Radiation Protection Dosimetry (2012), Vol. 148, No. 1, pp. 20 – 33 Advance Access publication 17 February 2011

doi:10.1093/rpd/ncq599

USE OF A DUAL-LABELLED OLIGONUCLEOTIDE AS A DNA DOSEMETER FOR RADIOLOGICAL EXPOSURE DETECTION

*Corresponding author: [email protected] Received October 15 2010, revised December 6 2010, accepted December 27 2010 A reporter molecule consisting of a synthetic oligonucleotide is being characterised for a novel damage detection scenario for its potential use as a field-deployable, personal deoxyribonucleic acid (DNA) dosemeter for radiation detection. This dosemeter is devoid of any biological properties other than being naked DNA and therefore has no DNA repair capabilities. It supports biodosimetry techniques, which require lengthy analysis of cells from irradiated individuals, and improves upon inorganic dosimetry, thereby providing for a more relevant means of measuring the accumulated dose from a potentially mixedradiation field. Radiation-induced single strand breaks (SSBs) within the DNA result in a quantifiable fluorescent signal. Proof of concept has been achieved over 250 mGy –10 Gy dose range in radiation fields from 60Co, with similar results seen using a linear accelerator X-ray source. Further refinements to both the molecule and the exposure/detection platform are expected to lead to enhanced levels of detection for mixed-field radiological events.

INTRODUCTION Although responding to a chemical, biological, radiological or nuclear (CBRN) incident, the level of exposure and health threat to first responders and other operational authorities must be appropriately managed. As a measure of the biological effect of radiation, dosimetry is used to evaluate, control and communicate the given health risk of the radiological event. Dosimetry for an radiological and nuclear event is difficult because of the unknown nature of the radiation field and the possibility of a mixedradiation field of different qualities (e.g. alpha, beta, gamma rays, X rays and neutrons). A deoxyribonucleic acid (DNA) dosemeter is therefore being developed to measure dose exposure to ionising radiation for all radiation types and for possible mixed-field applications. Two types of dosemeters, i.e., inorganic and biological dosemeters, are not always appropriate for this task. Inorganic dosemeters include gas ion chambers, thermoluminescence dosemeters (TLDs), radiographic or radiochromic film, diodes, MOSFETs, bubble chambers and tissue equivalent proportional counters. Typically, inorganic dosemeters measure a specific type of radiation with a given energy spectra or beam quality. The measured dose is converted to # Crown copyright 2011.

a dose-to-tissue value through pre-existing knowledge of interaction probabilities within the dosemeter. For instance, if the dosemeters were measuring the dose deposited from photons, the absorbed tissue dose would be calculated by scaling the measured dose by the ratio of the mass-energy absorption coefficients. This scaling term is a function of the incoming particle energy, making the calculation both particle-and energy-dependent, which is a limitation of most inorganic dosemeters. Typically, tissue-equivalent proportional counters are relatively large in size, which is a possible instrument for mixed-field dosimetry, but requires a complex lineal energy spectral analysis employing microdosimetry theory. Some inorganic dosemeters are considered tissueequivalent, implying that they have an effective atomic number similar to that of human tissue (Zeff ¼ 7.51). These include lithium fluoride TLDs (Zeff ¼ 8.14), radiochromic film (Zeff ¼ 6.98) and some proportional counters (Zeff ¼ 7.1)(1 – 3). As photon and electron interaction probabilities are similar among molecules with equal mean atomic numbers, tissueequivalent dosemeters have a wider energy range over which they are useful when compared with nontissue-equivalent dosemeters. Unfortunately, their

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T. Wood1, B. J. Lewis1,*, K. McDermott1, L. G. I. Bennett1, K. Avarmaa1, E. C. Corcoran1, D. Wilkinson2, A. Jones2, T. Jones2, E. Kennedy2, L. Prud’homme-Lalonde2, D. Boudreau3, J.-F. Gravel3, C. Drolet3, A. Kerr4, L. J. Schreiner4, J. R. M. Pierre5, R. Blagoeva5 and T. Veres6 1 Royal Military College of Canada, Kingston, Ontario, Canada K7K 7B4 2 Defence Research & Development Canada, Ottawa, Ontario, Canada K1A 0Z4 3 De´partement de chimie and Centre d’optique, photonique et laser (COPL), Universite´ Laval, Que´bec, Canada G1K 7P4 4 Cancer Centre of Southeastern Ontario, Kingston, Ontario, Canada K7L 5P9 5 Director General Nuclear Safety, Department of National Defence, Ottawa, Canada K1A 0K2 6 Institut des mate´riaux industriels, Conseil national de recherches Canada, Que´bec, Canada J4B 6Y4

DUAL-LABELLED OLIGONUCLEOTIDE DNA DOSEMETER

Figure 1. Schematic representation of the DNA molecule for the dosemeter. The quencher (Q) is 10 and 30 bases from the fluor reporter (R). Unlabelled, reporter only and dual-labelled oligos are shown.

