Biomolecular papain thin films grown by matrix assisted and conventional pulsed laser deposition: A comparative study

Biomolecular papain thin films grown by matrix assisted and conventional pulsed laser deposition: A comparative study
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  Biomolecular papain thin films grown by matrix assisted and conventionalpulsed laser deposition: A comparative study E. György, 1,2,a  A. Pérez del Pino, 3 G. Sauthier, 1 and A. Figueras 1 1 Consejo Superior de Investigaciones Cientificas, Centre d’Investigacions en Nanociència i Nanotecnologia,(CSIC-CIN2), Campus UAB, 08193 Bellaterra, Spain 2  National Institute for Lasers, Plasma, and Radiations Physics, P.O. Box MG 36, 76900 Bucharest V, Romania 3 Consejo Superior de Investigaciones Cientificas, Instituto de Cinecia de Materiales de Barcelona (CSIC- ICMAB), Campus UAB, 08193 Bellaterra, Spain  Received 5 August 2009; accepted 27 October 2009; published online 11 December 2009  Biomolecular papain thin films were grown both by matrix assisted pulsed laser evaporation  MAPLE   and conventional pulsed laser deposition   PLD   techniques with the aid of an UV KrF  (  =248 nm,    FWHM  20 ns   excimer laser source. For the MAPLE experiments the targetssubmitted to laser radiation consisted on frozen composites obtained by dissolving the biomaterialpowder in distilled water at 10 wt % concentration. Conventional pressed biomaterial powder targetswere used in the PLD experiments. The surface morphology of the obtained thin films was studiedby atomic force microscopy and their structure and composition were investigated by Fouriertransform infrared spectroscopy. The possible physical mechanisms implied in the ablationprocesses of the two techniques, under comparable experimental conditions were identified. Theresults showed that the growth mode, surface morphology as well as structure of the depositedbiomaterial thin films are determined both by the incident laser fluence value as well as targetpreparation procedure. ©  2009 American Institute of Physics .   doi:10.1063/1.3266670  I. INTRODUCTION The development of new biomaterial immobilizationtechniques is of key importance for the design and fabrica-tion of next generation biomedical devices as miniaturizedbiosensors and biochips, 1–3 biocompatible coatings for medi-cal implants and tissue engineering, 4–7 or controlled drugdelivery systems. 8,9 As known, conventional techniques aremultistep procedures of high cost, work only for specificorganic compounds, and in most cases involve toxic chemi-cal substances.Biomaterial transfer requires, besides the precise controlover the amount of the immobilized material and its surfacemorphology, also the maintenance of the molecular func-tions, the chemical composition, and structure of moleculesafter the immobilization process. Besides, immobilizationtechniques must be versatile enough for the deposition of uniform and adherent coatings of any organic compoundwith an accurate thickness control.Methods involving laser radiation could become an in-teresting alternative to conventional techniques, able to fulfillthese requirements. Indeed, by laser techniques the amountof material evaporated and deposited on the substrate surfacecan be precisely controlled by the number and/or intensity of the laser pulses used for the irradiation of the targets. Anadditional advantage of the laser techniques relies on the factthat practically any kind of substrate materials can be used,without any pretreatment procedure. 10–12 Despite the fact thatthe high intensity laser pulses are destructive for organic ma-terials, laser deposition techniques were already tested fororganic polymers 13–17 and also biomaterials, as horseradishperoxidase and insulin, 18 bovine serum albumin 19,20 pepsine, 21 silk fibroin 22,23 or fibrinogen blood proteins. 24 In this work we focused our attention on papain, aprotein-cleaving enzyme derived from papaya plant. Thefunctions of papain are well known, being employed to treatulcers, reduce fever after surgery, is active against gram-positive and gram-negative bacteria, and has beneficial ef-fects in systemic enzyme therapy in oncology. 25 This work isa continuation of our previous investigation regarding theeffect of biomaterial concentration in the frozen compositetarget on the papain thin films growth mode and surface mor-phology at a fixed incident laser fluence value. 