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.
Highlights
Genetic analysis is a complicated procedure involving three steps: (1) DNA extraction from raw biological samples, (2) sequence amplification with the real-time polymerase chain reaction (PCR) and (3) separation and selection of DNA for testing
electrowetting- on-dielectric (EWOD) Extraction to find the efficiency of cell-free DNA (cf-DNA) extraction procedure in EWOD platform, we initially performed extraction of designed-DNA in conventional way
On repeating experimental analysis we found that the conventional extraction yielded an extraction 14.8 percentages; later we tried the same on a EWOD system, for which the extraction yielded 23 percentages
Summary
Genetic analysis is a complicated procedure involving three steps: (1) DNA extraction from raw biological samples, (2) sequence amplification with the real-time polymerase chain reaction (PCR) and (3) separation and selection of DNA for testing. Much test sample and reagent and an enormous working laboratory or spaces are required for DNA extraction with a conventional method To overcome these limitations, we applied a lab-on-a-chip (LOC), which is a device that integrates one or several laboratory functions on a single integrated circuit (commonly called a chip) of size only mm[2] to a few cm[2] to achieve automation and high-throughput screening. There is no need of pump or valve as in typical microfluidic systems This simplicity and portability makes a EWOD-based DMF system widely popular in biomedical or chemical fields as a powerful platform for sample preparation. With this method, we have the possibility to perform on-chip all fundamental fluidic operations (droplet dispensing from a reservoir, transport, merging, mixing and splitting) using an array of electrodes. We used a standard curve analysis to achieve a quantitative analysis of unknown cf-DNA samples
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