A novel approach for this molecular-based dosemeter is the use of a dual-labelled oligodeoxyribonucleotide molecule (Figure 1) to measure small DNA fragments potentially with increased sensitivity. The dual-labelled, single-stranded DNA (oligonucleotide) is readily synthesised using industry-standard b-cyanoethyl-phosphoramidite chemistry. The current construct is representative of tissue but does not include other cellular components. The oligonucleotide is obtained from Biosearch Technologies Inc., Navato, CA. Initial experiments relied on a dual-labelled oligo of 10, 20 or 30 nucleotides (bases) long with a 50 covalently linked fluorescent chromophore (reporter or R) and a 30 covalently linked quencher molecule (Q). The construct comes synthesised with end labels using standard phosphoramidite chemistry. In particular, the monophosphate thyamine strand of a 10base form (i.e. dT(10)) has a chain-strand length of ˚ , compared with an overall length of 100 A ˚ for 60 A the complete construct. The molecular weights of the components of this particular construct are: 3200 amu for the dT(10) strand, 1180 amu for the fluorescein amidite (FAM) fluorescent chromosphore and 555 amu for the Black Hole QuencherTM complex (BHQ). The intact, full length dual-labelled oligodeoxynucleotide has a very low intrinsic fluorescent signal due to the Fo¨rster’s resonance energy transfer (FRET) between the fluorophore and the quencher, as depicted in Figure 2. Here FRET is a fluorescence resonance or electronic energy transfer process between two chromophores, where a donor chromophore, initially in its electronic excited state, may transfer energy to an acceptor chromophore (in proximity of typically less than 10 nm) through a non-radiative dipole –dipole coupling as detailed in ‘Theory of resonance energy transfer’. With radiation, the phosphodiester backbone can break within the single-stranded DNA, giving rise to a spatial separation of the intra-molecular fluor and quencher molecules, thereby disrupting the 21

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OVERALL METHODOLOGY

range does not span all possible energies and particle types as summarised by the International Atomic Energy Agency: ‘No dosimeter is water or tissueequivalent for all radiation beam qualities’ (in reference to X ray, gamma and neutron exposures)(4). In comparison, biological dosemeters use cellular response mechanisms to determine absorbed dose. Using tissue as a dosemeter eliminates the need to convert between measured dose and tissue dose, making biological dosemeters useful for all radiation qualities. However, the complicated nature of a cell could inhibit the sensitivity of these dosemeters (largely due to DNA repair mechanisms). A typical biological dosemeter is useful above a relatively high dose threshold of 0.2 Gy, or 0.5 –1 Gy in emergency triage scenarios (comparable with a LD50 of 3 –4 Gy)(5). Two common techniques of measuring dose with biological dosemeters are dicentric assay and the micronuclei assay, both being the consequence of chromosomal aberrations(6). A chromosomal aberration is formed when a cell is irradiated during early interphase causing DNA breaks. After synthesis, the damaged chromosomes form dicentrics and/or overlapping rings (chromosome aberrations) that are visible under a microscope(7). The number of aberrations per cell is dose-dependent. While inorganic dosemeters are typically capable of sensitive low-dose measurement, they are not always able to measure dose exposure for all radiation types. In contrast, biological dosemeters can accurately measure received doses from all radiation types; however, their sensitivity and the analysis time and equipment required for readout limit their use as personal monitoring devices. The proposed DNA dosemeter combines the advantages of both inorganic and biological dosimetry, and aims to accurately measure dose exposure for all radiation types with a lower-detection sensitivity of several 10’s of mGy up to 10 Gy in a compact device for personal use. This paper reviews the recent development of the DNA dosemeter and initial testing with the construct in low linear energy transfer radiation fields.

T. WOOD ET AL.

fluorescence quenching mechanism. A fluorescent signal is now produced, providing evidence that a single strand break (SSB) in the DNA molecule has occurred (Figure 3). This technique specifically measures the first SSB in a given strand and not any subsequent ones in the strand. However, since multiple breaks are rarer events, it is expected that the number of SSBs can be reasonably represented by the fluorescence signal.

the donor molecule (D) to the acceptor molecule (A) is given by   1 Ro 6 ð1Þ to R where to is the radiative lifetime of the donor and R is the intermolecular separation distance. The radiative lifetime can be indirectly measured via the empirical fluorescence lifetime t ¼ to fD, where fD is the fluorescence yield. Ro is known as the ‘Fo¨rster parameter’, which characteriszes the strength of the dipole –dipole contributions to energy transfer. Selecting a donor with a short lifetime can increase the efficiency of energy transfer. The parameter Ro contains information about the spectral overlap between the donor emission and acceptor absorption spectra (DNA dosemeter construct). The R 26 term is the reason that FRET is dubbed a ‘spectroscopic ruler’, i.e. when two FRET chromophores double their separation distance, FRET efficiency drops to 1/64th of the initial state. Hence, the detector configuration needs to be considered when testing the experimental efficiency of the system so that optimisation of the donor– acceptor pair, as well as the distance between them, will help lower the limit of detection (LOD). PDA ¼