26 The main purpose of the present research is to elucidatethe role of the target preparation procedures, frozen compos-ite, or conventional powder pressing, as well as incident laserfluence on the thin films surface morphology, avoiding thedecomposition of the processed biomaterial. Our aim is toidentify the physical phenomena implied in the ablation pro-cess during the two approach, matrix assisted pulsed laserevaporization   MAPLE   and conventional pulsed laser depo-sition   PLD  , and to establish the optimum conditions whichlead to the deposition of uniform and continuous thin films,with chemical composition and structure identical to the basematerial used for the targets’ preparation. II. EXPERIMENTAL The thin film growth experiments were performed insidea stainless steel deposition chamber. A pulsed UV KrF    =248 nm,    FWHM  25 ns,    =10 Hz   COMPexPro 205Lambda Physik excimer laser source was used for the irra- a  Author to whom correspondence should be addressed. Tel.:  34 93 581 3725. FAX:  34 93 581 37 17. Electronic mail: JOURNAL OF APPLIED PHYSICS  106 , 114702   2009  0021-8979/2009/106  11   /114702/6/$25.00 © 2009 American Institute of Physics 106 , 114702-1 Author complimentary copy. Redistribution subject to AIP license or copyright, see  diations. Before each deposition experiment, the irradiationchamber was evacuated down to a residual pressure of 1.6  10 −3 Pa. This pressure value was maintained during thepapain thin film growth experiments.The composite MAPLE targets were prepared by dis-solving papain in distilled water at 10 wt % papain concen-tration. The obtained solutions were frozen before startingthe experiments and kept frozen during the irradiation circu-lating liquid nitrogen inside a special, double wall targetholder. The complete MAPLE workstation was purchasedfrom SURFACE. For the growth of the thin films by conven-tional PLD the targets were prepared from powders of papainby pressing at 3 MPa.The laser beam was focused onto the target surface witha 30 cm focuse depth   FD   SUPRASIL 2 lens placed outsidethe deposition chamber. To avoid significant morphologicalchanges upon irradiation, the laser beam scanned the targets’surface at a constant velocity of 2 mm/s. The irradiated  XY  surface area was 1  1 cm 2 . The angle between the laserbeam and the target surface was chosen of 45°. For the depo-sition of each film we applied 1.5  10 4 subsequent laserpulses, for the MAPLE technique and 500 pulses in case of conventional PLD. The laser pulses succeeded each otherwith a repetition rate of 10 Hz. The laser fluence value inci-dent on the target surface was chosen in the range of   0.2–0.6   J / cm 2 .The glass substrates were placed parallel to the target ata separation distance of 5 cm. Prior to introduction inside thedeposition enclosure the substrates were carefully cleaned inultrasonic bath in acetone. During the irradiation the sub-strates were kept at room temperature.The surface morphology and growth mode of the depos-ited papain thin films were investigated by atomic force mi-croscopy   AFM   in acoustic   dynamic   configuration with a5100 atomic force microscopy atomic force microscopy/ scanning probe microscopy   AFM   /   SPM   apparatus fromAgilent Technologies. The AFM measurements were studiedwith  WSXM  software from Nanotec Electronica. The obtainedthin films chemical composition and bonding states betweenthe elements were studied by Fourier transform infraredspectroscopy   FTIR   in the wave number range 4000−500 cm −1 , using a 4 cm −1 resolution Perkin Elmer Inc.,Spectrum one, Wellesley, MA, USA apparatus. III. RESULTS AND DISCUSSIONSA. Morphological characterization of immobilizedpapain Figures 1  a  –1  d   show the top view   a  –  d   and tilted  e  –  h   AFM images of thin films obtained from frozencomposite targets containing 10 wt % papain concentrationat increasing incident laser fluence values, 0.2   Figs. 1  a   and1  e  , 0.4   Figs. 1  b   and 1  f   , and 0.6 J / cm 2  Figs. 1  c   and1  g   as well as from pressed powder target at 0.6 J / cm 2  Figs. 1  d   and 1  h   laser fluence value. As can be observed,at the lowest, 0.2 J / cm 2 laser fluence the deposited filmssurface is characterized by a uniform morphology   Fig. 1  a  .The higher magnification tilted AFM image   Fig. 1  e   re-veals that the film is constituted by tens of nanometer-sizedclusters. Indeed, from the corresponding local height histo-gram an average height of about 30 nm can be estimated,with a very narrow distribution range   Fig. 1  i  . With theincrease in the incident laser fluence, the smooth surface FIG. 2. Surface profiles of thin films obtained by the irradiation of the 10wt % concentration frozen composite targets at   a   0.2,   b   0.4, and   c  0.6 J / cm 2 incident laser fluence.FIG. 1. Top view   a  –  d   and tilted   e  –  h   AFM images, as well as localheight histograms   i  –  l   of the thin films obtained by the irradiation of 10wt % concentration frozen composite   a  –  c  ,   e  –  g  ,   i  –  k   and pressedpowder   d  ,   h  , and   l   targets at   a  ,   e  , and   i   0.2,   b  ,   f   , and    j   0.4,and   c  ,   d  ,   g  ,   h  ,   k  , and   l   0.6 J / cm 2 incident laser fluence. The topview and tilted AFM images correspond to 5  5 and 1  1    m 2 surfaceareas, respectively. 114702-2 György  et al.  