Research ArticlePeanut Detection Using Droplet Microfluidic Polymerase ChainReaction Device

Shou-Yu Ma,1,2 Yu-Cheng Chiang,3 Chia-Hsien Hsu,4 Jyh-Jian Chen,5 Chin-Chi Hsu,6

An-Chong Chao,1 and Yung-Sheng Lin 1

1Department of Chemical Engineering, National United University, Miaoli, Taiwan2Ph.D. Program in Tissue Engineering and Regenerative Medicine, National Chung Hsing University, Taichung, Taiwan3Department of Food Nutrition and Health Biotechnology, Asia University, Taichung, Taiwan4Institute of Biomedical Engineering and Nanomedicine, National Health Research Institutes, Miaoli, Taiwan5Department of Biomechatronics Engineering, National Pingtung University of Science and Technology, Pingtung, Taiwan6Department of Mechanical Engineering, National United University, Miaoli, Taiwan

Correspondence should be addressed to Yung-Sheng Lin; linys@nuu.edu.tw

Received 24 December 2018; Revised 28 February 2019; Accepted 17 March 2019; Published 2 May 2019

Guest Editor: Jesus R. Millan-Almaraz

Copyright © 2019 Shou-Yu Ma et al. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

In this study, we integrated genetic detection for polymerase chain reaction (PCR) with microfluidics technology for the detectionof peanut DNA. A cross-junction microchannel was used to induce emulsion droplets of water in oil for PCR on a chip. Comparedwith the single-phase flow, the emulsion droplet flow exhibited a 7.24% lower evaporation amount and prevented air bubblegeneration. PCR results of the droplet microfluidic PCR chip for peanut DNA fragment detection was verified by comparisonwith a commercial PCR thermal cycler and increased fluorescence intensity in SYBR Green reagent-based PCR. Moreover, PCRon the microfluidic PCR chip was successful for sesame, Salmonella spp., and Staphylococcus aureus. The droplet microfluidicPCR device developed in this study can be applied for peanut detection in the context of food allergy.

1. Introduction

Food allergy is a critical public health problem affecting chil-dren and adults [1]. Allergy to peanuts is one of the mostcommon food allergies [1–3]. Most allergic reactions to pea-nuts are immunoglobulin E-mediated reactions that maylead to serious anaphylaxis [4]. Because of the risk of severeallergic reactions and possible death and the lack of effectivetreatments for food allergy [1, 5], the best method for foodallergy amelioration is strict avoidance of the offendingfoods [6].

Compared with macroscopic equivalents in polymerasechain reaction (PCR) systems, the microfluidic PCR devicehas several advantages, such as reduced sample and reagentconsumption; these advantages enable inexpensive systemoperation and facilitate small thermal mass, low thermalinertia, and rapid heat transfer, improving the efficiency ofPCR amplification [7]. Microfluidic PCR devices have

attracted substantial attention [8, 9] and are widely used invarious domains, such as biology, chemistry, medicine,forensic science, food technology, and environmental science[10]. Microfluidic PCR devices can be classified into twotypes, namely, single-phase microfluidic PCR and dropletmicrofluidic PCR. Droplet microfluidic systems using twoimmiscible fluids have emerged as a promising tool. Thesesystems reduce analysis times, improve sensitivity, lowerdetection limits, increase high-throughput screening, andenhance operational flexibility [11–13]. Compared withsingle-phase microfluidic PCR, droplet microfluidic PCRexhibits greater ability to prevent biological or chemicaladsorption on microchannel surfaces, which otherwisecauses microfluidic PCR inhibition and carryover contami-nation [14]. Several studies of on-chip integration for dropletmicrofluidic PCR have been well reviewed [14].

Pan et al. [15] integrated multichamber PCR and mul-tichannel separation for parallel genetic analysis to detect

HindawiJournal of SensorsVolume 2019, Article ID 4712084, 9 pageshttps://doi.org/10.1155/2019/4712084

the hepatitis B virus, Mycobacterium tuberculosis, andgenotype the human leucocyte antigen. Wang et al. [16]developed an oscillatory-flow multiplex PCR in a capillarymicrofluidic channel for simultaneous determination ofSalmonella enterica, Escherichia coli O157:H7, and Listeriamonocytogenes. Cai et al. [17] used a microfluidic systemthat integrated dielectrophoresis with chip-based multiplexarray PCR for the rapid identification of pathogens includ-ing Pseudomonas aeruginosa, Staphylococcus aureus, andEscherichia coli O157:H7 in complex physiological matri-ces. Tachibana et al. [18] developed an autonomous dis-posable plastic microfluidic PCR chip controlled throughthe 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 abottom-well microfluidic chip with an infrared excitedtemperature control method and fluorescence codetectionof three PCR products for human papilloma virus. Jeonget al. [20] developed a disposable PCR chip from a poly-meric film to reduce thermal mass, and Escherichia coligenomic DNA was amplified.