Theory of resonance energy transfer

DNA dosemeter construct The DNA molecule is a single strand of thymine bases labelled with a chromophore on each end. As mentioned, a fluorescence complex (FAM) (i.e. reporter R molecule in Figure 2) is covalently bound to the DNA’s 50 end, and acts as the donor molecule in the FRET process. The DNA’s 30 end is labelled with a (BHQ) (i.e. quencher Q in Figure 2), which act as the acceptor molecule. When excited, the FAM transfers energy to the BHQ through FRET

Figure 2. Fluorescence quenching mechanism of the duallabelled oligo. The reporter (R) fluorescence is quenched via FRET by the quencher (Q).

Figure 3. Radiation introduces SSBs in the DNA so that the fluor/quencher pairing is disrupted by spatial separation. The quenching effect on the fluor is disabled and fluorescence is now reported from the broken DNA molecule.

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The DNA dosemeter is based on resonance energy transfer (RET), by which the molecular electronic excitation energy of a donor molecule (D) is redistributed to neighbouring acceptor molecules (A) through non-radiative dipole –dipole interactions. RET occurs in many photosensitive and energy harvesting systems following ultraviolet (UV)/visible light excitation including, for example, photosynthetic chlorophyll molecules(8). FRET is based on the RET process with nanometre distances between the chromophore donor and acceptor molecules. As given by Equation (A.26) (see derivation in Appendix A), the probability of energy transfer from

DUAL-LABELLED OLIGONUCLEOTIDE DNA DOSEMETER

occurring after radiation-induced damage is not precisely zero as some FAM chromophores may drift close enough to secondary BHQ chromophores for FRET to occur (i.e. with a probability PD – 2). Figure 5 depicts four possible scenarios following exposure to ionising radiation: (i) the molecule is undamaged and FRET occurs, (ii) the molecule is undamaged and FRET does not occur, (iii) the molecule is damaged and FRET occurs and (iv) the molecule is damaged and FRET does not occur. If the system contains N DNA molecules and the number of damaged molecules is proportional to the absorbed dose D, with a proportionality constant b, the number of undamaged and damaged molecules (Nu and Nd, respectively) can be expressed as: Nu ¼ Nð1  bDÞ

ð2aÞ

Nd ¼ N bD

ð2bÞ

where b will depend on the target size (i.e. DNA length) and the free radical concentration in the cellular environment. The number of molecules in each of the four scenarios in Figure 5 is N1 ¼ Nð1  bDÞPDA

ð3aÞ

N2 ¼ Nð1  bDÞð1  PDA Þ

ð3bÞ

N3 ¼ N bDPD2

ð3cÞ

Figure 4. Emission and Absorption Spectral Properties of the FAM and BHQ molecules. Data image obtained from BioSearch Technologies(14).

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with a fraction dependent on the overlap of donor emission and acceptor absorption spectra, the lifetime of the BHQ donor, and the distance R between the chromophores (Theory of resonance energy transfer). Figure 4 shows the normalised absorption and emission spectra for the FAM and BHQ chromophores. The FAM emission spectrum represents the term fD in Equation. (A.25) of Appendix A and has a maximum intensity at 520 nm. The BHQ absorption spectrum represents sA(E)/smax, where s(E) is the absorbance cross section as a function of energy, and smax is the maximum cross section at 535 nm. The FAM molecule has significant spectral overlap with the BHQ absorption spectrum; however, the BHQ spectrum is not as sharp as that of the FAM and is blurred into higher and lower wavelengths reducing the maximum value of PD – A in Equation (1). The thymine strand tethers the FAM donor and BHQ acceptor chromophores close together, allowing the molecules to undergo FRET. As discussed in ‘Overall methodology’, ionising radiation breaks the DNA strand and the chromophores drift apart until they reach an equilibrium state. Due to the R 26 energy transfer dependence, PD – A approaches zero as the FAM and BHQ chromophores separate. In this case, the molecular electronic excitation energy of the donor FAM chromophore is converted to a visible photon with the emission spectrum in Figure 4. However, the probability of FRET

T. WOOD ET AL.

N4 ¼ N bDð1  PD2 Þ

scattered photons from the sample container or surrounding environment. Once a photon is emitted, it will either undergo photoelectric absorption by a surrounding BHQ molecule or escape from the container. Accordingly, the probability of a photon being detected Pdet is determined by the probability that a signal photon is emitted Pg in Equation (5), and the probability of escape from the container Pesc:

ð3dÞ

Scenerios 2 and 4 lead to the emission of a photon, where the total number of photons released (Ng) in Figure 5 is Ng ¼ N2 þ N4 ¼ Ngo þ N aD

