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Spectroscopy and non-linear optical properties of DNA - Bilberry complex
Optical Materials 100 (2020) 109669
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Spectroscopy and non-linear optical properties of DNA - Bilberry complex �duret¸u a, François Kajzar a, b Ana-Maria Manea-Saghin a, *, Carla-Cezarina Pa a b
Faculty of Applied Chemistry and Materials Science, University POLITEHNICA of Bucharest, Polizu Street No 1, 011061, Bucharest, Romania Laboratoire de Chimie, CNRS, Universit�e Claude Bernard, ENS-Lyon, 46 All�ee d’Italie, 69364, Lyon cedex 07, France
A R T I C L E I N F O
A B S T R A C T
Keywords: Deoxyribonucleic acid (DNA) Bilberry extract (BBE) DNA-BBE complex Third harmonic generation Optical damage threshold
Natural extracts are getting more attention due to the fast degradability rate and low toxicity. In this paper the obtained results for the complex based on deoxyribonucleic acid biopolymer with bilberry (BBE) natural extract are presented and discussed. The interaction between the two materials (DNA-BBE complex) was evaluated by measuring the linear optical properties using UV–Vis and fluorescence spectral methods. Furthermore, the DNABBE complex was used for non-linear optical (NLO) properties investigations of the obtained thin films on glass substrate by spin-coating technique. Also, the optical damage threshold of the studied DNA-BBE complexes was measured in order to check their potentiality and interest for application in photonics.
1. Introduction Bilberry (Vaccinium myrtillus L.) is a fruit known also as European blueberry [1], huckleberry or whortleberry [2]. It grows abundantly in the Northern and Eastern areas of Europe [1], usually on acid soils, from marginal forests up to high altitude [3]. In bilberries fruit, anthocyanins represent about 90% of the total phenolic compounds [1]. The bilberry extract has beneficial effects as antioxidant, hypoglycemic and lipid-lowering agent [4]. Usually, it contains phenolic acids, tannins, flavonols (catechins, quercetin), resveratrol, hydroquinone, thiamin, vitamin C and mostly anthocyanosides, which are flavonoid derivatives of anthocyanins [1,2,4]. Anthocyanins are powerful antioxidants, water-soluble pigments that belong to the flavonoids class [5,6] and are naturally synthesized in the epidermal tissue. They confer the red, blue and purple colors to the berries [5,7]. Also, anthocyanines have pro tective role against cold stress, but they are sensible to environmental factors such as temperature or light that may affect their stability [8]. The most commonly occuring anthocyanidins are delphinidins, cyani dins, malvidins and petunidins [1], (Fig. 1). Synthetic polymers are subjected to slow degradation rates. Thus to avoid the pollution it is wishful to use natural materials such as bio polymers [9]. One of the main categories of biopolymers are poly nucleotides like DNA and RNA which are long chain polymers composed of 13 or more nucleotide monomers [10,11]. In previous studies, good optical properties were exhibited when DNA was used in combination with natural extracts [9]. The non-linear optical (NLO) properties of thin films are usually investigated using the Third Harmonic Generation
[12–21]. In this paper we report on preparation and characterization of a new biomaterial in view of its application in photonics. The composite ma terial was obtained by doping, in solution, the DNA extract with bilberry natural extract. It was characterized by UV–Vis spectroscopy and fluo rescence studies to evaluate its linear optical properties. Furthermore, the nonlinear optical (NLO) properties characterization of functional ized thin films was performed by the optical third harmonic generation (THG) measurements at 1064.2 nm fundamental wavelength. The op tical damage threshold measurements were also performed in order to establish the applicability of DNA-BBE thin films in photonics. 2. Materials, equipment and thin films processing A low molecular mass deoxyribonucleic acid, purchased from Sigma Aldrich Company, was used in this study. The bilberry natural hydro alcoholic extract used for this study had a concentration of approxi mately 50% and was received from Hofigal S.A. The UV–Vis linear optical absorption spectra were recorded using 1 cm quartz cuvettes, in 200–800 nm spectral range, on an instrument from Thermo Scientific, Evolution 220 model, with integrated Insight v. 2.3.345 Software. The optical fluorescence spectra were recorded with a spectrofluorimeter instrument from Jasco, FP-6500 model. The same bandwidth of 10 nm (ex)/10 nm (em) was used for all the registered spectra. Thin films were prepared by spin-coating technique on glass substrate using a spincoater from Laurell Technologies Corporation, model WS-400B-6NPP/ LITE. Their thickness was determined by the profilometry technique
* Corresponding author. E-mail address: [email protected] (A.-M. Manea-Saghin). https://doi.org/10.1016/j.optmat.2020.109669 Received 7 November 2019; Received in revised form 7 January 2020; Accepted 8 January 2020 Available online 16 January 2020 0925-3467/© 2020 Elsevier B.V. All rights reserved.
