Microchip-Based Macroporous Silica Sol−Gel Monolith for Efficient Isolation of DNA from Clinical Samples

Abstract

Effective microchip extraction of deoxyribonucleic acid (DNA) from crude biological matrixes has been demonstrated using silica beads or hybrid phases composed of beads and sol-gel. However, the use of monolithic sol-gels alone for extraction of human genomic DNA has been more difficult to define. Here we describe, for the first time, the successful use of monolithic tetramethyl orthosilicate-based sol-gels for effective micro-solid-phase extraction (muSPE) of DNA in a glass microchip format. A functional monolithic silica phase with micrometer-scale pores in the silica matrix resulted from addition of poly(ethylene glycol), a poragen, to the precursor mixture. This allowed a monolithic sol-gel bed to be established in a microchip channel that provided large surface area for DNA extraction with little flow-induced back pressure. DNA extraction efficiencies for simple systems (lambda-phage DNA) were approximately 85%, while efficiencies for the reproducible extraction of human genomic DNA from complex biological matrixes (human blood) were approximately 70%. Blockage of the sol-gel pores by components in the lysed blood was observed in repeat extraction on a single device as a decrease in the extraction efficiency. The developed muSPE protocol was further evaluated to show applicability to clinical samples and bacterial cultures, through extraction of PCR-amplifiable DNA.

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.…”

Droplet enhanced microfluidic-based DNA purification from bacterial lysates via phenol extraction

“…More recently, other phases have been investigated to reduce the non-specific binding of proteins and increase the loading capacity of the solid support. Some examples are monolithic silica phases (Bhattacharyya and Klapperich 2008;Kulinski et al 2009;Wen et al 2007;Wu et al 2006), porous silicon (Chen et al 2008), and chitosan coatings (Hagan et al 2009), which reported for obtaining extraction efficiencies within a range of 70 to 85%. Nevertheless, some preliminary phenol extraction studies using microfluidics have also been conducted by examining organic-aqueous interface stabilization (Reddy and Zahn 2005), experimental and computational evaluation of droplet-based flows (Mao et al 2006), electrohydrodynamic instability (EHD) mixing (Zahn and Reddy 2006), and diffusion limited approaches to protein and DNA partitioning (Morales and Zahn 2008).…”

Extraction of genomic DNA using a new amino silica monolithic column

“…The requirement of frits or weirs for the immobilization of particles also increased the difficulty in fabrica-tion. To circumvent this challenge, Wu et al [13] employed a tetramethylortho silicate-based monolith for effective DNA extraction without a frit or weir. Wen et al [14] developed a tetramethyl orthosilicate-grafted monolithic solid-phase in a capillary-based extraction system, which was further modified for microchip-based extraction [15].…”