ð4Þ

The second relation is derived using Equations (3b) and (d). The quantity Ngo ¼ N(12PD – A) is the number of photons emitted before exposure and a ¼ b(PD – A 2PD – 2) is the rate of change in the photon count with dose in units of Gy21. Equation (4) can also be expressed in terms of the probability that a molecule will emit a photon following a radiation dose D:

Pdet ¼ Pg Pesc 1det

ð6Þ

Here 1det is the detector efficiency, which depends on the geometry of the system and the photomultiplier gain. For example, the probability of escape from a detector with a cylindrical geometry is(9):

Ng ð5Þ ¼ Pg0 þ aD; N where Pgo is the probability that a non-irradiated molecule will emit a photon. Pg ¼

Pesc ¼

System efficiency and signal-to-noise ratio The lower LOD will be determined by the efficiency of the system and the signal-to-noise ratio (SNR). The noise is a function of system scatter and electronic noise. To maximise the SNR, the photon counter should not receive a large amount of

a a i 2a h a a a 2 K1 I1 þ K0 I0 1 3l l l l l l a a K1 ða=lÞK1 ða=lÞ þ  K0 I1 a=l l l a a þ K1 I0 ð7Þ l l

which depends on the ratio of the radius of the cylinder a to the mean free pathlength l for photon absorption. For this given geometry, modified Bessel functions of the first (K) and second kind (I ), arise. 24

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Figure 5. Possible scenarios for FRET or photon emission after sample has been exposed to ionising radiation.

DUAL-LABELLED OLIGONUCLEOTIDE DNA DOSEMETER

spectrum peaked at 2 MeV and with a maximum photon energy of 6 MeV. The samples were irradiated in phantom to ensure full dose buildup and backscatter in the dose range from 100 mGy to 10 Gy.

The signal strength (S) (as a count rate per Gy of radiation) is given as S ¼ NPesc 1det

dPg dD

ð8Þ

EXPERIMENTAL RESULTS AND DISCUSSION

which is evaluated using Equation (5) as: S ¼ N bðPDA  PD2 ÞPesc 1det   b Ro 6 N Pesc 1det to R

ð9Þ

The parameter b implicitly depends on the DNA length and hence the separation distance R (see Figure 1). For the second equality in Equation (9), it is assumed that PD – 2 is negligible. On the other hand, from Equation (8), the signal response is proportional to: (i) the number of target DNA molecules N (i.e. sample concentration), (ii) the Fo¨rster parameter Ro and (iii) the probability of escape Pesc. However, it is inversely proportional to the radiative lifetime to as well as the distance R. Hence, since the overall dependence of the signal strength is in opposite directions for these combined parameters as a function of R, the dosemeter signal can be optimised through experimental testing (see Experimental results and discussion). Sample irradiation All oligonucleotide molecules were purchased from Biosearch Technologies Inc., Novato, CA. The oligonucleotide samples were prepared for irradiation by suspension in ultrapure water at a desired concentration at Defence Research & Development Canada, Ottawa (DRDC-O). From this solution, samples of 150 ml were pipetted into a 96-well plate with a non-binding surface. The samples were then irradiated at 18 Gy h – 1 with a 60Co source. The fluorescence analysis was performed using a BioTeK Synergy HT Multimode Microplate at DRDC-O. Irradiations were performed with 60Co gamma rays and X rays from a medical linear accelerator. The 60Co gamma beam at the DRDC-O can be considered a monoenergetic field of 1.25 MeV photons (resulting from beta decay to an excited state of 60Ni and subsequent release of two gamma rays of energy 1.17 and 1.33 MeV). Additional experiments with X rays using a linear accelerator (linac) were conducted at the Cancer Centre of Southeastern Ontario (CCSEO) for a reproducibility check and to investigate the lower LOD. The linac accelerates electrons through a 6 MV potential to attain an energy of 6 MeV at the end of the accelerating waveguide. At that point the electrons bombard a tungsten target, creating a spectrum of X rays through bremsstrahlung radiation with a 25

