Fluorescence quenching of fulvic acids by fullerene in water
Highlights
► Fluorescence of fulvic acids can be quenched by nanoscaled-fullerene-aggregates. ► Static quenching was the main fluorescence quenching mechanism. ► Association constants were estimated with fluorescence quenching titration. ► Hydrophobic and π–π interactions control the interaction.
Introduction
Fullerene has shown promising applications in catalytic conversion, medical therapeutics, and nonlinear optics since its discovery in 1985 (Lyon et al., 2006). Fullerene is similar in structure to graphite, and its composition of stacked sheets that make it extremely hydrophobic, with a tendency to aggregate and deposit in polar solvents because of strong van der Waals forces. The solubility of fullerene is less than 10−9 mg/l in water and fullerene powder alone has no antibacterial properties (Fortner et al., 2005). However, fullerene is often required to be dispersed before potential engineering applications (Lyon et al., 2006; Yamakoshi et al., 1994). The dispersion methods commonly involve dissolving fullerene in certain kinds of solvents (e.g., dimethyl sulfoxide, polyvinylpyrrolidone, tetrahydrofuran, and toluene) that are subsequently replaced by water (Klavins and Ansone, 2010; Lyon et al., 2006). The fullerene water suspension contains nanoscaled-fullerene-aggregates (nC60), whose morphological properties differ from those of dissolved fullerene in the relevant solvent (Lyon et al., 2006). The nC60 forms are the most environmentally relevant forms of fullerene in the event of a spill of either fullerene powder or dissolved fullerene in water (Fortner et al., 2005; Lyon et al., 2006). Furthermore, the nC60 forms are toxic to micro-organisms, aquatic organisms and even animal/human cell lines (Fortner et al., 2005; Lyon et al., 2006). Therefore, it is necessary to investigate the fates and behaviors of fullerene in natural environments.
Humic substances are a poly dispersed mixture of organic macromolecules with a variety of aromatic and aliphatic blocks and functional groups occurring in water, soil and sediment (Kumke et al., 1998a,b; Wu and Xing, 2009; Zafiriou et al., 1984; Zepp et al., 1987). Humic substances can be operationally separated into fulvic acid (FA; soluble at all pH values), humic acid (soluble in alkaline media and insoluble in acidic media), and humin (insoluble at all pH values), according to the water solubility (Bai et al., 2008a,b; Wu et al., 2001; Wu and Xing, 2009). FAs play key roles in affecting speciation, toxicity and transport of inorganic and organic compounds (such as Cu2+, Hg2+, PAHs and carbamazepine) in aquatic environments due to the strong interactions between them (Cabaniss, 2011; Fang et al., 1998; Kumke et al., 1998b; Ohno et al., 2008; Wu et al., 2004). It would be a priority to examine the interactions between nC60 and FAs in cases of fullerene contaminating aquatic environments. Currently, little is known about the interaction mechanisms and possible roles of FAs in controlling the environmental and biogeochemical fate of fullerene.
Molecular fluorescence quenching titration has been widely employed to provide useful information regarding the interactions between humic substances and trace contaminants since the 1980s (Bai et al., 2008a,b; Fang et al., 1998; Gauthier et al., 1986; Kumke et al., 1998a,b; Lu and Jaffe, 2001; Ryan and Weber, 1982; Wu et al., 2001, 2004). This method was also satisfactorily used to study the interactions between fullerene and PAHs (Datta and Mukherjee, 2006) as well as fullerene and humic substances in organic solvents (Klavins and Ansone, 2010; Manciulea et al., 2009). The interactions between humic substances and nC60 in water have not yet been reported.
