Microfluidic-Based DNA Purification in a Two-Stage, Dual-Phase Microchip Containing a Reversed-Phase and a Photopolymerized Monolith

Abstract

In this report, we show that a novel capillary-based photopolymerized monolith offering unprecedented efficiency (approximately 80%) for DNA extraction from submicroliter volumes of whole blood (Wen, J.; Guillo, C.; Ferrance, J. P.; Landers, J. P. Anal. Chem. 2006, 78, 1673-1681) can be translated to microfluidic devices. However, owing to the large mass of protein present in blood, both DNA binding capacity and extraction efficiency were significantly decreased when extraction of DNA was carried out directly from whole blood (38+/-1%). To circumvent this, a novel two-stage microdevice was developed, consisting in a C18 reversed-phase column for protein capture (stage 1) in series with a monolithic column for DNA extraction (stage 2). The two-stage, dual-phase design improves the capability of the monolith for whole blood DNA extraction by approximately 100-fold. From a 10-microL load of whole blood containing 350 ng of DNA, 99% (340+/-10 ng) traverses the C18 phase while approximately 70% (1020+/-45 ug) of protein is retained. A total of 240+/-2 ng of DNA was eluted from the second-stage monolith, resulting in an overall extraction efficiency of 69+/-1%. This provided not only an improvement in extraction efficiency over other chip-based DNA extraction solid phases but also the highest extraction efficiency reported to-date for such sample volumes in a microfluidic device. As an added bonus, the two-stage, dual-phase microdevice allowed the 2-propanol wash step, typically required to remove proteins from the DNA extraction phase for successful PCR, to be completely eliminated, thus streamlining the process without affecting the PCR amplifiability of the extracted DNA.

Purification of Nucleic Acids in Microfluidic Devices

“…The vast majority (83%) of the extracted DNA was eluted in 8 µL elution buffer, a slightly larger elution volume than that of the two-stage dual-phase microdevice. 28 Though this device has not addressed the purification of human gDNA, which is much larger than bacterial gDNA, this work demonstrated the potential of these devices for accepting real clinical samples.…”

Rapid microfluidic solid-phase extraction system for hyper-methylated DNA enrichment and epigenetic analysis

“…[12][13][14][15] There have been many reports of lSPE DNA extraction. [12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27] Previous reports describe DNA lSPE systems with integrated polymerase chain reaction 21,22 and electrophoretic separation. [23][24][25] Furthermore, DNA lSPE has been applied to forensics, where samples are typically very dilute.…”

Polymer monolith‐integrated multilayer poly(dimethylsiloxane) microchip for online microextraction and capillary electrophoresis

“…However, in the case of analysis of individual analytes with low concentration in complex sample matrices, MCE is often preceded by appropriate pretreatment procedures [3]. Various pretreatment methods, including purifying and preconcentration [4,5], filtering [6], digesting [7,8] and cell culture [9], have been developed in microfluidic systems.…”