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In these experiments, the control and dual-labelled oligodeoxynucleotides were exposed to gamma radiation of 1, 5, 10 and 100 Gy. Photo-bleaching effects were investigated and are found to be insignificant compared with the number of strand breaks that directly occur by radiation. A linear increase in fluorescence from the 0 to 1 mM DNA concentrations at 100 Gy was observed as shown in Figure 6. A standard curve for the FAM fluorescence was determined by using a 0–1 mM concentration range of 50 -FAM-dT(10)-30 in water. Using serial dilutions of 50 -FAM-dT(10)-30 , the LOD was determined to be 3500 pg ml21 or 0.01 mM using 150 ml for the Bio-Mek Synergy HT Fluorescence Spectrometer. Further experiments were performed using higher DNA concentrations since more DNA targets are expected to yield a greater sensitivity in accordance with the theory of ‘System efficiency and signal-tonoise ratio’. Thus, a 66.6 mM DNA concentration was considered with dual-labelled 10, 20 and 30mers. These samples were exposed to gamma radiation of up to 1 Gy (in 0.1 Gy increments). However, no detectable signal was observed in this dose range (data not shown). A slight decline in fluorescence due to photo-bleaching effects(10), with repeated spectrometer scans, also occurred in these experiments. The 50 -FAM-dT(10)-BHQ1-30 , 50 FAM-dT(20)-BHQ1-30 and 50 -FAM-dT(30)-BHQ130 at 66.6 mM were then exposed to higher levels of gamma radiation at 10, 50 and 100 Gy, where doses greater than 10 Gy were detected in all oligodeoxynucleotides (data not shown). A more sensitive fluorescence spectrometer was designed and constructed by the Centre d’optique, photonique et laser (COPL) to detect smaller changes in fluorescence. This development proved to be significant for lower dose-detection capability. The fluorimeter (Figure 7) was initially calibrated with aqueous fluorescein isothiocyanate (FITC) and FAM-dT(10) solutions contained in disposable, low-cost plastic cuvettes (Eppendorf Uvettes). Figure 8 shows that the calculated LOD3s is 13 pM (1.310211M) for aqueous FITC solutions and 23 pM (2.310211 M) for FAM-labelled oligos, the latter being at least three orders of magnitude more sensitive than the commercial Bio-Mek Synergy HT Spectrometer used at DRDC-O for the initial series of experiments. The detection platform developed by COPL provides: (i) a better mechanical stability allowing for a lower measurement variability from sample-to-sample

T. WOOD ET AL.

where Io is the incoming beam intensity. No significant degradation in the BHQ molecule was observed for a dose up to 5 Gy. The mean free path (l ) of the 535-nm photon in the BHQ solution can be determined from the expression

and over several hours of operation; (ii) a customised user interface (written in LabView#) that is more efficient and accurate with respect to instrumental parameter control and data collection/archiving (i.e. pre-defined data sampling time, automatic calculation and storage of mean, standard deviation on measured signal, excitation laser power setting/ control, automated fluorescence filter selection and experimental parameter settings logged in a separate data file for traceability). Irradiated samples for the 0.5 mM 50 -FAMdT(10)-BHQ1-30 indicated that 1 Gy is now detectable with this configuration. Such testing at a higher DNA concentration of 1 mM of 50 -FAM-dT(10)BHQ1-30 showed that the fluorescence signal is at the top end of the dynamic fluorescence range of the FAM-Fluorimeter. Subsequently, three replica aliquots of the DNA molecule 50 -FAM-dT(10)-BHQ130 were prepared at 0.5 mM concentrations in ddH2O. These DNA samples were independently exposed to a 60Co source over a dose range of 100 mGy– 10 Gy, and assayed for fluorescence using 50 ml samples in the FAM fluorimeter. A distinct linear increase in fluorescence with absorbed dose was observed as shown in Figure 9. The detection response was corrected for a loss of absorbance of the BHQ (Figure 10) and fluorescence of the FAM reporter (Figure 11) molecules due to the possible destruction of these large molecules by radiation. The experiment in Figure 10 investigated the absorbance characteristics of the BHQ following exposure. The absorbance (A) was calculated by passing a 535-nm photon beam through an 8 mM BHQ solution and measuring the transmitted beam intensity (I ) via:   I A ¼  log Io

l¼

d A lnð10Þ

ð11Þ

where d is the distance travelled by the photon beam (¼1 cm). For an average measured absorption fraction of 0.227, a mean free path of 1.91 cm is evaluated from Equation (11) for the given system. This determination is required for the calculation of the escape probability (Pesc) in Equation (7) for this containment system. The degradation of the fluorescence response with dose is illustrated in Figure 11 for the FAM serial dilution. These results also showed no significant degradation with a dose up to 1 Gy. The results of these experiments were important to validate the theoretical model of the DNA dosemeter as described in ‘Overall methodology’. The dosemeter was found to have a linear signal response with dose in Figure 9, as predicted in Equation (9). These experiments were able to detect dose levels as low as 100 mGy. Since further testing showed no significant degradation of the BHQ or FAM molecules with dose, the change in fluorescence signal can therefore be attributed to the breakage of the DNA strands (and not due to a change in fluorescence properties for those parameters in Equation (5)). The sensitivity of the device is directly related to the LOD for the fluorescent signal. This work has particularly shown that the dosemeter response yields 1.21023 SSBs.Gy21.base pair (bp)21 (Figure 9), or equivalently 1 SSB in 800 DNA molecules at a dose of 1 Gy.

ð10Þ 26

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Figure 6. Comparison of 50 -FAM-dT(10)-BHQ1-30 fluorescence over 1, 5, 10 and 100 Gy gamma exposure.

