material and methods 2.1. Materials PCR reagents such as Taq polymerase, PCR buffer solution, and dNTPs were purchased from Promega. Bovine serum albumin (BSA; V fraction), bis[3-(trimethoxysilyl)propyl]amine (bis-TPA), and (tridecafluoro-1,1,2,2-tetrahydrooctyl)-triethoxysilane (FTES) were purchased from Sigma. TAE buffer (50) was purchased from Biosesang. A 100 bp DNA size marker was purchased from Takara and agarose powder was purchased from BioShop. Ethidium bromide (EtBr) (Loading STAR) was purchased from DyneBio. Poly(dimethylsiloxane) (PDMS) (Sylgard 184) and prepolymer were purchased from Dow Corning. An AccuPrep GMO DNA extraction kit was purchased from Bioneer for the extraction of genomic DNA
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from plants. A PC substrate with a thickness of 2 mm was purchased from Goodfellow. Ethanol (94%) and isopropyl alcohol (IPA) (99.5%) were purchased from Daejung Chemical & Metals. GM soybeans were kindly donated by the National Academy of Agricultural Science, Korea. 2.2. Microdevice design and fabrication Fig. 1A shows the concept for performing a rapid identification of GM soybeans using the PC microdevice having serpentine microchannel engraved using a computer numerical control (CNC) milling machine. The width, depth, and total length of the microchannel were designed to be 300 mm, 100 mm, and 210 cm, respectively, comprising 30 thermal cycles. The overall footprint of the microdevice was 50 40 mm. In this experiment, twotemperature PCR was performed by conducting annealing and extension at the same temperature (Nakayama et al., 2006; Sun, Yamaguchi, Ishida, Matsuo, & Misawa, 2002). The inlet and outlet ports (d ¼ 2 mm) were punched using a drilling machine. Silicone tubes (i.d. 1 mm, o.d. 2 mm) were inserted into the ports and were adhered to the substrate using PDMS glue. 2.3. Bonding strategy Fig. 1BeD shows the procedures for the hydrophilic and subsequent hydrophobic modifications of the PC surface and PCePC bonding following hydrophilic modification. In brief, the PC substrate was cleaned by sonication in water for 10 min, dipped into the IPA solution and shaken for 5 s at room temperature, and dried. Then, the PC surface was allowed to react with a 5% (v/v) bis-TPA solution in ethanol at room temperature for 1 min (Fig. 1B). Two PC substrates were embossed at 130 C under 0.1 MPa for 30 min (Fig. 1C). In this way, the PCePC assembly was realized under relatively mild conditions, i.e., using a lower temperature and pressure than typically applied in thermal bonding (Jang, Park, & czyk, Wegrzyn, Jankowski, Dabrowski, & Lee, 2014; Ogon Garstecki, 2010). After assembly, the microchannel was further treated with an ethanolic solution of FTES at room temperature for 1 h (Fig. 1D) (Jang, Park, & Lee, 2014). To examine the optimum condition for hydrophobic modification of the surface, the concentration of FTES were varied (1%, 3%, 5%, 10%, and 15%) (Jia, Fang, & Fang, 2004; Matinlinna, Areva, Lassila, & Vallittu, 2004). 2.4. Surface characterization 2.4.1. Contact angle measurement The water contact angles were measured by the sessile drop technique using a Phoenix 300 contact angle measuring system (Surface Electro Optics, Korea). The results were analyzed using the ImagePro 300 software. The measurements were performed five times and averaged. 2.4.2. FTIR analysis FTIR analyses were conducted using an FT/IR 4100 spectrometer (JASCO, Japan). Transmission data were acquired in the range of 4000e400 cm1 (100 scans at a 2 cm1 resolution). 2.5. Bond strength analysis The pull strength of the bonded PC assemblies (2 2 cm) was measured using a texture analyzer (QTS 25, Brookfield, Middleboro, MA). After coating both substrates with 5% bis-TPA, two PC substrates were partially assembled with an overlap length of 2 mm or an overlap area of 40 mm2. Bonding was performed at 130 C under 0.1 MPa for 30 min. Holes were punched on both PC substrates
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Fig. 1. (A) Overall concept for performing rapid identification of GM soybeans using flow-through PC microdevice having a serpentine microchannel. (B) Hydrophilic coating of PC through bis-TPA treatment. (C) PCePC bonding realized by embossing two bis-TPA-coated PC substrates at 130 C under 0.1 MPa. (D) Hydrophobic coating of PC microchannel through FTES treatment of the bis-TPA-coated PC surface.
using a drilling machine. Thick twine was inserted into the holes, and the bonded PC assemblies were pulled apart at a speed of 100 mm min1. The experiments were repeated five times to confirm bonding reproducibility. A leak test was performed by injecting an ink solution into the PC microchannel at flow rates of 0.45, 4.5, 45, and 90 mL min1 using a syringe pump (KDS 200, KD Scientific, New Hope, PA). 2.6. Temperature measurement The PC substrate was placed on two copper heating blocks. The temperature of one heating block was controlled at 95 C for denaturation, and that of the other heating block was controlled at 56 C for annealing/extension of the nucleic acid. The temperature was measured using an infrared (IR) camera (FLIR Thermovision A320). A black insulating tape was adhered to the top surface of the PC substrate for a precise measurement of the temperature. More than ten spots were randomly selected for measurement, and the temperatures were evaluated using an image analyzer (Thermal CAM Researcher 2.8). 2.7. Evaluation of the stability of sample flow inside a microchannel The PC microdevice was placed on two cooper heating blocks, whose temperatures were controlled at 95 C for denaturation and 56 C for annealing/extension of the nucleic acid. The sample was introduced into the microchannel using a syringe pump, and the flow rate was controlled at 2.5 mL min1 throughout the experiment. The flow stability of the sample inside the microchannel before and after the hydrophobic modification was recorded by measuring sample residence time in each thermal cycle. 2.8. DNA extraction DNA was extracted from the leaves of both GM and non-GM soybeans. First, the leaves were washed using 94% ethanol, and
then ground to a fine powder using liquid nitrogen. Then, the genomic DNA of the soybean was extracted using a plant genomic DNA extraction kit. The powder was incubated in 400 mL of plant lysis buffer to break the cellulose component of the cell wall. Subsequently, 20 mL of proteinase K was added into the tube, which was then incubated for 10 min at 60 C for protein precipitation. Afterward, the mixture was centrifuged at 12,000 rpm for 5 min, and the supernatant containing the extracted DNA was transferred to a new tube to precipitate the DNA using 100 mL of IPA. This mixture was passed through a binding column and the adsorbed DNA was thoroughly washed by centrifugation at 8,000 rpm, and then finally eluted using an elution buffer by centrifugation at 8,000 rpm for 1 min. The eluted DNA solution was stored at 4 C before use. 2.9. Flow-through PCR on a PC microdevice The microchannel formed on the PC microdevice was first coated with 1.5 mg mL1 BSA solution for 1.5 h at room temperature to prevent nonspecific adsorption of the PCR reagent on the wall of the microchannel. The PCR reagent (25 mL) contained the DNA template, the reaction buffer, 0.2 mM of dNTP mixture, 1.5 mg mL1 of BSA, 1 mM each of forward and reverse primers, and 0.25 U mL1 of Taq polymerase. BSA was also added to the sample solution to thoroughly passivate the inner wall of the microchannel as the sample moved through the microchannel. The genomic DNA of GM and non-GM soybeans were used as PCR templates. The primer sequences designed for the amplification of the 234 and 504 bp gene fragments within the 35S promoter were as follows: 50 ATC CCT TAC GTC AGT GGA GAT A 30 (forward) and 50 GCC CAG CTA TCT GTC ACT TTA T 30 (reverse). The primer sequences designed for the amplification of the 261 bp gene fragments within the bar gene were as follows: 50 ACT TCA GCA GGT GGG TGT A 30 (forward) and 50 ACC ACT ACA TCG AGA CAA GC 30 (reverse). For comparison, twotemperature PCR (Lopez & Prezioso, 2001; Schaerli et al., 2009; Wu, Kang, & Lee, 2011; Wu & Lee, 2011; Wu, Trinh, & Lee, 2012)
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Fig. 2. (A) Water contact angles measured on a pristine and a 5% bis-TPA-coated PC. (B) Water contact angles measured after further treatment with FTES for 1 h on the bis-TPAcoated PC.
