Peanut Detection Using Droplet Microfluidic Polymerase Chain Reaction Device

Department of Chemical Engineering, National United University, Miaoli, Taiwan Ph.D. Program in Tissue Engineering and Regenerative Medicine, National Chung Hsing University, Taichung, Taiwan Department of Food Nutrition and Health Biotechnology, Asia University, Taichung, Taiwan Institute of Biomedical Engineering and Nanomedicine, National Health Research Institutes, Miaoli, Taiwan Department of Biomechatronics Engineering, National Pingtung University of Science and Technology, Pingtung, Taiwan Department of Mechanical Engineering, National United University, Miaoli, Taiwan


Introduction
Food allergy is a critical public health problem affecting children and adults [1].Allergy to peanuts is one of the most common food allergies [1][2][3].Most allergic reactions to peanuts are immunoglobulin E-mediated reactions that may lead to serious anaphylaxis [4].Because of the risk of severe allergic reactions and possible death and the lack of effective treatments for food allergy [1,5], the best method for food allergy amelioration is strict avoidance of the offending foods [6].
Compared with macroscopic equivalents in polymerase chain reaction (PCR) systems, the microfluidic PCR device has several advantages, such as reduced sample and reagent consumption; these advantages enable inexpensive system operation and facilitate small thermal mass, low thermal inertia, and rapid heat transfer, improving the efficiency of PCR amplification [7].Microfluidic PCR devices have attracted substantial attention [8,9] and are widely used in various domains, such as biology, chemistry, medicine, forensic science, food technology, and environmental science [10].Microfluidic PCR devices can be classified into two types, namely, single-phase microfluidic PCR and droplet microfluidic PCR.Droplet microfluidic systems using two immiscible fluids have emerged as a promising tool.These systems reduce analysis times, improve sensitivity, lower detection limits, increase high-throughput screening, and enhance operational flexibility [11][12][13].Compared with single-phase microfluidic PCR, droplet microfluidic PCR exhibits greater ability to prevent biological or chemical adsorption on microchannel surfaces, which otherwise causes microfluidic PCR inhibition and carryover contamination [14].Several studies of on-chip integration for droplet microfluidic PCR have been well reviewed [14].
Pan et al. [15] integrated multichamber PCR and multichannel separation for parallel genetic analysis to detect the hepatitis B virus, Mycobacterium tuberculosis, and genotype the human leucocyte antigen.Wang et al. [16] developed an oscillatory-flow multiplex PCR in a capillary microfluidic channel for simultaneous determination of Salmonella enterica, Escherichia coli O157:H7, and Listeria monocytogenes.Cai et al. [17] used a microfluidic system that integrated dielectrophoresis with chip-based multiplex array PCR for the rapid identification of pathogens including Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli O157:H7 in complex physiological matrices.Tachibana et al. [18] developed an autonomous disposable plastic microfluidic PCR chip controlled through the capillary flow of the reagent in the microchannel for the quantitative detection of Escherichia coli.Liu et al. [19] introduced a PCR platform that integrated a bottom-well microfluidic chip with an infrared excited temperature control method and fluorescence codetection of three PCR products for human papilloma virus.Jeong et al. [20] developed a disposable PCR chip from a polymeric film to reduce thermal mass, and Escherichia coli genomic DNA was amplified.
To date, most microfluidic PCR devices have been developed for pathogen detection.However, microfluidic PCR devices have rarely been used for foodborne allergen detection, including detection of the DNA fragments of peanut species.Therefore, this study is aimed at developing a droplet

Chip Design.
A droplet microfluidic PCR chip with a cross-junction microchannel was constructed from a glass slide substrate (length/width/depth: 76 × 52 × 1 mm 3 ).The droplet microfluidic PCR chip was composed of two parts, namely, the upstream cross-junction microchannel and the downstream serpentine microchannel.As shown in Figure 1, the cross-junction microchannel was used for droplet formation and serpentine microchannel for droplet transportation at three temperatures.The middle channel and the two side channels were used at the cross-junction microchannel to inject dispersed and continuous phases, respectively.The cross-junction width was 200 μm, and the other microchannel was 300 μm.Thirty-five repeating cycles  3 Journal of Sensors took place in the serpentine microchannel, and the total length of the microchannel in the droplet microfluidic PCR chip was 261.7 cm.

