Lewis A. Marshall

Purification of Nucleic Acids from Whole Blood Using Isotachophoresis

We present and demonstrate a novel technique for the purification of nucleic acids from biological samples using isotachophoresis (ITP). We demonstrate a simple and rapid method to achieve ITP-based extraction, preconcentration, and purification of DNA from nanoliter volumes of whole blood. We show that ITP purification yields genomic DNA samples which can be quantitated with fluorescence measurements and are immediately compatible with polymerase chain reaction (PCR) (e.g., a PCR-friendly solution free of significant inhibitors). We hypothesize ITP purification is applicable to processing of a wide range of complex biological samples.

Purification of nucleic acids using isotachophoresis.

Reviewed are methods of nucleic acid (NA) extraction and sample preparation using an electrophoretic purification and focusing method called isotachophoresis (ITP). ITP requires no special surface chemistries or geometric structures, and can be achieved in a compact system with no moving parts. ITP is also compatible with a wide range of samples and lysing methods. Described are general principles of ITP, considerations around the application of ITP to biological samples (e.g., blood, urine and saliva), ITP electrolyte design considerations for fast and selective NA purification, and examples of ITP compatible lysing methods. Several of the challenges associated with purification of NAs are presented as well as methods to address these. Lastly, specific examples of lysing methods and ITP chemistries are described for purification of NA including host and pathogenic DNA, pathogenic rRNA, and host micro-RNA from complex sample matrices.

Integrated Printed Circuit Board Device for Cell Lysis and Nucleic Acid Extraction

Preparation of raw, untreated biological samples remains a major challenge in microfluidics. We present a novel microfluidic device based on the integration of printed circuit boards and an isotachophoresis assay for sample preparation of nucleic acids from biological samples. The device has integrated resistive heaters and temperature sensors as well as a 70 μm × 300 μm × 3.7 cm microfluidic channel connecting two 15 μL reservoirs. We demonstrated this device by extracting pathogenic nucleic acids from 1 μL dispensed volume of whole blood spiked with Plasmodium falciparum. We dispensed whole blood directly onto an on-chip reservoir, and the systems integrated heaters simultaneously lysed and mixed the sample. We used isotachophoresis to extract the nucleic acids into a secondary buffer via isotachophoresis. We analyzed the convective mixing action with micro particle image velocimetry (micro-PIV) and verified the purity and amount of extracted nucleic acids using off-chip quantitative polymerase chain reaction (PCR). We achieved a clinically relevant limit of detection of 500 parasites per microliter. The system has no moving parts, and the process is potentially compatible with a wide range of on-chip hybridization or amplification assays.

Extraction of DNA from Malaria-Infected Erythrocytes Using Isotachophoresis

We demonstrate a technique for purification of nucleic acids from malaria parasites infecting human erythrocytes using isotachophoresis (ITP). We release nucleic acids from malaria-infected erythrocytes by lysing with heat and proteinase K for 10 min and immediately, thereafter, load sample onto a capillary device. We study the effect of temperature on lysis efficiency. We also implement pressure-driven counterflow during ITP extraction to extend focusing time and increase nucleic acid yield. We show that the purified genomic DNA samples are compatible with polymerase chain reaction (PCR) and demonstrate a clinically relevant limit of detection of 0.5 parasites per nanoliter using quantitative PCR.

On-Chip Separation and Analysis of RNA and DNA from Single Cells

The simultaneous analysis of RNA and DNA of single cells remains a challenge as these species have very similar physical and biochemical properties and can cross-contaminate each other. Presented is an on-chip system that enables selective lysing of single living cells, extraction, focusing, and absolute quantification of cytoplasmic RNA mass and its physical separation from DNA in the nucleus using electrical lysing and isotachophoresis (ITP). This absolute quantitation is performed without enzymatic amplification in less than 5 min. The nucleus is preserved, and its DNA fluorescence signal can be measured independently. We demonstrate the technique using single mouse lymphocyte cells, for which we extracted an average of 14.1 pg of total RNA per cell. We also demonstrate correlation analysis between the absolute amount of RNA and relative amount of DNA, showing heterogeneity associated with cell cycles. The technique is compatible with fractionation of DNA and RNA and with downstream assays of each.

An injection molded microchip for nucleic acid purification from 25 microliter samples using isotachophoresis.

We present a novel microchip device for purification of nucleic acids from 25μL biological samples using isotachophoresis (ITP). The device design incorporates a custom capillary barrier structure to facilitate robust sample loading. The chip uses a 2mm channel width and 0.15mm depth to reduce processing time, mitigate Joule heating, and achieve high extraction efficiency. To reduce pH changes in the device due to electrolysis, we incorporated a buffering reservoir physically separated from the sample output reservoir. To reduce dispersion of the ITP-focused zone, we used optimized turn geometries. The chip was fabricated by injection molding PMMA and COC plastics through a commercial microfluidic foundry. The extraction efficiency of nucleic acids from the device was measured using fluorescent quantification, and an average recovery efficiency of 81% was achieved for nucleic acid masses between 250pg and 250ng. The devices were also used to purify DNA from whole blood, and the extracted DNA was amplified using qPCR to show the PCR compatibility of the purified sample.

Simultaneous Purification and Fractionation of Nucleic Acids and Proteins from Complex Samples Using Bidirectional Isotachophoresis

We report on our efforts to create an on-chip system to simultaneously purify and fractionate nucleic acids and proteins from complex samples using isotachophoresis (ITP). We have developed this technique to simultaneously extract extracellular DNA and proteins from human blood serum samples and deliver these to two separate output reservoirs on a chip. The purified DNA is compatible with quantitative polymerase chain reaction (qPCR), and proteins can be extracted so as to exclude albumin, the most abundant protein in serum. We describe significant remaining challenges in making this bidirectional method a robust and efficient technique. These challenges include managing channel surface adsorption of proteins, identifying the cause of observed reductions in low molecular weight proteins, and dealing with nonspecific binding of proteins and DNA.