was carried out using a thermal cycler (Bio-Rad C1000, Bio-Rad). The amplifications of both the 35S promoter sequences and the bar genes were carried out for 30 cycles, with denaturation performed at 95 C for 30 s and annealing/extension performed at 56 C for 30 s. The initial denaturation step was conducted at 95 C for 3 min and the final extension was carried out at 56 C for 5 min. The flow rate of the sample was controlled at 2.5 mL min1. The results were analyzed using agarose gel electrophoresis. The target amplicons were stained with EtBr and detected using a Gel Doc EZ system (Bio-Rad). 3. Results and discussion 3.1. Hydrophilic/hydrophobic modification of the PC surface Fig. 2 shows the results of the water contact angle measurements performed on a flat PC surface after hydrophilic and hydrophobic modifications. The water contact angle of pristine PC was measured to be 82.6 ± 0.6 ; this value decreased to 56.2 ± 0.4 after treatment with 5% bis-TPA (Fig. 2A). This result demonstrated that modification with bis-TPA makes the PC surface hydrophilic (Jang, Park, & Lee, 2014). After treatment with 5% bis-TPA, the PC surface was further treated with various concentrations (1%, 3%, 5%, 10%, and 15%) of FTES in ethanol, and the resulting water contact angles were shown in Fig. 2B. The water contact angles gradually increased with the increasing concentration of FTES up to a maximum water contact angle of 109.9 ± 1.2 for 5% FTES, which is in agreement with previous studies that report contact angle values of approximately 113 e123 for a surface coated with fluorinated alkoxysilane layers (Yang et al., 2002; Zhang & Xing, 2007). When the concentration of FTES increased further, however, the water contact angle of the substrate decreased slightly probably because of the self-aggregation of FTES in the solution. Based on this result, we selected the FTES concentration of 5% for subsequent experiments.
linkage appeared at an intensity of approximately 834 cm1 _ (Bloxham, Eicher-Lorka, Jakubenas, & Niaura, 2002). After treatment with 5% FTES on the surface coated with 5% bis-TPA, a new peak representative of CeF bonds appeared at 1378 cm1. 3.3. Bonding performance Fig. 4 shows the results of bond strength measurements when two PC substrates, both coated with 5% bis-TPA at room temperature for 1 min, were embossed at 130 C under 0.1 MPa for 30 min. The pull strength was measured to be approximately 529.7 kPa. This value was comparable to the previously reported bond czyk et al., 2010) for PC strengths of approximately 550 kPa (Ogon and 552 kPa (Shah et al., 2006) for PMMA, both obtained by solvent-assisted bonding. Fig. 4A shows the bonded PCePC assembly with an overlap length of 2 mm, equivalent to an overlap area of 40 mm2. Fig. 4B shows a photo taken at the moment of substrate detachment, and Fig. 4C shows a photo of the PC substrates after detachment. The yellow rectangles (in the web version) show the area of contact, which became opaque because of the chemical modification of the surface. The bond strength was further examined by performing a highthroughput leak test inside a microchannel with a total internal
3.2. FTIR analysis Fig. 3 shows the results of FTIR analyses performed on pristine PC, PC treated with 5% bis-TPA, and PC treated with 5% bis-TPA followed by 5% FTES. As shown in Fig. 3, the obtained spectrum of pristine PC sample displayed one peak with the intensity of 1725 cm1 which corresponded to the stretching vibration band of C]O bonds, and another peak corresponding to that of CeO bonds (from around 1320 to 1000 cm1) (Sinha, 2012). After treatment with 5% bis-TPA at room temperature, an additional peak representative of CeSi
Fig. 3. FTIR spectra of the PC surface before and after modification with hydrophilic (5% bis-TPA) and hydrophobic (FTES) coatings.
