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Effects of gold nanoparticles on photophysical behaviour of chlorophyll and pheophytin
Journal Pre-proof Effects of gold nanoparticles on photophysical behaviour of chlorophyll and pheophytin A.V. Mezacasa (Conceptualization)Data analysis) (Writing - review and editing), A.M. Queiroz, D.E. Graciano, M.S. Pontes, E.F. Santiago (Conceptualization)Data analysis Writing review and editing), I.P. OliveiraData analysis) (Writing - review and editing), AJ Lopez, G.A. Casagrande (Conceptualization)Data analysis), M.D. Scherer (Funding acquisition), D.D. dos ReisData analysis), S.L. Oliveira (Conceptualization)Data analysis) (Writing review and editing), A.R.L. CairesData analysis) (Funding acquisition) (Writing - review and editing)
PII:
S1010-6030(19)30850-0
DOI:
https://doi.org/10.1016/j.jphotochem.2019.112252
Reference:
JPC 112252
To appear in:
Journal of Photochemistry & Photobiology, A: Chemistry
Received Date:
21 May 2019
Revised Date:
3 November 2019
Accepted Date:
16 November 2019
Please cite this article as: Mezacasa AV, Queiroz AM, Graciano DE, Pontes MS, Santiago EF, Oliveira IP, Lopez A, Casagrande GA, Scherer MD, dos Reis DD, Oliveira SL, Caires ARL, Effects of gold nanoparticles on photophysical behaviour of chlorophyll and pheophytin, Journal of Photochemistry and amp; Photobiology, A: Chemistry (2019), doi: https://doi.org/10.1016/j.jphotochem.2019.112252
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Effects of gold nanoparticles on photophysical behaviour of chlorophyll and pheophytin A.V. Mezacasa1,2, A.M. Queiroz1,2, D.E. Graciano1,2, M.S. Pontes3, E.F. Santiago3, I.P. Oliveira4; AJ Lopez5, G.A. Casagrande2, M.D. Scherer2, D.D. dos Reis2, S.L. Oliveira2, A.R.L. Caires2,6,* Faculty of Exact Science and Technology, Federal University of Grande Dourados, Dourados, Brazil.
2
Institute of Physics, Federal University of Mato Grosso do Sul, Campo Grande, Brazil.
3
Natural Resources Study Center, State University of Mato Grosso do Sul, Dourados, Brazil.
4
Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil.
5
Institute of Chemistry, University of Campinas, Campinas, SP, Brazil.
6
School of Life Sciences, University of Essex, Colchester, UK.
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Graphical-abstract
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AuNPs strongly alter the optical features of Chlorophyll and Pheophytin Plasmon resonance contributed for fluorescence enhancement in both pigments Pheo exhibited fluorescence quenching in solutions with high AuNPs concentration Affinity of Chl and Pheo with AuNPs was determined Photoinduced electron transfer from Chl and Pheo to AuNPs surface plays a key role in the pigments-NPs interaction
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Highlights
ABSTRACT
The increasing application of engineering nanoparticles (ENPs) in various commercial products justifies the need for studying their impact on living organisms and environment. The interaction between gold nanoparticles (AuNPs) with different diameters (5, 10, and 20 nm) and two
photosynthetic pigments chlorophyll (Chl) and pheophytin (Pheo) was investigated using optical techniques and computational simulations. UV-Vis absorption, steady-state fluorescence, and timeresolved fluorescence showed that AuNPs affected the optical features of the studied photosynthetic pigments. Moreover, the optical and computational results show that an analogous Pheo molecule (chlorin) was less susceptible than an analogous chlorophyll molecule (Mg-chlorin) to interact with AuNPs. Therefore, AuNPs interact with both Chl and Pheo so that they may change the operation of the plant photosystems in case of uptake and internalisation in plants.
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*Corresponding author. E-mail: [email protected].
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Keywords: Chlorophyll; Pheophytin; Gold nanoparticle; Optical analysis; Computational simulation.
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1 Introduction
In recent years, materials in the nanometric scale have been investigated by different research
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areas and applied in commercial products because of their distinctive optical, electrical, and mechanical properties [1–4]. As a result, the utilisation of new products containing nanoparticles (NPs)
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has increased exponentially, becoming imperative studies on the social and environmental consequences [5,6]. The scarcity of information about the harmful effects of nanotechnological innovations on the environment and human health suggests greater prudence [7,8]. Gold nanoparticles (AuNPs) are among the most used nanomaterials to produce functional
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electrical coatings, improve the redox activity in photochemical and electrochemical applications by interacting with organic molecules, and other applications [9–12]. The successful use of AuNPs can be easily understood by the fact that they have unique optical and electronic characteristics based on the existence of the surface plasmon resonance (SPR) [13–16]. The choice of biological models and experimental techniques to investigate the effects induced by NPs on living organisms and environment constitutes a challenge considering the dependence of
the physicochemical properties of the NPs on composition, size, shape, and concentration, as well as the complexity of the interactions between them and organisms [5,17,18]. Plants are the basis of both the ecosystem and food chain, and they are submitted continuously to materials released and accumulated in the environment by human activities; so it is crucial to test the NPs effects on them, especially on their photosynthetic pigments [19–24]. The NPs impacts on photosynthetic pigmentslight interaction may alter the plant life cycle and productivity because the pigments play a crucial role in both harvesting light and photosynthesis [22–27]. For instance, AuNPs may either positively or
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negatively modify the photosynthetic performance, i.e. the quantum yield of the photosynthetic activity, depending on two competing processes, namely light absorption enhancement of the photosynthetic pigments because of the SPR effect that intensifies the local field around them [21] and
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charge (or energy) transfer from the excited photosynthetic pigments to the metal nanoparticle surface [12,22].
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Among all the photosynthetic pigments, chlorophyll a (Chl a) is the main pigment responsible
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for photosynthesis, and its fluorescence has been extensively used to monitor the physiological status of plants. Alterations in the operation of the photosystems I and II (PSI and PSII) impact the Chl a fluorescence [28–30]. Chl a is composed of a chlorin pigment with a magnesium ion at the centre of
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the aromatic plane, and it contains a methyl group (-CH3) as the substituent at carbon C-3 [31,32]. Pheophytin a (Pheo a) is another relevant photosynthetic pigment in the reaction centre (RC) of PSII. It has a similar chemical structure of the Chl a, but without the magnesium ion at the centre of the
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chlorin ring [33]. Structurally, in the RC of PSII, four central chlorin pigments are arranged in two symmetry-related branches (D1 and D2 polypeptides): D1 and D2 share a dimer (P680) of Chl a molecule (P680D1 and P680D2), and each branch is also composed of one monomeric Chl a (Chl a D1 or Chl a D2) and one Pheo a (Pheo a D1 or Pheo a D2) [34,35]. Although recent studies have reported that AuNPs can be uptaken, translocated to the stems and leaves, and accumulated by plants [36–42], there is still a lack of data about the AuNPs-
photosynthetic pigments interaction and the influence of AuNPs on the photosynthetic capacity of plants. Based on that, the present study investigated the interaction of two photosynthetic pigments (Chl and Pheo molecules), extracted from green leaves of Vicia faba, with AuNPs of different sizes and concentrations by using UV-Vis absorption, steady-state and time-resolved fluorescence, along with computational simulations of the interactions between the photosynthetic pigments and AuNPs surface.