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278 nm and 309 nm were identified and are assigned to a multitude of phenolic compounds such as hydroxybenzoic acids [2] and cyanidins from the class of anthocyanins [24], respectively to flavanols and fla vonols [2]. The optical absorption spectra of the bilberry extract shows a maximum absorption peak at 283 nm, where the DNA exhibits a weak absorbance. Due to the interaction between DNA and the bilberry extract a small bathochromic shift is observed, as shown in Fig. 2 for a bilberry extract concentration of 0.25%. The spectra of an aqueous bilberry extract solution was added as insertion in Fig. 2 in order to better visualize the shifts that may occur after the addition of the bilberry extract into the DNA solution. 3.2. Fluorescence spectra The emission fluorescence spectra were recorded for the DNA-BBE complex solutions and are presented in Fig. 3. Three maximum ab sorption peaks were identified at 316 nm, 429 nm and 627 nm, respectively. The emission peak at 316 nm could be assigned to cyanidin [25], the peak at 429 nm to pelargonidin [26] and the peak at 627 nm to pyrano-cyanidin or to pyrano-malvidin [27]. A slight increase of the fluorescent intensity signal was observed from DNA-BBE1 to DNA-BBE3 solutions, then from DNA-BBE3 to DNA-BBE5 solutions a slight decrease of the fluorescent intensity signal was noticed. This quenching effect could be assessed to the molecular aggregation at higher concentrations that leads to a decrease of the fluorescence signal [28]. The excitation spectra for the DNA-BBE1 solution were recorded at two different wavelengths and are presented in Fig. 4. From the spectra three maximum absorption peaks were identified at and 230 nm, 278 nm and 309 nm, respectively, which correspond with the maximum absorption peaks found in the UV–Vis spectra . Emission spectra presented in Fig. 5 were recorded using the same concentrations of 0.06% and 0.12% of bilberry extract (BBE) and bilberry extract in DNA (DNA-BBE) solution of 0.05 g/L concentration. It was observed an increase of the fluorescence intensity for the DNA so lution but also a small decrease of the DNA-BBE complex compared with the bilberry extract in bidistilled water for the same concentrations. This may be due to the molecular agreggation of the DNA extract with the bilberry extract due to the formation of DNA-BBE complex [29]. DNA-BBE complex may significantly contribute to the formation of molecular aggregates therefore could influence the fluorescence [30].
Fig. 1. Basic chemical structures of anthocyanins (reproduced from Ref. [6]).
using DEKTAK 120 model profilometer of KLA Tencor. Third harmonic generation and the optical damage threshold measurements were per formed using a Neodymium doped Yttrium Aluminium Garnet (Nd:YAG) laser at 1064.2 nm fundamental wavelength with 10 Hz frequency and 6 ns pulse duration. 2.1. Preparation of DNA-BBE complex solutions A stock solution of DNA extract was prepared in distilled water in a 0.05 g/L concentration. This solution was further used for preparation of DNA-BBE complex dilutions using different concentrations of bilberry extract. The linear optical absorption spectra of the solutions were recorded from the most diluted to more concentrated using abbrevia tions from DNA-BBE1 to DNA-BBE5 at bilberry extract concentrations of: 0.06% (BBE1), 0.12% (BBE2), 0.25% (BBE3), 0.50% (BBE4) and 0.74% (BBE5). All of them were prepared at room temperature (25 � C).