DUAL-LABELLED OLIGONUCLEOTIDE DNA DOSEMETER

The results from the two experiments at DRDC-O and CCSEO are compared in Figure 12 over the dose range of 0–1 Gy. Both sets of results confirm the linear relationship between fluorescence signal and dose as predicted with Equation (9). From a regression analysis with 95 % confidence limits, the slopes from these experiments give similar dose response of the fluorescence between the cobalt experiment (i.e. 3.07+0.24102 photons.s21.mGy21) and linac experiment (i.e. 2.62+0.17102 photons.s21. mGy21). However, the intercepts are statistically different from each other, which represent the amount of background signal present in the system (see

System efficiency and signal-to-noise ratio). This signal is due to the portion of the molecules not undergoing FRET, (1 2 Pg), scatter off of the containment system and other background interferences. The COPL-designed fluorimeter allowed a reduction in the background signal from scatter in between these sets of experiments as evident in the figure. A statistical analysis was further performed to determine the lower level of detection with error bars for one standard deviation as shown in Figure 12. A significance test ( p , 0.05) between the control (0 Gy) and a given measured data point was considered with a standard one-way analysis of variance analysis 27

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Figure 7. A customised fluorimeter to measure FAM fluorescence of the dual-labelled oligos in 50 ml solution using disposable Eppendorf Uvettew Sample Cuvettes. Excitation : ArþCW Laser, 488 nm (typical excitation power¼30 uW); M1,2, turning mirrors; PM, power monitor; VNDF, variable neutral density filter; A1,2, apertures; BD , beam dump; S, sample holder, 1 cm pathlength; L1,2, lenses; F1 , bandpass filter, Semrock FF01-528/38; PMT, photon-counting PMT, Hamamatsu H7421-40.

T. WOOD ET AL.

Figure 9. Dual-labelled oligo dose response curve at 0.5 mM irradiated with 0 –10.0 Gy gamma rays (corrected for FAM and BHQ destruction). The experiment was performed in triplicate. An error propagation was performed for correction of these data.

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Figure 8. Calibration curve of FAM-labelled oligo and aqueous FITC in 50 ml Eppendorf Uvettes. Error bars are shown for the individual data points.

DUAL-LABELLED OLIGONUCLEOTIDE DNA DOSEMETER

Figure 11. FAM fluorescence response with dose. The experiment was performed in triplicate and the error bars are for one standard deviation.

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Figure 10. BHQ absorbance with dose. The experiment was performed in triplicate and the error bars are for one standard deviation.

T. WOOD ET AL.

followed by a Dunnett’s post hoc test. This analysis showed that for the 60Co experiment (n ¼ 3), the 250 mGy data point is the first population (dose) to be statistically different from the control (0 Gy), while for the linac experiment (n ¼ 6), the 373.3 mGy data point is the first statistically different point. Hence, the lower level of detection based on this statistical analysis is 250 mGy. This dosemeter is currently at the sensitivity of cytogenetic techniques (0.1–0.2 Gy) but it is expected that further optimisation of the exposure/detection platform is expected to result in an even lower LOD (see Concluding remarks).

in ‘System efficiency and signal-to-noise ratio’ to lower the SNR for the system, which includes the DNA length and solution concentration. Further, experiments will be conducted to investigate the response of other acceptor and donor molecules to ascertain if the FAM and BHQ pair are ideal for the proposed DNA dosemeter construct. The dosemeter will also be tested in different radiation fields to determine its response and particle independence, including a greater range of photon energies and other particle types (e.g. electrons, neutrons, protons and alpha particles and heavy ions) as well as its use for mixed-radiation fields. This work describes a preliminary study on the feasibility and development of a prototype DNA dosemeter. Current work is also focusing on the development of a field-deployable unit (with reader); however, further investigation is needed for its practical use in the field. In particular, additional study is required with regards to: (i) the life-span and stability of the dual-labelled oglionucleotide construct over long periods of time; (ii) its application in a wet versus dry form; (iii) effects of microbial contamination and (iv) the influence of temperature. Once completely developed, this novel technology may be useful for military and counter-terrorism activities where complicated fields are present. However, the device needs to be tested for radiation of different quality and in mixed fields. The

CONCLUDING REMARKS In summary, the results suggest that this very simple reporter-target-quencher DNA molecule 50 -FAMdT(X)-BHQ1-30 (where X 10) has great potential as an accurate and quantitative means of measuring radiation dose via single-stranded DNA breaks. Radiation-induced SSBs within the DNA result in a quantifiable fluorescent signal. Proof of concept has been achieved over 250 mGy –10 Gy dose range in radiation fields from 60Co, with similar results seen using a linear accelerator X-ray source. Further refinements to both the molecule and the exposure/ detection platform may lead to a lower LOD. Moreover, future experimental work is planned to determine optimal values of the various parameters 30

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Figure 12. Fluorescence response of the DNA dosemeter with dose (the error bars are for one standard deviation).

