Eur. Phys. J. D (2014) 68: 333 DOI: 10.1140/epjd/e2014-40845-8

X-ray radiation of poly-L-arginine hydrochlorideand multilayered DNA-coatings

Agnieszka Stypczynska, Tony Nixon and Nigel Mason

Eur. Phys. J. D (2014) 68: 333DOI: 10.1140/epjd/e2014-40845-8

Regular Article

THE EUROPEANPHYSICAL JOURNAL D

X-ray radiation of poly-L-arginine hydrochlorideand multilayered DNA-coatings�

Agnieszka Stypczynska1,a, Tony Nixon2, and Nigel Mason1

1 The Open University, Department of Physical Sciences, Walton Hall, Milton Keynes, MK7 6AA, UK2 The Open University, Engineering and Innovation, Walton Hall, Milton Keynes, MK7 6AA, UK

Received 31 December 2013 / Received in final form 1 April 2014Published online 3 November 2014 – c© EDP Sciences, Societa Italiana di Fisica, Springer-Verlag 2014

Abstract. The aim of this work was to determine the chemical changes induced in thin films of the drypolypeptide poly-L-arginine hydrochloride and its mixture with calf thymus deoxyribonucleic acid (DNA)during 5 h of soft X-ray exposure. The physical and chemical effects of the soft X-ray irradiation werestudied using X-ray Photoelectron Spectroscopy (XPS). Analysis of O1s, N1s and C1s features in XPSspectra reveals the existence of several routes of radiation-induced decomposition and shows quantitativeand qualitative changes.

1 Introduction

Ionizing radiation used in X-ray imaging, radiotherapyand positron emission tomography produces a range ofstructural and chemical modifications within biologicaltissue. In order to understand such modifications (and anylong term consequences to the health of the patient), itis necessary to quantify the underlying physical, chemicaland biological effects of such ionizing radiation on the cellswithin the tissue. A large number of studies have focusedon investigating the interaction of high energy photons(X-ray, UV) [1–4] and various types of particulate radia-tion (ions and electrons) [5–8] with the deoxyribonucleicacid (DNA) molecule, which has been shown to be themost radiation sensitive of the cellular biomolecules. How-ever, to date, there have been fewer studies of how suchDNA damage is modified by the presence of surroundingspecies, particularly proteins. As a first step in exploringhow the presence of proteins and amino acids influencesthe damage in cellular DNA, the authors have investi-gated the modifications induced in dry biomolecular filmsof poly-L-arginine hydrochloride and its complex with calfthymus DNA.

Amino acids are known to fulfill an important role inthe human body, because they are the building blocks ofany biomolecular system being bounded by the amide link-age in peptides and proteins of living organisms and regu-lators of body functions. Moreover, they are componentsof skin, muscles, tendons, nerves and blood, also enzymes,

� Contribution to the Topical Issue “Nano-scale Insights intoIon-beam Cancer Therapy”, edited by Andrey V. Solov’yov,Nigel Mason, Paulo Limao-Vieira and Malgorzata Smialek-Telega.

a e-mail: a.stypczynska@open.ac.uk

antibodies and many hormones. The content of a proteinand its sequence of amino acids are determined in the gene;furthermore, when the functionality of particular aminoacid or protein is distorted it leads to consequences foran entire living organism [9,10]. Due to their sensitivityto ionizing radiation they are widely used in mutagene-sis and radiation protection studies as convenient models.However, quantification of the chemical changes inducedwithin the amino acid structure by ionizing radiation re-mains to be understood.