To date, most microfluidic PCR devices have been devel-oped for pathogen detection. However, microfluidic PCRdevices have rarely been used for foodborne allergen detec-tion, including detection of the DNA fragments of peanutspecies. Therefore, this study is aimed at developing a droplet

Sample inlet

Oil inlet

300 µm200 µm

200 µm

Flow direction

Outlet

Flow direction

200 µm

Flow direction

Side channel

Middle channel

Side channel

(a)

Flow direction

Flow direction

Flow direction

Outlet (PCR product)

Dispersed phase(PCR mixture reagents)

Continuous phase(mineral oil)

Droplet

Annealing60°C

Extension70°C

Denature95°C

(b)

Figure 1: Microchannel of droplet microfluidic PCR chip: (a) photograph and (b) schematic.

2 Journal of Sensors

microfluidic PCR device for peanut detection in the contextof food allergy.

2. Materials and Method

2.1. Chemicals. SU-8 negative tone photoresists and SU-8developers were purchased from MicroChem (Newton,MA, USA). Polydimethylsiloxane (PDMS) prepolymer(Sylgard 184) and a curing agent were purchased from DowCorning (Midland, MI, USA). ProTaq DNA polymerase,ProZyme PCR buffer (10x buffer; 100mM Tris-HCl, pH8.8at 25°C; 15mM MgCl2; 500mM KCl; and 1% Triton X-100), TAE buffer, Bio-100 Mass DNA ladder, and 6x loadingbuffer (30% w/v glycerol, 0.25% w/v bromophenol blue, and0.25% w/v xylene cyanol) were purchased from Bio-Protech(Taipei, Taiwan). dNTP mixtures of 10mM were purchasedfrom Genomics (Taipei, Taiwan). FastStart Universal SYBRGreen Master Mix was purchased from Roche Diagnostics

(Mannheim, Germany). Mineral oil (M3519-1L) and Sigma-cote were purchased from Sigma-Aldrich (St. Louis, MO,USA). Thermochromic pigments were obtained from TaipeiNew Prismatic (Taipei, Taiwan).

2.2. Chip Design. A droplet microfluidic PCR chip with across-junction microchannel was constructed from a glassslide substrate (length/width/depth: 76 × 52 × 1mm3). Thedroplet microfluidic PCR chip was composed of two parts,namely, the upstream cross-junction microchannel and thedownstream serpentine microchannel. As shown inFigure 1, the cross-junction microchannel was used fordroplet formation and serpentine microchannel for droplettransportation at three temperatures. The middle channeland the two side channels were used at the cross-junctionmicrochannel to inject dispersed and continuous phases,respectively. The cross-junction width was 200μm, and theother microchannel was 300μm. Thirty-five repeating cycles

Spin coating SU-8 on Siwafer and soft baking toevaporate solvent

Ultraviolet exposure

Development and hardbaking to completecross-linking

Post-exposure bake

Silanization treatment

Pouring PDMS onthe SU-8 mold

Detachment

Punching the ports

Bonding PDMS andglass slide

Glass slidePDMS

Mask

Soft SU-8 50Hard SU8-50

Si wafer Silanes

Figure 2: Flow diagram of microfluidic chip fabrication.

3Journal of Sensors

took place in the serpentine microchannel, and the totallength of the microchannel in the droplet microfluidic PCRchip was 261.7 cm.