DUAL-LABELLED OLIGONUCLEOTIDE DNA DOSEMETER 8. Andrews, D. L. Mechanistic principles and applications of resonance energy transfer. Can. J. Chem. 86(9), 855–870(16) (2008). 9. Case, K. M., De Hoffman, F. and Placzek, G. Introduction to the Theory of Neutron Diffusion. Los Alamos Scientific Laboratory (1953). 10. Benchaib, A., Delorme, R., Pluvinage, M., Bryon, P. A. and Souchier, C. Evaluation of five green fluorescence-emitting streptavidin-conjugated fluorochromes for use in immunofluorescence microscopy. Histochem. Cell Biol. 106, 253–256 (1996). 11. Takada, M., Lewis, B. J., Boudreau, M., Al Anid, H. and Bennett, L. G. I. Modelling of aircrew radiation exposure from galactic cosmic rays and solar particle events. Radiat. Prot. Dosim. 124(4), 289– 318 (2007). 12. Struve, W. S. Theory of electronic energy transfer. Anoxygenic Photosynthetic Bacteria. 2, 297–313 (1995). 13. Gottfried, K. Quantum Mechanics Vol: I Fundamentals. Benjamin (1966). 14. Biosearch Technologies Inc, Novato, CA (2010).

ACKNOWLEDGEMENTS

APPENDIX A. DERIVATION FOR THE PROBABILITY OF RESONANCE ENERGY TRANSFER

The authors acknowledge discussions with C. Vachon (National Research Council—Industrial Materials Institute).

The classical electrostatic potential energy U of a two point charge system (q1, q2) where the charges are separated by a distance (r) is given by the Coulomb energy:

FUNDING This work was sponsored by the Chemical, Biological, Radiological-Nuclear and Explosives Research Technology Initiative (CRTI), project # 06-0186RD.

U¼

q1 q2 r

ðA:1Þ

For a system of two molecules (donor D and acceptor A), the potential energy is given by the pair-wise summation over all nuclear and electronic charges:

REFERENCES 1. Kron, T., Butson, M., Wong, T. and Metcalfe, P. Readout of thermoluminescance dosimetry chips using a contact planchet heater. Aust. Phys. Eng. Med. 16, 137 –142 (1993). 2. Soares, C. G. Radiochromic film dosimetry. Radiat. Meas. 41, 100– 116 (2007). 3. Zhou, D., Semones, E., Gaza, R. and Weyland, M. Radiation measured with TEPC and CR-39 PNTDs in low earth orbit. Adv. Space Res. 40(11), 1571–1574 (2007). 4. International Atomic Energy Agency. Radiation oncology physics handbook. IAEA, Pub 1196 STI/PUB/ 1196. Available from: http://www-naweb.iaea.org/ nahu/dmrp/syllabus.shtm (2005). 5. International Atomic Energy Agency. Biodosimetry application in radiation emergencies: a manual. Technical Report 405. IAEA (2001). 6. McNamee, J. P., Flegal, F. N., Greene, H. B., Marro, L. and Wilkins, R. C. CBMN: validation of the cytokinesis-block micronucleus (CBMN) assay for use as a triage biological dosimetry tool (2009). Radiat. Prot. Dosim. 135(4) 232– 242 (2009). 7. Bender, M. A. Chromosome aberrations in irradiated human subjects. Ann. N.Y. Acad. Sci. 114, 249– 251 (1964).

Um ¼

X X qi qj i

j

rij

ðA:2Þ

Equation (A.2) can be broken into the molecular self energy (Us) of the donor (D) and acceptor (A), and the intermolecular interaction energy (Ui) between D and A: Um ¼ Us þ Ui Us ¼

Ui ¼

X qi qj X qi qj þ r r i;j[D ij i;j[A ij X qi qj i[D j[A

rij

ðA:3Þ ðA:4Þ

ðA:5Þ

In a FRET system, the intermolecular separation (R) is large compared with the molecular size and there is no significant overlap of the electronic wave functions. Under this condition, it is convenient to 31

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dosemeter could also find broad application for personnel working in nuclear plants or research, medical facilities and industrial settings where personal dosimetry is required for routine monitoring in support of radiation protection requirements. There could also be additional applications in the aerospace industry for aircrew radiation exposure assessment (11), and space exploration where questions regarding the cumulative effect of radiation are critical for envisaging long-term missions in space(3). This technology also lends itself to UV monitoring, which has applications in the water purification industry, occupational/ recreational settings and for the monitoring of UV levels for prevention of sunburns (which could lead to possible skin cancers). Additionally, this dosemeter could be used as a research tool as the basis for an assay to test putative radio protective agents/antioxidants since there is no DNA repair mechanism involved with this device.