J. Appl. Phys.  106 , 114702   2009  Author complimentary copy. Redistribution subject to AIP license or copyright, see  morphology of the films changes gradually. The depositedmaterial consists of large particulates   Figs. 1  b  , 1  f   , 1  c  ,and 1  g  . Moreover, the diameter and height of the largeparticulates increase with the increase in the laser fluence,reaching hundreds of nm at the highest, 0.6 J / cm 2 laser flu-ence   Figs. 1  c  , 1  g  , and 1  k  .In the followings, we compared the surface morphologyof the thin films obtained from the frozen composite targetswith that obtained by the irradiation of a pressed powdertarget of papain   Figs. 1  d  , 1  h  , and 1  l  . After only 500laser pulses   as compared to 1.5  10 4 pulses used for thedepositions from the frozen composite targets   large par-ticles, with diameters exceeding 1    m were observed on thesurface of the films, composed by small particles with amaximum height of around 200 nm   Fig. 1  l  .The surface profiles of the depositions obtained fromfrozen composite targets at increasing laser fluence valuesare presented in Fig. 2. The profile of the deposition obtainedat 0.2 J / cm 2 fluence is smooth and uniform over the wholescanned area   curve a  . Conversely, at higher, 0.4 and0.6 J / cm 2 laser fluence large particulates appear on the sur-face. Both diameters and heights of the particulates increasewith the increase in the laser fluence. The profile of the depo-sition obtained from the pressed powder target   Fig. 3   showsan uniform surface but also the presence of large particulates,with dimensions in the micrometer range.The root mean square   rms   surface roughness of thedeposited thin films was calculated from the AFM data. Thesurface roughness increases gradually from about 9 nm forthe film deposited at the lowest, 0.2 J / cm 2 to about 170 nmfor the film deposited at 0.6 J / cm 2 laser fluence. Similar, oreven higher roughness values were reported also for organicthin films deposited by MAPLE technique. 13,27 At the samelaser fluence the roughness of the film obtained from pressedpowder target is less, around 70 nm, as compared to thedepositions obtained from the frozen composite targets.However, we recall that the number of laser pulses was re-duced significantly in case of the deposition from the pressedpowder target, precisely in an attempt to reduce the surfaceroughness and avoid the formation of large particulates. Ac-cording to our previous studies the roughness of the filmobtained from the pressed powder target is several timeslarger as compared to the films obtained from the frozencomposite targets at identical laser fluence value and thesame number of pulses. 26 B. Structural characterization of immobilized papain In Fig. 4 we present the FTIR spectra corresponding tothe glass substrates, as well as to the papain powder used forthe preparation of the targets. As can be observed, at wavenumber values higher than 1200 cm −1 the FTIR spectrum of the glass substrates does not contain any significant featuresand, consequently, it is suitable for the study of the charac-teristic absorption bands of the deposited papain thin films.The spectrum of the papain powder shows the characteristicbands at 3400−3258 cm −1 due to N u H stretching of sec-ondary N-substituted amides, the bands around 2968−2859 cm −1 due to C u H stretching, the band at1640 cm −1 of the amide I region due to C v O stretching of the carboxilate anion, the band at 1533 cm −1 of the amide IIregion attributed to N u H deformation and C u N stretch-ing, the band around 1400 cm −1 due to C u H deformationof alkyl chains of amino acids, and the bands at 1125−1040 cm −1 due to C u S stretching of sulfides anddisulfides. 28,29 It is well known that the absorption peak po-sitions of the amide bands are quite sensitive to the second-ary structure of proteins. 30 As can be observed in Fig. 5, the FTIR spectra of thefilms deposited by MAPLE technique are similar in the in- FIG. 3. Surface profile of the thin film obtained by the irradiation of pressedpowder target at 0.6 J / cm 2 incident laser fluence.FIG. 4. FTIR spectra of the   a   glass substrate and   b   base papain powder used for the targets’ preparation. 114702-3 György  et al.  J. Appl. Phys.  106 , 114702   2009  Author complimentary copy. Redistribution subject to AIP license or copyright, see  vestigated experimental conditions   Fig. 5  I  . They repro-duce all the characteristic bands of the papain powder and noshifts in the bands were observed. This result confirms thatthe deposited thin films preserved the primary structure of the base material used for the targets preparation. Moreover,the absence of any shift in amide bands in the spectra of thefilms compared to the spectrum of the nonirradiated basematerial indicates that the secondary structure was preservedduring the laser immobilization process. These features rep-resent an evidence for the effectiveness of the frozen com-posite target preparation procedure used in the MAPLE tech-nique. Nevertheless, the absorption band intensity decreasewith the increase in the incident laser fluence   comparecurves a–c in Fig. 5  I  . The decrease in the