In this approach, X-ray Photoelectron Spectroscopy(XPS) has been applied to investigate transformations inthin films of the polypeptide poly-L-arginine hydrochlo-ride and its mixture with calf thymus DNA, which aregenerated in specific way to represent samples with nostructural and free water. XPS also provides informationabout the molecular environment which complements thequantitative determination of elemental surface composi-tion [11–13]. XPS has also been applied to study aminoacids and peptides in the form of thick powder coat-ings [14–17] and crystallized layers from aqueous solu-tions [18]. In all cases it was observed that such moleculesundergo chemical transformations under prolonged or in-tense exposure to X-rays. Radiation-induced modifica-tions of DL-lysine were noted by Bozack et al. [18], whoconcluded that the changes were due to photolytic (ratherthan thermal) decomposition. Bozack et al. also hypothe-sized that such changes occurred by decarboxylation ofthe compound, preceded by the formation of a neutralmolecule from a zwitterion and the loss of a hydrogenatom from the -COOH group. However, the results ofZubavichus et al. [15] did not confirm this hypothesis.Rather, in the case of alanine, serine, cysteine, asparticacid and asparagine, they showed that the protonation of-COOH group is not consistent with deprotonation of the

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Fig. 1. An example of general structure of α-amino acid basedon arginine, with the amino group on the left and the car-boxyl group on the right (drawn in ACD/Chem Sketch free-ware, ACDLABS 11.0).

-NH2 group. Moreover, it was reported that modificationsoccur in the form of dehydration, decarboxylation, decar-bonylation, deamination and desulfurization. A strong in-dication of chemical degradation of glycine after X-rayradiation was also observed by Tzvetkov and Netzer [19],with the identification of decomposition products such asCO2, H2, H2O and NH2. Their results showed that thetwo N1s components change significantly and in oppositedirection, with a declining intensity of NH+

3 and increasingintensity of the neutral amino group.

The different structural and chemical modificationsthat can be induced by non-monochromatic X-rays in nu-cleobases and in the DNA helix has been reported byPtasinska et al. [20], where decreases in the peak areasfor oxygen and phosphorous in C-OH and the phosphategroup, respectively, showed an induced break in the back-bone of deoxyribonucleic acid, while the reductions ob-served in the intensity of the amino and carbonyl groupswere assigned to the fragmentation of nucleobases. Previ-ous studies also reported 1s core electron binding energiesfor specific elements of different purine or/and pyrimidinenitrogenous bases, e.g., O1s spectra were revealed for cy-tosine [21] and thymine [21], N1s for adenine [22] and C1sfor condensed phase of all nucleobases [17,23].

XPS is also an attractive tool for the investigation ofadsorbed biological material on solid surfaces [24], provid-ing information on the chemical composition of such filmsand it has proven to be a valuable technique for exploringthe immobilization of biologically interacting molecules,e.g., hemoglobin, on a solid surface (with important clini-cal applications, especially in implantology). XPS studieshave also explored the kinetic mechanisms of protein films(e.g., the pioneering work of Ratner et al. [25–27]). Ex-ploring the relationship between the surface chemistry ofsynthetic materials and their biological interactions withcell components, allows predictions about the nature ofadsorbed compounds.

2 Experimental section and methods

2.1 Preparation of poly-L-arginine hydrochloride films

Commercially available powder of poly-L-arginine hy-drochloride (Fig. 1) (15 000–70000 Daltons, purchasedfrom Sigma-Aldrich) was used without further purifica-tion in these experiments. Poly-L-arginine hydrochloridewas dissolved in autoclaved water at a stock concentrationof 1 mg/ml. Before sample deposition, all the substrates

were cleaned by inserting them into a “Piranha solution”of 30% hydrogen peroxide and 98% sulphuric acid for halfan hour. Piranha solution must be handled with care asit is a well known oxidizer, which removes most organicmatter and also hydroxylates the surface, making it ex-tremely hydrophilic. Subsequently, the silicon substrateswere washed in high quality ultra-pure water and driedusing a pressurized nitrogen stream. Then a 20 μl dropof the prepared solution was placed onto a chemicallycleaned and dried silicon substrate (0.7 × 0.7 cm) pur-chased from Compart Technology LTD, giving the aver-age sample thickness of 0.3 μm, within 24% error, whichwas left to dry in a fume cupboard for 60 min.