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2 Materials and methods 2.1 Gold nanoparticles
AuNPs with nominal diameters of 5, 10, and 20 nm were purchased from Sigma-Aldrich
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(Brazil). They were dispersed as colloidal particles in solutions of 100 mL comprising approximately:
carbonate, and 0.02% sodium azide.
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2.2 Chlorophyll and pheophytin extraction
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0.01% HAuCl4 suspended in 0.01% tannic acid with 0.04% trisodium citrate, 0.26 mM potassium
Chl and Pheo were extracted from Vicia faba (L.). It has been often used as a model system for
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plant physiological studies [43]. Leaves were collected from the plant grown in a greenhouse, cut into small pieces, and added to the methanol P.A. in the proportion of 15:100 (m/v) for 72 h at 5°C. Chl extract purification was performed by using the procedure adapted from Moreira et al. [44] and Khalyfa
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et al. [45]. The separation of the pigments was made by the process adapted from Maestrin et al. [46] and Filho et al. [47]. Pheo was obtained following the method reported by Moreira et al. [44] and Gerola [48]. The total chlorophyll concentration [Chl a + b] for methanolic extraction was determined from the molecular absorption spectra based on the Arnon method adapted by Porra [49] (Equation 1): [𝐶ℎ𝑙 𝑎 + 𝑏] = 24.23 𝐴652 + 3.26 𝐴665
Equation 1
Where A652 and A665 are the absorbances at 652 and 665 nm, respectively, and the constants 24.23 and 3.26 are the specific extinction coefficients of the chlorophyll at 652 and 665 nm, respectively. The Pheo concentration was determined by considering the initial concentration of the Chl in the solution. The pH of the Pheo and Chl extracts was adjusted to 7.0, before the preparation of the AuNPs-pigments solution. 2.3 AuNPs-pigments solution preparation
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The AuNPs-photosynthetic pigments interaction analysis was carried out using gold nanoparticles with diameters of 5, 10, and 20 nm. For each diameter, ten AuNPs concentrations (0.3, 0.6, 1.2, 2.4, 4.8, 9.6, 19.3, 38.6, 77.2, and 154.5 µM) were prepared in aqueous solutions based on the
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moles of gold atoms. A convenient volume of the methanolic solution of the photosynthetic pigments was added to the AuNPs solution to obtain a final photosynthetic pigments concentration of 0.5 μM in
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all solutions.
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2.4 Optical analysis
The UV-Vis absorption spectra were recorded in the 350-800 nm range with a
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spectrophotometer (USB 4000 FL, Ocean Optics®). Steady-state fluorescence was carried out using a spectrofluorimeter consisting of a diode laser as the excitation source (405 nm), a monochromator (USB 2000 FL, Ocean Optics®), and a Y-type optical fibre. Fluorescence spectra were obtained between 450 and 800 nm adopting the front-face geometry and a 10-mm quartz cuvette.
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Fluorescence decay measurements were performed with a confocal multiphoton LSM 780
Zeiss microscope equipped with Picoquant Fluortime system. It is composed of a 20x objective lens and single-photon avalanche diode (SPAD) detectors with the temporal resolution of 70 ps. Emission at 673 nm was collected when excited by a pulsed laser (Coherent) operating at 400 nm with a pulse width of 140 fs and 80-MHz repetition rate. All optical measurements were executed at room temperature.
2.5 Computational analysis First-principles calculations were performed to obtain a detailed picture of the pigmentsnanoparticle interaction at the atomic level. Calculations including geometry optimization, singlepoint calculations, and excitation energies for all molecules (chlorin, Mg-chlorin, and Au12 slab) and molecular complexes (chlorin/Au12 and Mg-chlorin/Au12 slab) were carried out by means of the density functional ωB97X [50], in a similar way as recently used to determine the adsorption
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energy of organic molecules on gold surfaces [51]. The positions of all unconstrained atoms of the molecules adsorbed on the gold surface were optimised until both the convergence criterion for force (0.0003 Hartree/Bohr) and energy change (10-8 Hartree) be achieved. Time-dependent density functional theory (TD-DFT) approach was employed to compute the excitation energies. Methanol
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was also employed as a solvent in our simulations by using the Polarizable continuum model (PCM)
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[52].
For chlorin and Mg-chlorin, the cc-pVDZ basis set was used. For Au12 slab, the LANL2MB
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was chosen. In the case of chlorin/Au12 slab and Mg-chlorin/Au12 slab complexes, the basis set was the LANL2MB for gold atoms and cc-pVDZ for the C, H, N, and Mg atoms. Counterpoise corrections
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were applied to the molecular complexes to correct basis set superposition error (BSSE) [53–55]. The electronic structure was calculated using the Gaussian 09 package [56]. Mulliken charge distribution of the chlorin-gold surface complexes was generated by VMD [57].
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3 Results and discussion 3.1 Optical analysis
Fig. 1 shows the UV-Vis absorption spectra of the Chl and Pheo molecules. Both spectra present similarities because they are porphyrins with the Soret band in the 390-440 nm range and the Q-bands between 500 and 670 nm. The Q-bands, Qx(0,0), Qx(1,0), Qy(0,0), and Qy(1,0) in Pheo is alike are
similar to the ones in free-base porphyrins [58]. The Q-bands are derived from the transitions between two π HOMO orbitals and two π* LUMO orbitals [58,59]. The presence of the Mg in the porphyrin’s
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centre favours the transitions related to the Soret and Qy(1,0) bands [46,58,59].
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Figure 1 - UV-Vis absorption spectra of the methanolic extracts of Chl and Pheo at 10.7 μM.
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Plasmon resonance absorption band in the 500-550 nm region – generated by the electron collective oscillation on the surface of AuNPs in the Chl and Pheo extracts – were observed (Fig. 2)
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[12]. The UV-Vis spectra of the Chl and Pheo solutions containing AuNPs with 5, 10, and 20-nm diameter at different concentrations (0-154.5 μM) are presented in Figs. S1 and S2 (supplementary
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materials), respectively.
Figure 2 - UV-Vis absorption spectra of (A) Chl and (B) Pheo extract at 0.5 μM in the absence and presence of AuNPs with 5-nm diameter and concentration of 154.5 μM.
Plasmon resonance absorbance increased with the AuNPs concentration in the presence of Chl or Pheo at 0.5 μM, as shown in Fig. 3 for the 5-nm AuNPs. Besides, the plasmon resonance absorbance presented two linear regimes in the 0-10 and 10-155 μM in the presence of both photosynthetic pigments. However, Chl and Pheo extracts exhibited different increase rates, indicating that the collective oscillation of the electrons on the surface of AuNPs was affected by the Mg in the porphyrin
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ring. Similar absorbance behaviour at 520 nm versus nanoparticle concentration was also seen for the
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AuNPs with 10 and 20-nm diameter (Fig. S3, supplementary materials).
Figure 3 - Plasmon absorbance at 520 nm as a function of the 5-nm AuNPs concentration in the
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presence of (A) chlorophyll and (B) pheophytin at 0.5 μM. The lines are guides to the eyes.