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Fig. 4. Bond strength analysis. (A) Experimental setup for performing the pull test. (B) A photo showing the moment of substrate detachment. (C) A photo showing the bonded area (40 mm2) after the detachment. (D) A photo showing the result of the leak test.
volume of approximately 45 mL. The flow rates of the ink injection were varied at 0.45, 4.5, 45, and 90 mL min1, which correspond to per-minute injection volumes of 10, 100, 1000, and 2000 times, respectively, of the total internal volume of the microchannel used. The results showed that the microdevice fabricated by the suggested bonding method could withstand extremely high rates of liquid introduction, up to per-minute injection volumes 2000 times larger than the total internal volume of the microchannel, without leakage (Fig. 4D). A movie clip demonstrating the leak test is shown in Movie S1 in the Supplementary Information. Supplementary video related to this article can be found at http://dx.doi.org/10.1016/j.foodcont.2015.04.014. 3.4. Stability of liquid flow inside a hydrophobic PC microchannel Fig. 5 shows the stability of the liquid flow inside the microchannel before and after tuning the wettability of the microchannel into hydrophobic. As shown in Fig. 5A, the flow rate of the PCR reagent became more stable upon hydrophobic modification of the microchannel, whereas it fluctuated irregularly after each cycle of the amplification process when the microchannel remained hydrophilic. It was previously reported that the flow rate of the PCR
reagent inside a microchannel notably affects the efficiency of PCR performance, particularly when flow-through PCR is conducted (Li, Xing, & Zhang, 2009). It was also reported that the slip velocity on hydrophobic surfaces results in a significant drag reduction in microchannel flows (Choi, Westin, & Breuer, 2003; Kim & Kim, 2002; Tretheway & Meinhart, 2002). In addition to obtaining a stable liquid flow, bubble formation was also suppressed inside the hydrophobic microchannel. Fig. 5B and C shows the photos of the microdevices having hydrophilic and hydrophobic serpentine microchannels, respectively. As shown in Fig. 5B, sample plug became disconnected under actual heated condition inside the hydrophilic microchannel, whereas a continuous sample plug was maintained inside the hydrophobic microchannel as shown in Fig. 5C. These results verify that hydrophobic treatment of microchannel is necessary to realize a stable liquid flow when performing flow-through PCR. 3.5. Amplification of GM soybean using a thermal cycler It is important to determine the initial DNA concentration that can be used to perform flow-through PCR, because the adsorption of biomolecules onto the inner surface of a microchannel can have a
Fig. 5. Liquid fluctuation phenomena demonstrated inside the PC microchannel under actual heated conditions before and after coating the bis-TPA-coated channel surfaces with FTES.
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Zhang, Xing, & Xu, 2007) inside the microchannel. Fig. 6B shows the relative intensity scales of the target amplicons obtained in Fig. 6A, analyzed using the Image Lab 4.0 software. 3.6. Temperature control and flow-through PCR inside the PC microdevice
Fig. 6. Results of DNA amplification when the concentrations of DNA templates were varied. (A) Results of agarose gel electrophoresis. Lanes 1e7 show target amplicons when the concentrations of DNA templates were 1.0, 0.7, 0.5, 0.4, 0.2, 0.1, and 0.08 ng mL1, respectively. Lane M shows the 100 bp DNA size marker. (B) Relative intensity scales of the target amplicons obtained in (A).
high possibility of inhibiting PCR amplification (Zhang, Xing, & Xu, 2007). Before performing flow-through PCR, we conducted an experiment to find the lowest concentration of the DNA template which can still give detectable amplicon, by conducting PCR using a thermal cycler. Fig. 6A shows the results of agarose gel electrophoresis after the amplification of 35S promoter sequences when the concentrations of the DNA templates were varied from 1.0 to 0.08 ng mL1. Although the lowest DNA concentration detected by agarose gel electrophoresis was 0.08 ng mL1, the suggested DNA concentration for the best performance of flow-through PCR was 0.5 ng mL1 owing to capillary adsorption (Li, Xing, & Zhang, 2009;
Fig. 7A shows the overall experimental setup for performing a flow-through PCR. As shown in Fig. 7A, a piece of PC was placed on two copper heating blocks, one for denaturation and the other for annealing/extension. A black insulating tape was adhered to the PC surface for a precise measurement of the temperature using an IR camera. The exact placement of the PC microdevice was decided based on the desired residence time in each temperature zone. The temperatures for denaturation and annealing/extension were controlled at 95 ± 0.5 C and 56 ± 0.3 C, respectively (Fig. 7B), for both 35S promoter sequences and bar gene. The homogeneity of the temperature distribution was measured by the coefficients of variation (CV), which were 0.6% (n ¼ 10) for denaturation and 0.3% (n ¼ 10) for annealing/extension, demonstrating that discrete temperatures were obtained for each copper heating block. Because the thermal conductivity of PC is low (0.19e0.22 W K1 m1), we can assume that the temperature inside the microchannel is almost identical to the measured surface temperature of PC because heat will not easily dissipate outside the PC microchannel. The surface temperature was stabilized within 10 min after heating, and a homogenous temperature was maintained over 1 h (data not shown), which is sufficient time to complete one PCR experiment. Fig. 7C shows the results of the flow-through PCR for the detection of 35S promoter sequences. Lanes 1 and 2 show the results of positive and negative control experiments, respectively, performed using a thermal cycler when GM and non-GM soybeans were used as templates. Lanes 3 and 4 show the results of positive and negative control experiments, respectively, performed using a PC microdevice when GM and non-GM soybeans were used as templates, demonstrating successful amplification of the 35S promoter sequences on chip. The total running time was approximately 30e35 min. Fig. 7D shows the results of the flow-through PCR for the detection of the bar gene. Lanes 1 and 2 show the results of positive and negative control experiments, respectively,
Fig. 7. (A) Experimental setup for performing flow-through PCR. (B) Temperature measurement of the PC microdevice obtained using an IR camera. (C) Results of the amplification of the 234 and 504 bp gene fragments from the 35S promoter sequences. Lanes 1 and 2 show the amplification results using a thermal cycler. Lanes 3 and 4 show the amplification results using the PC microdevice. (D) Results of the amplification of the 261 bp gene fragments from the bar gene. Lanes 1 and 2 show the amplification results using a thermal cycler. Lanes 3 and 4 show the amplification results using the PC microdevice. Lane M shows the 100 bp DNA size marker.
