A microfabricated nucleic acid purification device for influenza genotyping.

Influenza virus is a common human and animal pathogen that is capable of causing pandemics among human populations. In addition to an annual vaccine and some antiviral drugs, rapid monitoring of changes in the virus in animal and human populations is necessary as another means of defense. Advances in microfluidics and micro total analytical systems have facilitated the development of microfabricated devices capable of complex biological assays that could be used for influenza genotyping. A necessary component of such a device is a nucleic acid purification unit. Here we demonstrate a design method and a novel microfabricated device for use in nucleic acid purification based on nucleic acid adsorption to colloidal silica. The design method is based on a pressure equation for flow through packed beds. It allows estimation of key design parameters needed for device optimization, such as channel dimensions, liquid flow rates, sample volume and the amount of silica needed. The necessary experiments to develop the design method are presented, and primarily pertain to adsorption and elution characteristics of nucleic acids on silica surfaces. The novel nucleic acid purification microchip is a device fabricated from poly(dimethylsiloxane), and utilizes a packed bed of paramagnetic silica particles that are immobilized by an external magnetic field. Device fabrication and assembly are simple. No curing steps to immobilize the packed bed are necessary. Purifications with the device take approximately 30 min. DNA recovery efficiencies up to 50 percent are possible with a loading mass of 1 ng. The packed bed is replaceable. A single device can be used for multiple purification runs. Application of the design method and the microfabricated purification device are demonstrated for a biological matrix consisting of an extract of Madin Darby Canine Kidney cells infected with influenza A virus. Both the design method and purification device will be applied to integration of the nucleic acid purification and detection steps in a portable microfluidic influenza genotyper.Ph.D.Applied SciencesChemical engineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/126798/2/3276269.pd

Low concentration DNA extraction and recovery using a silica solid phase

DNA extraction from clinical samples is commonly achieved with a silica solid phase extraction column in the presence of a chaotrope. Versions of these protocols have been adapted for point of care (POC) diagnostic devices in miniaturized platforms, but commercial kits require a high amount of input DNA. Thus, when the input clinical sample contains less than 1 μg of total DNA, the target-specific DNA recovery from most of these protocols is low without supplementing the sample with exogenous carrier DNA. In fact, many clinical samples used in the development of POC diagnostics often exhibit target DNA concentrations as low as 3 ng/mL. With the broader goal of improving the yield and efficiency of nucleic acid-based POC devices for dilute samples, we investigated both DNA adsorption and recovery from silica particles by using 1 pg- 1 μg of DNA with a set of adsorption and elution buffers ranging in pH and chaotropic presence. In terms of adsorption, we found that low pH and the presence of chaotropic guanidinium thiocyanate (GuSCN) enhanced DNA-silica adsorption. When eluting with a standard low-salt, high-pH buffer, > 70% of DNA was unrecoverable, except when DNA was initially adsorbed with 5 M GuSCN at pH 5.2. Unrecovered DNA was either not initially adsorbed or irreversibly bound on the silica surface. Recovery was improved when eluting with 95°C formamide and 1 M NaOH, which suggested that DNA-silica-chaotrope interactions are dominated by hydrophobic interactions and hydrogen bonding. While heated formamide and NaOH are non-ideal elution buffers for practical POC devices, the salient results are important for engineering a set of optimized reagents that could maximize nucleic acid recovery from a microfluidic DNA-silica-chaotrope system