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Public Security S&T Symposium 2009

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Presentation on theme: "Public Security S&T Symposium 2009"— Presentation transcript:

1 Public Security S&T Symposium 2009
CRTI RD Novel DNA-Based Radiological Dosimetry Technology Presented by: Isabelle Charlebois, CRI June 17th, 2009

2 Introduction and Objectives
ST IC The purpose of this project is to develop a personal and wearable dosimeter using a highly-innovative approach based on the specific recognition of DNA damage with a bio-sensitive polymer hybrid This three year project follows up on CRTI RD and CRTI RD The proposed research will seek to develop a two component fluidic device comprising:   1) a wearable sensor module consisting of a solid support harboring covalently bound known amounts of target nucleic acid of predefined length and having a specific terminal sequence tag 2) a portable detector module in which the wearable module will be inserted for detection of the cleaved nucleic acid by measuring the fluorescence emitted by the polymer-based sensor 2

3 Introduction - DNA Dosimeter Sensor Module
ST IC Introduction - DNA Dosimeter Sensor Module 20-base Tag Sequence Radiation 70-base oligonucleotide Break Anchor Released broken DNA Surface 3

4 Introduction - DNA Dosimeter Detector Module
ST IC Introduction - DNA Dosimeter Detector Module Released broken DNA FCR aggregates (nanometers) DNA probe Polymer Superlighting (nanometers) 4

5 Introduction - DNA Dosimeter Detector Module
ST IC Introduction - DNA Dosimeter Detector Module Released broken DNA FCR aggregates (nanometers) FCR aggregates (nanometers) Superlighting on beads Microbeads (micrometers) 5

6 Introduction - DNA Dosimeter Unique Features
ST IC Introduction - DNA Dosimeter Unique Features Detection of DNA damages in a synthetic system Compatible with all radiation types affecting human DNA No constraint or variables from other biological mechanisms such as DNA repair A novel detection technology Discovered and developed under previous CRTI grants Unique and sensitive signal amplification method This DNA dosimeter will help Canadian authorities in assessing health risks when faced with radiological or nuclear event 6

7 Sybr Green fluorescence
ST IC Results - DNA Dosimeter Development of 2 real-time PCR assays PCR-20 for tag detection, based on SybrGreen Tag 20 bases Released ssDNA Sybr Green fluorescence Real-time PCR Analysis Surface dsDNA only PCR-70 for 70 bases oligonucleotide detection, based on a Taqman probe Real-time PCR Analysis Fluorophore released Tag quencher 70 bases fluorophore Surface 7

8 Results - DNA Dosimeter Radiation Sensitivity of the DNA Target
ST IC Gamma-irradiation of unbound DNA in solution At 10 Gy, 35% of the DNA is not suitable for PCR amplification 8

9 Results - DNA Dosimeter Sensor module – Background DNA
ST IC Not all DNA stays on the surface (irradiated or not) First generation of sensor module = 0.01% of full-length DNA recovered (without irradiation) 0.01% of 1014 copies = 1010 copies Bound DNA Released broken DNA Surface + Background DNA 9

10 Results - DNA Dosimeter Sensor Module – Background DNA
ST IC Not all DNA stays on the surface (irradiated or not) First generation of sensor module = 0.01% of full-length DNA recovered (without irradiation) Improved procedures (chemical bounding, washing treatments, other type of exposure device) reduced background DNA up to % Link DNA surface Background Ratio (background/bounded DNA) EDC-NHS slides 2 x 1014* copies 2 x 109 copies 1/ 0.0001% Synthesis columns 1 x 1017 copies 5 x 109 copies 1/ % *DNA introduced at the beginning, not all DNA is bound to the surface, actual numbers are unknown 10

11 Results - DNA Dosimeter Irradiation Experiments at CERN
ST IC Mixed-field irradiation (≈ 5 Gy) at CERN facilities Increased Tag detection indicates DNA released upon irradiation 11

12 Results - DNA Dosimeter Detector Module
ST IC Design & fabrication of microfluidic detection module (beads manipulation, magnetic mixing & high performance capture unit) 1st prototype of microfluidic platform ready (manifold, electro-valves, software development) 12

13 Results – Detection Platform Prototype for sensitive detection of fluorophore-grafted beads
ST IC COPL’s detection platform: stand-alone (moves the optical head with respect to sample, images detection area, collects images/video files, detects fluorescence) Optical characterization of plastic materials that will be used for the microfluidic readout devices Development of novel fluorescence-based detection strategies for the total analysis of microvolumes at the single molecule level 13

14 Conclusions - DNA dosimeter
ST IC Sensor Module: Proof-of-concept Tools to quantify DNA DNA background requires improvement Detector Module: Microfluidic chips developed to confine fluorophore-grafted beads Detection platform is ready 14

15 Next Steps - DNA Dosimeter
ST IC Sensor Module: Validate improved exposure module for radiation detection Mixed-field radiation Specific radiation (gamma, neutrons) Develop post-irradiation treatments to reduce contribution from DNA background Detector Module: Design a simple fluidic interface to connect microfluidic detection chips with detection platform Develop / optimize detection strategies for fluorophore-grafted beads confined into microfluidic particle traps Validate AOB detection in microflluidic chips on the detection platform Integrate other modules with the detection platform Evaluate the radiation dose range covered by the detection approach and determine its applicability to targeted irradiation fields 15

16 CRTI 06-0186RD – DNA dosimeter A multidisciplinary team!
ST IC Teodor Veres Industrial Materials Institute (IMI) National Research Council Canada Materials / Nanofabrication / Microfluidic Dr. Michel G. Bergeron Centre de recherche en infectiologie (CRI) CHUQ Research Center Molecular Biology Brent Lewis / Les G. I. Bennett Royal Military College (RMC) Physical Dosimetry / Irradiations Mario Leclerc Research Centre on Advanced Materials (CERMA) Laval University Advanced Materials / Photoactive Polymers Diana Wilkinson Defense R&D Canada-Ottawa (DRDC) Biological Dosimetry Denis Boudreau Centre on Optics, Photonics and Lasers (COPL) Laval University Optics / Photonics / Analytical Chemistry Martin Pierre Director General Nuclear Safety (DGNS) Irradiations 16

17 Merci Thank you Questions?
ST IC Merci Thank you Questions? 17

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