2.2 Preparation of layer by layer samples of drypoly-L-arginine hydrochloride/DNA

The fabrication of multilayered coatings of poly-L-argininehydrochloride/DNA was performed by electrostatic self-assembly (ESA), also called layer by layer assembly, usingcalf thymus DNA (purchased from Sigma-Aldrich) as theanionic polyelectrolyte and poly-L-arginine as the cationicpolyelectrolyte. Multilayers can be generated through theadsorption of oppositely charged polyelectrolytes via elec-trostatic interactions. Previously cleaned and dried siliconsubstrates were immersed gently in an aqueous solutionof poly-L-arginine hydrochloride (0.1 mg/ml) for 30 min.This time interval is required for the adsorption of thefirst cationic polyelectrolyte layer onto the substrate [28].Afterwards the wafer was washed in ultra-pure waterfor 5 min and dried with a nitrogen stream. Such rinsingof the substrate removes weakly bonded molecules. Thesubstrate was then submerged into a calf thymus DNA so-lution (1 mg/ml) for 7 min and again cleaned in ultra-purewater for 5 min before being dried with gaseous nitrogen.In this way a poly-L-arginine and DNA first bilayer wasgenerated, the process was then repeated until 5 double-layers were formed.

2.3 XPS apparatus

X-ray photoelectron spectra were recorded within a load-locked Kratos XSAM 800 apparatus equipped with a dualanode X-ray source, a hemispherical electrostatic electronenergy analyzer and a channeltron electron multiplier. Thespectra were obtained using Mg Kα (1253.6 eV) radiationin the medium pass energy and fixed analyzer transmis-sion mode, also known as constant analyzer energy. Fora Mg anode the conversion to X-rays is estimated to bearound 0.5% giving a total X-ray emission of 1.3 W. Thesamples were approximately 1.5 cm± 0.3 cm from the an-ode. This figure is an estimate because the anode of theX-ray gun is offset and the exact location of the activearea of the anode can only be estimated since the unit issealed. Assuming X-rays are uniformly distributed (a rea-sonable assumption given that the X-rays are producedvery close to the surface of the anode) this gives a flu-ence of approximately 60 mW cm−2 ± 40 mW cm−2. It is

Eur. Phys. J. D (2014) 68: 333 Page 3 of 10

also worth noting that this is not a monochromatic sourcebut includes other weak lines and bremsstrahlung, whichcontribute around 15% [29] of the total X-ray flux. Thefigure of 60 mW cm−2 should be taken as an indicationof the order of magnitude since it has not been verified.The entry slits of the spectrometer were set to 4 mm giv-ing a resolution of approximately 1.1 eV. The high mag-nification analyzer mode was chosen to collect electronsfrom the smallest possible area on the specimen, approx-imately 4 mm2. O1s, N1s and C1s high resolution spec-tra were recorded in energy steps of 0.1 eV. The X-raygun was operated at 13 kV and 20 mA, the source power(260 W) was kept relatively low to minimize sample heat-ing. The typical pressure in the sample analysis chamberwas 10−7–10−8 Pa (10−9–10−10 mbar), as read on a cali-brated ion gauge. Due to charging effects during X-ray ex-posure, the energy axis of XPS spectra is usually shifted toC1s binding energy. Therefore, all spectra were calibratedusing the C1s photoelectron component peak, correspond-ing to the standard hydrocarbon binding energy for C-Cand C-H species of 285 eV [30]. Preparation of thin filmson grounded substrates using silver conductive paint re-sulted in only very weak effects of charging being observedin the present studies.