Fig. 4 shows the fluorescence spectra of the photosynthetic pigments under 405-nm excitation
in the presence of AuNPs with different diameters and concentrations. Both Photosynthetic pigments exhibited the usual spectrum of the porphyrin in the region of 660 to 710 nm [58,59]. Fluorescence enhancement of the Chl molecules was AuNPs content-dependent. This increase was originated by the
phenomenon known as metal-enhanced fluorescence (MEF), which is related to the plasmon resonance responsible for raising the incident electric field felt by Chl nearby the metal surfaces of AuNPs [60,61]. The total surface area increases as nanoparticle concentration increase for a given NP concentration, enhancing the plasmon resonance absorption and, consequently, the fluorescence intensity. MEF was also identified for the Pheo molecules in solutions with a low concentration of AuNPs (up to 19.3 μM). For AuNPs concentrations higher than 38.6 μM in the Pheo solution, fluorescence suppression was verified, suggesting that the Pheo molecules were either attached to the
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metal surface or close to it, allowing the electron transfer from excited Pheo to the AuNPs surface [3,12,22,62–64]. Further, both phenomena fluorescence enhancement and suppression were AuNPs diameter-dependent, given that the total metal surface area depends on both nanoparticle diameter and
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concentration.
Figure 4 - (A), (B), and (C) Fluorescence spectra of Chl and (D), (E), and (F) Pheo solutions with 5, 10, and 20-nm AuNPs, respectively, at concentrations of 0.3, 0.6, 1.2, 2.4, 4.8, 9.6, 19.3, 38.6, 77.2, and 154.5 μM. Samples were excited at 405-nm. The solid (black) lines correspond to the Chl and Pheo emission in the absence of AuNPs. The short dash-dot (blue) lines indicate the enhancement of the Chl and Pheo
emission in the presence of AuNPs. The short-dot (red) lines describe the suppression of the Chl and Pheo
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emission in the presence of AuNPs.
Figure 5 shows the fluorescence decay curves of Chl and Pheo when nearby AuNPs. The fluorescence lifetime was determined by a bi-exponential fitting and the average lifetime (𝜏𝑚 ) was calculated using Equation 2. 𝜏𝑚 =
(𝐴1 𝜏12 )+(𝐴2 𝜏22 )
Equation 2
(𝐴1 𝜏1 )+(𝐴2 𝜏2 )
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Where 𝜏1 and 𝜏2 are first and second decay times, respectively, and 𝐴1 and 𝐴2 their pre-
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exponential factors (amplitudes), respectively.
Figure 5 – (A) Chl and (B) Pheo fluorescence decay curves in the absence and presence of 5, 10, and 20-nm AuNPs. The average lifetime (𝜏𝑚 ) of (C) Chl and (D) Pheo as a function of the AuNPs surface area. The measurements were made for the Chl and Pheo solutions at 0.5 μM and AuNPs (5, 10, and 20 nm) at 4.2 μM. The samples were excited using a 405-nm laser.
The Chl fluorescence lifetime of around 5.06 ns was not altered for the AuNPs (Fig. 5C). Differently, the Pheo fluorescence lifetime decreased as a function of the availability of the AuNPs surface. The interaction between Chl and AuNPs responsible for the fluorescence enhancement involves the Chl in its ground state. On the other hand, photoinduced electron transfer from excited Pheo to the AuNPs surface may justify both the lifetime decrease and fluorescence quenching.
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Undoubtedly, the presence or absence of the Mg in the porphyrin´s centre plays a fundamental role
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in the pigments/AuNPs interplay.
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3.2 Computational analysis
A computational analysis of the affinity of the chlorin and Mg-chlorin for AuNPs was
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performed, accounting the interaction between free-base (H2C) and magnesium (MgC) chlorin
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with a gold surface (Fig. 6).
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Figure 6: Structures and atom numbering of (A) chlorin and (B) Mg-chlorin.
The structures of the chlorin and Mg-chlorin were deformed upon adsorption to the Au12
slab. The changes observed in bond distances before and after interaction with the gold surface are shown in Table 1. The mean and standard deviation of the change by taking into account all bond distances before and after adsorption was of 0.015 ± 0.012 Å and 0.019 ± 0.025 Å for chlorin and
Mg-chlorin, respectively. The maximum bond deformation was of 0.043 (C18-C19 bond) and 0.083 Å (C17- N24 bond) for chlorin and Mg-chlorin, respectively. This result indicates that Mgchlorin experiences a large deformation.
Table 1: Calculated atom distances of chlorin and Mg-chlorin before and after adsorption on the gold surface in a vacuum. Only changes in distance above 0.02 Å are shown. All values are in Å
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unit. Chlorin
Chlorin/Au
Atoms
Mg- Chlorin
Mg-Chlorin/Au
7-8
1.39996
1.43646
12-13
1.42134
1.46502
8-9
1.38525
1.35849
13-14
1.37457
9-10
1.42618
1.45329
17-18
10-11
1.37320
1.35313
11-12
1.43080
1.45513
18-19
1.39521
4-22
1.35019
7-22
1.36553
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1.37401
1.44138 1.35335
1.42208
1.44389
14-24
1.36499
1.31888
1.43849
17-24
1.36592
1.44850
1.39168
21-25
2.05500
2.11052
1.33580
22-25
2.07000
2.11244
23-25
2.05500
2.11900
24-25
2.10000
2.17300
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Atoms
Equation 3 defines the adsorption energy (𝐸𝑎𝑑𝑠 ). 𝐸𝑎𝑑𝑠 = 𝐸𝑠𝑦𝑠 − (𝐸𝑚𝑜𝑙 ∓ 𝐸𝑠𝑢𝑟 )
Equation 3
Where, 𝐸𝑚𝑜𝑙 , 𝐸𝑠𝑢𝑟 , and 𝐸𝑠𝑦𝑠 are the molecule, surface, and molecule-surface complex energies, respectively. Counterpoise correction was used to calculate the molecule-surface complex energies in order to avoid problems of basis set superposition [53–55]. The adsorption energies for chlorin and Mg-chlorin to the gold surface were calculated in a vacuum and methanol. The adsorption energies were of -235 and -312.38 kJ mol-1 for the chlorin/Au and chlorin-magnesium/Au complexes in a vacuum, respectively (Table 2). For the two complexes in methanol, the values
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were of -226.21 and -303.72 kJ mol-1. Lowest negative energies were observed in methanol
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because it stabilises the geometry of the chlorines and gold surface. A negative value of 𝐸𝑎𝑑𝑠 indicates that the adsorption is privileged, whereas a positive value means the opposite [65]. Thus,
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the chlorin adsorption on the gold surface is favoured by the presence of the Mg in the ring’s
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centre.
