Brian H. Augustine

Dynamic Solid Phase DNA Extraction and PCR Amplification in Polyester-Toner Based Microchip

A variety of substrates have been used for fabrication of microchips for DNA extraction, PCR amplification, and DNA fragment separation, including the more conventional glass and silicon as well as alternative polymer-based materials. Polyester represents one such polymer, and the laser-printing of toner onto polyester films has been shown to be effective for generating polyester-toner (PeT) microfluidic devices with channel depths on the order of tens of micrometers. Here, we describe a novel and simple process that allows for the production of multilayer, high aspect-ratio PeT microdevices with substantially larger channel depths. This innovative process utilizes a CO(2) laser to create the microchannel in polyester sheets containing a uniform layer of printed toner, and multilayer devices can easily be constructed by sandwiching the channel layer between uncoated cover sheets of polyester containing precut access holes. The process allows the fabrication of deep channels, with ~270 μm, and we demonstrate the effectiveness of multilayer PeT microchips for dynamic solid phase extraction (dSPE) and PCR amplification. With the former, we found that (i) more than 65% of DNA from 0.6 μL of blood was recovered, (ii) the resultant DNA was concentrated to greater than 3 ng/μL (which was better than other chip-based extraction methods), and (iii) the DNA recovered was compatible with downstream microchip-based PCR amplification. Illustrative of the compatibility of PeT microchips with the PCR process, the successful amplification of a 520 bp fragment of λ-phage DNA in a conventional thermocycler is shown. The ability to handle the diverse chemistries associated with DNA purification and extraction is a testimony to the potential utility of PeT microchips beyond separations and presents a promising new disposable platform for genetic analysis that is low cost and easy to fabricate.

Preliminary analysis of polyhydroxyalkanoate inclusions using atomic force microscopy

Atomic force microscopy analysis of polyhydroxyalkanoate (PHA) inclusions isolated from sonicated Ralstonia eutropha cells revealed that they exhibit two types of surface structure and shape; rough and ovoid, or smooth and spherical. Smooth inclusions possessed linear surface structures that were in parallel arrays with 7-nm spacing. Occasionally, cracks or fissures could be seen on the surface of the rough inclusions, which allowed a measurement of approximately 4 nm for the thickness of the boundary layer. When the rough inclusions were imaged at higher resolution, globular structures, 35 nm in diameter, having a central pore could be seen. These globular structures were connected by a network of 4-nm-wide linear structures. When the inclusions were treated with sodium lauryl sulfate, the boundary layer of the inclusion deteriorated in a manner that would be consistent with a lipid envelope. When the boundary layer was largely gone, 35-nm globular disks could be imaged laying on the surface of the filter beside the inclusions. These data have facilitated the development of a preliminary model for PHA inclusion structure that is more advanced than previous models.

PhaP Is Involved in the Formation of a Network on the Surface of Polyhydroxyalkanoate Inclusions in Cupriavidus necator H16

Polyhydroxyalkanoate (PHA) inclusions are polymeric storage inclusions formed in some bacterial species when carbon levels are high but levels of another essential nutrient, such as nitrogen, are low. Though much is known about PHA synthesis, little is known about inclusion structure. In this study, atomic force microscopy (AFM) was employed to elucidate the structure of PHA inclusions at the nanoscale level, including the characterization of different layers of structure. AFM data suggest that underneath the inclusion envelope, there is a 2- to 4-nm-thick network layer that resides on top of a harder layer that is likely to be a crystalline lamellar polymer. The network is comprised of approximately 20-nm-wide linear segments and junctions that are typically formed by the joining of three to four of the linear segments. In some cases, approximately 50-nm globular structures that are raised approximately 1 to 2 nm above the network are present at the junctions. These globular structures always have a central pore that is approximately 15 nm in diameter. To determine if the major surface protein of PHA inclusions, PhaP, is involved in the structure of this network, inclusions from Cupriavidus necator H16 DeltaphaP were examined. No network structure was detected. Instead, apparently random globular structures were found on the surfaces of the inclusions. When PhaP levels were reconstituted in this strain by the addition of phaP on a plasmid, the network was also reconstituted, albeit in a slightly different arrangement from that of the wild-type network. We conclude that PhaP participates in the formation of the inclusion network.

Wetting properties induced in nano-composite POSS-MA polymer films by atomic layer deposited oxides

Due to their unique properties, nano-composite polyhedral oligomeric silsequioxane (POSS) copolymer films are attractive for various applications. Here we show that their natural hydrophobic character can become hydrophilic when the films are modified by a thin oxide layer, up to 8 nm thick, prepared using atomic layer deposition. A proper choice of the deposition temperature and thickness of the oxide layer are required to achieve this goal. Unlike other polymeric systems, a marked transition to a hydrophilic state is observed with oxide layers deposited at increasing temperatures up to the glass transition temperature (∼110 °C) of the POSS copolymer film. The hydrophilic state is monitored through the water contact angle of the POSS film. Infrared absorbance spectra indicate that, in hydrophilic samples, the integral of peaks corresponding to surface Al–O (hydrophilic) is significantly larger than that of peaks linked to hydrophobic species.

Improving the adhesion of Au thin films onto poly(methyl methacrylate) substrates using spun-cast organic solvents

Polymeric substrates are becoming more widely used in a variety of technologies such as flat panel displays, biosensors, and photovoltaic devices. The advantages of polymeric substrates include improved manufacturability, lower cost of fabrication, lower processing temperatures, and overall thermal budget. A critical processing step is the deposition of metal thin films onto the polymeric substrate to fabricate electrodes and interconnecting wires. These components are essential in sensors, catalysts, photonics, polymer electronics, micro total analysis systems, and microelectrodes. Vapor deposited gold Au thin films are widely used in many of these technologies. The material properties that make Au useful include its corrosion resistance, high infrared reflectivity, and outstanding electrical and thermal conductivities 11% and 34% better than Al, respectively . Unfortunately, Au is a relatively inert metal that has notoriously poor adhesion to polymers. Process engineers have developed extensive methods to deposit Au interconnects and electrodes in silicon-based microelectronics and microelectromechanical systems by using a vapor deposited adhesion layer. Typically, this layer is produced by deposition of a reactive metal such as Cr or Ti, which can form a chemical bond with polar atoms on the surface. The adhesion layer is generally thin 5 nm and is deposited prior to the Au film without breaking vacuum so that the surface of the adhesion film does not oxidize. This produces a thin metal film that is conformal and well bonded to the Si, SiO2, or other inorganic substrates.

Use of in-situ atomic force microscopy to monitor the biodegradation of polyhydroxyalkanoates (PHAs)

Thin films of a co-polymer mixture of poly 3-hydroxybutyrate and poly 3-hydroxyvalerate P(3HB-3HV) were spun-cast onto glass slides resulting in 35 nm thick layers with a spherulitic microstructure. An untyped strain of Streptomyces sp. bacteria was isolated from soil samples, and its PHA depolymerase was used to degrade the P(3HB-3HV) thin films. Both ex-situ and in-situ atomic force microscopy (AFM) biodegradation studies were performed to determine the kinetics of the biodegradation over the course of three hours at room temperature. Ex-situ AFM was performed in Tapping Mode and in-situ AFM was performed in the PHA depolymerase using contact mode AFM in the liquid cell, allowing for the real-time analysis of P(3HB-3HV) biodegradation. Biodegradation is observed uniformly throughout the surface, and can be observed within 30 min. of depolymerase exposure. In-situ AFM analysis yields a linear degradation rate as a function of time, while the ex-situ study suggests a more complex kinetics.