The acquisition time was chosen as a compromise be-tween the signal-to-noise ratio in the recorded spectra andthe rate of radiation-induced decomposition. In order tomonitor any changes, a series of spectra were recorded con-tinuously for 5 h. At least four cycles with time irradiationfrom 0 to 5 h were recorded for each chemical elementregion for a poly-L-arginine hydrochloride sample andDNA-bi-layer samples – [poly-L-arginine/DNA]5. Aftercalibration, individual elements were identified then back-ground subtracted using a Shirley approximation [31,32].For each element, discrete peak energies were assignedbased on identified chemical shifts [14] then Gaussianpeaks were deconvoluted using a Marquardt-Levenberg al-gorithm (provided in CasaXPS (Computer Aided SurfaceAnalysis for X-ray Photoelectron Spectroscopy) [31,32] acommercial software package) to define amplitudes andfull widths at half maximum (FWHM).

3 Results and discussion

3.1 XPS characterization of poly-L-arginine

The α-amino acids, of which arginine (Fig. 1) is one,are molecules containing both amine and carboxyl func-tional groups. In this structure the amine group occupiesa position on the α-carbon atom of the carboxyl group,and the functional group is an organic substituent suchas H, CH3 or CH2OH. Arginine consists of a 4-carbonaliphatic straight chain, the distal end is capped by a com-plex guanidinium group (H2N-C=NH). The guanidiniumgroup is positively charged in neutral, acidic and evenmost basic environments, and thus imparts basic chem-ical properties to arginine. Conjugation between the dou-ble bond and the nitrogen lone pairs results in the positive

Fig. 2. XPS wide spectrum of a pristine poly-L-arginine hydro-chloride sample, showing the main spectral features.

charge being delocalized, which enables the formation ofmultiple hydrogen bonds.

An XPS survey spectrum of arginine was recorded asa control to test the sample coverage on the silicon sub-strate and to monitor for the presence of any contami-nants (Fig. 2). This spectrum shows Na1s, O1s, N1s, C1sand Cl2s, Cl2p photoelectron features and also the C, Nand O Auger electron peaks (visible at binding energy be-tween 745 and 1000 eV). In addition another peak is ob-served at a binding energy of 202 eV, which is attributedto ejection of photoelectrons from chlorine Cl2p. This peakwas anticipated because the monitored sample containeda hydrochloride salt.

The experimental binding energies for each chemicalelement obtained from the present spectra agree quitewell with those evaluated by Clark et al. for the polypep-tides [33]. The spectra were calibrated using the valuespresented by Bomben and Dev [14] whose assignment andinterpretation are consistent with the zwitterionic formu-lation for solid-state α-amino acids. In the present worka typical FWHM value was taken to be 1–4 eV, which isstandard for biological material.

High resolution spectra of poly-L-arginine are shown inFigure 3, each of the fitted Gaussian peaks in the spectracan be assigned to distinct functional groups. The mea-sured XPS values of O1s, N1s, and C1s binding energiesfor pristine poly-L-arginine are presented in Table 1. Thehigh resolution spectrum for the O1s shows two peaks.The higher binding energy component at 533 eV (peak 1)is assigned to the hydroxyl group (-OH) arising from ad-sorbed water created from hydrochloric acid. The lowerenergy component observed at 531.5 eV (peak 2) is as-signed to the carbonyl (-C=O) and the carboxyl group(-COOH).

The N1s spectra reveal two structures correspondingto a protonated (peak 1) and an unprotonated (peak 2)NH2 group. The NH+

3 group is the most pronounced inthe spectrum, which confirms that the zwitterionic state is

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Table 1. XPS measured values of O1s, N1s, and C1s binding energies for pristine poly-L-arginine.

CompoundBinding energy (eV)

O-OH O-C=O N-NH3+ N-NH2 C-C=O C-CN C-C-C-, -C-Hpoly-L-arginine 533 531.5 400 399 288.7 286.1 285

Fig. 3. High resolution spectra of O1s, N1s and C1s regionsfor poly-L-arginine recorded for a pristine sample: measuredresults (solid curve) and fitting results (dashed curves). The1s peaks were deconvoluted using a linear superposition of afew Gaussian components. Peak labelling: (a) O1s – alcohol(1), carbonyl/carboxyl groups (2); (b) N1s – protonated aminogroup (1), unprotonated amino group (2); (c) C1s – carbonyl(1), carbon bond to nitrogen (2), hydrocarbon/carbon bond tocarbon (3).

dominant. This is in agreement with other XPS studies forseveral amino acids containing NH2 groups [15,16,18,19].