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performed using a thermal cycler when GM and non-GM soybeans were used as templates. Lanes 3 and 4 show the results of positive and negative control experiments, respectively, performed using a PC microdevice when GM and non-GM soybeans were used as templates, demonstrating successful amplification of the bar gene on chip. The modified hydrophobic microchannel surface stabilized the movement of the solution inside the microchannel, and significantly reduced air bubble generation under actual heated condition. These results prove the reliability of the PC microdevice for gene amplification and pave the way for utilizing the miniaturized platform for rapid identification of GM products. 4. Conclusions In this study, flow-through PCR was successfully demonstrated using PC microdevices for rapid identification of GM soybean, by amplifying 35S promoter sequences and bar gene. The time required for target gene amplification using a microdevice was less than 35 min, resulting in approximately 3-fold faster amplification than when a conventional thermal cycler was used. Hydrophilic modification of the PC surface facilitated device bonding under relatively mild conditions, and subsequent hydrophobic treatment of the microchannel after device assembly ensured a stable flow of the sample inside the microchannel, even under heated condition. The PC microdevice can become an inexpensive but powerful portable tool to realize the simple manipulation of one of the most time-consuming and labor-intensive laboratory operations, PCR, and could pave the way for the fast analysis of health-threatening GMOs on a miniaturized platform. Acknowledgments This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (NRF2014R1A1A3051319). This work was also supported by the Public Welfare & Safety Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (NRF-2012M3A2A1051681). References Belgrader, P., Elkin, C. J., Brown, S. B., Nasarabadi, S. N., Langlois, R. G., Milanovich, F. P., et al. (2003). A reusable flow-through polymerase chain reaction instrument for the continuous monitoring of infectious biological agents. Analytical Chemistry, 75, 3446e3450. _ Bloxham, S., Eicher-Lorka, O., Jakubenas, R., & Niaura, G. (2002). Surface-enhanced Raman spectroscopy of ethanethiol adsorbed at copper electrode. Chemija, 13, 185e189. Brett, G. M., Chambers, S. J., Huang, L., & Morgan, M. R. A. (1999). Design and development of immunoassays for detection of proteins. Food Control, 10, 401e406. Burns, M. A., Mastrangelo, C. H., Sammarco, T. S., Man, F. P., Webster, J. R., Johnson, B. N., et al. (1996). Microfabricated structures for integrated DNA analysis. Proceedings of the National Academy of Sciences of the United States of America, 93, 5556e5561. Chen, J., Wabuyele, M., Chen, H., Patterson, D., Hupert, M., Shadpour, H., et al. (2005). Electrokinetically synchronized polymerase chain reaction microchip fabricated in polycarbonate. Analytical Chemistry, 77, 658e666. Chen, Y.-H., Wang, W.-C., Young, K.-C., Chang, T.-T., & Chen, S.-H. (1999). Plastic microchip electrophoresis for analysis of PCR products of hepatitis C virus. Clinical Chemistry, 45, 1938e1943. Choi, C.-H., Westin, K. J. A., & Breuer, K. S. (2003). Apparent slip flows in hydrophilic and hydrophobic microchannels. Physics of Fluids, 15, 2897e2902. Chow, A. W. (2002). Lab-on-a-chip: opportunities for chemical engineering. American Institute of Chemical Engineers Journal, 48, 1590e1595. Dona, A., & Arvanitoyannis, I. S. (2009). Health risks of genetically modified foods. Critical Reviews in Food Science and Nutrition, 49, 164e175. Dong, W., Yang, L., Shen, K., Kim, B., Kleter, G. A., Marvin, H. J., et al. (2008). GMDD: a database of GMO detection methods. BMC Bioinformatics, 9, 260. Felton, M. J. (2003). Lab on a chip: poised on the brink. Analytical Chemistry, 75, 505Ae508A.
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Contents lists available at ScienceDirect
Food Control journal homepage: www.elsevier.com/locate/foodcont
Miniaturized polymerase chain reaction device for rapid identification of genetically modified organisms Minh Luan Ha a, Nae Yoon Lee a, b, * a b
Department of BioNano Technology, Gachon University, 1342 Seongnam-daero, Sujeong-gu, Seongnam-si, Gyeonggi-do 461-701, Republic of Korea Gachon Medical Research Institute, Gil Medical Center, Inchon 405-760, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 January 2015 Received in revised form 6 April 2015 Accepted 8 April 2015 Available online 1 May 2015
In this study, a polycarbonate (PC) microdevice functioning as a reactor for the polymerase chain reaction (PCR) was fabricated for rapid identification of genetically modified organisms (GMOs). The PC microdevice was fabricated by first modifying its surface with an amine-functionalized alkoxysilane, namely bis[3-(trimethoxysilyl)propyl]amine (bis-TPA), to obtain a hydrophilic surface. Coating of bis-TPA on PC was realized by forming a urethane linkage between the amine terminals of the bis-TPA with the carbonate backbone of PC by aminolysis. This surface enabled the thermal bonding of two PC substrates at a relatively low temperature and atmospheric pressure, thereby maintaining the structure of the microchannel in high resolution. Next, the surface of the microchannel was further treated with a fluorosilane, namely tridecafluoro-(1,1,2,2-tetrahydrooctyl)-triethoxysilane (FTES), to obtain a hydrophobic surface inside the microchannel. This modification was realized by the hydrolysis and subsequent condensation of the alkoxy terminals of both bis-TPA and FTES to form a robust siloxane (SieOeSi) bond. The hydrophobic microchannel improved the PCR performance by stabilizing the fluid flow, particularly under heated conditions, when the flow-through PCR was conducted on a microdevice. Using the microdevice, the 35S promoter sequences and bar gene, which are commonly used for the identification of GMOs, were successfully amplified, resulting in the detection of 234 and 504 bp gene fragments for the 35S promoter sequences and 261 bp gene fragment for the bar gene from the genomic DNA extracted from the leaves of GM soybean. This process took approximately 30e35 min, which was approximately 3-fold faster than when using a conventional thermal cycler. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Genetically modified organism (GMO) Soybean Polycarbonate (PC) microdevice Surface hydrophilic/hydrophobic modification Flow-through polymerase chain reaction (PCR) 35S promoter bar gene
1. Introduction Genetically modified organisms (GMOs) are defined as organisms whose genetic materials have been altered by genetic engineering. The emergence of GMO products has brought numerous benefits such as the enhancement of crop productivity and the increase in nutritional content (Gachet, Martin, Vigneau, & Meyer, 1999). However, besides the above-mentioned advantages, GMOs also pose potential risks to human health (Dona & Arvanitoyannis, 2009). Currently, several methods are available for the identification of GMOs, such as protein-based and nucleotide-based amplification techniques, among which are enzyme-linked immunosorbent assay (ELISA), lateral flow strip, polymerase chain reaction (PCR), western blot analysis, as well as the detection of
* Corresponding author. Tel.: þ82 31 750 8556; fax: þ82 31 750 8774. E-mail addresses: [email protected] (M.L. Ha), [email protected] (N.Y. Lee). http://dx.doi.org/10.1016/j.foodcont.2015.04.014 0956-7135/© 2015 Elsevier Ltd. All rights reserved.
specific promoter and terminator sequences (Brett, Chambers, Huang, & Morgan, 1999; Dong et al., 2008; Liu et al., 2004; Miraglia et al., 2004; Permingeat, Reggiardo, & Vallejos, 2002; Rudi, Rud, & Holck, 2003; Ujhelyi et al., 2008; Xiao et al., 2012; Zhang & Guo, 2011). Among the aforementioned methods, nucleotide-based amplification by polymerase chain reaction (PCR) is the most commonly adopted method worldwide (Meyer, 1999; Pauli et al., 2001). Although PCR is one of the most timeconsuming and labor-intensive processes for genetic analyses, however, this method is indispensable for enhancing the sensitivity of target detection. It has been reported that a large number of GMOs share the same promoter of the 35S subunit of ribosomal RNA of cauliflower mosaic virus (P35S) (Lin, Chiang, & Shih, 2001; Liu, Xing, Shen, & Zhu, 2005), which can be used as a common marker for the detection of GMOs. Besides the 35S promoter sequences, bar gene, which is originally derived from the soil bacterium Streptomyces hygroscopicus and used to engineer herbicideresistant plants, was used to identify GMOs.
