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Spectroscopic measurements of interactions between hydrophobic 1-pyrenebutyric acid and silver colloidal nanoparticles
Accepted Manuscript Title: Spectroscopic measurements of interactions between hydrophobic 1-pyrenebutyric acid and silver colloidal nanoparticles Author: Nguyen Hoang Ly Thanh Danh Nguyen Thanh Lam Bui Sangyeop Lee Jaebum Choo Sang-Woo Joo PII: DOI: Reference:
S0927-7757(17)30001-8 http://dx.doi.org/doi:10.1016/j.colsurfa.2017.01.001 COLSUA 21265
To appear in:
Colloids and Surfaces A: Physicochem. Eng. Aspects
Received date: Revised date: Accepted date:
15-11-2016 28-12-2016 3-1-2017
Please cite this article as: Nguyen Hoang Ly, Thanh Danh Nguyen, Thanh Lam Bui, Sangyeop Lee, Jaebum Choo, Sang-Woo Joo, Spectroscopic measurements of interactions between hydrophobic 1-pyrenebutyric acid and silver colloidal nanoparticles, Colloids and Surfaces A: Physicochemical and Engineering Aspects http://dx.doi.org/10.1016/j.colsurfa.2017.01.001
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Spectroscopic measurements of interactions between hydrophobic 1pyrenebutyric acid and silver colloidal nanoparticles
Nguyen Hoang Lya, Thanh Danh Nguyena,b, Thanh Lam Buia,b, Sangyeop Leec, Jaebum Chooc, SangWoo Jooa,b,*
a
Department of Chemistry, Soongsil University, Seoul 156-743, Korea
b
Department of Information Communication, Materials, Chemistry Convergence Technology, Soongsil University, Seoul 156-743, Korea
c
Department of Bionano Engineering, Hanyang University, Sa-1-dong 1271, Ansan 426-791, South Korea
AUTHOR EMAIL ADDRESS: [email protected]
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Graphical abstract
Fluorescence Intensity (a. u.)
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Free Pyrene acids Pyrene acid-AgNPs
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400000
200000
0
PyC
PyA
PyB
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Highlights
Hydrophobic pyrene acids were chemically interacted with Ag nanoparticles.
1-Pyrene acids were found to adsorb on silver via the COO - bonds.
Fluorescence quenching behaviors of pyrene acids were compared on Ag.
Glutathione-triggered desorption of pyrene acids were observed in cancer cells.
1-Pyrenebutyrate showed stronger binding than 1-pyrenecarboxlyate on Ag.
Abstract We compared the adsorption and desorption of 1-pyrenecarboxylic acid (PyC), 1-pyreneacetic acid (PyA), and 1-pyrenebutyric acid (PyB) on silver nanoparticles (AgNPs) via interfacial spectroscopic tools to study the role of the aliphatic units between pyrene and carboxylic group. The negative surface charges of AgNPs at ca. –51 mV shifted up to –11 mV, after adsorbing hydrophobic pyrene compounds. The three pyrene acid compounds appeared to adsorb onto AgNPs via their carboxylate units by referring to the observation of the broad (COO-) bands at 1380–1410 cm-1 in the Raman spectra. X-ray photoelectron spectroscopy (XPS) also supported the C-O species in the C1s region. AgNPs were found to efficiently quench the fluorescence of the three pyrene acid adsorbates. The highest Stern–Volmer constant of PyA may be due to the largest overlap integral with the surface Plasmon absorption band of AgNPs. The butyric unit was expected to lead a stronger binding on Ag, as suggested by density functional theory (DFT) calculations. The adsorbed pyrene acid compounds appeared to be released from AgNPs by thiol-containing glutathione (GSH). PyB with the butyric group exhibited larger quenched fluorescence intensities and smaller released amounts than PyC and PyA in aqueous solutions and A549 cancer cells. Our study will be helpful in designing pyrene-based fluorescence sensors in cellular imaging. Keywords: Fluorescence quenching; Raman spectroscopy; Density functional theory; 1-Pyrenebutyric acid; Silver nanoparticles; Intracellular release 3
1. Introduction The estimation of hydrophobic forces has been given much attention due to its perspective in colloidal science [1]. In past decades, surface-enhanced Raman scattering (SERS), has proved to be a versatile interfacial spectroscopic tool for analyzing pollutants and biological samples, due to its enormous enhancements occurring on noble metal nanostructures [2-4]. On the other hand, the nanostructure materials can act as efficient quenching platforms of fluorophores [5-10]. Pyrene as a hydrophobic organic ligand [11] exhibits strong fluorescence, which can be utilized as ionic and molecular sensors [12,13]. Since metal nanoparticles can quench the fluorescence of organic adsorbates, the Stern–Volmer plot yields the quenching coefficients of metal nanoparticles [14]. A recent study shows that aminopyrene exhibited its higher quenching value than pyrene and aminomethylpyrene, when adsorbed on 2-21 nm size silver nanoparticles (AgNPs) via both Förster resonance energy transfer (FRET) and electron transfer [15]. Considering that the carboxylic acids bind strongly on Ag surfaces [16,17], this anchoring group may lead interfacial chemical bonding to result in efficient quenching behaviors. Since pyrene compounds are bio-accumulative organic pollutants with carcinogenic properties [18], understanding their interfacial behaviors, and developing methods for their detection, should be given significant attention within the environmental sciences and analytical methodologies. By manipulating the distances from the metal surfaces, controlled quenching behaviors were observed in the nanometal energy transfer mechanisms (NSET) [10]. The benzyl moieties of aromatic adsorbates achieve better ordering structures, yielding sp3 hybridization due to the methylene group rather than the linear sp hybridization for the sulfur atom [19,20]. Previous UV-Vis absorption and SERS study of benzyl thiol with a methylene (CH2) group can order better than thiolphenol without the methylene group [21]. Considering that the fabrication of wellordered self-assembled monolayers on metal surfaces is significant for designing better molecular electronic devices and superior functions in materials [22], the spectroscopic characterization of the role
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of the methylene group on such surfaces would assist the development of robust layers. Desorption of the organic adsorbates can be easily demonstrated by thiol-containing materials such as glutathione, which exist at as high concentration as a few mM [23]. In the recent SERS and fluorescence studies of drug conjugates on gold nanoparticles (AuNPs), the adsorbates were found to release efficiently under intracellular conditions [24]. Despite the previous sulfur- and nitrogencontaining drugs, the release of the carboxylate adsorbates on AgNPs has not been fully investigated to date. In this regard, we conducted experiments comparing the fluorescence-quenching behaviors of the three aromatic adsorbates of 1-pyrenecarboxylic acid (PyC), 1-pyreneacetic acid (PyA), and 1pyrenebutyric acid (PyB) on AgNPs. Our study will be helpful for developing guidelines on metal nanoparticle/pyrene-complex fluorescence sensors by better understanding their interfacial phenomena
2. Experimental Section 2.1. Sample preparations PyC (97%) and PyB (97%) were purchased from Sigma-Aldrich (St. Louis, USA), whereas PyA (97%) was obtained from Santa Cruz Biotechnology (Dallas, USA). L-Glutathione reduced (GSH) (98.0%) was purchased from Sigma-Aldrich (St. Louis, USA). Due to the poor solubility of pyrene in aqueous solutions, ethanol was introduced for the well dispersiblity. Citrate-reduced AgNPs were chemically synthesized according to the recipe of Lee and Meisel [25]. To obtain the fluorescence emission spectra, pyrene acid (2.5 x 10-3 M, 0.4 µL), AgNP solution (0.604 nM, 100.0 µL) and ethanol (899.6 µL) were decanted into a 1.5 mL Eppendorf tube and stirred. The mixture remained stable for over 5 min at room temperature. Subsequently, the fluorescence spectra of pyrene acid-AgNP solution (1.0 x 10-6 M of pyrene acid, 1000.0 µL) were recorded. To check the GSH-induced release for the pyrene acid-AgNP complex, 0.4 µL of pyrene acid was self-assembled with 100.0 µL of the AgNP solution for 5 min. Subsequently 2.0 µL of GSH (2.0 M) was added and reacted for 14 h. The mixture was diluted with 897.6 µL of ethanol and the fluorescence spectra of this solution were recorded one 5
more time. For the Stern–Volmer measurements, we had to increase the concentration of the AgNP solution up to 60.4 nM by means of centrifugation, precipitation, and redispersion. In the experiments of SERS, UV-Vis, fluorescence, X-ray photoelectron, and in vitro measurements, the prepared AgNPs (0.604 nM) were used without changing the concentrations. The precision and accuracy of our Rainin pipette (Oakland, USA) measurements were 0.012 and 0.024 L, respectively. The statistical range due to errors in volume was estimated to be as high as 6~12 %.