3.3. Third harmonic generation The nonlinear optical (NLO) properties of the studied thin films were determined by the optical third harmonic generation (THG) technique using the procedure elaborated by Kajzar et al. [31] (see also Kajzar) [32]. The light source is a Quantel Brillant model Q–switched neo dymium doped yttrium aluminium garnet (Nd:YAG) laser, operating at 1064.2 nm fundamental wavelength with 6 ns pulse duration and 10 Hz operation rate. Details of the experimental procedure are given in Ref. [32]. The technique allows to determine the very fast (in atosecond range), electronic origin, third-order NLO susceptibility χ ð3Þ ð 3ω; ω; ω; ωÞ. In this experiment the generated third harmonic beam intensity I3ω ðθÞ is collected in function of the incidence angle θ. The experiment is computer controlled. The obtained dependence is then fitted by the theoretical one [31]. Fig. 6 shows an example of the measured I3ω ðθÞ variation with the incidence angle for the substrate alone (a) and for substrate þ thin film assembly (b), respectively. The I3ω spectra are obtained by rotating the sample around an axis coinciding with the beam propagation direction and perpendicular to it. The observed non zero minima in Fig. 6(b) show thin film contri bution, varying monotonically with the incidence angle due to the thin film thickness much less than the coherence length. Points in Fig. 6 show the measured values while continuous line the calculated ones. A very good agreement between the theoretical and the measured values is observed. The THG measurements on substrate
2.2. Thin films preparation Thin films were prepared on glass substrate using the spin-coating technique. A two consecutive step program was used for deposition. The first step (40 s at 500 rpm) allowed a regular spreading of the so lution on the glass substrate, while the second one (60 s at 1000 rpm) was conducted for its drying. The prepared thin films were additionally dried in the oven at 60 � C for 1 h to remove the residual solvent. 3. Results and discussion 3.1. UV-VIS absorption spectra The UV–Vis absorption spectra of the solutions, prepared using different concentrations of bilberry extract in 0.05 g/L DNA solution are displayed in Fig. 2. The spectra present a specific absorption peak for the DNA at 260 nm [22,23]. Also, for the bilberry extract specific peaks at 2
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Optical Materials 100 (2020) 109669
Fig. 2. UV–Vis absorption spectra of the solutions prepared using DNA, bilberry extract and DNA-BBE complex.
Fig. 3. Emission spectra of solutions prepared using the DNA-BBE complex at: (a) λex ¼ 278 nm and (b) λex ¼ 309 nm.
Fig. 4. Excitation spectra of DNA-BBE1 complex solution at: (a) λem ¼ 316 nm and (b) λem ¼ 429 nm.
alone, done at the same conditions as for the thin film þ substrate as sembly allow their calibration. It is achieved when fitting the I3ω ðθÞ dependence for the film þ substrate spectra by varying the ratio of the corresponding (C) and glass (G) NLO susceptibilities to get the same
scale factor as for the glass plate measurements. � ð3Þ ρ ¼ χ ð3Þ C ð 3ω; ω; ω; ωÞ χ G ð 3ω; ω; ω; ωÞ
(1)
The measured ratios ρ for the studied films are listed in Table 1. For 3
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Optical Materials 100 (2020) 109669
the used experimental set up can be found in Ref. [36]. The measured values of optical damage thresholds for the studied materials are listed in Table 2 and compared with those measured for two biopolymers: DNA and CTMA as well as for synthetic polymers polycarbonate (PC) and poly (ethylene glycol) (PEG) [36]. If already for DNA and collagen the measured values are about one order of magnitudes larger than those observed for the synthetic polymers, they are still larger for the studied complexes. It shows that the addition of bilberry extract stabilizes the complex and make it resisting to higher laser light intensities than the matrix alone. This influence of the bilberry extract is seen also on the dependence of the optical damage threshold on the bilberry extract Table 1 BBE concentration variation of the ratios of THG susceptibility of the biopolymer complex DNA-BBE to that of glass substrate as well as the absolute values cali brated with cubic susceptibility of glass substrate. Material
Thickness (μm)
Refractive index nω
Refractive index n3ω
χð3Þ p ð
3ω; ω;
ω; ωÞ=χð3Þ G ð
3ω; ω; ω; ωÞ/χ(3) G (-3ω;ω,ω,ω)
Fig. 5. Emission spectra of DNA, BBE and DNA-BBE complex at λex ¼ 278 nm.
thin film refractive indices we used the values reported for DNA-CTMA by Grote et al. [33]. For calibration we used for glass χ ð3Þ ð 3ω; ω; ω; ωÞ susceptibility the value reported by Morichere et al. [34], which we updated for the new value for silica recently proposed by Gubler and Bosshard [35]. With that χ G ð 3ω; ω; ω; ωÞ value we obtained Table 1 the last colon of the THG susceptibilities for the studied films. They are 25–34 times larger than the reference silica χ ð3Þ ð 3ω; ω; ω; ωÞ susceptibility. For an NLO material with a large transparency range (cut off at ca 400 nm) it is a remarquable value. ð3Þ
3.4. Optical damage threshold
χ(3)(3ω;ω,ω,ω) ∙x 10 (esu)
14
DNABBE3 DNABBE4 DNABBE5 Silica
0.12
1.488
1.512
17.5
51.5
0.08
1.488
1.512
21
61.7
0.12
1.488
1.512
23
67.6
1008
1.44967
1.45051
Glass plate PMMA
1175
1.50664
1.538204
0.68 � 0.7 [35] 1 [34]
1.488
1.512
4.5
2.0 � 0.2 [35] 2.94 � 0.28 [34] 4.5
Table 2 Optical damage thresholds of DNA-BBE complexes.