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With the development of microfluidic technology, many researchers have endeavored to miniaturize the PCR process (Chow, 2002; Felton, 2003; Stone & Kim, 2001). Among the many biological reactions with miniaturization potential, flow-through PCR on a miniaturized platform significantly facilitated the speed of DNA amplification owing to the high surface-to-volume ratio, smaller thermal capacity, and larger heat transfer rate realized inside the microchannel. Moreover, this technology can take advantage of a reduced use of sample and reagents, potential for parallel reactions, and enhanced portability. For this reason, many researchers have attempted to develop microfluidic PCR devices (Belgrader et al., 2003; Burns et al., 1996; Chen, Wang, Young, Chang, & Chen, 1999; Lagally, Emrich, & Mathies, 2001; Lagally, Medintz, & Mathies, 2001; Matsubara et al., 2005; Nakano et al., 1994; Yang et al., 2002), which have been applied in the fields of biology, chemistry, medicine, forensic science, food technology, and environmental science. Polycarbonate (PC) has been widely utilized as the material of choice for fabricating microdevices because of its high impact resistance, low moisture absorption, relatively high glass transition temperature (Tg), and optical transparency (Chen et al., 2005; Liu et al., 2001; Yang et al., 2002; Zhang, Xu, Ma, & Zheng, 2006). Among these advantages, the high Tg (~145 C) of PC, in particular, makes it suitable for withstanding the reaction temperatures as high as 95 C that are required for the denaturation of nucleic acids. In this study, we fabricated a PC microdevice to perform flowthrough PCR for rapid identification of GM soybeans. To facilitate the thermal bonding of two PC substrates without deformation or collapse of the microchannel, the PC surface was first hydrophilically modified. The hydrophilic modification of PC was easily realized by forming a urethane linkage employing amine-terminated chemicals because the amine functionality can react with the carbonate backbone of PC by aminolysis (Wu & Lee, 2014; Zhang, Trinh, Yoo, & Lee, 2014). Oxidation of the alkoxy terminals of the amine-terminated chemicals on both PC surfaces ensures bonding through the formation of robust siloxane bonds (SieOeSi) under relatively mild conditions such as atmospheric pressure and a temperature lower than the Tg of PC. After assembling two PC substrates, the hydrophilic PC surface inside the microchannel was further modified with a hydrophobic fluorosilane (Wildes et al., 1999). Hydrolysis and condensation between the alkoxy terminals of the fluorosilane and the hydrophilically modified PC enabled the hydrophobic modification of the microchannel. This hydrophobic coating of PC ensured stable fluid flow inside the microchannel under heated conditions (Jankowski, Ogonczyk, Kosinski, Lisowski, & Garstecki, 2011). This device was adopted to perform flow-through PCR on a microdevice for the detection of GM soybeans, paving the way for the development of a portable platform for the rapid identification of GM products. 2. Material and methods 2.1. Materials PCR reagents such as Taq polymerase, PCR buffer solution, and dNTPs were purchased from Promega. Bovine serum albumin (BSA; V fraction), bis[3-(trimethoxysilyl)propyl]amine (bis-TPA), and (tridecafluoro-1,1,2,2-tetrahydrooctyl)-triethoxysilane (FTES) were purchased from Sigma. TAE buffer (50) was purchased from Biosesang. A 100 bp DNA size marker was purchased from Takara and agarose powder was purchased from BioShop. Ethidium bromide (EtBr) (Loading STAR) was purchased from DyneBio. Poly(dimethylsiloxane) (PDMS) (Sylgard 184) and prepolymer were purchased from Dow Corning. An AccuPrep GMO DNA extraction kit was purchased from Bioneer for the extraction of genomic DNA
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from plants. A PC substrate with a thickness of 2 mm was purchased from Goodfellow. Ethanol (94%) and isopropyl alcohol (IPA) (99.5%) were purchased from Daejung Chemical & Metals. GM soybeans were kindly donated by the National Academy of Agricultural Science, Korea. 2.2. Microdevice design and fabrication Fig. 1A shows the concept for performing a rapid identification of GM soybeans using the PC microdevice having serpentine microchannel engraved using a computer numerical control (CNC) milling machine. The width, depth, and total length of the microchannel were designed to be 300 mm, 100 mm, and 210 cm, respectively, comprising 30 thermal cycles. The overall footprint of the microdevice was 50 40 mm. In this experiment, twotemperature PCR was performed by conducting annealing and extension at the same temperature (Nakayama et al., 2006; Sun, Yamaguchi, Ishida, Matsuo, & Misawa, 2002). The inlet and outlet ports (d ¼ 2 mm) were punched using a drilling machine. Silicone tubes (i.d. 1 mm, o.d. 2 mm) were inserted into the ports and were adhered to the substrate using PDMS glue. 2.3. Bonding strategy Fig. 1BeD shows the procedures for the hydrophilic and subsequent hydrophobic modifications of the PC surface and PCePC bonding following hydrophilic modification. In brief, the PC substrate was cleaned by sonication in water for 10 min, dipped into the IPA solution and shaken for 5 s at room temperature, and dried. Then, the PC surface was allowed to react with a 5% (v/v) bis-TPA solution in ethanol at room temperature for 1 min (Fig. 1B). Two PC substrates were embossed at 130 C under 0.1 MPa for 30 min (Fig. 1C). In this way, the PCePC assembly was realized under relatively mild conditions, i.e., using a lower temperature and pressure than typically applied in thermal bonding (Jang, Park, & czyk, Wegrzyn, Jankowski, Dabrowski, & Lee, 2014; Ogon Garstecki, 2010). After assembly, the microchannel was further treated with an ethanolic solution of FTES at room temperature for 1 h (Fig. 1D) (Jang, Park, & Lee, 2014). To examine the optimum condition for hydrophobic modification of the surface, the concentration of FTES were varied (1%, 3%, 5%, 10%, and 15%) (Jia, Fang, & Fang, 2004; Matinlinna, Areva, Lassila, & Vallittu, 2004). 2.4. Surface characterization 2.4.1. Contact angle measurement The water contact angles were measured by the sessile drop technique using a Phoenix 300 contact angle measuring system (Surface Electro Optics, Korea). The results were analyzed using the ImagePro 300 software. The measurements were performed five times and averaged. 2.4.2. FTIR analysis FTIR analyses were conducted using an FT/IR 4100 spectrometer (JASCO, Japan). Transmission data were acquired in the range of 4000e400 cm1 (100 scans at a 2 cm1 resolution). 2.5. Bond strength analysis The pull strength of the bonded PC assemblies (2 2 cm) was measured using a texture analyzer (QTS 25, Brookfield, Middleboro, MA). After coating both substrates with 5% bis-TPA, two PC substrates were partially assembled with an overlap length of 2 mm or an overlap area of 40 mm2. Bonding was performed at 130 C under 0.1 MPa for 30 min. Holes were punched on both PC substrates
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Fig. 1. (A) Overall concept for performing rapid identification of GM soybeans using flow-through PC microdevice having a serpentine microchannel. (B) Hydrophilic coating of PC through bis-TPA treatment. (C) PCePC bonding realized by embossing two bis-TPA-coated PC substrates at 130 C under 0.1 MPa. (D) Hydrophobic coating of PC microchannel through FTES treatment of the bis-TPA-coated PC surface.
