Student Abstracts: Biology at ORNL

Detection of Cardiac Tissue Damage Using a Cantilever-based Biosensor. . LESLIE COOK (Davidson College, Davidson, NC 28036) PANOS DATKSOS (Oak Ridge National Laboratory, Oak Ridge, TN 37831) .
Detection of cardiac tissue damage currently involves detection of marker molecules released by the damaged cardiac cells, for example, myoglobin and troponin. The level of marker biomolecules present in the blood stream is usually determined using an antibody-based ELISA (enzyme-linked immunosorbent assay). Recent developments in biosensor research have shown that microcantilever-based sensors have the potential to show greater sensitivity than current ELISA techniques. Greater sensitivity for biomarker detection could result in earlier detection and treatment for cardiac patients. Troponin is a protein cardiac marker that is only released into the bloodstream upon damage to cardiac cells. We propose to develop an assay for troponin molecules by immobilizing monoclonal antibodies to troponin on microcantilevers using a specific orientation approach. Antibodies will be covalently crosslinked to microcantilevers using PDP-Hydrazide, which is reactive towards oxidized sugar and carboxylic acid groups on the Fc region of IgG antibodies. Functionalized cantilevers will then be exposed to varying concentrations of antigen (troponin) under flow conditions. Cantilever deflection (molecular interaction) will be measured using a position sensitive detector. Immobilization chemistry will be checked using contact angle measurements. Microcantilever technology will be important in detecting low levels of biomolecules and will facilitate early detection and early treatment of myocardial infarction. It also has great potential for low-level molecule detection in other areas of medical and environmental research.

Effect of Oxygen on Hydrogen Production in Wild type and Mutant Algae. SARA FALL (Syracuse University, Syracuse, NY 13210) JAMES W. LEE (Oak Ridge National Laboratory, Oak Ridge, TN 37831) .
As petroleum reserves are depleted at an alarming rate, scientists have realized the need to discover novel sources of renewable energy. Both mutant and wild types of the Chlamydomonas algae may be a novel source of hydrogen for energy purposes. In determining whether or not algae may in fact be used as an energy source, several environmental factors that may effect the photosynthetic pathway of the organism must be considered. Current thinking in photosynthesis supports the theory that oxygen may in fact inhibit the production of hydrogen. Therefore, it is necessary to conduct experiments on the effect of oxygen on hydrogen production. This can be accomplished by monitoring the hydrogen production of various types of algae in a dual flow reactor system. A solution of algae and minimal media is put in to reaction vessels and research grade helium and a helium-oxygen mixture of gases are run through the system by a computer-controlled flow meter. The hydrogen production is then measured. Hydrogen production increased following exposure to oxygen. CO2 was introduced into the system to test for a possible back mutation. The algae did not fix CO2. This could mean that RuBisco is not the site where O2 enters the photosynthetic pathway.

Evaluation of Nanofiber Structures for Molecular Assembly. LAURA LENN (Presbyterian College, Clinton, SC 29325) MITCH DOKTYCZ (Oak Ridge National Laboratory, Oak Ridge, TN 37831) .
Single-walled carbon nanotubes (SWNTs) and multiwalled carbon nanofibers are exciting molecular wires that exhibit phenomenal electrical and mechanical properties. High quality and high purity SWNTs are grown by pulsed laser ablation and isolated by multiple acid treatments and heating; vertically aligned carbon nanofibers (VACNFs) are grown using a plasma enhanced chemical vapor deposition (PECVD) process using a lithographically defined catalyst to initiate growth. Self-assembly of these structures is key in producing multi-component functional structures for applications in electronics and biomedicine. To address self-assembly, we are applying biomedical approaches and molecular biology tools and procedures in an effort to create complex multi-component structures. Molecular biology techniques, such as chemical labeling, characterization, and functionalization of the SWNTs and VACNFs are being investigated. Immobilization of biomolecules on carbon nanotubes by functionalizing the sidewalls is being pursued. Efforts have focused on attaching biomolecules, such as DNA and proteins, to the sides of the nanotubes and nanofibers. Characterization of these hybrid structures is by gel electrophoresis and fluorescence microscopy.

