include "../../../../includes/header.php"; ?>
Oak Ridge National Laboratory, Oak Ridge, TN 37831-6101, U.S.A.
This is an abstract for a presentation given at the
Sixth Foresight Conference on Molecular Nanotechnology.
There will be a link from here to the full article when it is available on the web.
This project involves the development of unique nanosystems that may revolutionize many areas of scientific research and technology development of nanofunctional devices. The initial focus area for this interdisciplinary research involving both experimental and computational approaches is optical nanowires and molecular switches for use in advanced biomedical, and environmental technologies.
The nanosystem (NS) technology being developed in this work will involve integration of various disciplines, combining the specificity of near-field laser probes, the sensitivity of electro-optic detection schemes, the miniaturization of integrated circuits, the exploration/design capabilities of computational chemical physics and the sophistication of silicon chip technology. Systems to be developed and investigated will include optical waveguides that have dimensions close to or less than the wavelength in the UV-visible range, i.e., "optical nanowires". Theoretical and computational modeling of light propagation and optical switching within various nanosystem designs will be performed to validate the experimental results. The key questions about the properties and potential applications of these devices are very amenable to the simulation methodology previously developed in our laboratory.
Recently we have successfully developed a nanosensor equipped with antibody-based bioprobe capable of monitoring biochemicals of single cells. Protocol for Nanoprobe Investigation of Single cells involved Clone 9 cells, a rat liver epithelial cell line grown in Ham's F-12 medium and supplemented with 5% fetal bovine serum and an additional 1mM glutamine. Cell cultures were routinely grown in a water jacketed cell culture incubator at 37% in an atmosphere of 5% CO2 in air. In preparation for an experiment, 1 x 105 cells in 5 mLs of medium were seeded into 60 mm dishes. The growth of the cells was monitored daily by microscopic observation, and when the cells reached a state of confluence of 50-60%, benzo [a] pyrene tetrol (BPT) was added and left in contact with the cells for 18 hours (i.e. overnight). The growth conditions were chosen so that the cells would be in log phase growth during the chemical treatment, but would not be so close to confluence that a confluent monolayer would form by the termination of the chemical exposure. Benzo [a] pyrene tetrol, a biomarker of DNA damage associated with exposure to the carcinogen benzo[a]pyrene, was prepared as a 1mM stock solution in reagent grade methanol and further diluted in reagent grade ethanol (95%) prior to addition to the cells. The final concentration of BPT in the culture medium of the dish was 1 x 10-7 M and the final alcohol concentration (combination of methanol and ethanol) was 0.1%.
Following chemical treatment, the medium containing BPT was aspirated and replaced with standard growth medium, prior to the nanoprobe procedure. Interrogation of single cells for the presence of BPT was carried out using antibody nanoprobes. These antibody nanoprobes were prepared from quartz optical fibers which were pulled in a fiber puller to extremely small dimensions (10-100 nm range). Subsequently, antibodies to BPT were covalently attached to the tips of the fibers, using procedures developed in our laboratory. The fibers were then coated with silver, so as to prevent light from emerging anywhere along the length of the fiber except the tip, where the antibodies are located.
The experimental setup used to probe single cells was readily adapted to this purpose from a standard micromanipulation/ microinjection apparatus. A Nikon Diaphot 300 inverted microscope with Diaphot 300/Diaphot 200 Incubator, to maintain the cell cultures at ~ 37° C on the microscope stage, was used for these experiments. The micro manipulation equipment used consisted of three dimensional manipulators for coarse adjustment and three dimensional hydraulic micromanipulators for final movements. The optical fiber nanoprobe was mounted on a micropipette holder. To record the fluorescence of BPT molecules binding to antibodies at the fiber tip, a Hamamatsu PMT detector assembly (HC125-2) was mounted in the front port of the Diaphot 300 microscope, and fluorescence was collected via this optical path.
Investigation of BPT in single cells using the nanoprobe was carried out in the following way. A culture dish of cells was placed on the pre-warmed microscope stage, and the nanoprobe, mounted on the micropipette holder, was moved into position (i.e. in the same plane of the cells), using bright field microscopic illumination, so that the tip was outside the cell to be probed. The magnification (total ) was usually 400x. All room and microscope light was extinguished, the laser shutter was opened, and laser light was allowed to illuminate the optical fiber. Usually, if the silver coating on the nanoprobe was appropriate, no light or only a faint glow at the tip could be seen on the nanoprobe. A reading was taken with the nanoprobe outside the cell and the laser shutter was closed. The nanoprobe was then moved into the cell, inside the cell membrane and extending a short way into the cytoplasm, but care was taken not to penetrate the nuclear envelope. The laser was triggered, and readings were taken as a function of time that the nanoprobe was inside the cell.