A very important parameter determining applicability of a given material in photonics, particularly in nonlinear optics, is its optical damage threshold. It is the maximum light intensity which a given dielectric material withstands. The laser pulses with intensities higher than this threshold one causes a permanent damage to the material. The optical damage threshold in the studied DNA-BBE thin films was measured with a nanosecond Q switched Nd:YAG laser operating at 1064.2 nm wavelength, with 6 ns pulse duration and 10 Hz operating frequency. The chosen laser wavelength is in the transparency range of the studied material, absorption induced damage is excluded. Details of
Sample
Beam Energy (mJ)
Optical damage threshold (GW/cm2)
DNA-BBE1 DNA-BBE2 DNA-BBE3 DNA-BBE4 DNA-BBE5 DNA Collagen PC PEG
33.7 52.9 57.8 60.2 67.9
15.5 24.3 26.5 27.6 31.2 5.30 4.40 0.30 0.78
Fig. 6. Calculated (solid line) and measured third harmonic generation intensity dependence on the incidence angle for glass substrate alone (a) and for the substrate þ thin film of DNA-BBE3 complex. 4
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content (Table 2). It increases with its increase. The values listed in Table 2 correspond to the specific experimental conditions, i.e. the fundamental wavelength of 1064.2 nm, repetition rate of 10 Hz and pulse duration of 6 ns. An increasing tendency for optical damage threshold with concen tration of bilberry extract is noticed, what support the potential of such composite biomaterials for biophotonic applications.
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4. Conclusions In this study we show that novel all biodegradable, originating from renewable ressources, non toxic, photonic materials can be obtained. These materials exhibit a high optical damage threshold, larger than the synthetic polymers. They can be processed into good optical quality thin films by the solution casting method, what is very important for appli cation in integrated optics. The photosensitive, nature made, molecule bilberry exhibits blue fluorescence showing potential of application in blue light emitting devices as well as in blue lasers. The obtained thin films show also interesting third order NLO properties. Thus, the ob tained materials present an attractive alternative for synthetic ones for application in photonics. Both DNA and bilberry are ionic compounds opposing charged. Thus the observed shift of bilberry optical absorption spectrum when added to DNA may be well accounted for the electrostatic interaction between them. The shift is increasing with the increasing dopant content, as ex pected. The fluorescece intensity increases also when passing from DNABBE1 to DNA-BBE3 solutions. For solutions with a higher bilberry con tent (DNA-BBE3 to DNA-BBE5) a slight decrease of the fluorescence intensity was noticed, this quenching effect being well accounted for the aggregation process, as it is usually observed at higher luminophore contents. We observe also very large optical damage threshold for studied complexes. They are even larger than that measured for DNA alone and are more than one order of magnitudes larger than measurd for synthetic polymers PC and PEG. It makes that these materials can be used in photonics, even if the light intensities are very high. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgement The authors acknowledge the financial support of UEFISCDI organ ism, under Contract Number 7/2018, Code Project PN-III-P1-1.1-PD2016-0580, BIO-COL-DNA. References [1] L. Zoratti, H. Klemettil€ a, L. Jaakola, Bilberry (Vaccinium myrtillus L.) ecotypes, in: M.S.J. Simmonds, V.R. Preedy (Eds.), Nutritional Composition of Fruit Cultivars, Academic Press - Elsevier, 2016, pp. 83–99, https://doi.org/10.1016/B978-0-12408117-8.00004-0. [2] W.-k. Chu, S.C.M. Cheung, R.A.W. Lau, I.F.F. Benzie, Bilberry (Vaccinium myrtillus L.), in: I.F. F Benzie, S. Wachtel-Galor (Eds.), Herbal Medicine: Biomolecular and Clinical Aspects, second ed., CRC Press/Taylor & Francis, Boca Raton (FL), 2011, pp. 55–71. [3] R. Nestby, D. Percival, I. Martinussen, N. Opstad, J. Rohloff, The European blueberry (Vaccinium myrtillus L.) and the potential for cultivation. A review, Eur. J. Plant Sci. Biotechnol. 5 (2011) 5–16. [4] C. Ulbricht, E. Basch, S. Basch, S. Bent, H. Boon, D. Burke, D. Costa, C. Falkson, N. Giese, M. Goble, S. Hashmi, S. Mukarjee, G. Papaliodis, E. Seamon, S. TanguayColucci, W. Weissner, J. Woods, An evidence-based systematic review of bilberry (Vaccinium myrtillus) by the natural standard research collaboration, J. Diet. Suppl. 6 (2009) 162–200, https://doi.org/10.1080/19390210902861858. [5] P. Ongkowijoyo, D.A. Luna-Vital, E.G. de Mejia, Extraction techniques and analysis of anthocyanins from food sources by mass spectrometry: an update, Food Chem. 250 (2018) 113–126, https://doi.org/10.1016/j.foodchem.2018.01.055.