using a drilling machine. Thick twine was inserted into the holes, and the bonded PC assemblies were pulled apart at a speed of 100 mm min1. The experiments were repeated five times to confirm bonding reproducibility. A leak test was performed by injecting an ink solution into the PC microchannel at flow rates of 0.45, 4.5, 45, and 90 mL min1 using a syringe pump (KDS 200, KD Scientific, New Hope, PA). 2.6. Temperature measurement The PC substrate was placed on two copper heating blocks. The temperature of one heating block was controlled at 95 C for denaturation, and that of the other heating block was controlled at 56 C for annealing/extension of the nucleic acid. The temperature was measured using an infrared (IR) camera (FLIR Thermovision A320). A black insulating tape was adhered to the top surface of the PC substrate for a precise measurement of the temperature. More than ten spots were randomly selected for measurement, and the temperatures were evaluated using an image analyzer (Thermal CAM Researcher 2.8). 2.7. Evaluation of the stability of sample flow inside a microchannel The PC microdevice was placed on two cooper heating blocks, whose temperatures were controlled at 95 C for denaturation and 56 C for annealing/extension of the nucleic acid. The sample was introduced into the microchannel using a syringe pump, and the flow rate was controlled at 2.5 mL min1 throughout the experiment. The flow stability of the sample inside the microchannel before and after the hydrophobic modification was recorded by measuring sample residence time in each thermal cycle. 2.8. DNA extraction DNA was extracted from the leaves of both GM and non-GM soybeans. First, the leaves were washed using 94% ethanol, and
then ground to a fine powder using liquid nitrogen. Then, the genomic DNA of the soybean was extracted using a plant genomic DNA extraction kit. The powder was incubated in 400 mL of plant lysis buffer to break the cellulose component of the cell wall. Subsequently, 20 mL of proteinase K was added into the tube, which was then incubated for 10 min at 60 C for protein precipitation. Afterward, the mixture was centrifuged at 12,000 rpm for 5 min, and the supernatant containing the extracted DNA was transferred to a new tube to precipitate the DNA using 100 mL of IPA. This mixture was passed through a binding column and the adsorbed DNA was thoroughly washed by centrifugation at 8,000 rpm, and then finally eluted using an elution buffer by centrifugation at 8,000 rpm for 1 min. The eluted DNA solution was stored at 4 C before use. 2.9. Flow-through PCR on a PC microdevice The microchannel formed on the PC microdevice was first coated with 1.5 mg mL1 BSA solution for 1.5 h at room temperature to prevent nonspecific adsorption of the PCR reagent on the wall of the microchannel. The PCR reagent (25 mL) contained the DNA template, the reaction buffer, 0.2 mM of dNTP mixture, 1.5 mg mL1 of BSA, 1 mM each of forward and reverse primers, and 0.25 U mL1 of Taq polymerase. BSA was also added to the sample solution to thoroughly passivate the inner wall of the microchannel as the sample moved through the microchannel. The genomic DNA of GM and non-GM soybeans were used as PCR templates. The primer sequences designed for the amplification of the 234 and 504 bp gene fragments within the 35S promoter were as follows: 50 ATC CCT TAC GTC AGT GGA GAT A 30 (forward) and 50 GCC CAG CTA TCT GTC ACT TTA T 30 (reverse). The primer sequences designed for the amplification of the 261 bp gene fragments within the bar gene were as follows: 50 ACT TCA GCA GGT GGG TGT A 30 (forward) and 50 ACC ACT ACA TCG AGA CAA GC 30 (reverse). For comparison, twotemperature PCR (Lopez & Prezioso, 2001; Schaerli et al., 2009; Wu, Kang, & Lee, 2011; Wu & Lee, 2011; Wu, Trinh, & Lee, 2012)
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Fig. 2. (A) Water contact angles measured on a pristine and a 5% bis-TPA-coated PC. (B) Water contact angles measured after further treatment with FTES for 1 h on the bis-TPAcoated PC.
