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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