2.2 Physical characterization Quasi-electrostatic light scattering (QELS) measurements to monitor size aggregation were performed using an Otsuka ELS Z2 model. Absorbance spectra of the AgNP solution were taken using a Mecasys 3220 spectrophotometer (Daejeon, Korea). The TEM image of the AgNP particles was obtained using a JEOL 1010 microscope. Raman spectra were obtained using a Renishaw RM 1000 microscope. Raman and infrared spectra were obtained using a Renishaw RM1000 microspectrometer equipped with a 633 nm HeNe laser (Melles Griot Model 25 LHP 928) and an air-cooled Ar-ion laser (Melles Griot Model 35-LAP-431-220) for 488 nm and 514 nm and Thermo 6700 spectrophotometer. Density functional theory (DFT) calculations were performed to estimate the binding energies and vibrational assignments. The optimized geometries of free and adsorbed PyC on six Ag cluster atoms were obtained at the levels of B3LYP/6-31G++(d,p) and B3LYP/LANL2DZ using a Gaussian 09 program [26].
2.3 Cell culture for intracellular release of pyrene carboxylates from AgNPs A549 (ATCC® CCL-185™) were provided from Seoul National University. A549 cells were cultured in Roswell Park Memorial Institute (RPMI) medium (Welgene, Daegu, Korea) with 10% fetal bovine serum (Welgene) and 1% antibiotic-antimycotic solution (Sigma, St. Louis, MO, USA). Cells
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were grown at 37˚C in humidified air, containing 5% CO2. To take fluorescent images of A549 living cells, the cells were seeded onto glass bottom confocal dishes at a density of 5 × 10 4 cells in 2 mL of RPMI medium and incubated for approximately 24 h at 37°C in 5% CO2. The cells were washed with Dulbecco's phosphate-buffered saline (DPBS) solution at pH 7.4, added with 2 mL of fresh media and 1.0 × 10-5 M (final concentration) of pyrene acid (or pyrene acid-adsorbed AgNP solution), then incubated for either 2 h or overnight (14 h). The fluorescence images were performed after washing the sample-treated cells with DPBS. The cells were kept in 2 mL fresh medium for living and imaged under an Olympus IX-71 fluorescence microscope using excitation wavelength of filter Olympus BP330-385 nm.
3. Results and discussion 3.1. Adsorption of pyrene acid compounds on AgNPs. Figure 1 illustrates the molecular structures of PyC, PyA, and PyB anchoring on AgNPs via the carboxylate groups. It must be mentioned that PyA and PyB have the methylene and the butyl group, respectively. PyC, however, does not have any aliphatic group. The morphologies of AgNPs were characterized by TEM images, which showed the average diameter to be ~38.5 nm as shown in Figure 2(b) and (c). The hydrodynamic diameters of the pristine and the pyrene acid-adsorbed AgNPs were measured to be 51.2 (±2.1) nm and 123.2 (±7.0)~133.3(±8.0) nm, respectively, indicating the size aggregation, according to the QELS measurements. The surface charges of pristine AgNPs were measured to be –51 mV. These values became changed to –14.5 mV ~ –11.0 mV, after adsorption of the hydrophobic pyrene acid compounds. Considering the negatively charged surfaces, it is expected that the carboxylic groups of PyC, PyA, and PyB would become deprotonated on colloidal silver. Although an excessive amount of ethanol may induce the aggregation of AgNPs, we could not observe the solvent-induced coagulation in our experiments. Although an excessive amount of ethanol may induce the aggregation of AgNPs, we could not
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observe the solvent-induced coagulation in our experiments. To obtain the hydrodynamic diamters and surface potential values, pyrene acid (2.5 x 10 -3 M, 0.4 µL), AgNP solution (100.0 µL) and distilled water (899.6 µL) were decanted into an 1.5 mL tube and stirred in aqueous conditions as a control test. The increase of the hydrodynamic diameters along with the decrease of the negative value of the surface change indicated the coagulation in the first stage. According the previous study of the DLVO theory, the zeta potential had more positive values after introducing the layers [34]. The increase of the hydrodynamic diameters along with the decrease of the negative value of the surface change indicated the coagulation in the first stage. It is possible that the deposition of hydrophobic pyrene on the surface of AgNPs forms a multi-layer structure [35].
3.2. Raman and infrared spectra of pyrene acid-adsorbed AgNPs Although carboxylic acids are assumed to bind to Ag surfaces via their COO- form [36], the interfacial structures of pyrene acids are not well known. To further investigate the interfacial structures of the three pyrene acid compounds on AgNPs, we employed surface Raman spectroscopic tools as summarized in Figure 3(a). The Raman spectra of pyrene were previously reported on gold nanoparticles by means of SERS [27]. Vibrational assignment of Raman spectra were referred to in the previous literatures [28-33]. The DFT calculation predicted the C=O band at 1708 cm-1 in the free state of PyC and the (COO-) band at 1332 cm-1 for the adsorbed state on six Ag cluster atoms, as illustrated in Figure 3(a). The C=O band was thought to disappear on Ag. Referring to the previous works on the electromagnetic effects, the surface orientations may be estimated by calculating the enhancement factors [37]. Despite our attempt to use the near-infrared laser excitation at 1064 nm in the NR spectrum, such strong fluorescence of PyC, PyA and PyB may prevent from the quality spectra in order to estimate the intrinsic NR spectrum. Due to the strong fluorescence of the pyrene, it was difficult to obtain the normal Raman (NR) spectra of PyC, PyA and PyB. On AgNPs, PyC exhibited several vibrational bands with relatively stronger intensities due to its shorter distances 8
from Ag metal surfaces. The SERS spectrum of PyC, PyA, and PyB on AgNPs using 633 nm revealed four main bands, at 1238, 1385, 1595 and 1624 cm−1; their assignments are listed in Table 2. The different excitation wavelengths at 488 and 514 nm would give similar behaviors. The dissimilar band intensities at 1595 and 1624 cm−1 suggest a charge transfer reaction on Ag surfaces. The SERS features of PyA and PyB were found to be much weaker due to the presence of methylene and butyl groups on AgNP surfaces. Although they are not shown here, the infrared absorption spectra of the three pyrene acid compounds on AgNPs have revealed the COO- band at 1380–1410 cm−1. XPS spectra in Figure 3(c) and (d) also exhibited the atomic compositions and possible chemical bonds on surfaces. The deconvoluted bands at 284.54, 286.05, 288.21, and 290.45 eV can be ascribed to the C1s modes corresponding to the (C-C), (C-O), (C=O), and (C-O) modes, respectively [38]. Referring to the stronger intensity of the C-O mode at 286.05 eV than that of the C=O mode at 288.21 eV, the C-O species appeared to remain mostly present on AgNPs, which is consistent with the COOband in the Raman and infrared spectra. After characterization of the pyrene acids on Ag surfaces, we performed the fluorescence quenching study.