Determination of Microsatellite Marker Polymorphisms on Chromosome Chr) 15 Between C57BL/6J (B6) and 129X1/SvJ Strains of Inbred Mice. MATTHEW MILLUS (Southwestern Community College, Chula Vista, CA 91915) DR. YUN YOU (Oak Ridge National Laboratory, Oak Ridge, TN 37831) .
Microsatellites, known as simple-sequence repeats (SSRs) or simple sequence length polymorphisms (SSLPs), are short, repetitive DNA sequences. They consist of 2 or 4 base pairs repeated 10 to 100 times that are flanked by unique sequences. They have been found throughout the genome of different inbred mouse strains. The most common SSRs found in the mouse genome are comprised of a CA dimer repeated in tandem. They are highly polymorphic in the number of repeating units among different inbred mouse strains, and are useful for genotyping and chromosome mapping. SSR length data exists for many different strains of inbred mice, only scattered data was available for the 129 strains at present. Polymorphisms on Chr 15 from 26.4cM to 55.7cM (centiMogan) were analyzed between B6 and 129X1/SvJ strain of inbred mice utilizing Chr 15 SSR markers. A hybrid F1 (C57BL/6J X 129X1/SvJ)embryonic stem (ES) cell line was used to confirm results and to identify any preferential PCR (polymerase chain reaction) amplification of B6 or 129X1/SvJ DNA. The results of the PCRs were visualized by ethidium bromide following agarose gel electrophoresis. 49 markers were tested, 15 demonstrated polymorphisms between B6 and 129X1/SvJ strains, 2 failed to produce results and the remaining 32 do not indicate polymorphisms on agarose gel. Data will be subsequently used to map deletions on the distal half of mouse Chromosome 15. A DNA targeting vector for the calcium channel beta subunit 3 (Cacnb3, xx cM on Chr 15) was developed to create deletion complexes centered at the Cacnb3 locus on the distal portion of Chr 15. Polymorphic markers tested above will be used to determine the size of deletions.

Development of Cantilever Based Biosensor for Cardiac Marker Detection. ARNAB MUKHERJEE (George Washington University, Washington, DC 20052) T. Thundat (Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831).. ARNAB MUKHERJEE (George Washington University, Washington, DC 20052) THOMAS THUNDAT (Oak Ridge National Laboratory, Oak Ridge, TN 37831) .
Interactions between biological molecules are of vital interest to many scientific and technological fields. Through the use of gold-coated silicon cantilevers, under flow, both physical mass loading and specific interactions between biological molecules can be detected. Using the highly specific interaction between biotin and neutravidin, a model system was developed for functionalizing the cantilever surfaces. This model was then tested using the cardiac marker myoglobin and myoglobin monoclonal antibodies. Antibodies (biotinylated goat antibody (IgG class); myoglobin antibodies) interact to differing cross-linkers such as DTSSP attached to gold-coated cantilevers; cantilevers are then exposed to neutravidin and myoglobin, respectively, under flow conditions. The specific properties of the cross-linker used can effect the orientation of the antibody and, consequently, the degree of interaction between the immobilized antibody and its antigen. Contact angle measurements were also used as a qualitative technique in the verification of the presence of the cross-linkers and immobilized antibody on the gold surface. Biomolecule interaction is measured as deflection of the cantilever through the use of a position sensitive detector. Interaction of myoglobin with myoglobin monoclonal antibody results in a net negative deflection. Interaction of myoglobin with monoclonal antibody will be quantified in order to achieve a nanogram order of sensitivity. These sensors show great potential for expanding the detection limits of marker biological molecules in both the medical and environmental disciplines.

Development of an Automated DNA Characterization Procedure for Use in DNA Microarray Preparation. REBECCA PARSLEY (Pellissippi State Technical Community College, Knoxville, TN 37933) MITCH DOKTYCZ (Oak Ridge National Laboratory, Oak Ridge, TN 37831) .
Detection and quantification of small amounts of DNA, such as PCR products, are extremely important in a wide variety of biological applications. A problem frequently encountered while attempting a gene expression analysis or the quantitation of a PCR amplification yield is the unreliable automation of experiments. The inaccurate data occurs because there are often variances in the amounts and/or concentrations of the samples. Therefore, an automated quantitation of probes for use in DNA microarrays was attempted using a Packard MultiPROBE II EX (MPII) robotic liquid handling system and a Perkin Elmer HT Soft 7000 Plus Bio Assay Reader. A standard curve that was comprised of known concentrations of DNA was first obtained through hand pipetting. This standard curve was then prepared using automated procedures on the MPII with a known amount of a fluorescent intercalating dye called picogreen. Precise readings of the liquid's fluorescence yielded a standard curve. Refinement of the procedure produced a reliable standard curve that allows for the determination of PCR products? concentrations by correlating the fluorescent readings with those of the standards. This achievement was significant in that the automated quantitation of the PCR amplification yields will allow for the rapid characterization of the large numbers of PCR products needed to prepare high density DNA arrays.