In this study, we integrated genetic detection for polymerase chain reaction (PCR) with microfluidics technology for the detection of peanut DNA. A cross-junction microchannel was used to induce emulsion droplets of water in oil for PCR on a chip. Compared with the single-phase flow, the emulsion droplet flow exhibited a 7.24% lower evaporation amount and prevented air bubble generation. PCR results of the droplet microfluidic PCR chip for peanut DNA fragment detection was verified by comparison with a commercial PCR thermal cycler and increased fluorescence intensity in SYBR Green reagent-based PCR. Moreover, PCR on the microfluidic PCR chip was successful for sesame,
Food allergy is a critical public health problem affecting children and adults [
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 [
Pan et al. [
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 microfluidic PCR device for peanut detection in the context of food allergy.
SU-8 negative tone photoresists and SU-8 developers were purchased from MicroChem (Newton, MA, USA). Polydimethylsiloxane (PDMS) prepolymer (Sylgard 184) and a curing agent were purchased from Dow Corning (Midland, MI, USA). ProTaq DNA polymerase, ProZyme PCR buffer (10x buffer; 100 mM Tris-HCl, pH 8.8 at 25°C; 15 mM MgCl2; 500 mM KCl; and 1% Triton X-100), TAE buffer, Bio-100 Mass DNA ladder, and 6x loading buffer (30%
A droplet microfluidic PCR chip with a cross-junction microchannel was constructed from a glass slide substrate (length/width/depth:
Microchannel of droplet microfluidic PCR chip: (a) photograph and (b) schematic.
The SU-8 microfluidic chip mold was fabricated using photolithography (Figure
Flow diagram of microfluidic chip fabrication.
Figure
Schematic of the heating platform.
The chip was baked in an oven at 100°C for 8 h to render the PDMS microchannel surface hydrophobic [
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,
Primer sequences of samples.
Sample | Target gene | Primer sequence (5 |
---|---|---|
Peanut | Internal transcribed spacer 1 (ITS1) | 5 |
5 | ||
Sesame | 2S albumin | 5 |
5 | ||
Random DNA fragment | 5 | |
5 | ||
hsp | 5 | |
5 |
Figure
IR thermal image of the microfluidic chip including three regions with different temperatures.
Thermochromic pigment with reversible colors at different temperatures in the microchannel.
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
Flow conditions of different samples in evaporation experiments.
Case | Sample | Flow rate ( |
Regressed mass flow rate (mg/min) | |
---|---|---|---|---|
Main microchannel for continuous phase | Side microchannel for dispersed 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 |
Figure
Emulsion droplet generated at the cross-flow microchannel in the microfluidic chip.
Figure
(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.
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) [
Figure
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
Two-temperature PCR was also performed in the droplet microfluidic PCR chip.
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,
There is no data availability in this paper.
The sponsors had no role in the study design; the collection, analyses, or interpretation of data; the writing of the manuscript; or the decision to publish the results.
The authors declare no conflict of interest.
This work was supported by the Ministry of Science and Technology, Taiwan.