The possible fluorescence quenching mechanisms include dynamic, static, and a combination of dynamic and static quenching (Kumke et al., 1998a; Pan et al., 2012). The dynamic quenching agent provides a non-radiative route for the loss of the excited state energy during the lifetime of an excited state of fluorescer (Pan et al., 2012). Static quenching however is a process where non-fluorescent complexes are formed. Both mechanisms were reported for different compounds quenched by fullerene (Datta and Mukherjee, 2006; Klavins and Ansone, 2010; Manciulea et al., 2009). For example, dynamic quenching is the main mechanism involved when anthracene is quenched by fullerene (Datta and Mukherjee, 2006), while the static quenching process is involved for humic substances quenched by fullerene in organic mediums (Klavins and Ansone, 2010). Therefore, the discussion on the quenching mechanism involved in water is still open. For mono-dispersal systems, the linear Stern–Volmer plot could easily be indicative of a single mechanism of fluorophores with equal accessibility to the quencher, and a combination of two quenching mechanisms typically produces a nonlinear plot (Bai et al., 2008a,b; Klavins and Ansone, 2010). Diffusion rates and dynamic collision rates increase with increasing temperature. In contrast, complex formation strength tends to decrease with increasing temperature. Thus, fluorescence quenching increases under dynamic quenching, but decreases under static quenching as temperature increases. The temperature-dependant method can simply be used to investigate the quenching mechanisms, by comparing the quenching extent at various temperatures. Dynamic quenching reduces the average lifetime of the fluorophore while static quenching does not have the same effect. Therefore, the fluorescence lifetime measurement is the most commonly used method to identify the quenching mechanism (Marwani et al., 2007, 2009). Thus, the quenching mechanisms can be confirmed by: (a) exploring the curvature with a Stern–Volmer plot, (b) comparing the association constant with an extremely efficient quencher, (c) investigating temperature dependence of fluorescence titration, and (d) fluorescence lifetime measurement (Bai et al., 2008a,b; Marwani et al., 2007, 2009). Because of the complicated composition and structure of humic substances, as well as the unknown process of their interactions with nC60, all four methods were performed to investigate the main quenching mechanisms.
The objective of the present study was to investigate the interactions between FAs and nC60 and its affecting factors, e.g., FA concentration levels, pH values and temperatures. Using the fluorescence quenching method, temperature-dependent experiments, and fluorescence lifetime measurements, association constants between FAs and nC60 were carried out and estimated, and the interaction mechanisms between fulvic acid and nC60 are discussed.
Section snippets
Reagents and materials
Fullerene with a purity of 99.9% was obtained from Puyang Yongxin Fullerene Technology Co., Ltd. (Puyang City, China). The nC60 suspension was produced using the method recommended by Andrievsky et al. (1995) and Lyon et al. (2006). Using this method, about 5 ml of 1.0 g/l fullerene in toluene was added to about 500 ml pure water. The layered mixture was sonicated with a sonifier cell disruptor (Ultrasonic Processor, Auto Science, Canada) at 70–90 W for 15 min intervals (allowing the machine to
Results and discussion
The three-dimensional excitation–emission matrixes indicated that the major fluorescence peaks occurred at excitation/emission wavelengths of 280–400 nm/380–500 nm for both AFA and BFA. Both peaks are referred to as humic-like fluorescence (Kumke et al., 1998a,b; Zafiriou et al., 1984; Zepp, 1978; Zepp and Schlotzhauer, 1983; Zepp et al., 1985). The maximal fluorescence intensities measured with emission fluorescence spectra showed a significant linear relationship (p < 0.01) with FA
Conclusion
Overall, the intrinsic fluorescence of FAs could be significantly quenched by nC60, while the location of main fluorescence peaks remained constant with nC60 added, indicating the unchanged conformation and rigidity of FAs during titration. The static quenching mechanism proved to be the predominant mechanism for FA quenching with nC60 by obtaining the significant linear coefficient of the Stern–Volmer model, higher log K compared with extremely efficient quencher, temperature-dependent
Acknowledgments
The authors are grateful for the financial support from China's National Basic Research Program (2008CB418200) and the National Natural Science Foundation of China (40903036 and 41173084), KYZX (2010KYYW04), BARD (IS-4353-10), and USDA Hatch program (MAS00978).
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