Microfluidic Chip Fabrication.
The SU-8 microfluidic chip mold was fabricated using photolithography (Figure 2).The negative photoresist SU-8 50 was spin coated onto a 4-inch silicon wafer at 1930 rpm for 30 s and soft baked at 95 °C for 22 min.A mask was applied on the aligner for 11.06 s (365 mJ/cm 2 ) of ultraviolet exposure, and the pattern was transferred to SU-8 50.Postexposure baking was performed at 65 °C for 1 min and again at 95 °C for 6 min.The unexposed SU-8 50 was dissolved through treatment with SU-8 developer solution for 6.8 min.Then, the SU-8 master mold was rinsed in isopropyl alcohol and hard-baked at 150 °C to complete the cross-linking.Thereafter, the PDMS prepolymer and curing agent were mixed in a weight ratio of 10 : 1 (w/w) and degassed.The PDMS mixture was poured into the fabricated SU-8 master mold.After thermal curing at 65 °C for 2 h, the PDMS replica was detached from the SU-8 master mold, and the ports were punched for the sample input and output.Finally, the PDMS replica and glass substrate were bonded through oxygen plasma treatment of the microfluidic PCR chip.
2.4.Temperature-Controlled Heating Platform.Figure 3 depicts the designed heater assembly.The heating platform used Bakelite (210 mm × 297 mm × 40 mm) as the platform substrate and a 0.1 mm glass fiber board as a heat-insulating layer to reduce heat conduction and heat convection interference in each heater.Two flat electrothermal aluminum plate heaters (60 mm × 42 mm × 4 mm) were used for DNA denaturation (95 °C) and primer annealing (60 °C).A third electrothermal aluminum block heater (40 mm × 30 mm × 8 mm) with an adjustable height was used for DNA extension (70 °C).Aluminum blocks (40 mm × 25 mm × 9 5 mm) were used as the heat transfer medium.A thermocouple was inserted into the blocks for temperature measurement.An iron fixture and a bottom heat-resistant sponge were used to maintain complete contact between the droplet microfluidic PCR chip and the heating platform.The temperaturecontrolled feedback system consisted of a proportionalintegral-derivative controller (Mac 10D, Shimax, Akita, Journal of Sensors Japan), and a solid state relay (KD20C25AX, Kytech, Taoyuan, Taiwan) was used to maintain stable temperatures for three heating regions in the PCR.
2.5.On-Chip PCR.The chip was baked in an oven at 100 °C for 8 h to render the PDMS microchannel surface hydrophobic [21].Subsequently, the Sigmacote surfactant was introduced into the entire microchannel to form a hydrophobic film on the microchannel surface.The PCR adhesive sealing film (Microseal B, Bio-Rad, Hercules, CA, USA) was applied on the PDMS surface of the microfluidic chip to prevent evaporation of PCR mixture reagents.For strong contact, a thermal conductive sheet was placed between the microfluidic chip and the heating platform.
To demonstrate the performance of the droplet microfluidic PCR device, a DNA template of peanut species was used for amplification by the PCR.In addition, this chip was verified by DNA of sesame, Salmonella spp., and Staphylococcus aureus, with the specific target genes denoted in Table 1.PCR mixture for DNA amplification contained 2.5 mM dNTPs, 2 U ProTaq DNA polymerase in the Pro-Zyme PCR buffer (10x buffer), 10 μM of each primer, and the template DNA.The PCR mixture reagents and mineral oil were simultaneously injected into the microfluidic chip from the dispersed phase inlet and continuous phase inlet through two independent syringe pumps (NE-300, New Era Pump Systems Inc., USA).Finally, the amplified product was collected in an Eppendorf tube from the outlet, and the amplification product and mineral oil were separated using a microcentrifuge.The amplified product in the bottom layer of the Eppendorf tube was drawn and analyzed on a 2% agarose gel using electrophoresis (Mupid-2 mini gel electrophoresis system, Cosmo Bio, Tokyo, Japan).