The carbon peak can be fitted with three compositepeaks. The peak at 288.7 eV is assigned to the carbon inthe carboxyl group (peak 1), while the peak at 286.1 eVis attributed to a carbon bond to nitrogen (peak 2). An-other peak is observed at 285 eV, which corresponds tohydrocarbon and backbone carbon (peak 3). The fittingof three such carbon peaks is in agreement with the ex-perimental data presented by Bomben and Dev [14] forarginine hydrochloride.

Fig. 4. High resolution XPS spectra of O1s (a), N1s (b) andC1s (c) regions for pristine poly-L-arginine sample (solid curve)and after 5 h of uninterrupted X-ray irradiation (dashed curve).

3.2 Compositional changes of poly-L-argininehydrochloride induced by X-rays

To explore the changes induced by X-ray exposure thesamples were irradiated under vacuum by the Mg Kα(1253.6 eV) source for 5 h and XPS spectra wererecorded throughout the irradiation period. The effect ofsuch long term exposure to X-ray irradiation on poly-L-arginine molecule is shown in Figure 4 (pristine-solidcurve, irradiated-dashed curve). For each of these threemain chemical elements (O1s, N1s and C1s) a decrease inthe peak intensity is noticeable. This may be ascribed todecomposition of the investigated samples due to X-raysrather than evaporation of amino acid. However, in orderto rule out any evaporation effects poly-L-arginine sam-ples were introduced to the UHV chamber and left for 5 hafter which high resolution spectra were recorded for O1s,N1s and C1s. These spectra did not reveal any changesfor poly-L-arginine hydrochloride and therefore it can be

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Fig. 5. The effect of X-ray exposure time upon thechange in abundance of chemical groups of poly-L-argininehydrochloride.

assumed that evaporation effects are negligible in this ex-periment.

In order to unravel chemical modifications in the ana-lyzed sample, the percentage amount of chemical groupswas plotted as a function of irradiation time as shownin Figure 5. Significant variations in the total oxygen con-centration are observed; the relative amount of oxygen de-creasing by about 15% in first hour of the irradiation andthereafter gradually declining by another 9% in the sec-ond hour. The percentage assigned to -OH (peak 1) and-C=O (peak 2) seem to decrease as a function of time.The loss of -OH indicates the possible detachment of wa-ter molecules related to -C-OH bond scission such that thehydroxyl groups, both at the carbon atoms in the side ofchains and in the carboxyl groups, are lost by the irradia-tion process, which agrees with other amino acid decom-position studies [15,16]. The loss of -C=O indicates strongdegradation of the amino acid by about 14% in the firsthour of exposure.

As depicted in Figure 5, the total concentration ofnitrogen in poly-L-arginine hydrochloride also decreases.Nitrogen can exist as both protonated NH+

3 and unproto-nated NH2 sites. The time evolution of areas for the twospectral features for N1s seems to complement one an-other in that the one ascribed to the protonated (peak 1)decreased during first hour of irradiation, while the unpro-

Fig. 6. Proposed reaction scheme for poly-L-arginine decayunder continuous X-ray exposure.

tonated (peak 2) increased. This suggests the possibility

of proton loss (NH+3

−H+−−−→ NH2).In the case of carbon the percentage amount for three

components (alcohol – peak 1, carbon bond to nitrogen– peak 2 and hydrocarbon/backbone carbon – peak 3) isplotted in Figure 5. The total carbon concentration wasobserved to decrease and eventually plateau after a pe-riod of 3 h. The percentage of -C=O and -C-C-, -C-Hrepresenting peaks 1, 3, respectively, decreases, while thepercentage of -CN (peak 2) increases during the first hourin the present studies, this suggests decarboxylation anddecomposition as a consequence of X-ray exposure.