𝐸𝑎𝑑𝑠 accounts the distortion (𝐸𝑑𝑖𝑠 ) and interaction (𝐸𝑖𝑛𝑡 ) energies (Equation 4) [65]:
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𝐸𝑎𝑑𝑠 = 𝐸𝑑𝑖𝑠 + 𝐸𝑖𝑛𝑡
Equation 4
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𝑠𝑢𝑟 𝑚𝑜𝑙 Where 𝐸𝑑𝑖𝑠 is the sum of the distortion energy of the surface (𝐸𝑑𝑖𝑠 ) and molecule (𝐸𝑑𝑖𝑠 ) when the
adsorption is completed (Equation 5):
𝑠𝑢𝑟 𝑚𝑜𝑙 𝐸𝑑𝑖𝑠 = 𝐸𝑑𝑖𝑠 + 𝐸𝑑𝑖𝑠
Equation 5
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Both chlorin and Au12 slab were allowed to relax in x, y and z directions during the optimisation process. Consequently, the two structures experienced molecular distortions 𝑚𝑜𝑙 compared to their optimal geometry. The distortion energies of the chlorines (𝐸𝑑𝑖𝑠 ) and Au12 𝑠𝑢𝑟 slab (𝐸𝑑𝑖𝑠 ) were calculated as the difference between the energies of the geometry in the optimised
complex and its optimised geometry using
𝑚𝑜𝑙 𝑠𝑢𝑟 𝐸𝑑𝑖𝑠 = 𝐸𝑚𝑜𝑙/𝑐𝑜𝑚𝑝𝑙𝑒𝑥 − 𝐸𝑚𝑜𝑙 and 𝐸𝑑𝑖𝑠 =
𝐸𝑠𝑢𝑟/𝑐𝑜𝑚𝑝𝑙𝑒𝑥 − 𝐸𝑠𝑢𝑟 , where 𝐸𝑚𝑜𝑙/𝑐𝑜𝑚𝑝𝑙𝑒𝑥 , 𝐸𝑠𝑢𝑟/𝑐𝑜𝑚𝑝𝑙𝑒𝑥 , 𝐸𝑚𝑜𝑙 , and 𝐸𝑠𝑢𝑟 are the energies related to
the geometry of the pigment in the optimised complex, the geometry of the Au12 slab in the optimised complex, the optimised geometry of the pigment and the optimised geometry of the Au12 slab, respectively. The 𝐸𝑑𝑖𝑠 for chlorin and Mg-chlorin upon adsorption on the gold surface are of 23.28 and 62.73 kJ mol-1 in a vacuum, respectively, (Table 2). The positive energies point out that the structure of both molecules in the presence of the surface is less favourable compared to the
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optimised structures in a vacuum. 𝐸𝑑𝑖𝑠 for Mg-chlorin on the surface is accomplished by a
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significant molecular distortion compared to the adsorption of the chlorin. Differently, the distortion energies are negative for chlorin, and Mg-chlorin adsorbed on the gold surface in
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methanol, indicating that the complex formation is favoured. 𝐸𝑖𝑛𝑡 was also determined by taking
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the difference between the adsorption and distortion energy (Equation 4). The results demonstrate that the interaction between Mg-chlorin and gold surface is more advantageous than one involving
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chlorin (Table 2).
The Mulliken charges of the complexes reveal that a charge transfer mechanism from the
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chlorin molecules to the gold surface takes place during the interaction (Table 2). The magnitude of the charge transfer is higher for the interaction between Mg-chlorin and gold surface. Therefore, the charge transfer process plays an essential role during chlorines and gold surface interaction
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and may be a possible cause of the difference observed between the pigments and gold surface.
Table 2: Adsorption, distortion, and interaction energy of chlorin and Mg-chlorin upon adsorption on the gold surface. Mulliken charges of each monomer in the optimised complex is also shown.
Eads*
Molecule
Eint*
Eads
Eint
Edis
Esurdis
Emoldis
Au charges/e-
Pigment charges/e-
-0.59
+0.59
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(*) The energies were calculated using counterpoise correction and are expressed in kJ mol-1.
+1.55
Vacuum -176.40
-199.68
-235.62
-258.9
23.28
-2.27
25.55
Mg-Chlorin
-185.21
-247.94
-312.38
-375.11
62.73
-9.55
72.28
Methanol
-1.55
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Chlorin
---
---
-226.21
-212.60
-13.61
-38.10
24.49
-0.59
+0.59
Mg-Chlorin
---
---
-303.72
-301.38
-2.34
-48.11
45.77
-1.43
+1.43
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Chlorin
Finally, the simulation results also demonstrated that the calculated electronic spectra of
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chlorin and Mg-chlorin in a vacuum presented two main peaks (Figure 7A and B, respectively). The calculated absorption peaks are higher in energy when compared to the experimental ones [66]. However, the energy difference between these peaks, which are in the order of 220-260 nm, is similar to the experimentally obtained. The use of methanol in the calculations provoked a
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redshift (an energy decrease). However, it was unable to improve the correspondence among the simulated and experimental excitation energies (Figure S4A and S4B). Nevertheless, the shape of the calculated electronic spectra is similar to the experimental. This result confirms that the used density functional in our calculations can reproduce the electronic spectra with a systematic error, describing the energy shift to lower values [67]. The calculations of the electronic spectra of the chlorin/Au and Mg-chlorin/Au complexes are presented in Figs. 7C and D, respectively. The
interaction between the chlorines molecules and the gold surface led to the formation of additional absorption peak (compared to the calculated spectra of isolated pigments). These peaks are located at about 395 and 379 nm for the chlorin/Au and Mg-chlorin/Au complexes in a vacuum, respectively. Similar peaks were also observed for the complexes in methanol (Figure S4C and S4D). Molecular orbitals results also show charge transfer from the chlorines molecules to the gold surface are related to the electronic transitions involved in these absorption peaks (Figure S5, S6,
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S7, and S8). In a vacuum, the electronic transitions responsible for the charge transfer phenomena
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in Mg-chlorin and gold surface are HOMO-3 → LUMO [2.95%], HOMO-1 → LUMO+2 [2.08%] and HOMO-1 → LUMO+3 [2.26%] (Figure S6). For the interaction involving chlorin and gold
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surface, the transition HOMO-3 → LUMO+4 [2.25%] is the unique responsible for charge transfer
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(Figure S5). Therefore, charge transfer is stronger for the interaction involving the Mg-chlorin and gold than chlorin and gold, following the calculated adsorption energies and the Mulliken charge
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analysis.
Figure 7: Calculated electronic spectra of (A) chlorin, (B) Mg-chlorin, (C) chlorin/Au12 slab complex, and (D) Mg-chlorin/Au12 slab complex in a vacuum. The calculated electronic transitions are shown as vertical black bars. The deconvoluted absorption spectra are displayed in red.
4 Conclusion The present study report that AuNPs alter the absorption and fluorescence of the Chl and Pheo molecules as a result of the plasmon resonance effect and photoinduced electron transfer. The fluorescence in Chl and Pheo changed as a function of the diameter and concentration of the
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Au nanoparticles. The Chl emission increased with the NPs concentration because of the raise of
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the total surface area and, consequently, of the plasmon resonance absorption. In turn, the Pheo fluorescence quenching in high AuNPs concentrations could be explained by the photoinduced
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electron transfer from excited Pheo to the AuNPs surfaces, considering the high affinity between them. Time-resolved fluorescence measurements and computational analyses imply that the Pheo-
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AuNPs interaction is more effective than one involving Chl molecules. In summary, AuNPs
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interact with two important photosynthetic pigments (Chl and Pheo) so that the impacts of these nanoparticles on the functioning of plant photosystems should not be ruled out.
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Author Contributions
“Conceptualization, A.V.M., G.A.C., E.F.S., S.L.O. and A.R.L.C.; Pigments’ extraction and preparation, A.V.M., A.M.Q., M.S.P., M.D.S. and D.E.G.; Optical measurements, A.V.M., D.E.G.
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and A.M.Q.; Computational simulations, I.O.P., A.J.L. and D.D.R.; Data analysis, A.R.L.C., A.V.M., I.O.P., G.A.C., D.D.R., S.L.O., and E.F.S.; Funding Acquisition, M.D.S. and A.R.L.C.; Writing & Editing, A.R.L.C., A.V.M., I.O.P., E.F.S., and S.L.O.”
Conflicts of interest There are no conflicts to declare.