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Spectroscopy and non-linear optical properties of DNA - Bilberry complex �duret¸u a, François Kajzar a, b Ana-Maria Manea-Saghin a, *, Carla-Cezarina Pa a b
Faculty of Applied Chemistry and Materials Science, University POLITEHNICA of Bucharest, Polizu Street No 1, 011061, Bucharest, Romania Laboratoire de Chimie, CNRS, Universit�e Claude Bernard, ENS-Lyon, 46 All�ee d’Italie, 69364, Lyon cedex 07, France
A R T I C L E I N F O
A B S T R A C T
Keywords: Deoxyribonucleic acid (DNA) Bilberry extract (BBE) DNA-BBE complex Third harmonic generation Optical damage threshold
Natural extracts are getting more attention due to the fast degradability rate and low toxicity. In this paper the obtained results for the complex based on deoxyribonucleic acid biopolymer with bilberry (BBE) natural extract are presented and discussed. The interaction between the two materials (DNA-BBE complex) was evaluated by measuring the linear optical properties using UV–Vis and fluorescence spectral methods. Furthermore, the DNABBE complex was used for non-linear optical (NLO) properties investigations of the obtained thin films on glass substrate by spin-coating technique. Also, the optical damage threshold of the studied DNA-BBE complexes was measured in order to check their potentiality and interest for application in photonics.
1. Introduction Bilberry (Vaccinium myrtillus L.) is a fruit known also as European blueberry [1], huckleberry or whortleberry [2]. It grows abundantly in the Northern and Eastern areas of Europe [1], usually on acid soils, from marginal forests up to high altitude [3]. In bilberries fruit, anthocyanins represent about 90% of the total phenolic compounds [1]. The bilberry extract has beneficial effects as antioxidant, hypoglycemic and lipid-lowering agent [4]. Usually, it contains phenolic acids, tannins, flavonols (catechins, quercetin), resveratrol, hydroquinone, thiamin, vitamin C and mostly anthocyanosides, which are flavonoid derivatives of anthocyanins [1,2,4]. Anthocyanins are powerful antioxidants, water-soluble pigments that belong to the flavonoids class [5,6] and are naturally synthesized in the epidermal tissue. They confer the red, blue and purple colors to the berries [5,7]. Also, anthocyanines have pro tective role against cold stress, but they are sensible to environmental factors such as temperature or light that may affect their stability [8]. The most commonly occuring anthocyanidins are delphinidins, cyani dins, malvidins and petunidins [1], (Fig. 1). Synthetic polymers are subjected to slow degradation rates. Thus to avoid the pollution it is wishful to use natural materials such as bio polymers [9]. One of the main categories of biopolymers are poly nucleotides like DNA and RNA which are long chain polymers composed of 13 or more nucleotide monomers [10,11]. In previous studies, good optical properties were exhibited when DNA was used in combination with natural extracts [9]. The non-linear optical (NLO) properties of thin films are usually investigated using the Third Harmonic Generation
[12–21]. In this paper we report on preparation and characterization of a new biomaterial in view of its application in photonics. The composite ma terial was obtained by doping, in solution, the DNA extract with bilberry natural extract. It was characterized by UV–Vis spectroscopy and fluo rescence studies to evaluate its linear optical properties. Furthermore, the nonlinear optical (NLO) properties characterization of functional ized thin films was performed by the optical third harmonic generation (THG) measurements at 1064.2 nm fundamental wavelength. The op tical damage threshold measurements were also performed in order to establish the applicability of DNA-BBE thin films in photonics. 2. Materials, equipment and thin films processing A low molecular mass deoxyribonucleic acid, purchased from Sigma Aldrich Company, was used in this study. The bilberry natural hydro alcoholic extract used for this study had a concentration of approxi mately 50% and was received from Hofigal S.A. The UV–Vis linear optical absorption spectra were recorded using 1 cm quartz cuvettes, in 200–800 nm spectral range, on an instrument from Thermo Scientific, Evolution 220 model, with integrated Insight v. 2.3.345 Software. The optical fluorescence spectra were recorded with a spectrofluorimeter instrument from Jasco, FP-6500 model. The same bandwidth of 10 nm (ex)/10 nm (em) was used for all the registered spectra. Thin films were prepared by spin-coating technique on glass substrate using a spincoater from Laurell Technologies Corporation, model WS-400B-6NPP/ LITE. Their thickness was determined by the profilometry technique
* Corresponding author. E-mail address: [email protected] (A.-M. Manea-Saghin). https://doi.org/10.1016/j.optmat.2020.109669 Received 7 November 2019; Received in revised form 7 January 2020; Accepted 8 January 2020 Available online 16 January 2020 0925-3467/© 2020 Elsevier B.V. All rights reserved.