Results and Discussion
3.1.Temperature Measurement.Figure 4 illustrates the temperature distribution of the glass substrate in the droplet microfluidic PCR chip placed on the heating platform through infrared thermography (TAS-G100EXD, NEC, Japan).Results indicated that the temperature distribution was divided into three uniform temperature regions, corresponding to DNA denaturation at 95 °C on the right side, extension at 70 °C in the middle region, and primer annealing at 60 °C on the left side.Furthermore, the temperature in the microchannel of the microfluidic chip was tested using a thermochromic pigment that exhibited a color change from black to colorless at temperatures above 70 °C.As shown in Figure 5, the thermochromic pigment in the microchannel was transparent on the right side for DNA denaturation, gray in the middle region for extension, and black on the left side for primer annealing.These results confirmed that a homogeneous temperature distribution was achieved for the PCR thermal conditions for DNA denaturation, primer annealing, and extension.

Evaporation of Water in the Microfluidic
Chip.An evaporation experiment was conducted to determine the degree of droplet microfluidic reducing evaporation in comparison with that in single-phase microfluidic PCR.Under identical temperature gradient conditions in PCR, the collected output flow from the droplet microfluidic chip was measured using a five-digit electronic balance for three cases, namely, water, mineral oil, and emulsion droplet (water droplet in mineral oil).For comparison, the flow rates of the dispersed and continuous phases in the emulsion droplet case were set identical to the water and mineral oil cases.Table 2 summarizes the experimental conditions and results.The regressed mass flow rates of water (case A), mineral oil (case B), and emulsion droplet (case C) in the microfluidic chip were 1.41, 1.50, and 3.02 mg/min, respectively.Assuming that no evaporation occurred for mineral oil, the mass flow rate of the water in the emulsion droplet was evaluated by subtracting the mass flow rate in case B from the mass flow rate in case C. The net mass flow rate of water in the emulsion droplet was 1.52 mg/min, which was higher than 1.41 mg/min in case A. This 0.11 mg/min difference (7.24%) was the evaporation reduction of water in droplet microfluidic PCR compared 3.3.DNA Amplification.Figure 6 illustrates droplet formation at the cross-junction microchannel photographed using high-speed digital cameras (Integrated Design Tools, Pasadena CA, USA) mounted on an inverted microscope (Nikon Eclipse Ti-E, Nikon, Kobe, Japan).The volumetric flow rate of the mineral oil was 2.500 μL/min, and that of PCR mixture reagents was 0.750 μL/min.Results indicated that the symmetric shear force created by the mineral oil flow gradually necked the flow of PCR mixture reagents at the crossjunction and then formed a spherical emulsion droplet in the broadened downstream microchannel as a result of interfacial tensions.The emulsion droplet moved toward the downstream serpentine microchannel for PCR. Figure 7(a) depicts the gel electrophoresis results for peanut products.The white pixels comprising the target band of the PCR product from the droplet microfluidic PCR chip was larger than the commercial PCR thermal cycler (GeneAmp PCR System 2720, Perkin Elmer, Branchburg, NJ, USA), and the peanut sample template DNA had no signal before PCR.These specific target bands were further analyzed using ImageJ software to quantify the average fluorescence intensity of white pixels (Figure 7(b)).As indicated in Figure 7(b), the fluorescence intensity of the PCR product from the droplet microfluidic PCR chip was approximately two times higher than the intensity from the commercial PCR thermal cycler.The higher fluorescence signal from the droplet microfluidic PCR chip may be attributed to the concentration of the PCR products, which in turn was a consequence of the small evaporation of flow solution.
In addition, a SYBR green reagent-based PCR was conducted in the droplet microfluidic PCR chip.The fluorescence intensity of the PCR product of peanut template DNA was determined using a previously developed compact fluorescent system with a fiber-coupled light-emitting diode (LED) [24].In this detection system, a 12 μL sample solution was excited using a 470 nm LED, and fluorescence with a wavelength between 505 and 545 nm was detected.Results revealed that the fluorescence signal of the environment background was 13.13 nW, the fluorescence signal of the peanut template DNA before PCR was 19.95 nW, and the fluorescence signal of the PCR product from the droplet microfluidic PCR chip increased to 57.85 nW.Therefore, both PCR mixture reagents and SYBR Green reagentbased PCR reagents confirmed that the droplet microfluidic PCR chip successfully conducted PCR to amplify peanut template DNA.