3.3. Fluorescence quenching of pyrene acid-assembled AgNPs The UV-Vis absorption spectrum of AgNPs showed a broad surface Plasmon band with a maximum of ~400 nm, as shown in Figure 4. The molecular extinction coefficients of pyrene compounds are reported to be 10000-35000 M-1 cm-1 in various solvent [39,40]. The fluorescence emission spectra of PyC, PyA, and PyB exhibited similar behaviors to the vibronic spectra of pyrene in previous studies [41,42]. The overlap integrals from the donor and acceptor in the energy transfer could be referred from the report [43]. Fluorescence quenching behaviours of pyrene acid-assembled AgNPs were recorded by measuring the fluorescence intensity of free pyrene acids and pyrene acid-adsorbed AgNPs. As shown in Figure 4, the excitation maximum of AgNPs had reached the wavelength of approximately 400 nm
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and the emission maxima of PyC, PyA and PyB had ranged in wavelength from 350 nm to 450 nm. The relative overlapping integral between the UV-Vis spectra of AgNPs and the fluorescence emission spectra of PyC, PyA and PyB were estimated to be 39.06, 44.68, and 37.90, respectively, which showed the largest overlapping area for PyA. The carboxyl distance from an Au atom was previously reported to be 4.69 Å [44]. The longest dimension of pyrene could be calculated as 9.2 Å [45]. Assuming the C-C distance of 1.54 Å, the molecular lengths of PyC, PyA, and PyB could be estimated as 15.4, 17.0, and 20.1 Å, respectively. Considering the longer distances of PyB from Ag surfaces, the fluorescence quenching by either NSET [11] or FRET [16] may be less efficient than those of PyC and PyA. This was recently discussed concerning the distance-dependent SERS and fluorescence quenching behaviors [46]. The concentration of the prepared AgNPs was estimated to be 0.604 nM according to the previous report [47]. After increasing the concentrations of AgNPs, the Stern–Volmer constants of PyC, PyA, and PyB were determined to be 2.4 (±0.3) × 108, 3.4 (±0.4) × 108, and 1.7 (±0.4) × 108 M-1, respectively. PyB showed the lowest fluorescence quenching efficiency as well as the smallest Stern– Volmer constant value in comparison to PyC and PyA. These values are comparable to the value 2.2 × 108 M-1 for 1-methylaminopyrene on gold nanoparticles [48]. It is noteworthy that PyA exhibits the highest quenching efficiency with the largest Stern– Volmer constant. This suggests that the inserted methylene CH2 group of PyA can play a certain role in the adsorption on Ag surfaces, although this is not definite, as in the case of thiophenol and benzylthiol [21]. Not only the higher integral overlap but also the better packing efficiencies on Ag surfaces may yield the highest fluorescence quenching value. To further estimate the energetic stabilities of PyC, PyA, and PyB on Ag, we performed the desorption experiments using GSH. We could not clearly observe different behaviors of PyC, PyA, and PyB, which would have similar molecular lengths under our experimental conditions. FRET mechanisms may not be efficient due to low concentrations of AgNPs under our experimental conditions. By introducing newly designed
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pyrene structures, we plan to study the distance R dependent quenching mechanisms of Förster resonance energy transfer (R-6) and Dexter electron transfer (e-kR).
3.4. GSH-induced desorption of pyrene acids assembled on AgNPs The thiol group of GSH can strongly bind to Ag (or Au) surfaces according to the previous reports, the carboylate groups of pyrene are to be easily replaced on Ag surfaces. Although not shown here, our DFT calculations also supported the strong binding of GSH on Ag (or Au). We found that the fluorescence intensities of the three compounds appeared to increase to support the release of the pyrene compound on Ag surfaces, presumably due to the substitution reaction of GSH. As shown in Figure 5, the GSH-induced desorption of pyrene acids on a surface of AgNPs exhibited an increase in the fluorescence intensities due to the removal of the quenching effects. We compared the fluorescence intensities of free pyrene acids to those of pyrene acid-adsorbed AgNPs, before and after overnight treatment with GSH for 14 h. To compare the fluorescence recovery effects of PyC, PyA and PyB on AgNPs under intracellular condition, 4 mM of GSH was externally supplied to the pyrene acid-adsorbed AgNP complexes. The decreased fluorescence intensities of pyrene acid compounds were recovered when GSH was added, as shown in Figure 5. It was found that PyB exhibited the weakest fluorescence intensity, whereas PyC and PyA showed similar quenching and recovery behaviors. This result may indicate that not the methylene CH2 group but the butyric group effectively quenches the fluorescence on Ag surfaces. In addition, the external GSH may not efficiently replace the PyB adsorbates due to the butyric acid group, which is the case for PyC and PyA. It is likely that the methylene CH2 group of PyA may not play a significant role in the fluorescence quenching behaviors on Ag surfaces. To check the release behaviors under the intracellular conditions, we performed live cell fluorescence imaging experiments.
3.5. Releases of pyrene acids from AgNPs via intracellular GSH in A549 cells
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It was found that pyrene acid-adsorbed AgNPs could enter the cells efficiently due to their hydrophobic and rather positive surface charged natures. The A549 cells were incubated for overnight (14 h) with free PyA and PyA-adsorbed AgNPs. After washing the sample-treated cells with DPBS, the fluorescence images were obtained using an inverted microscope with the excitation and emission filters for PyA. We found that the quenched fluorescence intensity (2 h) was recovered overnight (14 h), although it is not shown. Pyrene acid adsorbates appeared to desorb in a similar way from AgNPs in cytosols via the intracellular GSH and other cellular components. Under cellular conditions, we managed to differentiate the most quenching effect in PyB, as shown in Figure 6. Our interfacial study of PyC, PyA, and PyB may provide the hindsight to design the pyrene-based sensors in combination with the quenching nanoparticles.
4. Conclusions PyC, PyA, and PyB appeared to adsorb on negatively-charged AgNPs via their carboxylic groups from the prominent (COO-) band at 1380–1410 cm-1 by means of SERS spectroscopic tools. XPS also supported the abundance of the C-O species on Ag. TEM images and QELS examined the size distributions of ~50 nm AgNPs. The surface potential became positive after the adsorption of hydrophobic PyC, PyA, and PyB. By using concentration-dependent fluorescence measurements, the Stern–Volmer constants for the quenching efficiencies of AgNPs on PyC, PyA, and PyB were estimated to be 2.4 (±0.3) × 108, 3.4 (±0.4) × 108, and 1.7 (±0.4) × 108 M-1, respectively. The three pyrene acid adsorbates appeared to detach from AgNP surfaces via the externally-supplied GSH (4 mM) to show the fluorescence increase. PyB exhibited the highest quenching behaviors and the weakest fluorescence recovery after treatment with GSH.