Acknowledgements This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001. The authors acknowledge the financial support provided by the CAPES-PrInt funding program (grant numbers: 88887.353061/2019-00 and 88887.311920/2018-00) and the National Institute of Science and Technology of Basic Optics and
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Optics Applied to Life Science (grant number: 465360/2014-9). The authors are also grateful to the financial support provided by CNPq (309636/2017-5, and 304844/2018-7), FUNDECT
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(256/2016), CAPES (23104.040816/2018-41), and FAPESP (2017/02201-4). The authors also acknowledge Prof. Francisco E. G. Guimarães (IFSC/USP) for supporting the time-resolved
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fluorescence measurements.
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PII:
S1010-6030(19)30850-0
DOI:
https://doi.org/10.1016/j.jphotochem.2019.112252
Reference:
JPC 112252
To appear in:
Journal of Photochemistry & Photobiology, A: Chemistry
Received Date:
21 May 2019
Revised Date:
3 November 2019
Accepted Date:
16 November 2019
Please cite this article as: Mezacasa AV, Queiroz AM, Graciano DE, Pontes MS, Santiago EF, Oliveira IP, Lopez A, Casagrande GA, Scherer MD, dos Reis DD, Oliveira SL, Caires ARL, Effects of gold nanoparticles on photophysical behaviour of chlorophyll and pheophytin, Journal of Photochemistry and amp; Photobiology, A: Chemistry (2019), doi: https://doi.org/10.1016/j.jphotochem.2019.112252
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.
Effects of gold nanoparticles on photophysical behaviour of chlorophyll and pheophytin A.V. Mezacasa1,2, A.M. Queiroz1,2, D.E. Graciano1,2, M.S. Pontes3, E.F. Santiago3, I.P. Oliveira4; AJ Lopez5, G.A. Casagrande2, M.D. Scherer2, D.D. dos Reis2, S.L. Oliveira2, A.R.L. Caires2,6,* Faculty of Exact Science and Technology, Federal University of Grande Dourados, Dourados, Brazil.
2
Institute of Physics, Federal University of Mato Grosso do Sul, Campo Grande, Brazil.
3
Natural Resources Study Center, State University of Mato Grosso do Sul, Dourados, Brazil.
4
Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil.
5
Institute of Chemistry, University of Campinas, Campinas, SP, Brazil.
6
School of Life Sciences, University of Essex, Colchester, UK.
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Graphical-abstract
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AuNPs strongly alter the optical features of Chlorophyll and Pheophytin Plasmon resonance contributed for fluorescence enhancement in both pigments Pheo exhibited fluorescence quenching in solutions with high AuNPs concentration Affinity of Chl and Pheo with AuNPs was determined Photoinduced electron transfer from Chl and Pheo to AuNPs surface plays a key role in the pigments-NPs interaction
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Highlights
ABSTRACT
The increasing application of engineering nanoparticles (ENPs) in various commercial products justifies the need for studying their impact on living organisms and environment. The interaction between gold nanoparticles (AuNPs) with different diameters (5, 10, and 20 nm) and two
photosynthetic pigments chlorophyll (Chl) and pheophytin (Pheo) was investigated using optical techniques and computational simulations. UV-Vis absorption, steady-state fluorescence, and timeresolved fluorescence showed that AuNPs affected the optical features of the studied photosynthetic pigments. Moreover, the optical and computational results show that an analogous Pheo molecule (chlorin) was less susceptible than an analogous chlorophyll molecule (Mg-chlorin) to interact with AuNPs. Therefore, AuNPs interact with both Chl and Pheo so that they may change the operation of the plant photosystems in case of uptake and internalisation in plants.
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*Corresponding author. E-mail: [email protected].
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Keywords: Chlorophyll; Pheophytin; Gold nanoparticle; Optical analysis; Computational simulation.
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1 Introduction
In recent years, materials in the nanometric scale have been investigated by different research
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areas and applied in commercial products because of their distinctive optical, electrical, and mechanical properties [1–4]. As a result, the utilisation of new products containing nanoparticles (NPs)
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has increased exponentially, becoming imperative studies on the social and environmental consequences [5,6]. The scarcity of information about the harmful effects of nanotechnological innovations on the environment and human health suggests greater prudence [7,8]. Gold nanoparticles (AuNPs) are among the most used nanomaterials to produce functional
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electrical coatings, improve the redox activity in photochemical and electrochemical applications by interacting with organic molecules, and other applications [9–12]. The successful use of AuNPs can be easily understood by the fact that they have unique optical and electronic characteristics based on the existence of the surface plasmon resonance (SPR) [13–16]. The choice of biological models and experimental techniques to investigate the effects induced by NPs on living organisms and environment constitutes a challenge considering the dependence of
the physicochemical properties of the NPs on composition, size, shape, and concentration, as well as the complexity of the interactions between them and organisms [5,17,18]. Plants are the basis of both the ecosystem and food chain, and they are submitted continuously to materials released and accumulated in the environment by human activities; so it is crucial to test the NPs effects on them, especially on their photosynthetic pigments [19–24]. The NPs impacts on photosynthetic pigmentslight interaction may alter the plant life cycle and productivity because the pigments play a crucial role in both harvesting light and photosynthesis [22–27]. For instance, AuNPs may either positively or
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negatively modify the photosynthetic performance, i.e. the quantum yield of the photosynthetic activity, depending on two competing processes, namely light absorption enhancement of the photosynthetic pigments because of the SPR effect that intensifies the local field around them [21] and
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charge (or energy) transfer from the excited photosynthetic pigments to the metal nanoparticle surface [12,22].
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Among all the photosynthetic pigments, chlorophyll a (Chl a) is the main pigment responsible
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for photosynthesis, and its fluorescence has been extensively used to monitor the physiological status of plants. Alterations in the operation of the photosystems I and II (PSI and PSII) impact the Chl a fluorescence [28–30]. Chl a is composed of a chlorin pigment with a magnesium ion at the centre of
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the aromatic plane, and it contains a methyl group (-CH3) as the substituent at carbon C-3 [31,32]. Pheophytin a (Pheo a) is another relevant photosynthetic pigment in the reaction centre (RC) of PSII. It has a similar chemical structure of the Chl a, but without the magnesium ion at the centre of the
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chlorin ring [33]. Structurally, in the RC of PSII, four central chlorin pigments are arranged in two symmetry-related branches (D1 and D2 polypeptides): D1 and D2 share a dimer (P680) of Chl a molecule (P680D1 and P680D2), and each branch is also composed of one monomeric Chl a (Chl a D1 or Chl a D2) and one Pheo a (Pheo a D1 or Pheo a D2) [34,35]. Although recent studies have reported that AuNPs can be uptaken, translocated to the stems and leaves, and accumulated by plants [36–42], there is still a lack of data about the AuNPs-
photosynthetic pigments interaction and the influence of AuNPs on the photosynthetic capacity of plants. Based on that, the present study investigated the interaction of two photosynthetic pigments (Chl and Pheo molecules), extracted from green leaves of Vicia faba, with AuNPs of different sizes and concentrations by using UV-Vis absorption, steady-state and time-resolved fluorescence, along with computational simulations of the interactions between the photosynthetic pigments and AuNPs surface.
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2 Materials and methods 2.1 Gold nanoparticles
AuNPs with nominal diameters of 5, 10, and 20 nm were purchased from Sigma-Aldrich
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(Brazil). They were dispersed as colloidal particles in solutions of 100 mL comprising approximately:
carbonate, and 0.02% sodium azide.