A proposed decomposition scheme that is consistentwith the experimental data is depicted in Figure 6. Aminoacids are typical amphoteric compounds (they can reactas either a base or an acid), which in both solid and liq-uid states exist as a salt with a proton transferred froman acidic -COOH group to an alkaline -NH2 group. In the

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present case (the first step in Fig. 6), proton transfer takesplace from carboxyl group to the further localized aminosite. The reason behind this behavior is the higher alka-linity of the aforementioned group. The closer -NH2 groupis less alkaline due to the influence of the carboxyl groupwith strongly electronegative oxygen atoms.

Upon X-ray irradiation, as shown at the second stepin Figure 6, an ionic balance is achieved and the zwit-terionic form converts into a free form. It is expectedthat the cation to anion ratio will depend on the pHof the solution. Conjugate acid is the dominant formin the case of low pH values, while high values of pHare conducive to the existence of a conjugate base. Thisbehavior is suggested by the results depicted in Fig-ure 5, where the dehydrogenation of protonated aminogroup is observed due to X-ray irradiation. Decarboxyla-tion can then take place to form the radical of 1-amino-4-[(diaminomethyl)amino]butyl, which rapidly picks up ahydrogen atom to create the final product, N-(4-amino-butyl)methanetriamine.

Summarizing, after 5 h of irradiation the O1s bindingenergy signal decreases markedly in intensity and the C1scomponent, in the form of carboxyl group, nearly disap-pears. These observations (Fig. 5) are therefore in accordwith the loss of carbon dioxide from the film (Fig. 6).

3.3 XPS characterization of multilayeredDNA-coatings

The aim of the present studies was to identify patternsin the X-ray induced damage of DNA and its dry com-plexes with poly-L-arginine hydrochloride. Samples ofmultilayered coatings deposited onto a silicon substratewere prepared by ESA using DNA as the anionic poly-electrolyte and poly-L-arginine as cationic polyelectrolyteas described above. In this method dry polyelectrolytemultilayers can be generated through the adsorption ofoppositely charged polyelectrolytes via their electrostaticinteractions.

A survey spectrum of dry five double layered com-plexes is shown in Figure 7. This spectrum was recordedat the beginning of X-ray irradiation at a constant powerlevel of 260 W and reveals the O1s, N1s, C1s, P2s and P2pphotoelectron features. Additionally, well defined phos-phorous photoelectron features were detected albeit withrelatively small intensity. This can be explained by thechemical composition and atomic concentration of multi-layered samples [34].

In Figure 8 the major peaks present in the O1s, N1s,C1s and P2p spectra are assigned to DNA features. Tovisualize data, XPS measured values of O1s, N1s, C1s,and P2p binding energies for dry films of pristine [poly-L-arginine/DNA]5 are presented in Table 2. In the caseof oxygen, three peaks could be identified and are relatedto: alcohol (peak 1), carbonyl/carboxyl groups (peak 2),oxygen in the phosphate group (peak 3). The principalN1s core level peak in Figure 8b has a binding energybetween 402.5 and 397 eV, the higher energy componentis assigned to amino group and lower energy component

Fig. 7. The XPS survey spectra of dry multilayered DNAcoatings.

is related to imino site. The C1s photoelectron peak inFigure 8c may be deconvoluted to reveal four structures,which correspond to urea (peak 1), amide (peak 2), al-cohol/cyclic ether/carbon bond to nitrogen (peak 3) andhydrocarbon (peak 4). Main P2p core-level peak has abinding energy of 133.3 eV.

3.4 Compositional changes of multilayeredDNA-coatings induced by X-rays

Dry films of arginine/DNA bi-layers were irradiated undervacuum by the Mg Kα (1253.6 eV) X-ray source for 5 hand XPS spectra were monitored throughout the irradi-ation period. The changes induced by such radiation canbe readily seen by comparison of the XPS spectra beforeand after X-ray exposure (Fig. 9). However, it is recom-mended to continue the irradiation studies on DNA/aminoacid complexes, where greater statistical accuracy in thedata points can be achieved by repeating high resolutionirradiation measurements.