2.3. Microfluidic Chip Fabrication. The SU-8 microfluidicchip mold was fabricated using photolithography (Figure 2).The negative photoresist SU-8 50 was spin coated onto a4-inch silicon wafer at 1930 rpm for 30 s and soft bakedat 95°C for 22min. A mask was applied on the alignerfor 11.06 s (365mJ/cm2) of ultraviolet exposure, and thepattern was transferred to SU-8 50. Postexposure bakingwas performed at 65°C for 1min and again at 95°C for6min. The unexposed SU-8 50 was dissolved throughtreatment with SU-8 developer solution for 6.8min. Then,the SU-8 master mold was rinsed in isopropyl alcohol andhard-baked at 150°C to complete the cross-linking. There-after, the PDMS prepolymer and curing agent were mixedin a weight ratio of 10 : 1 (w/w) and degassed. The PDMSmixture was poured into the fabricated SU-8 master mold.After thermal curing at 65°C for 2 h, the PDMS replicawas detached from the SU-8 master mold, and the portswere punched for the sample input and output. Finally,

the PDMS replica and glass substrate were bondedthrough oxygen plasma treatment of the microfluidicPCR chip.

2.4. Temperature-Controlled Heating Platform. Figure 3depicts the designed heater assembly. The heating platformused Bakelite (210mm × 297mm × 40mm) as the platformsubstrate and a 0.1mm glass fiber board as a heat-insulatinglayer to reduce heat conduction and heat convection interfer-ence in each heater. Two flat electrothermal aluminum plateheaters (60mm × 42mm × 4mm) were used for DNA dena-turation (95°C) and primer annealing (60°C). A third electro-thermal aluminum block heater (40mm × 30mm × 8mm)with an adjustable height was used for DNA extension(70°C). Aluminum blocks (40mm × 25mm × 9 5mm) wereused as the heat transfer medium. A thermocouple wasinserted into the blocks for temperature measurement. Aniron fixture and a bottom heat-resistant sponge were used tomaintain complete contact between the droplet microfluidicPCR chip and the heating platform. The temperature-controlled feedback system consisted of a proportional-integral-derivative controller (Mac 10D, Shimax, Akita,

Plate heater

Block heaterBakelite Glass fiber

Aluminum block

Sponge

Microfluidic chip

Fixture

Aluminum block

Thermocouple

Microfluidic chip

Figure 3: Schematic of the heating platform.

Table 1: Primer sequences of samples.

Sample Target gene Primer sequence (5′ to 3′)

Peanut Internal transcribed spacer 1 (ITS1)5′-GAGTCCACAAACACCCGAGG-3′ (F)

5′-AGTCGTTCTTAACTCTTGTGGTCA-3′ (R)

Sesame 2S albumin5′-GTGCCGCTGTGAGGCCATT-3′ (F)5′-CTCGGAATTGGCATTGCTG-3′ (R)

Salmonella spp. Random DNA fragment5′-TTTGCGTTGCGTCTGTCC-3′ (F)5′-GCTTATCGTCTGCGGCTC-3′ (R)

Staphylococcus aureus hsp5′- ACAAATAATAAAGGTGGC-3′ (F)5′-TATCGCCAGTTTGTACTT-3′ (R)

4 Journal of Sensors

Japan), and a solid state relay (KD20C25AX, Kytech,Taoyuan, Taiwan) was used to maintain stable temperaturesfor three heating regions in the PCR.

2.5. On-Chip PCR. The chip was baked in an oven at 100°Cfor 8 h to render the PDMS microchannel surface hydropho-bic [21]. Subsequently, the Sigmacote surfactant was intro-duced into the entire microchannel to form a hydrophobicfilm on the microchannel surface. The PCR adhesive sealingfilm (Microseal B, Bio-Rad, Hercules, CA, USA) was appliedon the PDMS surface of the microfluidic chip to preventevaporation of PCR mixture reagents. For strong contact, athermal conductive sheet was placed between the microflui-dic chip and the heating platform.

To demonstrate the performance of the droplet micro-fluidic PCR device, a DNA template of peanut species wasused for amplification by the PCR. In addition, this chipwas verified by DNA of sesame, Salmonella spp., and Staph-ylococcus aureus, with the specific target genes denoted inTable 1. The PCR mixture for DNA amplification contained2.5mM dNTPs, 2U ProTaq DNA polymerase in the Pro-Zyme PCR buffer (10x buffer), 10μM of each primer, andthe template DNA. The PCR mixture reagents and mineraloil were simultaneously injected into the microfluidic chipfrom the dispersed phase inlet and continuous phase inletthrough two independent syringe pumps (NE-300, New EraPump Systems Inc., USA). Finally, the amplified productwas collected in an Eppendorf tube from the outlet, and theamplification product and mineral oil were separated usinga microcentrifuge. The amplified product in the bottom layerof the Eppendorf tube was drawn and analyzed on a 2% aga-rose gel using electrophoresis (Mupid-2 mini gel electropho-resis system, Cosmo Bio, Tokyo, Japan).