Such a nanosensor device could lead to the development of nanosensing systems that would have the capability to monitor biochemical processes in single cells for chemical and biological warfare early sensing and defense, or for the protection of the soldiers and the general population. The proposed NS devices will be the forerunner of hybrid opto-electronic devices, capable of multiple sensing functions at the molecular level inside human organs. Validation of the theoretical models in an actual biological system could be implemented by the development of a single gene "switch" which could operate either in the mode of an inducer or repressor to switch on or off the function of some well-characterized gene system. The proposed NS technology would allow the development of a new generation of integrated self-contained nanofunctional devices (ultimately down to molecular sizes) incorporating multiple end-point sensing elements, optical quantum detectors, and integrated electro-optic molecular switching capability.
Biosketch of Presenting Author: Tuan Vo-Dinh
Tuan Vo-Dinh (Ph.D. in Biophysical Chemistry (1975) is currently a Corporate Fellow and Group Leader of the Advanced Monitoring Development Group, Life Sciences Division at the Oak Ridge National Laboratory. Dr. Vo-Dinh has made major contributions to the development of the room temperature phosphorescence (RTP), synchronous luminescence (SL), and surface-enhanced Raman scattering (SERS) techniques for environmental and biomedical diagnostics. He has developed the surface-enhanced Raman gene (SERGen) probe technology and the DNA Biochip that can be used to detect DNA bio-targets rapidly, simply, and without the use of hazardous radioactive labels. The SERGen technique and DNA Biochip device can have applications in DNA sequencing and mapping, medical diagnosis of genetic-based cancer and other diseases, monitoring human exposure to infectious agents, and screening of blood supplies for pathogens. Integrating biotechnology, fiberoptics, laser techniques and molecular spectroscopy, he has developed unique antibody-based fiberoptic fluoroimmunosensors (FIS), nanosensors and nanoprobes which open new horizons to a fundamental technology of "smart catheter/sensor" for in vivo analysis. In collaboration with medical institutions, Dr. Vo-Dinh has developed a minimally invasive optical method for cancer diagnosis without biopsy.For his scientific achievements, Dr. Vo-Dinh has received various awards:
- 1997, BER-50 Award in Biological and Environmental Research, US Department of Energy
- 1996, RD-100 Award for Most Technologically Significant Advance in R&D (SERGen Probe)
- 1996, Inventor of the Year Award, Inventors Club of America
- 1996, Inventor of the Year Award, Tennessee Inventors Association
- 1995, Award for Excellence in Technology Transfer, Federal Laboratory Consortium (SERODS)
- 1994, RD-100 Award for Most Technologically Significant Advance in R&D (Spot Test for PCBs)
- 1993, Advanced Technology Award, Inventors Club of America (RTP Screening)
- 1992, RD-100 Award for Most Significant Advance in R&D (SERODS Data Storage Technology)
- 1992, Inventors International Hall of Fame Award, Inventors Club of America
- 1992, Thomas Jefferson Award, Martin Marietta Corporation
- 1992, Inventor of the Year Award, Oak Ridge National Laboratory (ORNL)
- 1989, Medal Languedoc-Rousillon Award (France)
- 1988, Gold Medal Award, Society for Applied Spectroscopy
- 1987, RD-100 Award, for Technological Advance in Industrial Research (FIS)
- 1986, Award for Excellence in Technology Transfer, Federal Laboratory Consortium
- 1984, Award for Excellence in Research, Health and Safety Research Division, ORNL
- 1981, RD-100 Award for Technological Advance in Industrial Research (PNA Dosimeter)
Dr. Vo-Dinh has authored over 200 publications in peer-reviewed scientific journals. He is the author and editor of 7 books, and hold 15 patents, five of which have been licensed to several companies for commercial development (Luminoscope, SERS Toxic Chemical Analyzer, SERODS optical data storage technology, synchronous luminescence, and optical biopsy for cancer detection). Dr. Vo-Dinh is a Fellow of the American Institute of Chemists, Topical Editor of the international journal Polycyclic Aromatic Compounds, and Associate Editor of ANALUSIS, an international journal on analytical chemistry. He has served on the Editorial and Advisory Board of Applied Spectroscopy, Talanta, and Spectrochimica Acta Reviews, and Biomedical Optics. He is the editor of the book series on Advances in Environmental and Process Control Technologies, and serves as the chairman for several international scientific organizations.
Dr. Tuan Vo-Dinh
Group Leader, Corporate Fellow
Advanced Monitoring Development Group, Oak Ridge National Laboratory
Oak Ridge, TN 37831-6101, U.S.A.
Tel: 423-574 6249; FAX: 423-576- 7651