The percentage amount for each functional group ofinvestigated elements (O1s, N1s, C1s, and P2p) is shownas a function of exposure time in Figure 10. Substantialchemical modifications in the total oxygen concentrationare observed; the relative amount of oxygen in dry [poly-L-arginine/DNA]5 drops by about 17% for 3 h of irradiationand thereafter remains relatively constant. The percent-age assigned to -OH (peak 1), -C=O (peak 2) and -PO3−

4(peak 3) seem to decrease as a function of time by 12%,16%, and 24%, respectively.

In the case of nitrogen, the total concentration alsodecreases by 9% over the irradiation time; the peak at-tributed to the amino group (peak 1) drops rapidly by 18%after one hour of exposure, and then decreases gradu-ally. The percentage amount of the lower binding energy,identified as imino group (peak 2), shows a substantialincrease of 19% over the irradiation time.

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Fig. 8. High resolution XPS spectra of O1s, N1s, C1s and P2p regions for dry [poly-L-arginine/DNA]5 sample: experimentaldata (solid curve) and fitting results (dashed curve). The 1s peaks were deconvoluted using linear superposition of a few Gaussiancomponents. Peak labelling: (a) O1s – alcohol (1), carbonyl/carboxyl groups (2), oxygen in phosphate group (3); (b) N1s –amino group (1), imino site (2); (c) C1s – urea (1), amide (2), alcohol/cyclic ether/carbon bond to nitrogen (3), hydrocarbon(4); P2p – the phosphate group (1).

Table 2. XPS measured values of O1s, N1s, C1s, and P2p binding energies for pristine [poly-L-arginine/DNA]5 dry.

CompoundBinding energy (eV)

O-OH O-C=O O-PO43- N-NH2 N=NH C-CO(NH2)2 C-CONH2 C-COH, -COR, -CH C-C-H P-PO43-[poly-L-arginine/

533 531.8 530.4 400 399 287.5 286.5 285 283.5 133.3DNA]5 dry

Fig. 9. High resolution XPS spectra of O1s (a), N1s (b), C1s (c) and P2p (d) regions for dry pristine [poly-L-arginine/DNA]5sample (solid curve) and after 5 h of uninterrupted X-ray irradiation (dashed curve).

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Fig. 10. The effect of X-ray exposure time upon the change in abundance of chemical groups of [poly-L-arginine/DNA]5.

During exposure to radiation, the total percentageamount of carbon as well as the peaks assigned to -CONH2

(peak 2), -C-H (peak 4) decrease by 14%, 73%, 64%, re-spectively. Substantial increase of 20% was detected forpeak corresponding to -CO(NH2)2 (peak 1), whereas per-centage amount for -COH, -COR, -C-H (peak 3) remainedconstant.

The time evolution for -PO3−4 component in [poly-

L-arginine/DNA]5 is presented in Figure 10d. The totalphosphorous concentration and the peak correspondingto phosphate group show only a small decrease (not morethan 11%).

The changes caused by X-ray irradiation were also in-vestigated for wet multilayered DNA coatings samples.The term “wet” sample therefore refers to the existence oftrapped water between layers. In spite of large statisticaluncertainties an attempt to fit Gaussians to the measuredpeaks (data not shown) was made and it was evident thatdamage introduced to wet films is more substantial thanthat to dry samples. It can consequently be concludedthat wet samples are more strongly affected by indirectdamage, where the hydrated cellular environment absorbsthe incident photon and highly reactive radicals attack thebiomacromolecules [35]. It should be highlighted that rad-icals are responsible for breaks of chemical bonds, produc-tion of chemical changes and initiation of chain of eventsthat result in biological damage. In the case of dry sam-ples, the direct action initiates ionization of those atomsthat are part of the irradiated molecules. It has been esti-mated that direct effects in vivo contribute to 40–50% ofthe DNA [36] damage and is produced by the formation ofholes (sites of one-electron loss) and excess electrons (sites

of one-electron gain) by ionization of the DNA salvationshell and subsequent transfer to the DNA itself [37].