Acknowledgement J. C. acknowledges the support by the National Research Foundation of Korea through Grant number 2009-00426. 12
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bis(2-ethylhexyl) sulfosuccinate solutions in n-decane as observed by photon-correlation spectroscopy and nonaqueous electrophoresis, Langmuir 30 (2014) 12729–12735. [36] S-W. Joo, S.W. Han, H.S. Han, K. Kim, Adsorption and stability of phthalic acid on aqueous silver colloidal surface, J. Raman Spectrosc. 31 (2000) 145–150. [37] E-O. Ganbold, D Kang, S-W. Joo, Raman spectroscopic study of 6-amino-7-deazapurine, Vib. Spectrosc. 80 (2015) 6–10. [38] J.F. Moulder, W.F. Stickle, P.E. Sobol, K.D. Bomben, Handbook of X-ray Photoelectron Spectroscopy; Physical Electronics, Inc. Eden Prairie, MN, (1992). [39] E. Manandhar, J.H. Broome, J. Myrick, W. Lagrone, P.J. Cragg, K.J. Wallace, A pyrene-based fluorescent sensor for Zn2+ ions: a molecular ‘butterfly’, Chem. Commun. 47 (2011) 8796–8798. [40] H. Siu, J. Duhame, Molar absorption coefficient of pyrene aggregates in water, J. Phys. Chem. B 112 (2008) 15301–15312. [41] G. Bains, A.B. Patel, V. Narayanaswami, Pyrene: a probe to study protein conformation and conformational changes. Molecules 16 (2011) 7909–7935. [42] K. Kalyanasundaram, J.K. Thomas, Environmental effects on vibronic band intensities in pyrene monomer fluorescence and their application in studies of micellar systems, J. Am. Chem. Soc. 99 (1977) 2039–2044. [43] D.W. Piston, G.-J. Kremers, Fluorescent protein FRET: the good,the bad and the ugly. Trends. Biochem. Sci. 32 (2007) 408–414. [44] F. Chen, X. Li, J. Hihath, Z. Huang, N. Tao, Effect of anchoring groups on single-molecule conductance: comparative study of thiol-, amine-, and carboxylic-acid-terminated molecules, J. Am. Chem. Soc. 128 (2006) 15874–15881. [45] J. Martins, E. Melo, Molecular mechanism of lateral diffusion of py10-PC and free pyrene in fluid DMPC bilayers, Biophys. J. 80 (2001) 832–840. [46] E-O. Ganbold, J. Yoon, D. Kim, S-W. Joo, Nonidentical intracellular drug release rates in Raman
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and fluorescence spectroscopic determination, PhysChemPhysChem 17 (2015) 3019–3023. [47] K. Kalishwaralal, S. BarathManiKanth, S.R.K. Pandian, V. Deepak, S. Gurunathan, Silver nanoparticles impede the biofilm formation by Pseudomonas aeruginosa and Staphylococcus epidermidis, Colloids Surf. B 79 (2010) 340–344. [48] S.K. Ghosh, A. Pal, S. Kundu, S. Nath, T. Pal, Fluorescence quenching of 1-methylaminopyrene near gold nanoparticles: size regime dependence of the small metallic particles, Chem. Phys. Lett. 395 (2004) 366–372.
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Figure captions Fig. 1. Interfacial structures of PyC, PyA, and PyB anchor on AgNPs via the carboxylate groups. PyA and PyB have the methylene and the butyric group, respectively. PyC does not have any aliphatic group (left). The atomic numbering of PyC on Ag6 for the vibrational assignments is based on potential energy distribution calculations (right), as listed in Table 2.
Fig. 2. (a) Representative TEM images of AgNPs. (b) A magnified view of a TEM image. (c) Hydrodynamic diameters (number distributions) and (d) surface charge changes after the adsorption of PyA, PyB, and PyC on AgNPs. Error bars showed the standard deviation after the three repetitive measurements.
Fig. 3. (a) DFT spectrum of PyC in its neutral state and the adsorbed state on Ag 6. SERS spectra of PyC, PyA, and PyB at 633 nm on AgNPs. (b) XPS spectra of AgNP-PyC. (c) The right image is a magnified view of the left figure from 280 to 294 eV for the C1s region.
Fig. 4. Stern–Volmer plots of the fluorescence intensities of (a) PyC, (b) PyA, and (c) PyB quenched by AgNPs. The measurements were performed three times to yield the error bars.
Fig. 5. GSH-induced release of (a) PyC, (b) PyB, and (c) PyA from AgNPs overnight (14 h). The concentration of GSH was 4 mM. The measurements were performed three times to yield the error bars.
Fig. 6. (a) Fluorescence microscopic images of the control (left), free PyC, PyA, and PyB (middle) in comparison to those adsorbed on AgNPs (right). All were incubated overnight. (b) Stick diagrams of fluorescence intensity of PyC, PyA, and PyB on AgNPs in A549 cells.
19
Fig. 1. Interfacial structures of PyC, PyA, and PyB anchor on AgNPs via the carboxylate groups. PyA and PyB have the methylene and the butyric group, respectively. PyC does not have any aliphatic group (left). The atomic numbering of PyC on Ag6 for the vibrational assignments is based on potential energy distribution calculations (right), as listed in Table 2.
20
(a)
(b)
(c)
(d)
20
10
0
AgNPs PyB-AgNPs
AgNPs PyB-AgNPs
6 Intensity
Differential Number (%)
30
4
2
50
100 150 Diameter (nm)
200
0 -200
-150
-100 -50 0 Zeta Potential (mV)
50
100
Fig. 2. (a) Representative TEM images of AgNPs. (b) A magnified view of a TEM image. (c) Hydrodynamic diameters (number distributions) and (d) surface charge changes after the adsorption of PyA, PyB, and PyC on AgNPs. Error bars showed the standard deviation after the three repetitive measurements.
21
(a)
1238
-
1595 1624
Ag-PyB
1595 1624
1385
Raman Intensity (Arbitr. Unit)
1238
(COO )
1595 1624
1385
1200 1216 1238
Ag-PyA
1675
1643
1332
1228 1260 1284
Ag-PyC
DFT-PyC-Ag
1000
1708
1621
1585
1362 1398
1193 1212 1232
6
DFT-PyC
1500
2000 -1
Wavenumber (cm ) (b)
(c)
30000
AgNPs-PyC
4000
O1s
C-O
Counts / s
Counts / s
C-C Ag3d
20000 C1s
Ag3p
3000
2000
10000 1000
Si2p Si2s
0 0
500 Binding Energy (eV)
1000
280
C=O
284 288 Binding Energy (eV)
C-O 292
Fig. 3. a) DFT spectrum of PyC in its neutral state and the adsorbed state on Ag 6. SERS spectra of PyC, PyA, and PyB at 633 nm on AgNPs. (b) XPS spectra of AgNP-PyC. (c) The right image is a magnified view of the left figure from 280 to 294 eV for the C1s region.
22
(a)
(b)
(c)
Fig. 4. Stern–Volmer plots of the fluorescence intensities of (a) PyC, (b) PyA, and (c) PyB quenched by AgNPs. The measurements were performed three times to yield the error bars.
23
Fluorescence (a.u.)
Relative Intensity (a.u.)
Free PyC GSH-AgNPs-PyC AgNPs-PyC
(a)
69.7 %
35.0 %
Free PyC
400 420 Wavelength (nm)
Free PyA GSH-AgNPs-PyA AgNPs-PyA
Fluorescence (a.u.)
(b)
360
380
400 420 Wavelength (nm)
Fluorescence (a.u.)
71.4 %
41.4 %
Free PyA
440 Free PyB GSH-AgNPs-PyB AgNPs-PyB
(c)
380
400 420 Wavelength (nm)
GSH-AgNPs-PyA
AgNPs-PyA
55.3 %
26.3 %
Free PyB
360
AgNPs-PyC
440
Relative Intensity (a.u.)
380
Relative Intensity (a.u.)
360
GSH-AgNPs-PyC
GSH-AgNPs-PyB
AgNPs-PyB
440
Fig. 5. GSH–induced release of (a) PyC, (b) PyB, and (c) PyA from AgNPs overnight (14 h). The concentration of GSH was 4 mM. The measurements were performed three times to yield the error bars. 24
(a)
(b)
Fluorescence Intensity (a. u.)
800000
Free Pyrene acids Pyrene acid-AgNPs
600000
400000
200000
0
PyC
PyA
PyB
Fig. 6. (a) Fluorescence microscopic images of the control (left), free PyC, PyA, and PyB (middle) in comparison to those adsorbed on AgNPs (right). All were incubated for overnight. (b) Stick diagrams of fluorescence intensity of PyC, PyA, and PyB on AgNPs in A549 cells.