Conclusion
This study developed a droplet microfluidic PCR device to amplify specific peanut DNA fragments for detection of foodborne allergens.The proposed droplet microfluidic PCR chip reduced the evaporation of the PCR reaction reagents to stabilize the fluid flow in the microchannel and thus improved the efficiency of PCR amplification compared with that of a single-phase microfluidic chip.The PCR product of the peanut template DNA from the droplet microfluidic PCR chip was verified by comparison with the commercial PCR thermal cycler and enhanced fluorescence in SYBR Green reagent-based PCR reaction.The developed device was also successfully used to amplify DNA for various species, including sesame, Salmonella spp., and Staphylococcus aureus.This droplet microfluidic PCR chip can be applied to increase the efficiency of DNA analyses for peanut detection in the context of foodborne allergy.

Figure 3 :
Figure 3: Schematic of the heating platform.

Figure 4 :Figure 5 :
Figure 4: IR thermal image of the microfluidic chip including three regions with different temperatures.

Figure 8
demonstrates the capability of the droplet microfluidic PCR chip in applications for various species, including 181 bp of ITS1 from peanut, 406 bp of Staphylococcus aureus, 146 bp of 2S albumin from sesame, and 237 bp of Salmonella spp.As shown in Figures 8(a) and 8(b), PCR products from the droplet microfluidic PCR chip were clearly visible, but those for template DNA were not.The smear of the target band for Staphylococcus aureus probably occurred because of a decrease in the ion concentration of electrophoresis buffer, presence of nuclease, or degraded gel during electrophoresis [25].

Figure 6 :
Figure 6: Emulsion droplet generated at the cross-flow microchannel in the microfluidic chip.

Figure 8 :
Figure 8: Gel electrophoresis of various species products.(a) Lane M: 100 bp ladder marker, lane 1: PCR product of peanut from the droplet microfluidic PCR chip, lane 2: peanut template DNA before PCR, lane 3: PCR product of Staphylococcus aureus from the droplet microfluidic PCR chip, lane 4: Staphylococcus aureus template DNA before PCR.(b) Lane M: 100 bp ladder marker, lane 1: PCR product of sesame from the droplet microfluidic PCR chip, lane 2: sesame template DNA before PCR.(c) Lane M: 100 bp ladder marker, lane 1: PCR product of Salmonella spp.from the droplet microfluidic PCR chip, lane 2: Salmonella spp.template DNA before PCR.

Figure 7 :
Figure 7: (a) Gel electrophoresis of peanut products.Lane M: 100 bp ladder marker, lane 1: PCR product from the commercial PCR thermal cycler, lane 2: peanut template DNA before PCR, lane 3: PCR product from the droplet microfluidic PCR chip.(b) Intensity comparison of the target bands.Peanut DNA fragment ITS1 was used as a template.

Table 1 :
Primer sequences of samples.

Table 2 :
Flow conditions of different samples in evaporation experiments.