T. WOOD ET AL.

expand the interaction energy term in a (1/R) power series because higher-ordered terms can be neglected: Ui ¼

1 X Un n¼1

Un ¼

¼

l, n1 X X

m ð1ÞlD ðn  1Þ!Qm lD ðDÞQlA ðAÞ

lD ¼0 m¼l,

½ðlD mÞ!ðlD þmÞ!ðlA mÞ!ðlA  mÞ!1=2

ðA:14Þ

where R is the vector between the centre of the D and A molecules, orientated along the z-axis. Equation (A.14) is further simplified when the electric dipole –dipole orientation factor is introduced:

ðA:6Þ

Rn

ˆ ˆ mD  mA  3ðmD  RÞðm A  RÞ 3 R

ˆ m ˆ ˆD m ˆ A  3ðm ˆ D  RÞð ˆ A  RÞ k¼m

ðA:15Þ

ðA:7Þ Ui ¼

Q1 1 ¼ Q01

mx  i my pffiffiffi 2

Q11 ¼ 

2p PD!A ¼  jkD AjUi jDA lj2 rðEÞ h

ðA:10Þ

Expanding Equation (A.7) leads to the following Un terms: U1 ¼ qD qA

ðA:11Þ

U2 ¼ qD mAz þ qA mDz

ðA:12Þ

jkD AjUI jDA lj2 ¼

k2 jkD jmD jDlj2 jkAjmA jA lj2 R6 ðA:18Þ

Integrating Equation (A.18) over all allowable energy states yields:

U3 ¼ qD Q02 ðAÞ þ qA Q02 ðDÞ

 ð ð 2pk2 1 PD!A ¼  6 dE d1A pA ð1A ÞjkAjmA jA lj2  h R gD gA ð  d1 D p D ð1 D ÞjkD jmD jDlj2

1 1 1  Q1 1 ðDÞQ1 ðAÞ  Q1 ðAÞQ1 ðDÞ

 2Q01 ðDÞQ01 ðAÞ

ðA:17Þ

where * represents an excited state, E is the initial donor excitation energy, r(E) is the energy density of allowable states and É is Plank’s constant. In the case of neutral donor and acceptor atoms, and considering only the first three leading terms of Equation (A.6), the bracket of Equation (A.17) is easily separable into donor and acceptor contributions(12):

ðA:9Þ

mx þ imy pffiffiffi 2

ðA:16Þ

In a RET system, the probability of energy transfer from the donor molecule (D) to the acceptor molecule (A) is given by Dirac’s Golden Rule(12):

ðA:8Þ

¼ mz

mD mA k R3

ðA:13Þ

For the case of neutral donor and acceptor atoms, qD ¼ qA ¼ 0, and the leading three Ui terms reduce to:

ðA:19Þ

1 1 1 0 0 Q1 1 ðDÞQ1 ðAÞQ1 ðAÞQ1 ðDÞ2Q1 ðDÞQ1 ðAÞ Ui ¼ R3

where 1 is the respective ground state energy of the molecules, p is the energy distribution, and g is the state degeneracies. The probability of photon emission (AD) by an excited donor D* is given by(12)

¼

1 2 ðmDx  imDy ÞðmAx  imAy Þ þ 12 ðmDx  imDy ÞðmAx  imAy Þ R3

¼

 2mDz mAz

4E 3 n3 AD ¼  3 3h c gD

mDx mAx þ mDy mAy  2mDz mAz R3

ð

p D ð1 D Þd1 D jkD jmD jDlj2

ðA:20Þ

and the donor’s radiative lifetime (to) is given by the 32

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The parameter l, is the lesser of lD and lA ¼ (n– 12lD), and Qm l is the tensor component of the electrostatic multipole operator(12, 13). For example, Qoo is the net molecular charge (q) of the molecule and Q1 is the linear Cartesian components of the electric dipole moment operator (m):

DUAL-LABELLED OLIGONUCLEOTIDE DNA DOSEMETER

inverse integral of AD over all energies:

to ¼ Ð

1 AD ðEÞdE

where v is angular frequency. Ro characterises the strength of the dipole –dipole contributions to the energy transfer:

ðA:21Þ



It follows that the donor contribution of Equation. (A.19) reduces to the following expression: ð d1 D p D ð1 D ÞjkD jmD jDlj2 ¼

 c3 g 3h D fD ðEÞ 4E 3 n3 to

Ro ¼

ðA:25Þ

  fD ðvÞsA ðvÞ 3 k 2 Ro 6 dv ; 2 to R v4 ðA:24Þ

ðA:26Þ

Here to is the radiative lifetime and Ro characterises the strength of the dipole –dipole contributions to energy transfer.

33

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PD!A

1=6 fD ðvÞsA ðvÞ d v v4

  1 Ro 6 P random ¼ z}|{ to R D ! A

Combining Equations (A.19), (A.22) and (A.23) yields the simplified probability expression: ð

ð

Equation (A.24) describes the probability of RET between an excited donor molecule and a ground acceptor molecule when the intermolecular separation is greater than the size of the molecules, and each molecule is charge neutral. The k 2 term represents the dipole – dipole orientation factor and averages to 2/3 when the system is composed of many randomly oriented donor– acceptor pairs, leading to

ðA:22Þ

where fD(E) is the normalised donor emission spectra. The molecular absorption cross section sA of the acceptor atom is given by the Einstein formula for electric dipole absorption: ð nE pA ð1A Þd1A jkAjmA jA lj2 ðA:23Þ sA ðEÞ ¼  3ch gA

9c4 k2 ¼ 8pn4 R6 to

3c4 4pn4