25
Table 1 Size and surface charges of AgNPs. The determined Stern-Volmer constants of PyC, PyA, and PyB on AgNPs. ___________________________________________________________________________________ Molecules
Binding energy (kJ/mol)
Diameters
Surface charges
(nm)
(mV)
Stern-Volmer constants (M-1)
___________________________________________________________________________________ AgNPs 51.2 (±2.1) -48(±5) PyC--Ag
150.62
123.2 (±7.0)
-15(±2)
2.4 (±0.3) × 108
PyA--Ag
151.88
128.6(±2.1)
-12(±3)
3.4 (±0.4) × 108
PyB--Ag
161.08
133.3(±8.0)
-11(±2)
1.7 (±0.4) × 108
26
Table 2 Spectral data and vibrational assignments for PyC, PyA, and PyB. VEDA Calculation of PyC-Ag6.
DFT
SERS Assignment
PyC
PyC-Ag6
PyC
1193
1228
1202
C-C-H bending + Ring stretching, (H24-C13-C11)(28%) + (C15-C14)(13%)
1212
1260
1216
C-C-H bending + Ring stretching, (H22-C6-C1)(11%) + (H28-C2C1)(20%) + (C3-C2)(11%)
1232
1284
1238
C-C-H bending, (H20-C7-C10)(12%) + (H22-C6-C1)(11%) + (H12-C11C13)(20%)
1398
1332
1385
s(COO-) stretching, (O26-C25)(29%) + (C25-C14)(13%) + (O27C25)(16%)
1585
1643
1595
Ring stretching, (C13-C11)(13%) + (C9-C8)(10%) + (C1-C6)(18%)
1621
1675
1624
C=C, (C7-C10)(22%) + (C16-C17)(18%) + (C7-C10)(21%) + (C16C17)(22%) + (C13-C11)(10%)
Abbreviations: δ; Torsion, ; stretching, ; in-plane bending, ; out-of-plane bending. Unit in cm1. No scale factor was applied. Atomic numbering depicted in Figure 1.
27
S0927-7757(17)30001-8 http://dx.doi.org/doi:10.1016/j.colsurfa.2017.01.001 COLSUA 21265
To appear in:
Colloids and Surfaces A: Physicochem. Eng. Aspects
Received date: Revised date: Accepted date:
15-11-2016 28-12-2016 3-1-2017
Please cite this article as: Nguyen Hoang Ly, Thanh Danh Nguyen, Thanh Lam Bui, Sangyeop Lee, Jaebum Choo, Sang-Woo Joo, Spectroscopic measurements of interactions between hydrophobic 1-pyrenebutyric acid and silver colloidal nanoparticles, Colloids and Surfaces A: Physicochemical and Engineering Aspects http://dx.doi.org/10.1016/j.colsurfa.2017.01.001
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Spectroscopic measurements of interactions between hydrophobic 1pyrenebutyric acid and silver colloidal nanoparticles
Nguyen Hoang Lya, Thanh Danh Nguyena,b, Thanh Lam Buia,b, Sangyeop Leec, Jaebum Chooc, SangWoo Jooa,b,*
a
Department of Chemistry, Soongsil University, Seoul 156-743, Korea
b
Department of Information Communication, Materials, Chemistry Convergence Technology, Soongsil University, Seoul 156-743, Korea
c
Department of Bionano Engineering, Hanyang University, Sa-1-dong 1271, Ansan 426-791, South Korea
AUTHOR EMAIL ADDRESS: [email protected]
1
Graphical abstract
Fluorescence Intensity (a. u.)
800000
Free Pyrene acids Pyrene acid-AgNPs
600000
400000
200000
0
PyC
PyA
PyB
2
Highlights
Hydrophobic pyrene acids were chemically interacted with Ag nanoparticles.
1-Pyrene acids were found to adsorb on silver via the COO - bonds.
Fluorescence quenching behaviors of pyrene acids were compared on Ag.
Glutathione-triggered desorption of pyrene acids were observed in cancer cells.
1-Pyrenebutyrate showed stronger binding than 1-pyrenecarboxlyate on Ag.
Abstract We compared the adsorption and desorption of 1-pyrenecarboxylic acid (PyC), 1-pyreneacetic acid (PyA), and 1-pyrenebutyric acid (PyB) on silver nanoparticles (AgNPs) via interfacial spectroscopic tools to study the role of the aliphatic units between pyrene and carboxylic group. The negative surface charges of AgNPs at ca. –51 mV shifted up to –11 mV, after adsorbing hydrophobic pyrene compounds. The three pyrene acid compounds appeared to adsorb onto AgNPs via their carboxylate units by referring to the observation of the broad (COO-) bands at 1380–1410 cm-1 in the Raman spectra. X-ray photoelectron spectroscopy (XPS) also supported the C-O species in the C1s region. AgNPs were found to efficiently quench the fluorescence of the three pyrene acid adsorbates. The highest Stern–Volmer constant of PyA may be due to the largest overlap integral with the surface Plasmon absorption band of AgNPs. The butyric unit was expected to lead a stronger binding on Ag, as suggested by density functional theory (DFT) calculations. The adsorbed pyrene acid compounds appeared to be released from AgNPs by thiol-containing glutathione (GSH). PyB with the butyric group exhibited larger quenched fluorescence intensities and smaller released amounts than PyC and PyA in aqueous solutions and A549 cancer cells. Our study will be helpful in designing pyrene-based fluorescence sensors in cellular imaging. Keywords: Fluorescence quenching; Raman spectroscopy; Density functional theory; 1-Pyrenebutyric acid; Silver nanoparticles; Intracellular release 3
1. Introduction The estimation of hydrophobic forces has been given much attention due to its perspective in colloidal science [1]. In past decades, surface-enhanced Raman scattering (SERS), has proved to be a versatile interfacial spectroscopic tool for analyzing pollutants and biological samples, due to its enormous enhancements occurring on noble metal nanostructures [2-4]. On the other hand, the nanostructure materials can act as efficient quenching platforms of fluorophores [5-10]. Pyrene as a hydrophobic organic ligand [11] exhibits strong fluorescence, which can be utilized as ionic and molecular sensors [12,13]. Since metal nanoparticles can quench the fluorescence of organic adsorbates, the Stern–Volmer plot yields the quenching coefficients of metal nanoparticles [14]. A recent study shows that aminopyrene exhibited its higher quenching value than pyrene and aminomethylpyrene, when adsorbed on 2-21 nm size silver nanoparticles (AgNPs) via both Förster resonance energy transfer (FRET) and electron transfer [15]. Considering that the carboxylic acids bind strongly on Ag surfaces [16,17], this anchoring group may lead interfacial chemical bonding to result in efficient quenching behaviors. Since pyrene compounds are bio-accumulative organic pollutants with carcinogenic properties [18], understanding their interfacial behaviors, and developing methods for their detection, should be given significant attention within the environmental sciences and analytical methodologies. By manipulating the distances from the metal surfaces, controlled quenching behaviors were observed in the nanometal energy transfer mechanisms (NSET) [10]. The benzyl moieties of aromatic adsorbates achieve better ordering structures, yielding sp3 hybridization due to the methylene group rather than the linear sp hybridization for the sulfur atom [19,20]. Previous UV-Vis absorption and SERS study of benzyl thiol with a methylene (CH2) group can order better than thiolphenol without the methylene group [21]. Considering that the fabrication of wellordered self-assembled monolayers on metal surfaces is significant for designing better molecular electronic devices and superior functions in materials [22], the spectroscopic characterization of the role
4
of the methylene group on such surfaces would assist the development of robust layers. Desorption of the organic adsorbates can be easily demonstrated by thiol-containing materials such as glutathione, which exist at as high concentration as a few mM [23]. In the recent SERS and fluorescence studies of drug conjugates on gold nanoparticles (AuNPs), the adsorbates were found to release efficiently under intracellular conditions [24]. Despite the previous sulfur- and nitrogencontaining drugs, the release of the carboxylate adsorbates on AgNPs has not been fully investigated to date. In this regard, we conducted experiments comparing the fluorescence-quenching behaviors of the three aromatic adsorbates of 1-pyrenecarboxylic acid (PyC), 1-pyreneacetic acid (PyA), and 1pyrenebutyric acid (PyB) on AgNPs. Our study will be helpful for developing guidelines on metal nanoparticle/pyrene-complex fluorescence sensors by better understanding their interfacial phenomena
2. Experimental Section 2.1. Sample preparations PyC (97%) and PyB (97%) were purchased from Sigma-Aldrich (St. Louis, USA), whereas PyA (97%) was obtained from Santa Cruz Biotechnology (Dallas, USA). L-Glutathione reduced (GSH) (98.0%) was purchased from Sigma-Aldrich (St. Louis, USA). Due to the poor solubility of pyrene in aqueous solutions, ethanol was introduced for the well dispersiblity. Citrate-reduced AgNPs were chemically synthesized according to the recipe of Lee and Meisel [25]. To obtain the fluorescence emission spectra, pyrene acid (2.5 x 10-3 M, 0.4 µL), AgNP solution (0.604 nM, 100.0 µL) and ethanol (899.6 µL) were decanted into a 1.5 mL Eppendorf tube and stirred. The mixture remained stable for over 5 min at room temperature. Subsequently, the fluorescence spectra of pyrene acid-AgNP solution (1.0 x 10-6 M of pyrene acid, 1000.0 µL) were recorded. To check the GSH-induced release for the pyrene acid-AgNP complex, 0.4 µL of pyrene acid was self-assembled with 100.0 µL of the AgNP solution for 5 min. Subsequently 2.0 µL of GSH (2.0 M) was added and reacted for 14 h. The mixture was diluted with 897.6 µL of ethanol and the fluorescence spectra of this solution were recorded one 5
more time. For the Stern–Volmer measurements, we had to increase the concentration of the AgNP solution up to 60.4 nM by means of centrifugation, precipitation, and redispersion. In the experiments of SERS, UV-Vis, fluorescence, X-ray photoelectron, and in vitro measurements, the prepared AgNPs (0.604 nM) were used without changing the concentrations. The precision and accuracy of our Rainin pipette (Oakland, USA) measurements were 0.012 and 0.024 L, respectively. The statistical range due to errors in volume was estimated to be as high as 6~12 %.