Previously reported results [20] revealed that the DNAmolecule is vulnerable upon X-ray and undergoes chem-ical and structural modifications. The XPS spectra forDNA have complicated shapes due to contribution of sev-eral functional groups and shake-up satellites. High reso-lution XPS spectra of the O1s, N1s, C1s, and P2p bandsrecorded throughout the irradiation study of deoxyribonu-cleic acid from calf thymus, indicated few processes, e.g.,dehydrogenation, oxygen loss, and formation of radicals,which lead to base damage and/or strand breaks in theDNA molecule.

Analysis of O, N regions within DNA showed a de-crease during X-ray exposure, mostly due to decom-position and desorption of small fragments. Significantchanges in the shape of the photoelectron peaks were alsoobserved for the carbon peak, which can be attributed toformation of new products during irradiation. Accordingto the studies of Akamatsu et al. [38] on degradation ofthymine by monochromatic ultrasoft X-rays the formationof a thymine allylic radical also takes place and might bea precursor of 5-formyl uracil and hydroxymethyl uracil.

4 Conclusion

An XPS investigation of poly-L-arginine and multilayeredDNA-coatings on a silicon substrate is reported in thispaper, the described work is a part of an ongoing projectto characterize the mechanisms by which ionizing radia-tion damages DNA and its surrounding molecules [20].

Eur. Phys. J. D (2014) 68: 333 Page 9 of 10

The aim of the present study was to check the influ-ence of X-ray radiation on biomolecules during 5 h ex-posure. The measured binding energies and determinedratios were investigated in terms of the chemistry of aminoacids. The results are consistent with the suggestion thatamino acids and their complexes with DNA form a con-venient modelling system in studies of radiation damage.The results showed that the chosen molecules are verysensitive to damage by soft X-ray exposure and undergomajor chemical modifications under such exposure. A de-tailed study of XPS spectra for particular elements beforeand after irradiation, has revealed a number of reactionpathways (e.g., proton transfer, decarboxylation), whichlead to base damage and/or strand breaks in the DNAmolecule and its analogues.

In the case of poly-L-arginine, changes in the XPSspectra after exposure were mostly due to fragmentationof the sample. The observed changes in the shape of pho-toelectron lines for C1s can be attributed to radiation in-duced rupture of structural bonds.

The changes caused by the soft X-ray irradiation werealso explored for dry multilayered DNA coatings sam-ples. It can be concluded that the observed changes in theshape of photoelectron spectral peaks for dry samples arecaused by the direct action of ionizing radiation (includ-ing secondary electron damage), which leads to ionizationof atoms that are part of the molecules studied.

The present work is one of what remains a limitednumber of explorations being pursued internationally andmany more experiments are still needed. For example,it would be useful to continue X-ray photon irradiationstudies to create a systematic database assembling bind-ing energies related to many more biologically significantmolecules (e.g., intercalants, radiosensitizers) since it iswell known that high energy photons create radiationdamage via secondary species including radicals, ions, andsecondary electrons. That is why for a deeper understand-ing of this type of damage, it is also necessary to perform aseries of experiments on the reactions induced by some ofthese secondary species, including the damage induced bythe abundant secondary low energy electrons (with detec-tion via e.g., agarose or polyacrylamide gel electrophore-sis (AGE or PAGE), high performance liquid chromatog-raphy (HPLC) and/or mass spectrometry). Additionally,it is recommended to continue the irradiation studies onDNA/amino acid complexes such that greater statisticalaccuracy in the data points can be achieved.

Agnieszka Stypczynska wishes to gratefully acknowledgeSylwia Ptasinska (University of Notre Dame, United States)and Ziad Francis (Saint Joseph University, Lebanon) for allfruitful discussions during the course of this work.

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