Structure and fabrication details of an integrated modularized microfluidic system

This article contains schemes, original experimental data and figures for an integrated modularized microfluidic system described in “An integrated microfluidic system for bovine DNA purification and digital PCR detection [1]”. In this data article, we described the structure and fabrication of the integrated modularized microfluidic system. This microfluidic system was applied to isolate DNA from ovine tissue lysate and detect the bovine DNA with digital PCR (dPCR). The DNA extraction efficiency of the microdevice was compared with the efficiency of benchtop protocol.


a b s t r a c t
This article contains schemes, original experimental data and figures for an integrated modularized microfluidic system described in "An integrated microfluidic system for bovine DNA purification and digital PCR detection [1]". In this data article, we described the structure and fabrication of the integrated modularized microfluidic system. This microfluidic system was applied to isolate DNA from ovine tissue lysate and detect the bovine DNA with digital PCR (dPCR). The DNA extraction efficiency of the microdevice was compared with the efficiency of benchtop protocol.
& 2015 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Subject area
Biology More specific subject area microfluidic Type of data

Value of the data
The structure and fabrication details of an integrated modularized microfluidic system are given. Amplification plots and DNA yields resulting from on-chip isolation can be compared with the benchtop procedure.
Data from qPCR were compared with data from dPCR.
1. Data, experimental design, materials and methods

Microdevice design and fabrication
Integration is increasingly recognized as an important technical challenge in lab-on-a-chip device. More efficient and lower cost integrated microfluidic systems decrease or eliminate reliance on traditional lab equipment. Chin et al. [2] integrated new procedures for manufacturing, fluid handling and signal detection in microdevice into a single 'mChip' assay to replicate all steps of ELISA. Easley et al. [3] developed an integrated microfluidic genetic analysis system that could extract and purify DNA from crude whole blood sample, carry out PCR-based amplification, following by separation and detection in a manner that allows for microliter samples to be screened for infectious pathogens with sample-in-answer-out results.
We have integrated a NA extraction module and a dPCR reaction module on this chip. As shown in Fig. 1, three washing buffer and sample lysate were preloaded in the Teflon tube one by one with air  (Fig. 1c). The NA capture zone was a teardrop-shaped three-way tube (inlet, outlet 1 and outlet 2) (Fig. 1d) with a sizeable magnet. The dPCR module (Fig. 1e) had a dPCR region with 650 reaction chambers (200 μm diameter and 230 μm high) in a square area (15.0 mm Â 15.0 mm) (Fig. 1f) and a μfilter with annular duct (200 mm in width, 40 mm in depth).
For the DNA isolation form tissue, we introduced 70% (v/v) ethanol, 70% (v/v) ethanol, buffer MP3, and lysate orderly by adjusting the knob of pipettor. As shown in Fig. 2, the volume of these reagents  and the space of two reagent segments could be controlled by the range that we adjusted the knob of pipettor.
Fluidic isolation of the NA extraction region and dPCR region was essential for the purposes of avoiding the incompatibility of the NA extraction with the amplification process. Because of the pull force from the negative pressure from the other side of the capture zone, the reagents could not seep into the channel that connected the dPCR module and NA extraction module, in case the reagents disturbed the subsequent dPCR reaction. As shown in Fig. 3, reagents solution were introduced into the NA isolation area from the Teflon tube by the syringe. The Mag-Bind particles were collected in the NA isolation area by the magnet, and the waste liquid was disposed into the syringe but the reagents did not seep into outlet 2. The μfilter provided negative pressure to sample loading and a syringe was used to pull the reagents for NA isolation. Then PCR mix was introduced into the NA isolation area from the Teflon tube, and took the DNA on the Mag-Bind particles into the dPCR part.

DNA extraction
The purification of NA from specimens was laborious and time-consuming until BOOM et al. [4] developed a rapid and simple method. Compared to conventional methods, the microdevices have shown advantages in low reagent and sample consumption, enhanced sensitivity, increased speed, etc. [5][6][7].
When 20 μL lysed sample with the Mag-Bind particles was pulled into the chip and passed through the capture zone by the negative pressure from other side, the Mag-Bind particles with NAs were collected by a magnet. Then the three sections of reagent in Teflon tube were pulled into the capture zone and washed away proteins and/or other contaminants on the Mag-Bind particles. Finally, DNA was eluted by PCR mix and introduced into the dPCR region for subsequent dPCR analysis.
But when the Mag-Bind particles were collected together and washed by 70% (v/v) ethanol, the Mag-Bind particles were clumped together and resisted washing and elution. We introduced a groove for magnet sliding above the NA capture zone to drive the magnetic particles movement around and improve the efficiency of washing and elution. The elution efficiency of PCR mix in a 0.2 mL tube and microdevice was determined by gel electrophoresis (Fig. 4). The genomic DNA and PCR results emerged in the same lane. The flow rates for wash reagents was 10 μL/min. Purified NAs obtained from benchtop and microdevice were quantified using real-time PCR with the Real Time PCR Bovine and Ovine DNA Detection Kit (TAKARA, RR913). Differences on quantified NA between benchtop and microdevice results were evaluated using the Mann-Witney U statistical test (SPSS 19.0, P ¼0.602 40.05) ( Table 1). The data showed that there was non-significant differences between onchip isolation compared to the benchtop procedure in DNA yields (Fig. 5). The bovine meat in different mixture with the ovine meat were detected by qPCR (Fig. 6).