2.2 Physical characterization Quasi-electrostatic light scattering (QELS) measurements to monitor size aggregation were performed using an Otsuka ELS Z2 model. Absorbance spectra of the AgNP solution were taken using a Mecasys 3220 spectrophotometer (Daejeon, Korea). The TEM image of the AgNP particles was obtained using a JEOL 1010 microscope. Raman spectra were obtained using a Renishaw RM 1000 microscope. Raman and infrared spectra were obtained using a Renishaw RM1000 microspectrometer equipped with a 633 nm HeNe laser (Melles Griot Model 25 LHP 928) and an air-cooled Ar-ion laser (Melles Griot Model 35-LAP-431-220) for 488 nm and 514 nm and Thermo 6700 spectrophotometer. Density functional theory (DFT) calculations were performed to estimate the binding energies and vibrational assignments. The optimized geometries of free and adsorbed PyC on six Ag cluster atoms were obtained at the levels of B3LYP/6-31G++(d,p) and B3LYP/LANL2DZ using a Gaussian 09 program [26].
2.3 Cell culture for intracellular release of pyrene carboxylates from AgNPs A549 (ATCC® CCL-185™) were provided from Seoul National University. A549 cells were cultured in Roswell Park Memorial Institute (RPMI) medium (Welgene, Daegu, Korea) with 10% fetal bovine serum (Welgene) and 1% antibiotic-antimycotic solution (Sigma, St. Louis, MO, USA). Cells
6
were grown at 37˚C in humidified air, containing 5% CO2. To take fluorescent images of A549 living cells, the cells were seeded onto glass bottom confocal dishes at a density of 5 × 10 4 cells in 2 mL of RPMI medium and incubated for approximately 24 h at 37°C in 5% CO2. The cells were washed with Dulbecco's phosphate-buffered saline (DPBS) solution at pH 7.4, added with 2 mL of fresh media and 1.0 × 10-5 M (final concentration) of pyrene acid (or pyrene acid-adsorbed AgNP solution), then incubated for either 2 h or overnight (14 h). The fluorescence images were performed after washing the sample-treated cells with DPBS. The cells were kept in 2 mL fresh medium for living and imaged under an Olympus IX-71 fluorescence microscope using excitation wavelength of filter Olympus BP330-385 nm.
3. Results and discussion 3.1. Adsorption of pyrene acid compounds on AgNPs. Figure 1 illustrates the molecular structures of PyC, PyA, and PyB anchoring on AgNPs via the carboxylate groups. It must be mentioned that PyA and PyB have the methylene and the butyl group, respectively. PyC, however, does not have any aliphatic group. The morphologies of AgNPs were characterized by TEM images, which showed the average diameter to be ~38.5 nm as shown in Figure 2(b) and (c). The hydrodynamic diameters of the pristine and the pyrene acid-adsorbed AgNPs were measured to be 51.2 (±2.1) nm and 123.2 (±7.0)~133.3(±8.0) nm, respectively, indicating the size aggregation, according to the QELS measurements. The surface charges of pristine AgNPs were measured to be –51 mV. These values became changed to –14.5 mV ~ –11.0 mV, after adsorption of the hydrophobic pyrene acid compounds. Considering the negatively charged surfaces, it is expected that the carboxylic groups of PyC, PyA, and PyB would become deprotonated on colloidal silver. Although an excessive amount of ethanol may induce the aggregation of AgNPs, we could not observe the solvent-induced coagulation in our experiments. Although an excessive amount of ethanol may induce the aggregation of AgNPs, we could not
7
observe the solvent-induced coagulation in our experiments. To obtain the hydrodynamic diamters and surface potential values, pyrene acid (2.5 x 10 -3 M, 0.4 µL), AgNP solution (100.0 µL) and distilled water (899.6 µL) were decanted into an 1.5 mL tube and stirred in aqueous conditions as a control test. The increase of the hydrodynamic diameters along with the decrease of the negative value of the surface change indicated the coagulation in the first stage. According the previous study of the DLVO theory, the zeta potential had more positive values after introducing the layers [34]. The increase of the hydrodynamic diameters along with the decrease of the negative value of the surface change indicated the coagulation in the first stage. It is possible that the deposition of hydrophobic pyrene on the surface of AgNPs forms a multi-layer structure [35].
3.2. Raman and infrared spectra of pyrene acid-adsorbed AgNPs Although carboxylic acids are assumed to bind to Ag surfaces via their COO- form [36], the interfacial structures of pyrene acids are not well known. To further investigate the interfacial structures of the three pyrene acid compounds on AgNPs, we employed surface Raman spectroscopic tools as summarized in Figure 3(a). The Raman spectra of pyrene were previously reported on gold nanoparticles by means of SERS [27]. Vibrational assignment of Raman spectra were referred to in the previous literatures [28-33]. The DFT calculation predicted the C=O band at 1708 cm-1 in the free state of PyC and the (COO-) band at 1332 cm-1 for the adsorbed state on six Ag cluster atoms, as illustrated in Figure 3(a). The C=O band was thought to disappear on Ag. Referring to the previous works on the electromagnetic effects, the surface orientations may be estimated by calculating the enhancement factors [37]. Despite our attempt to use the near-infrared laser excitation at 1064 nm in the NR spectrum, such strong fluorescence of PyC, PyA and PyB may prevent from the quality spectra in order to estimate the intrinsic NR spectrum. Due to the strong fluorescence of the pyrene, it was difficult to obtain the normal Raman (NR) spectra of PyC, PyA and PyB. On AgNPs, PyC exhibited several vibrational bands with relatively stronger intensities due to its shorter distances 8
from Ag metal surfaces. The SERS spectrum of PyC, PyA, and PyB on AgNPs using 633 nm revealed four main bands, at 1238, 1385, 1595 and 1624 cm−1; their assignments are listed in Table 2. The different excitation wavelengths at 488 and 514 nm would give similar behaviors. The dissimilar band intensities at 1595 and 1624 cm−1 suggest a charge transfer reaction on Ag surfaces. The SERS features of PyA and PyB were found to be much weaker due to the presence of methylene and butyl groups on AgNP surfaces. Although they are not shown here, the infrared absorption spectra of the three pyrene acid compounds on AgNPs have revealed the COO- band at 1380–1410 cm−1. XPS spectra in Figure 3(c) and (d) also exhibited the atomic compositions and possible chemical bonds on surfaces. The deconvoluted bands at 284.54, 286.05, 288.21, and 290.45 eV can be ascribed to the C1s modes corresponding to the (C-C), (C-O), (C=O), and (C-O) modes, respectively [38]. Referring to the stronger intensity of the C-O mode at 286.05 eV than that of the C=O mode at 288.21 eV, the C-O species appeared to remain mostly present on AgNPs, which is consistent with the COOband in the Raman and infrared spectra. After characterization of the pyrene acids on Ag surfaces, we performed the fluorescence quenching study.