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2.2 Chlorophyll and pheophytin extraction
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0.01% HAuCl4 suspended in 0.01% tannic acid with 0.04% trisodium citrate, 0.26 mM potassium
Chl and Pheo were extracted from Vicia faba (L.). It has been often used as a model system for
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plant physiological studies [43]. Leaves were collected from the plant grown in a greenhouse, cut into small pieces, and added to the methanol P.A. in the proportion of 15:100 (m/v) for 72 h at 5°C. Chl extract purification was performed by using the procedure adapted from Moreira et al. [44] and Khalyfa
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et al. [45]. The separation of the pigments was made by the process adapted from Maestrin et al. [46] and Filho et al. [47]. Pheo was obtained following the method reported by Moreira et al. [44] and Gerola [48]. The total chlorophyll concentration [Chl a + b] for methanolic extraction was determined from the molecular absorption spectra based on the Arnon method adapted by Porra [49] (Equation 1): [𝐶ℎ𝑙 𝑎 + 𝑏] = 24.23 𝐴652 + 3.26 𝐴665
Equation 1
Where A652 and A665 are the absorbances at 652 and 665 nm, respectively, and the constants 24.23 and 3.26 are the specific extinction coefficients of the chlorophyll at 652 and 665 nm, respectively. The Pheo concentration was determined by considering the initial concentration of the Chl in the solution. The pH of the Pheo and Chl extracts was adjusted to 7.0, before the preparation of the AuNPs-pigments solution. 2.3 AuNPs-pigments solution preparation
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The AuNPs-photosynthetic pigments interaction analysis was carried out using gold nanoparticles with diameters of 5, 10, and 20 nm. For each diameter, ten AuNPs concentrations (0.3, 0.6, 1.2, 2.4, 4.8, 9.6, 19.3, 38.6, 77.2, and 154.5 µM) were prepared in aqueous solutions based on the
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moles of gold atoms. A convenient volume of the methanolic solution of the photosynthetic pigments was added to the AuNPs solution to obtain a final photosynthetic pigments concentration of 0.5 μM in
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all solutions.
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2.4 Optical analysis
The UV-Vis absorption spectra were recorded in the 350-800 nm range with a
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spectrophotometer (USB 4000 FL, Ocean Optics®). Steady-state fluorescence was carried out using a spectrofluorimeter consisting of a diode laser as the excitation source (405 nm), a monochromator (USB 2000 FL, Ocean Optics®), and a Y-type optical fibre. Fluorescence spectra were obtained between 450 and 800 nm adopting the front-face geometry and a 10-mm quartz cuvette.
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Fluorescence decay measurements were performed with a confocal multiphoton LSM 780
Zeiss microscope equipped with Picoquant Fluortime system. It is composed of a 20x objective lens and single-photon avalanche diode (SPAD) detectors with the temporal resolution of 70 ps. Emission at 673 nm was collected when excited by a pulsed laser (Coherent) operating at 400 nm with a pulse width of 140 fs and 80-MHz repetition rate. All optical measurements were executed at room temperature.
2.5 Computational analysis First-principles calculations were performed to obtain a detailed picture of the pigmentsnanoparticle interaction at the atomic level. Calculations including geometry optimization, singlepoint calculations, and excitation energies for all molecules (chlorin, Mg-chlorin, and Au12 slab) and molecular complexes (chlorin/Au12 and Mg-chlorin/Au12 slab) were carried out by means of the density functional ωB97X [50], in a similar way as recently used to determine the adsorption
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energy of organic molecules on gold surfaces [51]. The positions of all unconstrained atoms of the molecules adsorbed on the gold surface were optimised until both the convergence criterion for force (0.0003 Hartree/Bohr) and energy change (10-8 Hartree) be achieved. Time-dependent density functional theory (TD-DFT) approach was employed to compute the excitation energies. Methanol
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was also employed as a solvent in our simulations by using the Polarizable continuum model (PCM)
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[52].
For chlorin and Mg-chlorin, the cc-pVDZ basis set was used. For Au12 slab, the LANL2MB
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was chosen. In the case of chlorin/Au12 slab and Mg-chlorin/Au12 slab complexes, the basis set was the LANL2MB for gold atoms and cc-pVDZ for the C, H, N, and Mg atoms. Counterpoise corrections
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were applied to the molecular complexes to correct basis set superposition error (BSSE) [53–55]. The electronic structure was calculated using the Gaussian 09 package [56]. Mulliken charge distribution of the chlorin-gold surface complexes was generated by VMD [57].
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3 Results and discussion 3.1 Optical analysis
Fig. 1 shows the UV-Vis absorption spectra of the Chl and Pheo molecules. Both spectra present similarities because they are porphyrins with the Soret band in the 390-440 nm range and the Q-bands between 500 and 670 nm. The Q-bands, Qx(0,0), Qx(1,0), Qy(0,0), and Qy(1,0) in Pheo is alike are
similar to the ones in free-base porphyrins [58]. The Q-bands are derived from the transitions between two π HOMO orbitals and two π* LUMO orbitals [58,59]. The presence of the Mg in the porphyrin’s
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centre favours the transitions related to the Soret and Qy(1,0) bands [46,58,59].
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Figure 1 - UV-Vis absorption spectra of the methanolic extracts of Chl and Pheo at 10.7 μM.
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Plasmon resonance absorption band in the 500-550 nm region – generated by the electron collective oscillation on the surface of AuNPs in the Chl and Pheo extracts – were observed (Fig. 2)
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[12]. The UV-Vis spectra of the Chl and Pheo solutions containing AuNPs with 5, 10, and 20-nm diameter at different concentrations (0-154.5 μM) are presented in Figs. S1 and S2 (supplementary
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materials), respectively.
Figure 2 - UV-Vis absorption spectra of (A) Chl and (B) Pheo extract at 0.5 μM in the absence and presence of AuNPs with 5-nm diameter and concentration of 154.5 μM.
Plasmon resonance absorbance increased with the AuNPs concentration in the presence of Chl or Pheo at 0.5 μM, as shown in Fig. 3 for the 5-nm AuNPs. Besides, the plasmon resonance absorbance presented two linear regimes in the 0-10 and 10-155 μM in the presence of both photosynthetic pigments. However, Chl and Pheo extracts exhibited different increase rates, indicating that the collective oscillation of the electrons on the surface of AuNPs was affected by the Mg in the porphyrin
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ring. Similar absorbance behaviour at 520 nm versus nanoparticle concentration was also seen for the
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AuNPs with 10 and 20-nm diameter (Fig. S3, supplementary materials).
Figure 3 - Plasmon absorbance at 520 nm as a function of the 5-nm AuNPs concentration in the
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presence of (A) chlorophyll and (B) pheophytin at 0.5 μM. The lines are guides to the eyes.
Fig. 4 shows the fluorescence spectra of the photosynthetic pigments under 405-nm excitation
in the presence of AuNPs with different diameters and concentrations. Both Photosynthetic pigments exhibited the usual spectrum of the porphyrin in the region of 660 to 710 nm [58,59]. Fluorescence enhancement of the Chl molecules was AuNPs content-dependent. This increase was originated by the
phenomenon known as metal-enhanced fluorescence (MEF), which is related to the plasmon resonance responsible for raising the incident electric field felt by Chl nearby the metal surfaces of AuNPs [60,61]. The total surface area increases as nanoparticle concentration increase for a given NP concentration, enhancing the plasmon resonance absorption and, consequently, the fluorescence intensity. MEF was also identified for the Pheo molecules in solutions with a low concentration of AuNPs (up to 19.3 μM). For AuNPs concentrations higher than 38.6 μM in the Pheo solution, fluorescence suppression was verified, suggesting that the Pheo molecules were either attached to the
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metal surface or close to it, allowing the electron transfer from excited Pheo to the AuNPs surface [3,12,22,62–64]. Further, both phenomena fluorescence enhancement and suppression were AuNPs diameter-dependent, given that the total metal surface area depends on both nanoparticle diameter and
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concentration.
