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. 2020 Jun 16;10(1):9708.
doi: 10.1038/s41598-020-66779-z.

Extraction of Cell-free Dna from An Embryo-culture Medium Using Micro-scale Bio-reagents on Ewod

Anand Baby Alias  1 , Cheng-En Chiang  1 , Hong-Yuan Huang  2   3 , Kai-Ti Lin  4 , Pei-Jhen Lu  5 , Yi-Wen Wang  5 , Tzu-Hui Wu  6 , Pei-Shin Jiang  6 , Chien-An Chen  6 , Da-Jeng Yao  7
Affiliations

Extraction of Cell-free Dna from An Embryo-culture Medium Using Micro-scale Bio-reagents on Ewod

Anand Baby Alias et al. Sci Rep. .

Abstract

As scientific and technical knowledge advances, research on biomedical micro-electromechanical systems (bio-MEMS) is also developing towards lab-on-a-chip (LOC) devices. A digital microfluidic (DMF) system specialized for an electrowetting- on-dielectric (EWOD) mechanism is a promising technique for such point-of-care systems. EWOD microfluidic biochemical analytical systems provide applications over a broad range in the lab-on-a-chip field. In this report, we treated extraction of cell-free DNA (cf-DNA) at a small concentration from a mouse embryo culture medium (2.5 days & 3.5 days) with electro-wetting on a dielectric (EWOD) platform using bio-reagents of micro-scale quantity. For such extraction, we modified a conventional method of genomic-DNA (g-DNA) extraction using magnetic beads (MB). To prove that extraction of cf-DNA with EWOD was accomplished, as trials we extracted designed-DNA (obtained from Chang Gung Memorial Hospital (CGMH), Taiwan which shows properties similar to that of cf-DNA). Using that designed DNA, extraction with both conventional and EWOD methods has been performed; the mean percentage of extraction with both methods was calculated for a comparison. From the cycle threshold (Ct) results with a quantitative polymerase chain reaction (q-PCR), the mean extraction percentages were obtained as 14.8 percent according to the conventional method and 23 percent with EWOD. These results show that DNA extraction with EWOD appears promising. The EWOD extraction involved voltage 100 V and frequency 2 kHz. From this analysis, we generated a protocol for an improved extraction percentage on a EWOD chip and performed cf-DNA extraction from an embryo-culture medium (KSOM medium) at 3.5 and 2.5 days. The mean weight obtained for EWOD-extracted cf-DNA is 0.33 fg from the 3.5-day sample and 31.95 fg from the 2.5-day sample. All these results will pave a new path towards a renowned lab-on-a-chip concept.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Comparison between average extraction percentage for conventional and EWOD methods of cf-DNA extraction.
Figure 2
Figure 2
Placement and steps of bio-reagents used for cf-DNA extraction with EWOD.
Figure 3
Figure 3
Typical Procedures of cf-DNA extraction with magnetic beads.
Figure 4
Figure 4
Steps for chip cleaning to remove residual cf-DNA from used EWOD chip.
Figure 5
Figure 5
Standard curve plot. Abscissal axis shows the Ct values obtained during q-PCR for various concentrations (ordinate axis) of samples.
Figure 6
Figure 6
Mean weight/fg of EWOD-extracted cf-DNA from an embryo-culture medium (2.5 days & 3.5 days).
Figure 7
Figure 7
Droplet Generation. (a) Reagent was placed on the reservoir electrode, (b) the generation electrode was turned ON, (c,d) Adjacent transport electrodes (based on droplet size requirement) the electrodes were turned ON as shown in figure (here two transportation electrodes turned ON), (e) Turn OFF all the transportation electrodes (except the end one) (d) Turn ON the reservoir electrode. Thus a droplet is generated at the end transportation electrode which is in ON state.
See this image and copyright information in PMC

References

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