was carried out using a thermal cycler (Bio-Rad C1000, Bio-Rad). The amplifications of both the 35S promoter sequences and the bar genes were carried out for 30 cycles, with denaturation performed at 95 C for 30 s and annealing/extension performed at 56 C for 30 s. The initial denaturation step was conducted at 95 C for 3 min and the final extension was carried out at 56 C for 5 min. The flow rate of the sample was controlled at 2.5 mL min1. The results were analyzed using agarose gel electrophoresis. The target amplicons were stained with EtBr and detected using a Gel Doc EZ system (Bio-Rad). 3. Results and discussion 3.1. Hydrophilic/hydrophobic modification of the PC surface Fig. 2 shows the results of the water contact angle measurements performed on a flat PC surface after hydrophilic and hydrophobic modifications. The water contact angle of pristine PC was measured to be 82.6 ± 0.6 ; this value decreased to 56.2 ± 0.4 after treatment with 5% bis-TPA (Fig. 2A). This result demonstrated that modification with bis-TPA makes the PC surface hydrophilic (Jang, Park, & Lee, 2014). After treatment with 5% bis-TPA, the PC surface was further treated with various concentrations (1%, 3%, 5%, 10%, and 15%) of FTES in ethanol, and the resulting water contact angles were shown in Fig. 2B. The water contact angles gradually increased with the increasing concentration of FTES up to a maximum water contact angle of 109.9 ± 1.2 for 5% FTES, which is in agreement with previous studies that report contact angle values of approximately 113 e123 for a surface coated with fluorinated alkoxysilane layers (Yang et al., 2002; Zhang & Xing, 2007). When the concentration of FTES increased further, however, the water contact angle of the substrate decreased slightly probably because of the self-aggregation of FTES in the solution. Based on this result, we selected the FTES concentration of 5% for subsequent experiments.
linkage appeared at an intensity of approximately 834 cm1 _ (Bloxham, Eicher-Lorka, Jakubenas, & Niaura, 2002). After treatment with 5% FTES on the surface coated with 5% bis-TPA, a new peak representative of CeF bonds appeared at 1378 cm1. 3.3. Bonding performance Fig. 4 shows the results of bond strength measurements when two PC substrates, both coated with 5% bis-TPA at room temperature for 1 min, were embossed at 130 C under 0.1 MPa for 30 min. The pull strength was measured to be approximately 529.7 kPa. This value was comparable to the previously reported bond czyk et al., 2010) for PC strengths of approximately 550 kPa (Ogon and 552 kPa (Shah et al., 2006) for PMMA, both obtained by solvent-assisted bonding. Fig. 4A shows the bonded PCePC assembly with an overlap length of 2 mm, equivalent to an overlap area of 40 mm2. Fig. 4B shows a photo taken at the moment of substrate detachment, and Fig. 4C shows a photo of the PC substrates after detachment. The yellow rectangles (in the web version) show the area of contact, which became opaque because of the chemical modification of the surface. The bond strength was further examined by performing a highthroughput leak test inside a microchannel with a total internal
3.2. FTIR analysis Fig. 3 shows the results of FTIR analyses performed on pristine PC, PC treated with 5% bis-TPA, and PC treated with 5% bis-TPA followed by 5% FTES. As shown in Fig. 3, the obtained spectrum of pristine PC sample displayed one peak with the intensity of 1725 cm1 which corresponded to the stretching vibration band of C]O bonds, and another peak corresponding to that of CeO bonds (from around 1320 to 1000 cm1) (Sinha, 2012). After treatment with 5% bis-TPA at room temperature, an additional peak representative of CeSi
Fig. 3. FTIR spectra of the PC surface before and after modification with hydrophilic (5% bis-TPA) and hydrophobic (FTES) coatings.
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Fig. 4. Bond strength analysis. (A) Experimental setup for performing the pull test. (B) A photo showing the moment of substrate detachment. (C) A photo showing the bonded area (40 mm2) after the detachment. (D) A photo showing the result of the leak test.
volume of approximately 45 mL. The flow rates of the ink injection were varied at 0.45, 4.5, 45, and 90 mL min1, which correspond to per-minute injection volumes of 10, 100, 1000, and 2000 times, respectively, of the total internal volume of the microchannel used. The results showed that the microdevice fabricated by the suggested bonding method could withstand extremely high rates of liquid introduction, up to per-minute injection volumes 2000 times larger than the total internal volume of the microchannel, without leakage (Fig. 4D). A movie clip demonstrating the leak test is shown in Movie S1 in the Supplementary Information. Supplementary video related to this article can be found at http://dx.doi.org/10.1016/j.foodcont.2015.04.014. 3.4. Stability of liquid flow inside a hydrophobic PC microchannel Fig. 5 shows the stability of the liquid flow inside the microchannel before and after tuning the wettability of the microchannel into hydrophobic. As shown in Fig. 5A, the flow rate of the PCR reagent became more stable upon hydrophobic modification of the microchannel, whereas it fluctuated irregularly after each cycle of the amplification process when the microchannel remained hydrophilic. It was previously reported that the flow rate of the PCR
reagent inside a microchannel notably affects the efficiency of PCR performance, particularly when flow-through PCR is conducted (Li, Xing, & Zhang, 2009). It was also reported that the slip velocity on hydrophobic surfaces results in a significant drag reduction in microchannel flows (Choi, Westin, & Breuer, 2003; Kim & Kim, 2002; Tretheway & Meinhart, 2002). In addition to obtaining a stable liquid flow, bubble formation was also suppressed inside the hydrophobic microchannel. Fig. 5B and C shows the photos of the microdevices having hydrophilic and hydrophobic serpentine microchannels, respectively. As shown in Fig. 5B, sample plug became disconnected under actual heated condition inside the hydrophilic microchannel, whereas a continuous sample plug was maintained inside the hydrophobic microchannel as shown in Fig. 5C. These results verify that hydrophobic treatment of microchannel is necessary to realize a stable liquid flow when performing flow-through PCR. 3.5. Amplification of GM soybean using a thermal cycler It is important to determine the initial DNA concentration that can be used to perform flow-through PCR, because the adsorption of biomolecules onto the inner surface of a microchannel can have a
Fig. 5. Liquid fluctuation phenomena demonstrated inside the PC microchannel under actual heated conditions before and after coating the bis-TPA-coated channel surfaces with FTES.
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Zhang, Xing, & Xu, 2007) inside the microchannel. Fig. 6B shows the relative intensity scales of the target amplicons obtained in Fig. 6A, analyzed using the Image Lab 4.0 software. 3.6. Temperature control and flow-through PCR inside the PC microdevice
Fig. 6. Results of DNA amplification when the concentrations of DNA templates were varied. (A) Results of agarose gel electrophoresis. Lanes 1e7 show target amplicons when the concentrations of DNA templates were 1.0, 0.7, 0.5, 0.4, 0.2, 0.1, and 0.08 ng mL1, respectively. Lane M shows the 100 bp DNA size marker. (B) Relative intensity scales of the target amplicons obtained in (A).