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278 nm and 309 nm were identified and are assigned to a multitude of phenolic compounds such as hydroxybenzoic acids [2] and cyanidins from the class of anthocyanins [24], respectively to flavanols and fla vonols [2]. The optical absorption spectra of the bilberry extract shows a maximum absorption peak at 283 nm, where the DNA exhibits a weak absorbance. Due to the interaction between DNA and the bilberry extract a small bathochromic shift is observed, as shown in Fig. 2 for a bilberry extract concentration of 0.25%. The spectra of an aqueous bilberry extract solution was added as insertion in Fig. 2 in order to better visualize the shifts that may occur after the addition of the bilberry extract into the DNA solution. 3.2. Fluorescence spectra The emission fluorescence spectra were recorded for the DNA-BBE complex solutions and are presented in Fig. 3. Three maximum ab sorption peaks were identified at 316 nm, 429 nm and 627 nm, respectively. The emission peak at 316 nm could be assigned to cyanidin [25], the peak at 429 nm to pelargonidin [26] and the peak at 627 nm to pyrano-cyanidin or to pyrano-malvidin [27]. A slight increase of the fluorescent intensity signal was observed from DNA-BBE1 to DNA-BBE3 solutions, then from DNA-BBE3 to DNA-BBE5 solutions a slight decrease of the fluorescent intensity signal was noticed. This quenching effect could be assessed to the molecular aggregation at higher concentrations that leads to a decrease of the fluorescence signal [28]. The excitation spectra for the DNA-BBE1 solution were recorded at two different wavelengths and are presented in Fig. 4. From the spectra three maximum absorption peaks were identified at and 230 nm, 278 nm and 309 nm, respectively, which correspond with the maximum absorption peaks found in the UV–Vis spectra . Emission spectra presented in Fig. 5 were recorded using the same concentrations of 0.06% and 0.12% of bilberry extract (BBE) and bilberry extract in DNA (DNA-BBE) solution of 0.05 g/L concentration. It was observed an increase of the fluorescence intensity for the DNA so lution but also a small decrease of the DNA-BBE complex compared with the bilberry extract in bidistilled water for the same concentrations. This may be due to the molecular agreggation of the DNA extract with the bilberry extract due to the formation of DNA-BBE complex [29]. DNA-BBE complex may significantly contribute to the formation of molecular aggregates therefore could influence the fluorescence [30].
Fig. 1. Basic chemical structures of anthocyanins (reproduced from Ref. [6]).
using DEKTAK 120 model profilometer of KLA Tencor. Third harmonic generation and the optical damage threshold measurements were per formed using a Neodymium doped Yttrium Aluminium Garnet (Nd:YAG) laser at 1064.2 nm fundamental wavelength with 10 Hz frequency and 6 ns pulse duration. 2.1. Preparation of DNA-BBE complex solutions A stock solution of DNA extract was prepared in distilled water in a 0.05 g/L concentration. This solution was further used for preparation of DNA-BBE complex dilutions using different concentrations of bilberry extract. The linear optical absorption spectra of the solutions were recorded from the most diluted to more concentrated using abbrevia tions from DNA-BBE1 to DNA-BBE5 at bilberry extract concentrations of: 0.06% (BBE1), 0.12% (BBE2), 0.25% (BBE3), 0.50% (BBE4) and 0.74% (BBE5). All of them were prepared at room temperature (25 � C).
3.3. Third harmonic generation The nonlinear optical (NLO) properties of the studied thin films were determined by the optical third harmonic generation (THG) technique using the procedure elaborated by Kajzar et al. [31] (see also Kajzar) [32]. The light source is a Quantel Brillant model Q–switched neo dymium doped yttrium aluminium garnet (Nd:YAG) laser, operating at 1064.2 nm fundamental wavelength with 6 ns pulse duration and 10 Hz operation rate. Details of the experimental procedure are given in Ref. [32]. The technique allows to determine the very fast (in atosecond range), electronic origin, third-order NLO susceptibility χ ð3Þ ð 3ω; ω; ω; ωÞ. In this experiment the generated third harmonic beam intensity I3ω ðθÞ is collected in function of the incidence angle θ. The experiment is computer controlled. The obtained dependence is then fitted by the theoretical one [31]. Fig. 6 shows an example of the measured I3ω ðθÞ variation with the incidence angle for the substrate alone (a) and for substrate þ thin film assembly (b), respectively. The I3ω spectra are obtained by rotating the sample around an axis coinciding with the beam propagation direction and perpendicular to it. The observed non zero minima in Fig. 6(b) show thin film contri bution, varying monotonically with the incidence angle due to the thin film thickness much less than the coherence length. Points in Fig. 6 show the measured values while continuous line the calculated ones. A very good agreement between the theoretical and the measured values is observed. The THG measurements on substrate
2.2. Thin films preparation Thin films were prepared on glass substrate using the spin-coating technique. A two consecutive step program was used for deposition. The first step (40 s at 500 rpm) allowed a regular spreading of the so lution on the glass substrate, while the second one (60 s at 1000 rpm) was conducted for its drying. The prepared thin films were additionally dried in the oven at 60 � C for 1 h to remove the residual solvent. 3. Results and discussion 3.1. UV-VIS absorption spectra The UV–Vis absorption spectra of the solutions, prepared using different concentrations of bilberry extract in 0.05 g/L DNA solution are displayed in Fig. 2. The spectra present a specific absorption peak for the DNA at 260 nm [22,23]. Also, for the bilberry extract specific peaks at 2
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Fig. 2. UV–Vis absorption spectra of the solutions prepared using DNA, bilberry extract and DNA-BBE complex.
