Category G. Enabling Sciences
G1. Development of an All-In-One, Portable Peptide Detection System Using Microchip Electrophoresis with Fluorescence Detection for the Study of Traumatic Brain Injury
Nathan Oborny1, David Scott2, Simon Pfeiffer3, Susan Lunte2
1Department of Bioengineering, University of Kansas, Lawrence, KS; 2Department of Chemistry, University of Kansas, Lawrence, KS; 3Institute for Analytical Chemistry, University of Regensburg, Germany
The overall goal of this work is to develop an integrated device that can be employed for bedside monitoring of biomarkers present following traumatic brain injury (TBI), in a hospital setting. These biomarkers include excitatory amino acids such as glutamate and aspartate, as well as neuropeptides such as dynorphin and enkephalin, which can be found in abnormal levels within the extracellular fluid of the brain following TBI. While methods exist currently to monitor many of these biomarkers, they typically lack temporal resolution due to the sample sizes needed combined with the slow speed at which samples can be collected. In an effort to decrease sample size, on-line microdialysis sampling coupled to microchip electrophoresis and fluorescence detection will be used.
Microchip electrophoresis (ME) allows for the miniaturization of separation-based sensors. This miniaturization can lead to decreased reagent volumes, increased separation efficiency and decreased costs . Coupling ME to microdialysis sampling can be highly advantageous for these reasons. However, while the separation process itself has decreased in size, most microfluidic systems still rely on large, expensive, benchtop equipment to operate the microchip and detect analytes of interest. Therefore, in an effort to decrease the footprint of the associated high voltage power supply and detection equipment allowing their placement in a hospital setting, this work has focused in the near term on constructing an inexpensive, portable, all-in-one fluorescence detection system. This system is capable of detecting nanomolar concentrations of fluorescently derivatized peptides and amino acids.
G2. Genome Sequencing Core Lab at KU-Lawrence
Wang, Xinkun1,2, Laura Rogers1,2, Erik Lundquist1,3, Sue Lunte1,4,5
1 Center for Molecular Analysis of Disease Pathways, 2 Higuchi Biosciences Center, 3 Department of Molecular Biosciences, 4 Department of Chemistry, 5 Department of Pharmaceutical Chemistry, University of Kansas
The Genome Sequencing Core (GSC) is one of three research core labs in the newly established NIH COBRE Center for Molecular Analysis of Disease Pathways (CMADP) at KU. The major mission of the GSC is to provide researchers with next-generation sequencing (NGS) technologies. NGS, carried out in a massively parallel fashion, has been revolutionizing bio-medical research and used in a growing list of applications. Projects supported by the GSC include de novo genome assembly, genome re-sequencing for identification of mutations and polymorphisms, transcriptome analysis (RNA-Seq), epigenomic and gene regulation studies such as ChIP-Seq, Methyl-Seq, small RNA discovery and analysis. The Genome Sequencing Core enhances the genomics infrastructure already at KU, in the KU Genomics Facility and the Natural History Museum first generation sequencing facility, by bringing the astronomically high-throughput Illumina HiSeq 2500 sequencing capabilities to researchers at KU-Lawrence and across Kansas and the region. This next-generation sequencer has the capacity to generate 3-6 billion reads of 100bp per run of two eight-lane flow cells (600Gb data). In its rapid mode, it can generate 1.2 billion reads of 150 bp per run on two two-lane flow cells (120Gb data) in 27 hours. To capture the full power of NGS, we provide a whole range of project support, from project consultation, sample QC, library construction, cluster generation, data generation, to preliminary data analysis. For latest pricing, current job queue, or other info, visit the Core’s website: http://www.gsc.ku.edu/.