high possibility of inhibiting PCR amplification (Zhang, Xing, & Xu, 2007). Before performing flow-through PCR, we conducted an experiment to find the lowest concentration of the DNA template which can still give detectable amplicon, by conducting PCR using a thermal cycler. Fig. 6A shows the results of agarose gel electrophoresis after the amplification of 35S promoter sequences when the concentrations of the DNA templates were varied from 1.0 to 0.08 ng mL1. Although the lowest DNA concentration detected by agarose gel electrophoresis was 0.08 ng mL1, the suggested DNA concentration for the best performance of flow-through PCR was 0.5 ng mL1 owing to capillary adsorption (Li, Xing, & Zhang, 2009;
Fig. 7A shows the overall experimental setup for performing a flow-through PCR. As shown in Fig. 7A, a piece of PC was placed on two copper heating blocks, one for denaturation and the other for annealing/extension. A black insulating tape was adhered to the PC surface for a precise measurement of the temperature using an IR camera. The exact placement of the PC microdevice was decided based on the desired residence time in each temperature zone. The temperatures for denaturation and annealing/extension were controlled at 95 ± 0.5 C and 56 ± 0.3 C, respectively (Fig. 7B), for both 35S promoter sequences and bar gene. The homogeneity of the temperature distribution was measured by the coefficients of variation (CV), which were 0.6% (n ¼ 10) for denaturation and 0.3% (n ¼ 10) for annealing/extension, demonstrating that discrete temperatures were obtained for each copper heating block. Because the thermal conductivity of PC is low (0.19e0.22 W K1 m1), we can assume that the temperature inside the microchannel is almost identical to the measured surface temperature of PC because heat will not easily dissipate outside the PC microchannel. The surface temperature was stabilized within 10 min after heating, and a homogenous temperature was maintained over 1 h (data not shown), which is sufficient time to complete one PCR experiment. Fig. 7C shows the results of the flow-through PCR for the detection of 35S promoter sequences. Lanes 1 and 2 show the results of positive and negative control experiments, respectively, performed using a thermal cycler when GM and non-GM soybeans were used as templates. Lanes 3 and 4 show the results of positive and negative control experiments, respectively, performed using a PC microdevice when GM and non-GM soybeans were used as templates, demonstrating successful amplification of the 35S promoter sequences on chip. The total running time was approximately 30e35 min. Fig. 7D shows the results of the flow-through PCR for the detection of the bar gene. Lanes 1 and 2 show the results of positive and negative control experiments, respectively,
Fig. 7. (A) Experimental setup for performing flow-through PCR. (B) Temperature measurement of the PC microdevice obtained using an IR camera. (C) Results of the amplification of the 234 and 504 bp gene fragments from the 35S promoter sequences. Lanes 1 and 2 show the amplification results using a thermal cycler. Lanes 3 and 4 show the amplification results using the PC microdevice. (D) Results of the amplification of the 261 bp gene fragments from the bar gene. Lanes 1 and 2 show the amplification results using a thermal cycler. Lanes 3 and 4 show the amplification results using the PC microdevice. Lane M shows the 100 bp DNA size marker.
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performed using a thermal cycler when GM and non-GM soybeans were used as templates. Lanes 3 and 4 show the results of positive and negative control experiments, respectively, performed using a PC microdevice when GM and non-GM soybeans were used as templates, demonstrating successful amplification of the bar gene on chip. The modified hydrophobic microchannel surface stabilized the movement of the solution inside the microchannel, and significantly reduced air bubble generation under actual heated condition. These results prove the reliability of the PC microdevice for gene amplification and pave the way for utilizing the miniaturized platform for rapid identification of GM products. 4. Conclusions In this study, flow-through PCR was successfully demonstrated using PC microdevices for rapid identification of GM soybean, by amplifying 35S promoter sequences and bar gene. The time required for target gene amplification using a microdevice was less than 35 min, resulting in approximately 3-fold faster amplification than when a conventional thermal cycler was used. Hydrophilic modification of the PC surface facilitated device bonding under relatively mild conditions, and subsequent hydrophobic treatment of the microchannel after device assembly ensured a stable flow of the sample inside the microchannel, even under heated condition. The PC microdevice can become an inexpensive but powerful portable tool to realize the simple manipulation of one of the most time-consuming and labor-intensive laboratory operations, PCR, and could pave the way for the fast analysis of health-threatening GMOs on a miniaturized platform. Acknowledgments This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (NRF2014R1A1A3051319). This work was also supported by the Public Welfare & Safety Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (NRF-2012M3A2A1051681). References Belgrader, P., Elkin, C. J., Brown, S. B., Nasarabadi, S. N., Langlois, R. G., Milanovich, F. P., et al. (2003). A reusable flow-through polymerase chain reaction instrument for the continuous monitoring of infectious biological agents. Analytical Chemistry, 75, 3446e3450. _ Bloxham, S., Eicher-Lorka, O., Jakubenas, R., & Niaura, G. (2002). Surface-enhanced Raman spectroscopy of ethanethiol adsorbed at copper electrode. Chemija, 13, 185e189. Brett, G. M., Chambers, S. J., Huang, L., & Morgan, M. R. A. (1999). Design and development of immunoassays for detection of proteins. Food Control, 10, 401e406. Burns, M. A., Mastrangelo, C. H., Sammarco, T. S., Man, F. P., Webster, J. R., Johnson, B. N., et al. (1996). Microfabricated structures for integrated DNA analysis. Proceedings of the National Academy of Sciences of the United States of America, 93, 5556e5561. Chen, J., Wabuyele, M., Chen, H., Patterson, D., Hupert, M., Shadpour, H., et al. (2005). Electrokinetically synchronized polymerase chain reaction microchip fabricated in polycarbonate. Analytical Chemistry, 77, 658e666. Chen, Y.-H., Wang, W.-C., Young, K.-C., Chang, T.-T., & Chen, S.-H. (1999). Plastic microchip electrophoresis for analysis of PCR products of hepatitis C virus. Clinical Chemistry, 45, 1938e1943. Choi, C.-H., Westin, K. J. A., & Breuer, K. S. (2003). Apparent slip flows in hydrophilic and hydrophobic microchannels. Physics of Fluids, 15, 2897e2902. Chow, A. W. (2002). Lab-on-a-chip: opportunities for chemical engineering. American Institute of Chemical Engineers Journal, 48, 1590e1595. Dona, A., & Arvanitoyannis, I. S. (2009). Health risks of genetically modified foods. Critical Reviews in Food Science and Nutrition, 49, 164e175. Dong, W., Yang, L., Shen, K., Kim, B., Kleter, G. A., Marvin, H. J., et al. (2008). GMDD: a database of GMO detection methods. BMC Bioinformatics, 9, 260. Felton, M. J. (2003). Lab on a chip: poised on the brink. Analytical Chemistry, 75, 505Ae508A.
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