Fig. 3. Emission spectra of solutions prepared using the DNA-BBE complex at: (a) λex ¼ 278 nm and (b) λex ¼ 309 nm.
Fig. 4. Excitation spectra of DNA-BBE1 complex solution at: (a) λem ¼ 316 nm and (b) λem ¼ 429 nm.
alone, done at the same conditions as for the thin film þ substrate as sembly allow their calibration. It is achieved when fitting the I3ω ðθÞ dependence for the film þ substrate spectra by varying the ratio of the corresponding (C) and glass (G) NLO susceptibilities to get the same
scale factor as for the glass plate measurements. � ð3Þ ρ ¼ χ ð3Þ C ð 3ω; ω; ω; ωÞ χ G ð 3ω; ω; ω; ωÞ
(1)
The measured ratios ρ for the studied films are listed in Table 1. For 3
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the used experimental set up can be found in Ref. [36]. The measured values of optical damage thresholds for the studied materials are listed in Table 2 and compared with those measured for two biopolymers: DNA and CTMA as well as for synthetic polymers polycarbonate (PC) and poly (ethylene glycol) (PEG) [36]. If already for DNA and collagen the measured values are about one order of magnitudes larger than those observed for the synthetic polymers, they are still larger for the studied complexes. It shows that the addition of bilberry extract stabilizes the complex and make it resisting to higher laser light intensities than the matrix alone. This influence of the bilberry extract is seen also on the dependence of the optical damage threshold on the bilberry extract Table 1 BBE concentration variation of the ratios of THG susceptibility of the biopolymer complex DNA-BBE to that of glass substrate as well as the absolute values cali brated with cubic susceptibility of glass substrate. Material
Thickness (μm)
Refractive index nω
Refractive index n3ω
χð3Þ p ð
3ω; ω;
ω; ωÞ=χð3Þ G ð
3ω; ω; ω; ωÞ/χ(3) G (-3ω;ω,ω,ω)
Fig. 5. Emission spectra of DNA, BBE and DNA-BBE complex at λex ¼ 278 nm.
thin film refractive indices we used the values reported for DNA-CTMA by Grote et al. [33]. For calibration we used for glass χ ð3Þ ð 3ω; ω; ω; ωÞ susceptibility the value reported by Morichere et al. [34], which we updated for the new value for silica recently proposed by Gubler and Bosshard [35]. With that χ G ð 3ω; ω; ω; ωÞ value we obtained Table 1 the last colon of the THG susceptibilities for the studied films. They are 25–34 times larger than the reference silica χ ð3Þ ð 3ω; ω; ω; ωÞ susceptibility. For an NLO material with a large transparency range (cut off at ca 400 nm) it is a remarquable value. ð3Þ
3.4. Optical damage threshold
χ(3)(3ω;ω,ω,ω) ∙x 10 (esu)
14
DNABBE3 DNABBE4 DNABBE5 Silica
0.12
1.488
1.512
17.5
51.5
0.08
1.488
1.512
21
61.7
0.12
1.488
1.512
23
67.6
1008
1.44967
1.45051
Glass plate PMMA
1175
1.50664
1.538204
0.68 � 0.7 [35] 1 [34]
1.488
1.512
4.5
2.0 � 0.2 [35] 2.94 � 0.28 [34] 4.5
Table 2 Optical damage thresholds of DNA-BBE complexes.