intensity of theFTIR bands suggests that the increase in the laser fluenceleads to a partial decomposition of the irradiated biomaterial,even if embedded in the frozen solvent matrix.On the other hand, in the spectrum of the films depositedby conventional PLD   Fig. 5  II  , some characteristic bandsof papain are missing, accompanied by the appearance of new bands. These features could be associated to decompo-sition, fragmentation, followed by new bonds formation of part of the biomaterial exposed to laser irradiation and trans-ferred to the substrate surface. IV. DISCUSSIONS We present in Table I a review concerning the rms sur-face roughness, average local height, nanometer-sized clus-ters, and large particulates mean diameters, as a function of incident laser fluence and the target preparation conditions.The different surface features of films deposited by con-ventional PLD and MAPLE techniques could be attributed tothe different laser-material interaction and ablation processestaking place in case of the frozen composite as well aspressed powder targets. As known, during conventional PLDthe large, micrometer-sized particulates observed in the sur-face of thin films are attributed to material expulsion directlyfrom the irradiated target surface under the laser generatedplasma recoil pressure, while the tens of nanometers dimen-sions nanoparticles could form by clusterization mechanismsduring the transit of the ablated material from the target to-ward the substrate, or on the substrate surface. 10–12 In our MAPLE experiments the used water solvent isweakly, while the biomaterial is highly absorbing at thewavelength of the incident laser radiation. In Fig. 6 wepresent the optical absorption spectrum of pure distilled wa-ter   curve a   used as solvent for the preparation of the com-posite targets. Indeed, the 248 nm laser wavelength is situ-ated at the bottom of the absorption spectrum. At a first sightwater is then an unsuitable matrix. We would like to noticethat this is the opposite situation of usual MAPLE experi-ments, where the solvents are chosen highly absorbing at theincident laser wavelength in order to protect the organic ma-terial and prevent its decomposition. However, as evidencedby our FTIR results, even in this configuration the trans-ferred biomaterial preserved its structure and composition. FIG. 5. FTIR spectra of    I   thin films obtained from 10 wt % concentration frozen composite targets and   a   0.2,   b   0.4 as well as   c   0.6 J / cm 2 incident laserfluence and   II   thin film obtained from pressed powder target and 0.6 J / cm 2 incident laser fluence.TABLE I. Thin films rms surface roughness, average local height  h , clustersmean diameter,  d  , and large particles mean diameters,  D , as a function of incident laser fluence and target preparation conditions. F  l  J / cm 2   Targetrms  nm  h  nm  d   nm   D  nm  0.2 10 wt % composite 9 24 25  ¯ 0.4 10 wt % composite 65 190 60 4000.6 10 wt % composite 170 444 80 7000.6 Pressed powder 70 180 50 1800 FIG. 6. Optical absorption spectra of    a   pure distilled water,   b   water ice,10 wt % concentration   c   liquid, and   d   frozen biomaterial solution. 114702-4 György  et al.  J. Appl. Phys.  106 , 114702   2009  Author complimentary copy. Redistribution subject to AIP license or copyright, see  However, since the irradiated matrix is not in liquid, butsolid phase, we measured the optical absorption spectra of water ice, as well as 10 wt % concentration liquid and frozenbiomaterial solution   curves b, c, and d in Fig. 6  . For theabsorption measurements we used MAPLE targets of 5 mmin thickness, prepared following the same procedure as forthe irradiation experiments. The absorption was found to behigher in ice as compared to water. Indeed, it is known thatice imperfections, entrapped air bubbles, cracks, and otherdefects, play an important role in light absorption and scat-tering processes. 31 Multiphoton absorption of light was reported for waterice matrix following 248 nm laser irradiation and was foundto lead to ejection of water molecules but with a low yield atlaser intensities comparable to those used in ourexperiments. 32 Electronic excitation of the matrix may beproduced as two-photon absorption process since the photo-electron emission threshold for ice is about 10 eV. 32–34 Con-versely, these processes were found to be dominant for laserintensities one to two orders of magnitude higher as com-pared to the present experiments. 33 At laser intensity valuessimilar to those used in this study, removal of water mol-ecules was attributed to excitons formation in the vicinity of defects by multiphoton absorption. Excitons dissipate theirenergy to weakly bound water molecules that desorb but theprocess stopped after several laser pulses. 