Figure 4 - (A), (B), and (C) Fluorescence spectra of Chl and (D), (E), and (F) Pheo solutions with 5, 10, and 20-nm AuNPs, respectively, at concentrations of 0.3, 0.6, 1.2, 2.4, 4.8, 9.6, 19.3, 38.6, 77.2, and 154.5 μM. Samples were excited at 405-nm. The solid (black) lines correspond to the Chl and Pheo emission in the absence of AuNPs. The short dash-dot (blue) lines indicate the enhancement of the Chl and Pheo
emission in the presence of AuNPs. The short-dot (red) lines describe the suppression of the Chl and Pheo
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emission in the presence of AuNPs.
Figure 5 shows the fluorescence decay curves of Chl and Pheo when nearby AuNPs. The fluorescence lifetime was determined by a bi-exponential fitting and the average lifetime (𝜏𝑚 ) was calculated using Equation 2. 𝜏𝑚 =
(𝐴1 𝜏12 )+(𝐴2 𝜏22 )
Equation 2
(𝐴1 𝜏1 )+(𝐴2 𝜏2 )
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Where 𝜏1 and 𝜏2 are first and second decay times, respectively, and 𝐴1 and 𝐴2 their pre-
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exponential factors (amplitudes), respectively.
Figure 5 – (A) Chl and (B) Pheo fluorescence decay curves in the absence and presence of 5, 10, and 20-nm AuNPs. The average lifetime (𝜏𝑚 ) of (C) Chl and (D) Pheo as a function of the AuNPs surface area. The measurements were made for the Chl and Pheo solutions at 0.5 μM and AuNPs (5, 10, and 20 nm) at 4.2 μM. The samples were excited using a 405-nm laser.
The Chl fluorescence lifetime of around 5.06 ns was not altered for the AuNPs (Fig. 5C). Differently, the Pheo fluorescence lifetime decreased as a function of the availability of the AuNPs surface. The interaction between Chl and AuNPs responsible for the fluorescence enhancement involves the Chl in its ground state. On the other hand, photoinduced electron transfer from excited Pheo to the AuNPs surface may justify both the lifetime decrease and fluorescence quenching.
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Undoubtedly, the presence or absence of the Mg in the porphyrin´s centre plays a fundamental role
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in the pigments/AuNPs interplay.
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3.2 Computational analysis
A computational analysis of the affinity of the chlorin and Mg-chlorin for AuNPs was
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performed, accounting the interaction between free-base (H2C) and magnesium (MgC) chlorin
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with a gold surface (Fig. 6).
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Figure 6: Structures and atom numbering of (A) chlorin and (B) Mg-chlorin.
The structures of the chlorin and Mg-chlorin were deformed upon adsorption to the Au12
slab. The changes observed in bond distances before and after interaction with the gold surface are shown in Table 1. The mean and standard deviation of the change by taking into account all bond distances before and after adsorption was of 0.015 ± 0.012 Å and 0.019 ± 0.025 Å for chlorin and
Mg-chlorin, respectively. The maximum bond deformation was of 0.043 (C18-C19 bond) and 0.083 Å (C17- N24 bond) for chlorin and Mg-chlorin, respectively. This result indicates that Mgchlorin experiences a large deformation.
Table 1: Calculated atom distances of chlorin and Mg-chlorin before and after adsorption on the gold surface in a vacuum. Only changes in distance above 0.02 Å are shown. All values are in Å
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unit. Chlorin
Chlorin/Au
Atoms
Mg- Chlorin
Mg-Chlorin/Au
7-8
1.39996
1.43646
12-13
1.42134
1.46502
8-9
1.38525
1.35849
13-14
1.37457
9-10
1.42618
1.45329
17-18
10-11
1.37320
1.35313
11-12
1.43080
1.45513
18-19
1.39521
4-22
1.35019
7-22
1.36553
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1.37401
1.44138 1.35335
1.42208
1.44389
14-24
1.36499
1.31888
1.43849
17-24
1.36592
1.44850
1.39168
21-25
2.05500
2.11052
1.33580
22-25
2.07000
2.11244
23-25
2.05500
2.11900
24-25
2.10000
2.17300
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18-19
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Atoms
Equation 3 defines the adsorption energy (𝐸𝑎𝑑𝑠 ). 𝐸𝑎𝑑𝑠 = 𝐸𝑠𝑦𝑠 − (𝐸𝑚𝑜𝑙 ∓ 𝐸𝑠𝑢𝑟 )
Equation 3
Where, 𝐸𝑚𝑜𝑙 , 𝐸𝑠𝑢𝑟 , and 𝐸𝑠𝑦𝑠 are the molecule, surface, and molecule-surface complex energies, respectively. Counterpoise correction was used to calculate the molecule-surface complex energies in order to avoid problems of basis set superposition [53–55]. The adsorption energies for chlorin and Mg-chlorin to the gold surface were calculated in a vacuum and methanol. The adsorption energies were of -235 and -312.38 kJ mol-1 for the chlorin/Au and chlorin-magnesium/Au complexes in a vacuum, respectively (Table 2). For the two complexes in methanol, the values
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were of -226.21 and -303.72 kJ mol-1. Lowest negative energies were observed in methanol
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because it stabilises the geometry of the chlorines and gold surface. A negative value of 𝐸𝑎𝑑𝑠 indicates that the adsorption is privileged, whereas a positive value means the opposite [65]. Thus,
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the chlorin adsorption on the gold surface is favoured by the presence of the Mg in the ring’s
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centre.
𝐸𝑎𝑑𝑠 accounts the distortion (𝐸𝑑𝑖𝑠 ) and interaction (𝐸𝑖𝑛𝑡 ) energies (Equation 4) [65]:
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𝐸𝑎𝑑𝑠 = 𝐸𝑑𝑖𝑠 + 𝐸𝑖𝑛𝑡
Equation 4
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𝑠𝑢𝑟 𝑚𝑜𝑙 Where 𝐸𝑑𝑖𝑠 is the sum of the distortion energy of the surface (𝐸𝑑𝑖𝑠 ) and molecule (𝐸𝑑𝑖𝑠 ) when the
adsorption is completed (Equation 5):
𝑠𝑢𝑟 𝑚𝑜𝑙 𝐸𝑑𝑖𝑠 = 𝐸𝑑𝑖𝑠 + 𝐸𝑑𝑖𝑠
Equation 5
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Both chlorin and Au12 slab were allowed to relax in x, y and z directions during the optimisation process. Consequently, the two structures experienced molecular distortions 𝑚𝑜𝑙 compared to their optimal geometry. The distortion energies of the chlorines (𝐸𝑑𝑖𝑠 ) and Au12 𝑠𝑢𝑟 slab (𝐸𝑑𝑖𝑠 ) were calculated as the difference between the energies of the geometry in the optimised
complex and its optimised geometry using
𝑚𝑜𝑙 𝑠𝑢𝑟 𝐸𝑑𝑖𝑠 = 𝐸𝑚𝑜𝑙/𝑐𝑜𝑚𝑝𝑙𝑒𝑥 − 𝐸𝑚𝑜𝑙 and 𝐸𝑑𝑖𝑠 =
𝐸𝑠𝑢𝑟/𝑐𝑜𝑚𝑝𝑙𝑒𝑥 − 𝐸𝑠𝑢𝑟 , where 𝐸𝑚𝑜𝑙/𝑐𝑜𝑚𝑝𝑙𝑒𝑥 , 𝐸𝑠𝑢𝑟/𝑐𝑜𝑚𝑝𝑙𝑒𝑥 , 𝐸𝑚𝑜𝑙 , and 𝐸𝑠𝑢𝑟 are the energies related to
the geometry of the pigment in the optimised complex, the geometry of the Au12 slab in the optimised complex, the optimised geometry of the pigment and the optimised geometry of the Au12 slab, respectively. The 𝐸𝑑𝑖𝑠 for chlorin and Mg-chlorin upon adsorption on the gold surface are of 23.28 and 62.73 kJ mol-1 in a vacuum, respectively, (Table 2). The positive energies point out that the structure of both molecules in the presence of the surface is less favourable compared to the
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optimised structures in a vacuum. 𝐸𝑑𝑖𝑠 for Mg-chlorin on the surface is accomplished by a
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significant molecular distortion compared to the adsorption of the chlorin. Differently, the distortion energies are negative for chlorin, and Mg-chlorin adsorbed on the gold surface in
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methanol, indicating that the complex formation is favoured. 𝐸𝑖𝑛𝑡 was also determined by taking
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the difference between the adsorption and distortion energy (Equation 4). The results demonstrate that the interaction between Mg-chlorin and gold surface is more advantageous than one involving
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chlorin (Table 2).