A very important parameter determining applicability of a given material in photonics, particularly in nonlinear optics, is its optical damage threshold. It is the maximum light intensity which a given dielectric material withstands. The laser pulses with intensities higher than this threshold one causes a permanent damage to the material. The optical damage threshold in the studied DNA-BBE thin films was measured with a nanosecond Q switched Nd:YAG laser operating at 1064.2 nm wavelength, with 6 ns pulse duration and 10 Hz operating frequency. The chosen laser wavelength is in the transparency range of the studied material, absorption induced damage is excluded. Details of
Sample
Beam Energy (mJ)
Optical damage threshold (GW/cm2)
DNA-BBE1 DNA-BBE2 DNA-BBE3 DNA-BBE4 DNA-BBE5 DNA Collagen PC PEG
33.7 52.9 57.8 60.2 67.9
15.5 24.3 26.5 27.6 31.2 5.30 4.40 0.30 0.78
Fig. 6. Calculated (solid line) and measured third harmonic generation intensity dependence on the incidence angle for glass substrate alone (a) and for the substrate þ thin film of DNA-BBE3 complex. 4
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content (Table 2). It increases with its increase. The values listed in Table 2 correspond to the specific experimental conditions, i.e. the fundamental wavelength of 1064.2 nm, repetition rate of 10 Hz and pulse duration of 6 ns. An increasing tendency for optical damage threshold with concen tration of bilberry extract is noticed, what support the potential of such composite biomaterials for biophotonic applications.
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4. Conclusions In this study we show that novel all biodegradable, originating from renewable ressources, non toxic, photonic materials can be obtained. These materials exhibit a high optical damage threshold, larger than the synthetic polymers. They can be processed into good optical quality thin films by the solution casting method, what is very important for appli cation in integrated optics. The photosensitive, nature made, molecule bilberry exhibits blue fluorescence showing potential of application in blue light emitting devices as well as in blue lasers. The obtained thin films show also interesting third order NLO properties. Thus, the ob tained materials present an attractive alternative for synthetic ones for application in photonics. Both DNA and bilberry are ionic compounds opposing charged. Thus the observed shift of bilberry optical absorption spectrum when added to DNA may be well accounted for the electrostatic interaction between them. The shift is increasing with the increasing dopant content, as ex pected. The fluorescece intensity increases also when passing from DNABBE1 to DNA-BBE3 solutions. For solutions with a higher bilberry con tent (DNA-BBE3 to DNA-BBE5) a slight decrease of the fluorescence intensity was noticed, this quenching effect being well accounted for the aggregation process, as it is usually observed at higher luminophore contents. We observe also very large optical damage threshold for studied complexes. They are even larger than that measured for DNA alone and are more than one order of magnitudes larger than measurd for synthetic polymers PC and PEG. It makes that these materials can be used in photonics, even if the light intensities are very high. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgement The authors acknowledge the financial support of UEFISCDI organ ism, under Contract Number 7/2018, Code Project PN-III-P1-1.1-PD2016-0580, BIO-COL-DNA. References [1] L. Zoratti, H. Klemettil€ a, L. Jaakola, Bilberry (Vaccinium myrtillus L.) ecotypes, in: M.S.J. Simmonds, V.R. Preedy (Eds.), Nutritional Composition of Fruit Cultivars, Academic Press - Elsevier, 2016, pp. 83–99, https://doi.org/10.1016/B978-0-12408117-8.00004-0. [2] W.-k. Chu, S.C.M. Cheung, R.A.W. Lau, I.F.F. Benzie, Bilberry (Vaccinium myrtillus L.), in: I.F. F Benzie, S. Wachtel-Galor (Eds.), Herbal Medicine: Biomolecular and Clinical Aspects, second ed., CRC Press/Taylor & Francis, Boca Raton (FL), 2011, pp. 55–71. [3] R. Nestby, D. Percival, I. Martinussen, N. Opstad, J. Rohloff, The European blueberry (Vaccinium myrtillus L.) and the potential for cultivation. A review, Eur. J. Plant Sci. Biotechnol. 5 (2011) 5–16. [4] C. Ulbricht, E. Basch, S. Basch, S. Bent, H. Boon, D. Burke, D. Costa, C. Falkson, N. Giese, M. Goble, S. Hashmi, S. Mukarjee, G. Papaliodis, E. Seamon, S. TanguayColucci, W. Weissner, J. Woods, An evidence-based systematic review of bilberry (Vaccinium myrtillus) by the natural standard research collaboration, J. Diet. Suppl. 6 (2009) 162–200, https://doi.org/10.1080/19390210902861858. [5] P. Ongkowijoyo, D.A. Luna-Vital, E.G. de Mejia, Extraction techniques and analysis of anthocyanins from food sources by mass spectrometry: an update, Food Chem. 250 (2018) 113–126, https://doi.org/10.1016/j.foodchem.2018.01.055.
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