35 Ionization breakdown in ice which could drive materialout of the ice by local excitation or heating, i.e., accelerationof seed electrons in defects and impurities to an avalancheionization process by inverse bremsstrahlung   IB   can bealso excluded. The reported threshold intensities are abouttwo orders of magnitude higher that those used in the presentstudy and the process is more efficient for IR than UV laserradiation due to the IB absorption law    2 . 36,37 Photothermal ablation processes, i.e., heating of the sub-strate beneath the frozen matrix, followed by heat conductioninto the overlying ice, leading to ablation, can be excluded aswell for targets of a few millimeter thicknesses. This processwas reported to be effective at laser intensity values compa-rable to those used in our studies, but for water ice thick-nesses of only 1000 Å. 38 It was found that substrate heatingvanishes completely as the thickness increases up to 2  10 6 Å. 32 Conversely, during the irradiation of the frozen biomate-rial solution nonhomogeneous absorption mechanisms couldtake place and stand at the srcin of the observed features. 39 The ablation process could be related to local overheating of absorbing areas constituted by biomolecules in the outmostsurface layer, heating the solvent in their vicinity. In vacuumconditions, the water solvent starts boiling just above itsmelting temperature, the vapors transporting the biomol-ecules toward the substrate surface. Even if the biomoleculesof the outmost surface layer are denaturated, the water vaporcan carry molecules from deeper zones, where the tempera-ture does not surpasses the denaturation temperature of thebiomaterial. 40 As a consequence, at low laser fluences, thesurface morphology, constituted by tens of nanometer-sizedparticulates can be assigned to surface evaporation and clus-terization. The process is similar until certain extend to theindirect adsorbate mediated absorption process described inRef. 41 for benzene-water mixture. The laser energy ab-sorbed by the benzene molecules is transferred to the watermolecules causing them to desorb from the frozen matrix.The presence of large, micrometer-sized particulates incase of MAPLE deposited organic thin films were related,according to theoretical predictions, to droplets ejection fromthe target surface as a result of explosive evaporation or spal-lation mechanisms, 42–44 termed also as “cold laser ablation.”Thus, the main part of the material may be ejected from thetarget as cold droplets from the molten solvent, from thezone below the outmost surface layer, under the action of therecoil pressure exerted by the ablation products. This abla-tion process could explain the increased surface roughness of the films deposited at higher laser fluences as well as thenonuniform surface morphology constituted by micrometer-sized conglomerates. V. CONCLUSIONS Biomolecular papain thin films were grown by matrixassisted and conventional PLD methods. The growth mode,surface morphology as well as composition of the depositedbiomaterial thin films were investigated and found to be de-fined both by the incident laser fluence value as well as targetpreparation procedure. The deposited thin films preserved thestructure of the base material used for the targets preparation,representing an evidence for the effectiveness of the frozencomposite target preparation procedure used in the MAPLEtechnique. Micrometer-sized particulates observed in case of thin films obtained from the pressed powder target couldsrcinate directly from the irradiated target surface expelledunder the laser generated plasma recoil pressure, while thetens of nanometers dimensions nanoparticles could form byclusterization mechanisms. Nonhomogeneous absorptionmechanisms could stand at the srcin of the observed fea-tures in the case of MAPLE deposited films. At low laserfluences, the mass ejection can be explained by surfaceevaporation. With the increase in the laser fluence hydrody-namic ablation mechanisms become predominant. The mate-rial may be ejected from the target as cold droplets from themolten solvent, not directly heated by the laser radiation,under the action of the recoil pressure exerted by the ablationproducts. ACKNOWLEDGMENTS The authors acknowledge with thanks the financial sup-port from the Spanish Ministry for Science and Innovationunder the Contract No. MAT2006-26534-E and the Roma-nian National University Research Council under the Con-tract IDEAS No. 652. 1 W. L. Xing and J. Cheng,  Biochips: Technology and Applications  Springer-Verlag, Heidelberg, 2003  . 2 S. Rodriguez-Mozaz, M. P. Marco, M. J. L. De Alda, and D. Barcelo, PureAppl. Chem.  76 , 723   2004  . 3 S. J. Dong and B. Q. Wang, Electroanalysis  14 , 7   2002  . 4 M. Martina and D. W. Hutmacher, Polym. Int.  56 , 145   2007  . 5 R. A. Stile and K. E. Healy, Biomacromolecules  2 , 185   2001  . 6 S. Sofia, M. B. McCarthy, G. Gronowicz, and D. L. Kaplan, J. Biomed.Mater. Res.  54 , 139   2001  . 114702-5 György  et al.  J. Appl. Phys.  106 , 114702   2009  Author complimentary copy. Redistribution subject to AIP license or copyright, see
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