3.3. Fluorescence quenching of pyrene acid-assembled AgNPs The UV-Vis absorption spectrum of AgNPs showed a broad surface Plasmon band with a maximum of ~400 nm, as shown in Figure 4. The molecular extinction coefficients of pyrene compounds are reported to be 10000-35000 M-1 cm-1 in various solvent [39,40]. The fluorescence emission spectra of PyC, PyA, and PyB exhibited similar behaviors to the vibronic spectra of pyrene in previous studies [41,42]. The overlap integrals from the donor and acceptor in the energy transfer could be referred from the report [43]. Fluorescence quenching behaviours of pyrene acid-assembled AgNPs were recorded by measuring the fluorescence intensity of free pyrene acids and pyrene acid-adsorbed AgNPs. As shown in Figure 4, the excitation maximum of AgNPs had reached the wavelength of approximately 400 nm
9
and the emission maxima of PyC, PyA and PyB had ranged in wavelength from 350 nm to 450 nm. The relative overlapping integral between the UV-Vis spectra of AgNPs and the fluorescence emission spectra of PyC, PyA and PyB were estimated to be 39.06, 44.68, and 37.90, respectively, which showed the largest overlapping area for PyA. The carboxyl distance from an Au atom was previously reported to be 4.69 Å [44]. The longest dimension of pyrene could be calculated as 9.2 Å [45]. Assuming the C-C distance of 1.54 Å, the molecular lengths of PyC, PyA, and PyB could be estimated as 15.4, 17.0, and 20.1 Å, respectively. Considering the longer distances of PyB from Ag surfaces, the fluorescence quenching by either NSET [11] or FRET [16] may be less efficient than those of PyC and PyA. This was recently discussed concerning the distance-dependent SERS and fluorescence quenching behaviors [46]. The concentration of the prepared AgNPs was estimated to be 0.604 nM according to the previous report [47]. After increasing the concentrations of AgNPs, the Stern–Volmer constants of PyC, PyA, and PyB were determined to be 2.4 (±0.3) × 108, 3.4 (±0.4) × 108, and 1.7 (±0.4) × 108 M-1, respectively. PyB showed the lowest fluorescence quenching efficiency as well as the smallest Stern– Volmer constant value in comparison to PyC and PyA. These values are comparable to the value 2.2 × 108 M-1 for 1-methylaminopyrene on gold nanoparticles [48]. It is noteworthy that PyA exhibits the highest quenching efficiency with the largest Stern– Volmer constant. This suggests that the inserted methylene CH2 group of PyA can play a certain role in the adsorption on Ag surfaces, although this is not definite, as in the case of thiophenol and benzylthiol [21]. Not only the higher integral overlap but also the better packing efficiencies on Ag surfaces may yield the highest fluorescence quenching value. To further estimate the energetic stabilities of PyC, PyA, and PyB on Ag, we performed the desorption experiments using GSH. We could not clearly observe different behaviors of PyC, PyA, and PyB, which would have similar molecular lengths under our experimental conditions. FRET mechanisms may not be efficient due to low concentrations of AgNPs under our experimental conditions. By introducing newly designed
10
pyrene structures, we plan to study the distance R dependent quenching mechanisms of Förster resonance energy transfer (R-6) and Dexter electron transfer (e-kR).
3.4. GSH-induced desorption of pyrene acids assembled on AgNPs The thiol group of GSH can strongly bind to Ag (or Au) surfaces according to the previous reports, the carboylate groups of pyrene are to be easily replaced on Ag surfaces. Although not shown here, our DFT calculations also supported the strong binding of GSH on Ag (or Au). We found that the fluorescence intensities of the three compounds appeared to increase to support the release of the pyrene compound on Ag surfaces, presumably due to the substitution reaction of GSH. As shown in Figure 5, the GSH-induced desorption of pyrene acids on a surface of AgNPs exhibited an increase in the fluorescence intensities due to the removal of the quenching effects. We compared the fluorescence intensities of free pyrene acids to those of pyrene acid-adsorbed AgNPs, before and after overnight treatment with GSH for 14 h. To compare the fluorescence recovery effects of PyC, PyA and PyB on AgNPs under intracellular condition, 4 mM of GSH was externally supplied to the pyrene acid-adsorbed AgNP complexes. The decreased fluorescence intensities of pyrene acid compounds were recovered when GSH was added, as shown in Figure 5. It was found that PyB exhibited the weakest fluorescence intensity, whereas PyC and PyA showed similar quenching and recovery behaviors. This result may indicate that not the methylene CH2 group but the butyric group effectively quenches the fluorescence on Ag surfaces. In addition, the external GSH may not efficiently replace the PyB adsorbates due to the butyric acid group, which is the case for PyC and PyA. It is likely that the methylene CH2 group of PyA may not play a significant role in the fluorescence quenching behaviors on Ag surfaces. To check the release behaviors under the intracellular conditions, we performed live cell fluorescence imaging experiments.
3.5. Releases of pyrene acids from AgNPs via intracellular GSH in A549 cells
11
It was found that pyrene acid-adsorbed AgNPs could enter the cells efficiently due to their hydrophobic and rather positive surface charged natures. The A549 cells were incubated for overnight (14 h) with free PyA and PyA-adsorbed AgNPs. After washing the sample-treated cells with DPBS, the fluorescence images were obtained using an inverted microscope with the excitation and emission filters for PyA. We found that the quenched fluorescence intensity (2 h) was recovered overnight (14 h), although it is not shown. Pyrene acid adsorbates appeared to desorb in a similar way from AgNPs in cytosols via the intracellular GSH and other cellular components. Under cellular conditions, we managed to differentiate the most quenching effect in PyB, as shown in Figure 6. Our interfacial study of PyC, PyA, and PyB may provide the hindsight to design the pyrene-based sensors in combination with the quenching nanoparticles.
4. Conclusions PyC, PyA, and PyB appeared to adsorb on negatively-charged AgNPs via their carboxylic groups from the prominent (COO-) band at 1380–1410 cm-1 by means of SERS spectroscopic tools. XPS also supported the abundance of the C-O species on Ag. TEM images and QELS examined the size distributions of ~50 nm AgNPs. The surface potential became positive after the adsorption of hydrophobic PyC, PyA, and PyB. By using concentration-dependent fluorescence measurements, the Stern–Volmer constants for the quenching efficiencies of AgNPs on PyC, PyA, and PyB were estimated to be 2.4 (±0.3) × 108, 3.4 (±0.4) × 108, and 1.7 (±0.4) × 108 M-1, respectively. The three pyrene acid adsorbates appeared to detach from AgNP surfaces via the externally-supplied GSH (4 mM) to show the fluorescence increase. PyB exhibited the highest quenching behaviors and the weakest fluorescence recovery after treatment with GSH.
