Copyright (c) 2012 All rights reserved. http://works.bepress.com/sam_nugen Recent documents in Sam R. Nugen en-us Mon, 05 Nov 2012 11:55:16 PST 3600 http://works.bepress.com/sam_nugen/5 http://works.bepress.com/sam_nugen/5 Fri, 20 Jan 2012 08:38:30 PST We developed a software program for the rapid selection of detection probes to be used in nucleic acid-based assays. In comparison to commercially available software packages, our program allows the addition of oligotags as required by nucleic acid sequence-based amplification (NASBA) as well as automatic BLAST searches for all probe/primer pairs. We then demonstrated the usefulness of the program by designing a novel lateral flow biosensor for Streptococcus pyogenes that does not rely on amplification methods such as the polymerase chain reaction (PCR) or NASBA to obtain low limits of detection, but instead uses multiple reporter and capture probes per target sequence and an instantaneous amplification via dye-encapsulating liposomes. These assays will decrease the detection time to just a 20 min hybridization reaction and avoid costly enzymatic gene amplification reactions. The lateral flow assay was developed quantifying the 16S rRNA from S. pyogenes by designing reporter and capture probes that specifically hybridize with the RNA and form a sandwich. DNA reporter probes were tagged with dye-encapsulating liposomes, biotinylated DNA oligonucleotides were used as capture probes. From the initial number of capture and reporter probes chosen, a combination of two capture and three reporter probes were found to provide optimal signal generation and significant enhancement over single capture/reporter probe combinations. The selectivity of the biosensor was proven by analyzing organisms closely related to S. pyogenes, such as other Streptococcus and Enterococcus species. All probes had been selected by the software program within minutes and no iterative optimization and re-design of the oligonucleotides was required which enabled a very rapid biosensor prototyping. While the sensitivity obtained with the biosensor was only 135 ng, future experiments will decrease this significantly by the addition of more reporter and capture probes for either the same rRNA or a different nucleic acid target molecule. This will lead to the possibility of detecting S. pyogenes with a rugged assay that does not require a cell culturing or gene amplification step and will therefore enable rapid, specific and sensitive onsite testing.

]]> Sam R. Nugen et al. http://works.bepress.com/sam_nugen/4 http://works.bepress.com/sam_nugen/4 Fri, 20 Jan 2012 08:37:29 PST Despite the recent advances in food pathogen detection, there still exist many challenges and opportunities to improve the current technology. Techniques such as immunomagnetic separation (IMS) and polymerase chain reaction (PCR) have paved the way for rapid and sensitive detection of foodborne pathogens, and advances in nanobiotechnology have allowed for miniaturization of devices. Collaborations between workers in the fields of engineering, nanotechnology and food science have introduced new lab-on-a-chip technologies permitting development of portable, hand-held biosensors for food pathogen detection. This report highlights examples within the current state of the art, and emphasizes areas in which further research is needed.

]]> Sam R. Nugen et al. http://works.bepress.com/sam_nugen/3 http://works.bepress.com/sam_nugen/3 Fri, 20 Jan 2012 08:36:37 PST We describe investigations toward a disposable polymer-based chip for the isolation of eukaryotic mRNA. This work focuses here on the improvement of the fabrication methods for rapid prototyping and the actual application at lowest RNA concentrations with total channel volumes of 3.5 μL. Messenger RNA isolation was achieved using paramagnetic oligo (dT)25 beads within a microfluidic channel which incorporated a sawtooth microstructured design to aid in mixing. The structures were shown to facilitate mixing beteen two fluids in parallel flow when compared to a channel without structures. The chip was fabricated by means of hot embossing poly(methyl methacrylate) (PMMA) using a copper master. Copper was used as the master material due to its excellent thermal, mechanical, and electroplating properties. Fabrication of the master consisted of the structuring of a polished copper plate using KMPR 1050 as an electroplating mold for forming the microchannel structures. The copper master was found to be much more robust than traditional silicon masters used for prototyping. The use of KMPR enabled the generation of high straight walls in contrast to SU-8 masters. In addition, embossing times were able to be decreased by a factor of 3 due to improved heat conduction and avoidance of a lengthy and delicate de-embossing step.