G3. Microfluidic-based Electrophoretic Separation of Calmodulin Binding Proteins
Thushara Samarasinghe, Susan M. Lunte, and Carey K. Johnson
Department of Chemistry, University of Kansas, Lawrence, KS
Calmodulin (CaM) is a ubiquitous Ca2+ signaling protein that regulates more than 100 different enzymes in many intracellular pathways. Investigation of this complex CaM-binding “interactome” requires a sensitive and, rapid screening mechanism. An assay will provide major CaM binding protein (CBP) profiles and possible correlations with cell states, diseases, cell development, and other cellular dynamics. The objective of this current work is to develop a highly sensitive fluorescence-based detection method coupled with prototype microfluidic separation assay for CBPs. AF647-labeled CaM and two standard proteins (BSA and concanavalin A) were separated to test the microchip platform. A photochemical bi-functional cross- linker was used to make a covalent link between AF647-labeled CaM and CBP to allow separation under denaturing conditions. Two CBPs, calcineurin (CN) and eNOS were used as model proteins and photo cross-linked with CaM using NHS-LC-Diazirine. CaM, CaM-CN and CaM-eNOS photoproducts were separated on a PDMS/Glass microchip with under conditions that suppress electro-osmotic flow.
G4. mRNA EXPRESSION OF CANDIDATE GENES FOR A PHEROMONAL DIFFERENCE BETWEEN DROSOPHILA SIMULANS AND D. SECHELLIA
Swartzlander, D.1, and Gleason, J.M.2.
1Department of Molecular Biosciences, University of Kansas, Lawrence, KS. 2Department of Ecology and Evolutionary Biology, University of Kansas, Lawrence, KS.
Courtship behavior in Drosophila includes chemosensory signals such as pheromones. Differential pheromone production between species can cause failure in mate recognition, thereby leading to reproductive isolation. Drosophila simulans and D. sechellia are reproductively isolated because of a pheromonal difference in the females. Based on quantitative trait loci (QTL) mapping of this difference, together with the knownpheromone biosynthesis pathway in D. melanogaster, we hypothesize that the genesdesatF, eloF, and additional candidate genes are responsible for differential pheromone production between D. simulans and D. sechellia. desatF is a desaturase that removes hydrogens from a carbon chain. eloF is an elongase that add carbons to a carbon chain. We predict that the candidate genes most likely to be involved in pheromone production differences will show an mRNA expression pattern similar to that of desatF and eloF between D. simulans and D. sechellia. To test our hypothesis, we identified candidate genes based on their QTL mapping locations as well as their known or predicted functions, and measured mRNA expression levels of each gene. Our results thus far show that only desatF and eloF have an mRNA expression pattern consistent with involvement in pheromone production differences between D. simulans and D. sechellia.
G5. Dual-Electrode Electrochemical Detection for Microchip Electrophoresis: Voltammetric Identification of Chemically Labile Species
1Pann Pichetsurnthorn, 2Dulan Gunasekara, 2Susan Lunte
1University of Kansas, Department of Chemical Engineering, Lawrence, KS
2University of Kansas, Department of Chemistry, Lawrence, KS
Reactive nitrogen species such as nitric oxide (NO.) and peroxynitrite (ONOO-) are chemically labile species that participate in oxidative stress and nitration/nitrosylation in vivo. These species have been implicated in several cardiovascular and neurodegenerative diseases. The short life time of these molecules makes them difficult to detect, often requiring indirect methods of analysis. Microchip electrophoresis coupled to amperometric detection (ME-EC) offers fast separations and sensitive detection- allowing these species to be characterized before significant degradation. Amperometric detection normally utilizes migration time to identify analytes in a sample. For complex samples such as cell lysates, analyte identification solely utilizing migration time becomes problematic when contamination protrudes. Therefore, a ME-EC method with dual electrodes was developed for identification of analytes by voltammetric characterization. Voltammetric information for analytes was obtained through a current ratio generated by employing two working electrodes in a series configuration. The current ratio can be unique to analytes with different half-wave potentials and deviations from such can imply impurities. The electrodes were integrated into a 5 cm simple “T” microchip. In this setup, the first electrode is in in-channel configuration while the second electrode is in end-channel configuration. Nitrite, tyrosine and hydrogen peroxide standards were used to optimize the system. Current ratios for these standards were generated by correcting sensitivity differences between two working electrodes. Applying this to commercially available peroxynitrite samples, it was found that test samples were contaminated with hydrogen peroxide, which is used in peroxynitrite synthesis. This method will be employed to identify RNS production in bulk cell lysates. The ultimate goal of this project is to identify the heterogeneity of reactive nitrogen species production in single cells.