3. Results and Discussion

3.1. Temperature Measurement. Figure 4 illustrates thetemperature distribution of the glass substrate in the dropletmicrofluidic PCR chip placed on the heating platformthrough infrared thermography (TAS-G100EXD, NEC,Japan). Results indicated that the temperature distributionwas divided into three uniform temperature regions, corre-sponding to DNA denaturation at 95°C on the right side,extension at 70°C in the middle region, and primer annealingat 60°C on the left side. Furthermore, the temperature in themicrochannel of the microfluidic chip was tested using athermochromic pigment that exhibited a color change fromblack to colorless at temperatures above 70°C. As shown inFigure 5, the thermochromic pigment in the microchannelwas transparent on the right side for DNA denaturation, grayin the middle region for extension, and black on the left sidefor primer annealing. These results confirmed that a homo-geneous temperature distribution was achieved for the PCRthermal conditions for DNA denaturation, primer annealing,and extension.

3.2. Evaporation of Water in the Microfluidic Chip. An evap-oration experiment was conducted to determine the degree ofdroplet microfluidic reducing evaporation in comparison

with that in single-phase microfluidic PCR. Under identicaltemperature gradient conditions in PCR, the collected outputflow from the droplet microfluidic chip was measured using afive-digit electronic balance for three cases, namely, water,mineral oil, and emulsion droplet (water droplet in mineraloil). For comparison, the flow rates of the dispersed and con-tinuous phases in the emulsion droplet case were set identicalto the water and mineral oil cases. Table 2 summarizes theexperimental conditions and results. The regressed mass flowrates of water (case A), mineral oil (case B), and emulsiondroplet (case C) in the microfluidic chip were 1.41, 1.50,and 3.02mg/min, respectively. Assuming that no evapora-tion occurred for mineral oil, the mass flow rate of the waterin the emulsion droplet was evaluated by subtracting themass flow rate in case B from the mass flow rate in case C.The net mass flow rate of water in the emulsion droplet was1.52mg/min, which was higher than 1.41mg/min in case A.This 0.11mg/min difference (7.24%) was the evaporationreduction of water in droplet microfluidic PCR compared

Annealing Extension Denature

Fixture

Glass substrate

Figure 4: IR thermal image of the microfluidic chip including threeregions with different temperatures.

Oil inlet

Oil inlet

Sample inlet

Outlet

Black Gray Colorless

Figure 5: Thermochromic pigment with reversible colors atdifferent temperatures in the microchannel.

5Journal of Sensors

with single-phase microfluidic PCR. Reduction of waterevaporation in droplet microfluidic PCR considerably pre-vented bubble generation and stabilized the flow condition.Thus, the design of the droplet microfluidic chip in this studyresolved the bubble problem observed in single-phase micro-fluidic PCR [22, 23].

3.3. DNA Amplification. Figure 6 illustrates droplet forma-tion at the cross-junction microchannel photographed usinghigh-speed digital cameras (Integrated Design Tools, Pasa-dena CA, USA) mounted on an inverted microscope (NikonEclipse Ti-E, Nikon, Kobe, Japan). The volumetric flow rateof the mineral oil was 2.500μL/min, and that of PCR mixturereagents was 0.750μL/min. Results indicated that the sym-metric shear force created by the mineral oil flow graduallynecked the flow of PCR mixture reagents at the cross-junction and then formed a spherical emulsion droplet inthe broadened downstreammicrochannel as a result of inter-facial tensions. The emulsion droplet moved toward thedownstream serpentine microchannel for PCR.

Figure 7(a) depicts the gel electrophoresis results for pea-nut products. The white pixels comprising the target band ofthe PCR product from the droplet microfluidic PCR chip waslarger than the commercial PCR thermal cycler (GeneAmpPCR System 2720, Perkin Elmer, Branchburg, NJ, USA),and the peanut sample template DNA had no signal beforePCR. These specific target bands were further analyzedusing ImageJ software to quantify the average fluorescenceintensity of white pixels (Figure 7(b)). As indicated inFigure 7(b), the fluorescence intensity of the PCR productfrom the droplet microfluidic PCR chip was approximatelytwo times higher than the intensity from the commercial

PCR thermal cycler. The higher fluorescence signal fromthe droplet microfluidic PCR chip may be attributed tothe concentration of the PCR products, which in turn wasa consequence of the small evaporation of flow solution.