Acknowledgement J. C. acknowledges the support by the National Research Foundation of Korea through Grant number 2009-00426. 12
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Figure captions Fig. 1. Interfacial structures of PyC, PyA, and PyB anchor on AgNPs via the carboxylate groups. PyA and PyB have the methylene and the butyric group, respectively. PyC does not have any aliphatic group (left). The atomic numbering of PyC on Ag6 for the vibrational assignments is based on potential energy distribution calculations (right), as listed in Table 2.
Fig. 2. (a) Representative TEM images of AgNPs. (b) A magnified view of a TEM image. (c) Hydrodynamic diameters (number distributions) and (d) surface charge changes after the adsorption of PyA, PyB, and PyC on AgNPs. Error bars showed the standard deviation after the three repetitive measurements.
Fig. 3. (a) DFT spectrum of PyC in its neutral state and the adsorbed state on Ag 6. SERS spectra of PyC, PyA, and PyB at 633 nm on AgNPs. (b) XPS spectra of AgNP-PyC. (c) The right image is a magnified view of the left figure from 280 to 294 eV for the C1s region.
Fig. 4. Stern–Volmer plots of the fluorescence intensities of (a) PyC, (b) PyA, and (c) PyB quenched by AgNPs. The measurements were performed three times to yield the error bars.
Fig. 5. GSH-induced release of (a) PyC, (b) PyB, and (c) PyA from AgNPs overnight (14 h). The concentration of GSH was 4 mM. The measurements were performed three times to yield the error bars.
Fig. 6. (a) Fluorescence microscopic images of the control (left), free PyC, PyA, and PyB (middle) in comparison to those adsorbed on AgNPs (right). All were incubated overnight. (b) Stick diagrams of fluorescence intensity of PyC, PyA, and PyB on AgNPs in A549 cells.
19
Fig. 1. Interfacial structures of PyC, PyA, and PyB anchor on AgNPs via the carboxylate groups. PyA and PyB have the methylene and the butyric group, respectively. PyC does not have any aliphatic group (left). The atomic numbering of PyC on Ag6 for the vibrational assignments is based on potential energy distribution calculations (right), as listed in Table 2.
20
(a)
(b)
(c)
(d)
20
10
0
AgNPs PyB-AgNPs
AgNPs PyB-AgNPs
6 Intensity
Differential Number (%)
30
4
2
50
100 150 Diameter (nm)
200
0 -200
-150
-100 -50 0 Zeta Potential (mV)
50
100
Fig. 2. (a) Representative TEM images of AgNPs. (b) A magnified view of a TEM image. (c) Hydrodynamic diameters (number distributions) and (d) surface charge changes after the adsorption of PyA, PyB, and PyC on AgNPs. Error bars showed the standard deviation after the three repetitive measurements.
21
(a)
1238
-
1595 1624
Ag-PyB
1595 1624
1385
Raman Intensity (Arbitr. Unit)
1238
(COO )
1595 1624
1385
1200 1216 1238
Ag-PyA
1675
1643
1332
1228 1260 1284
Ag-PyC
DFT-PyC-Ag
1000
1708
1621
1585
1362 1398
1193 1212 1232
6
DFT-PyC
1500
2000 -1
Wavenumber (cm ) (b)
(c)
30000
AgNPs-PyC
4000
O1s
C-O
Counts / s
Counts / s
C-C Ag3d
20000 C1s
Ag3p
3000
2000
10000 1000
Si2p Si2s
0 0
500 Binding Energy (eV)
1000
280
C=O
284 288 Binding Energy (eV)
C-O 292
Fig. 3. a) DFT spectrum of PyC in its neutral state and the adsorbed state on Ag 6. SERS spectra of PyC, PyA, and PyB at 633 nm on AgNPs. (b) XPS spectra of AgNP-PyC. (c) The right image is a magnified view of the left figure from 280 to 294 eV for the C1s region.
22
(a)
(b)
(c)
Fig. 4. Stern–Volmer plots of the fluorescence intensities of (a) PyC, (b) PyA, and (c) PyB quenched by AgNPs. The measurements were performed three times to yield the error bars.
23
Fluorescence (a.u.)
Relative Intensity (a.u.)
Free PyC GSH-AgNPs-PyC AgNPs-PyC
(a)
69.7 %
35.0 %
Free PyC
400 420 Wavelength (nm)
Free PyA GSH-AgNPs-PyA AgNPs-PyA
Fluorescence (a.u.)
(b)
360
380
400 420 Wavelength (nm)
Fluorescence (a.u.)
71.4 %
41.4 %
Free PyA
440 Free PyB GSH-AgNPs-PyB AgNPs-PyB
(c)
380
400 420 Wavelength (nm)
GSH-AgNPs-PyA
AgNPs-PyA
55.3 %
26.3 %
Free PyB
360
AgNPs-PyC
440
Relative Intensity (a.u.)
380
Relative Intensity (a.u.)
360
GSH-AgNPs-PyC
GSH-AgNPs-PyB
AgNPs-PyB
440
Fig. 5. GSH–induced release of (a) PyC, (b) PyB, and (c) PyA from AgNPs overnight (14 h). The concentration of GSH was 4 mM. The measurements were performed three times to yield the error bars. 24
(a)
(b)
Fluorescence Intensity (a. u.)
800000
Free Pyrene acids Pyrene acid-AgNPs
600000
400000
200000
0
PyC
PyA
PyB
Fig. 6. (a) Fluorescence microscopic images of the control (left), free PyC, PyA, and PyB (middle) in comparison to those adsorbed on AgNPs (right). All were incubated for overnight. (b) Stick diagrams of fluorescence intensity of PyC, PyA, and PyB on AgNPs in A549 cells.
25
Table 1 Size and surface charges of AgNPs. The determined Stern-Volmer constants of PyC, PyA, and PyB on AgNPs. ___________________________________________________________________________________ Molecules
Binding energy (kJ/mol)
Diameters
Surface charges
(nm)
(mV)
Stern-Volmer constants (M-1)
___________________________________________________________________________________ AgNPs 51.2 (±2.1) -48(±5) PyC--Ag
150.62
123.2 (±7.0)
-15(±2)
2.4 (±0.3) × 108
PyA--Ag
151.88
128.6(±2.1)
-12(±3)
3.4 (±0.4) × 108
PyB--Ag
161.08
133.3(±8.0)
-11(±2)
1.7 (±0.4) × 108
26
Table 2 Spectral data and vibrational assignments for PyC, PyA, and PyB. VEDA Calculation of PyC-Ag6.
DFT
SERS Assignment
PyC
PyC-Ag6
PyC
1193
1228
1202
C-C-H bending + Ring stretching, (H24-C13-C11)(28%) + (C15-C14)(13%)
1212
1260
1216
C-C-H bending + Ring stretching, (H22-C6-C1)(11%) + (H28-C2C1)(20%) + (C3-C2)(11%)
1232
1284
1238
C-C-H bending, (H20-C7-C10)(12%) + (H22-C6-C1)(11%) + (H12-C11C13)(20%)
1398
1332
1385
s(COO-) stretching, (O26-C25)(29%) + (C25-C14)(13%) + (O27C25)(16%)
1585
1643
1595
Ring stretching, (C13-C11)(13%) + (C9-C8)(10%) + (C1-C6)(18%)
1621
1675
1624
C=C, (C7-C10)(22%) + (C16-C17)(18%) + (C7-C10)(21%) + (C16C17)(22%) + (C13-C11)(10%)
Abbreviations: δ; Torsion, ; stretching, ; in-plane bending, ; out-of-plane bending. Unit in cm1. No scale factor was applied. Atomic numbering depicted in Figure 1.
27