]]> Sam R. Nugen et al. http://works.bepress.com/sam_nugen/2 http://works.bepress.com/sam_nugen/2 Fri, 20 Jan 2012 08:35:33 PST This paper discusses the design, microfabrication and use of an electrochemical biosensor based on a polymer substrate for cost effectiveness and disposability. As model analyte, amplified hsp70 mRNA from Cryptosporidium parvum was chosen. Microfluidic channels were fabricated in poly(methyl methacrylate) (PMMA) using hot embossing with a copper master. The electrochemical transducer, an interdigitated ultramicroelectrode array (IDUA) was also realized directly on the PMMA surface. First, the unstructured PMMA surface was UV functionalized. An 8 min UV treatment resulted in a carboxylic acid density of approximately 8 nmol/cm2 on the PMMA surface. The surface carboxylic acid groups were then conjugated to cystamine using water-soluble carbodiimide chemistry. Gold (200 nm) was then evaporated onto the thiol-functionalized surface. Using standard photolithography techniques, the IDUA containing 10 μm wide electrodes with 5 μm gaps was then formed followed by a gold etch. The PMMA surface containing the microchannel was subsequently bonded to the PMMA surface containing the IDUA using UV-assisted thermal bonding. The additional UV treatment also served to decrease the water contact angle of the surface from 62.5° ± 0.7° to 48.4° ± 0.2° thus, aiding with the capillary flow in the device. The hsp70 mRNA was isolated from C. parvum oocysts and amplified using nucleic acid sequence-based amplification (NASBA). The amplicon was detected in a sandwich hybridization assay with capture probe-coated superparamagnetic beads and reporter probe-tagged liposomes. The liposomes entrapped potassium ferro/ferrihexacyanide to enable amperometric quantification of the amplicon on the IDUA. Amplified mRNA from only 1 oocyst was detectable with this PMMA biosensor. The final detection device measured approximately 10 mm × 40 mm × 3 mm and contained two detection channels for dual analyses.

]]> Sam R. Nugen et al. http://works.bepress.com/sam_nugen/1 http://works.bepress.com/sam_nugen/1 Fri, 20 Jan 2012 08:34:18 PST A simple, membrane-strip-based lateral-flow (LF) biosensor assay and a high-throughput microtiter plate assay have been combined with a reverse transcriptase polymerase chain reaction (RT-PCR) for the detection of a small number (ten) of viable Mycobacterium (M.) avium subsp. paratuberculosis (MAP) cells in fecal samples. The assays are based on the identification of the RNA of the IS900 element of MAP. For the assay, RNA was extracted from fecal samples spiked with a known quantity of (101 to 106) MAP cells and amplified using RT-PCR and identified by the LF biosensor and the microtiter plate assay. While the LF biosensor assay requires only 30 min of assay time, the overall process took 10 h for the detection of 10 viable cells. The assays are based on an oligonucleotide sandwich hybridization assay format and use either a membrane flow through system with an immobilized DNA probe that hybridizes with the target sequence or a microtiter plate well. Signal amplification is provided when the target sequence hybridizes to a second DNA probe that has been coupled to liposomes encapsulating the dye, sulforhodamine B. The dye in the liposomes provides a signal that can be read visually, quantified with a hand-held reflectometer, or with a fluorescence reader. Specificity analysis of the assays revealed no cross reactivity with other mycobacteria, such as M. avium complex, M. ulcerans, M. marium, M. kansasii, M. abscessus, M. asiaticum, M. phlei, M. fortuitum, M. scrofulaceum, M. intracellulare, M. smegmatis, and M. bovis. The overall assay for the detection of live MAP organisms is comparatively less expensive and quick, especially in comparison to standard MAP detection using a culture method requiring 6-8 weeks of incubation time, and is significantly less expensive than real-time PCR.

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