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Public Security S&T Symposium 2009
CRTI 06-0186RD
Novel DNA-Based Radiological Dosimetry Technology
Presented by:Isabelle Charlebois, CRI
June 17th, 2009
Introduction and Objectives
• 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 02-0021RD and CRTI 03-0005RD
• 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
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Introduction - DNA Dosimeter Sensor Module
Surface
Anchor
70-baseoligonucleotide
20-baseTag
SequenceRadiation
Break
Released broken DNA
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FCR aggregates(nanometers)
PolymerDNA probe
Superlighting(nanometers)
Releasedbroken DNA
Introduction - DNA Dosimeter Detector Module
4
Introduction - DNA Dosimeter Detector Module
FCR aggregates(nanometers)
FCR aggregates(nanometers)
Microbeads(micrometers)
Releasedbroken DNA
Superlighting on beads
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Introduction - DNA DosimeterUnique 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
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ReleasedssDNA
Surface
Tag20 bases
Real-time PCRAnalysis
PCR-20 for tag detection, based on SybrGreen
Surface
Tag
PCR-70 for 70 bases oligonucleotide detection, based on a Taqman probe
Sybr Green fluorescenc
e
dsDNA only
fluorophore
quencher
Real-time PCRAnalysis
Fluorophore released
70 bases
Results - DNA DosimeterDevelopment of 2 real-time PCR assays
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Results - DNA DosimeterRadiation Sensitivity of the DNA Target
• Gamma-irradiation of unbound DNA in solution• At 10 Gy, 35% of the DNA is not suitable for PCR amplification
Effect of radiation on free oligonucleotide DNA in water (mean of 3 samples for each radiation doses)
0
500
1000
1500
2000
2500
3000
3500
4000
Radiation (Gray)
DN
A c
on
cen
trat
ion
(co
pie
s/µ
l)
O Gy
2,5 Gy
5 Gy
10 Gy
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Results - DNA DosimeterSensor module – Background DNA
• 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
Surface
BoundDNA
Released broken DNA
+ Background DNA
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Results - DNA DosimeterSensor Module – Background DNA
• 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 0.0000001%
Link DNA surface Background Ratio(background/bounded DNA)
EDC-NHS slides 2 x 1014* copies 2 x 109 copies 1/100 0000.0001%
Synthesis columns 1 x 1017 copies 5 x 109 copies 1/100 000 0000.0000001%
*DNA introduced at the beginning, not all DNA is bound to the surface, actual numbers are unknown
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Results - DNA DosimeterIrradiation Experiments at CERN
• Mixed-field irradiation (≈ 5 Gy) at CERN facilities• Increased Tag detection indicates DNA released upon
irradiation
DNA detected after irradiation at CERF (high dose)
0,00E+00
5,00E+08
1,00E+09
1,50E+09
2,00E+09
2,50E+09
3,00E+09
3,50E+09
4,00E+09
4,50E+09
no rad rad
AD
N (
cop
ies)
PCR 20
PCR 70
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Results - DNA DosimeterDetector Module
• 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)
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Results – Detection Platform Prototype for sensitive detection of fluorophore-grafted beads
O bjec
t ive
D etection plane
D etector
Fiber-coupled laser diode410 nm
C C D cam era
X YZ stages
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
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Conclusions - DNA dosimeter
• 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
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Next Steps - DNA Dosimeter• 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
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CRTI 06-0186RD – DNA dosimeterA multidisciplinary team!
Dr. Michel G. BergeronCentre de recherche en infectiologie (CRI)CHUQ Research CenterMolecular Biology
Teodor VeresIndustrial Materials Institute (IMI) National Research Council CanadaMaterials / Nanofabrication / Microfluidic
Mario LeclercResearch Centre on Advanced Materials (CERMA)Laval UniversityAdvanced Materials / Photoactive Polymers
Denis BoudreauCentre on Optics, Photonics and Lasers (COPL)Laval UniversityOptics / Photonics / Analytical Chemistry
Brent Lewis / Les G. I. BennettRoyal Military College (RMC)Physical Dosimetry / Irradiations
Diana WilkinsonDefense R&D Canada-Ottawa (DRDC)Biological Dosimetry
Martin PierreDirector General Nuclear Safety (DGNS)Irradiations
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Questions?
Merci
Thank you
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