The Mulliken charges of the complexes reveal that a charge transfer mechanism from the
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chlorin molecules to the gold surface takes place during the interaction (Table 2). The magnitude of the charge transfer is higher for the interaction between Mg-chlorin and gold surface. Therefore, the charge transfer process plays an essential role during chlorines and gold surface interaction
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and may be a possible cause of the difference observed between the pigments and gold surface.
Table 2: Adsorption, distortion, and interaction energy of chlorin and Mg-chlorin upon adsorption on the gold surface. Mulliken charges of each monomer in the optimised complex is also shown.
Eads*
Molecule
Eint*
Eads
Eint
Edis
Esurdis
Emoldis
Au charges/e-
Pigment charges/e-
-0.59
+0.59
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(*) The energies were calculated using counterpoise correction and are expressed in kJ mol-1.
+1.55
Vacuum -176.40
-199.68
-235.62
-258.9
23.28
-2.27
25.55
Mg-Chlorin
-185.21
-247.94
-312.38
-375.11
62.73
-9.55
72.28
Methanol
-1.55
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Chlorin
---
---
-226.21
-212.60
-13.61
-38.10
24.49
-0.59
+0.59
Mg-Chlorin
---
---
-303.72
-301.38
-2.34
-48.11
45.77
-1.43
+1.43
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Chlorin
Finally, the simulation results also demonstrated that the calculated electronic spectra of
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chlorin and Mg-chlorin in a vacuum presented two main peaks (Figure 7A and B, respectively). The calculated absorption peaks are higher in energy when compared to the experimental ones [66]. However, the energy difference between these peaks, which are in the order of 220-260 nm, is similar to the experimentally obtained. The use of methanol in the calculations provoked a
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redshift (an energy decrease). However, it was unable to improve the correspondence among the simulated and experimental excitation energies (Figure S4A and S4B). Nevertheless, the shape of the calculated electronic spectra is similar to the experimental. This result confirms that the used density functional in our calculations can reproduce the electronic spectra with a systematic error, describing the energy shift to lower values [67]. The calculations of the electronic spectra of the chlorin/Au and Mg-chlorin/Au complexes are presented in Figs. 7C and D, respectively. The
interaction between the chlorines molecules and the gold surface led to the formation of additional absorption peak (compared to the calculated spectra of isolated pigments). These peaks are located at about 395 and 379 nm for the chlorin/Au and Mg-chlorin/Au complexes in a vacuum, respectively. Similar peaks were also observed for the complexes in methanol (Figure S4C and S4D). Molecular orbitals results also show charge transfer from the chlorines molecules to the gold surface are related to the electronic transitions involved in these absorption peaks (Figure S5, S6,
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S7, and S8). In a vacuum, the electronic transitions responsible for the charge transfer phenomena
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in Mg-chlorin and gold surface are HOMO-3 → LUMO [2.95%], HOMO-1 → LUMO+2 [2.08%] and HOMO-1 → LUMO+3 [2.26%] (Figure S6). For the interaction involving chlorin and gold
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surface, the transition HOMO-3 → LUMO+4 [2.25%] is the unique responsible for charge transfer
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(Figure S5). Therefore, charge transfer is stronger for the interaction involving the Mg-chlorin and gold than chlorin and gold, following the calculated adsorption energies and the Mulliken charge
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analysis.
Figure 7: Calculated electronic spectra of (A) chlorin, (B) Mg-chlorin, (C) chlorin/Au12 slab complex, and (D) Mg-chlorin/Au12 slab complex in a vacuum. The calculated electronic transitions are shown as vertical black bars. The deconvoluted absorption spectra are displayed in red.
4 Conclusion The present study report that AuNPs alter the absorption and fluorescence of the Chl and Pheo molecules as a result of the plasmon resonance effect and photoinduced electron transfer. The fluorescence in Chl and Pheo changed as a function of the diameter and concentration of the
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Au nanoparticles. The Chl emission increased with the NPs concentration because of the raise of
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the total surface area and, consequently, of the plasmon resonance absorption. In turn, the Pheo fluorescence quenching in high AuNPs concentrations could be explained by the photoinduced
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electron transfer from excited Pheo to the AuNPs surfaces, considering the high affinity between them. Time-resolved fluorescence measurements and computational analyses imply that the Pheo-
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AuNPs interaction is more effective than one involving Chl molecules. In summary, AuNPs
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interact with two important photosynthetic pigments (Chl and Pheo) so that the impacts of these nanoparticles on the functioning of plant photosystems should not be ruled out.
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Author Contributions
“Conceptualization, A.V.M., G.A.C., E.F.S., S.L.O. and A.R.L.C.; Pigments’ extraction and preparation, A.V.M., A.M.Q., M.S.P., M.D.S. and D.E.G.; Optical measurements, A.V.M., D.E.G.
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and A.M.Q.; Computational simulations, I.O.P., A.J.L. and D.D.R.; Data analysis, A.R.L.C., A.V.M., I.O.P., G.A.C., D.D.R., S.L.O., and E.F.S.; Funding Acquisition, M.D.S. and A.R.L.C.; Writing & Editing, A.R.L.C., A.V.M., I.O.P., E.F.S., and S.L.O.”
Conflicts of interest There are no conflicts to declare.
Acknowledgements This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001. The authors acknowledge the financial support provided by the CAPES-PrInt funding program (grant numbers: 88887.353061/2019-00 and 88887.311920/2018-00) and the National Institute of Science and Technology of Basic Optics and
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Optics Applied to Life Science (grant number: 465360/2014-9). The authors are also grateful to the financial support provided by CNPq (309636/2017-5, and 304844/2018-7), FUNDECT
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(256/2016), CAPES (23104.040816/2018-41), and FAPESP (2017/02201-4). The authors also acknowledge Prof. Francisco E. G. Guimarães (IFSC/USP) for supporting the time-resolved
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fluorescence measurements.
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