In addition, a SYBR green reagent-based PCR was con-ducted in the droplet microfluidic PCR chip. The fluores-cence intensity of the PCR product of peanut templateDNA was determined using a previously developed compactfluorescent system with a fiber-coupled light-emitting diode(LED) [24]. In this detection system, a 12μL sample solutionwas excited using a 470nm LED, and fluorescence with awavelength between 505 and 545nm was detected. Resultsrevealed that the fluorescence signal of the environmentbackground was 13.13 nW, the fluorescence signal of thepeanut template DNA before PCR was 19.95 nW, and thefluorescence signal of the PCR product from the dropletmicrofluidic PCR chip increased to 57.85 nW. Therefore,both PCR mixture reagents and SYBR Green reagent-based PCR reagents confirmed that the droplet microflui-dic PCR chip successfully conducted PCR to amplify pea-nut template DNA.

Figure 8 demonstrates the capability of the dropletmicrofluidic PCR chip in applications for various species,including 181 bp of ITS1 from peanut, 406 bp of Staphylococ-cus aureus, 146 bp of 2S albumin from sesame, and 237 bp ofSalmonella spp. As shown in Figures 8(a) and 8(b), PCRproducts from the droplet microfluidic PCR chip were clearlyvisible, but those for template DNA were not. The smear ofthe target band for Staphylococcus aureus probably occurredbecause of a decrease in the ion concentration of electropho-resis buffer, presence of nuclease, or degraded gel duringelectrophoresis [25].

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

Case SampleFlow rate (μL/min)

Regressed mass flow rate(mg/min)

Main microchannel forcontinuous phase

Side microchannel fordispersed phase

A Water 0.750 0.750 1.41

B Mineral oil 2.333 0.083 1.50

C Emulsion droplet 2.416 1.500 3.02

Flow direction

Continuous phase (mineral oil)

Dispersed phase(PCR mixture reagents)

Continuous phase (mineral oil)

200 µm

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

6 Journal of Sensors

Two-temperature PCR was also performed in the dropletmicrofluidic PCR chip. Salmonella spp. was used as the targetgene, and the temperatures of DNA denaturation and primerannealing or extension amplification were set 94°C and 70°C,respectively. As shown in Figure 8(c), the target DNA was

successfully amplified for the 237 bp of Salmonella spp. How-ever, the PCR product displayed nonhomogeneity in thefluorescence signal. Therefore, the developed microfluidicPCR chip is feasible for amplification of DNA fragments ofvarious species, confirming that the droplet microfluidic

M 1 2 3 4

406 bp

181 bp

(a)

146 bp

1 2 M

(b)

237 bp

1 2 M

(c)

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

181 bp

A

M 1 32

B

(a)0

30

60

90

120

150

Inte

nsity

(a.u

.)

A B(b)

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

7Journal of Sensors

PCR chip is a potential diagnostic method for identifyingfood allergens such as peanut and sesame.

4. Conclusion

This study developed a droplet microfluidic PCR device toamplify specific peanut DNA fragments for detection offoodborne allergens. The proposed droplet microfluidicPCR chip reduced the evaporation of the PCR reactionreagents to stabilize the fluid flow in the microchannel andthus improved the efficiency of PCR amplification comparedwith that of a single-phase microfluidic chip. The PCRproduct of the peanut template DNA from the droplet micro-fluidic PCR chip was verified by comparison with thecommercial PCR thermal cycler and enhanced fluorescencein SYBR Green reagent-based PCR reaction. The developeddevice was also successfully used to amplify DNA for variousspecies, including sesame, Salmonella spp., and Staphylococ-cus aureus. This droplet microfluidic PCR chip can beapplied to increase the efficiency of DNA analyses for peanutdetection in the context of foodborne allergy.

Data Availability

There is no data availability in this paper.

Disclosure

The sponsors had no role in the study design; the collection,analyses, or interpretation of data; the writing of the manu-script; or the decision to publish the results.

Conflicts of Interest

The authors declare no conflict of interest.

Acknowledgments

This work was supported by the Ministry of Science andTechnology, Taiwan.

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