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Fenton reaction-triggered colorimetric detection of phenols in water samples using unmodified gold nanoparticles
Accepted Manuscript Title: Fenton reaction-triggered colorimetric detection of phenols in water samples using unmodified gold nanoparticles Author: Li-pei Zhang Yun-peng Xing Lan-hua Liu Xiao-hong Zhou Han-chang Shi PII: DOI: Reference:
S0925-4005(15)30665-1 http://dx.doi.org/doi:10.1016/j.snb.2015.11.083 SNB 19341
To appear in:
Sensors and Actuators B
Received date: Accepted date:
24-9-2015 18-11-2015
Please cite this article as: L.-p. Zhang, Y.-p. Xing, L.-h. Liu, X.-h. Zhou, H.-c. Shi, Fenton reaction-triggered colorimetric detection of phenols in water samples using unmodified gold nanoparticles, Sensors and Actuators B: Chemical (2015), http://dx.doi.org/10.1016/j.snb.2015.11.083 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.
Fenton reaction-triggered colorimetric detection of phenols in water samples using unmodified gold nanoparticles Li-pei Zhang, Yun-peng Xing, Lan-hua Liu, Xiao-hong Zhou*, Han-chang Shi*
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Abstract This work demonstrates a rapid and sensitive colorimetric detection of phenols by using single-stranded DNA (ssDNA)-regulated gold nanoparticles (AuNPs) as indicators. AuNPs can be stabilized in the presence of ssDNA through electrostatic repulsion, which prevents the salt-induced aggregation of AuNPs in solution. However, hydroxyl radicals (·OH) generated by the Fenton reaction can cleave the ssDNA on the nanoparticle surface into mono- or oligonucleotide fragments, disrupting AuNPs stability. Phenolic compounds are known to be capable of being degraded or oxidized by ·OH produced by Fenton reaction. Thus, phenols can effectively scavenge ·OH to avoid ssDNA cleavage, protecting AuNPs from salt-induced aggregation. The ability of phenols to scavenge ·OH provides a quantitative basis for phenol sensing. In this study, catechol and hydroquinone were selected as analytes and detected using the proposed ssDNA–AuNPs colorimetric probe. The detection sensitivities of the colorimetric sensor are 0.2–7.0 µM for catechol and 2.7–19 µM for hydroquinone. The proposed bioassay eliminates tedious sample pretreatment and offers favorable sensitivity and selectivity for targets in the presence of other investigated metal ions and organic molecules. The detection limits are 0.11 µM for catechol and 1.6 µM for hydroquinone, with relative standard deviations of 3.7% for catechol and 4.8% for hydroquinone. The recovery rate of catechol in real water samples ranges from 95% to 116%, confirming the application potential of the method to measure phenols in real samples.
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Keywords Gold nanoparticles, Fenton reaction, Phenol, Colorimetric sensor 1. Introduction Phenolic compounds are common industrial byproducts that are accumulated in the environment and the ecological food chain through consumption or uptake. Their presence in air, water, and food results in acute environmental problems and poses toxic risks to human health; for instance, chlorophenols and nitrophenols possess carcinogenic and immunosuppressive properties1, 2. As a result, both the European Union and the United States Environmental Protection Agency have included phenols in their lists of priority pollutants. The European Community set the maximum amount of phenols in wastewater at lower than 1 ppm3, 4. Various traditional analytical methods 1
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have been developed for phenolic compound detection; these methods include chromatography5, laboratory-based spectrophotometry6, immunochemical7 and enzyme-based electrochemical biosensing8. These conventional methods are accurate and have low detection limits but are time-consuming and usually require complicated sample pretreatment procedures. Therefore, developing simple, easy-to-operate, and cost-effective detection methods are critical to monitor phenolic compounds in actual conditions and evaluate the public risk of exposure to these compounds. Colorimetric biosensing using gold nanoparticles (AuNPs) as indicators have attracted considerable attention as a simple and rapid visual detection assay with a huge potential for field analysis9, 10. Previously reported colorimetric biosensors rely on AuNPs modification with specific ligands to realize the target-mediated cross-linking of AuNPs11. However, using unmodified AuNPs as indicators based on noncross-linking aggregation is convenient and cost effective because the elaborate and expensive synthesis of ligand-modified AuNPs is not required. For example, the randomly coiled single-stranded DNA (ssDNA) is selectively adsorbed onto the surface of AuNPs, leading to mono-dispersed AuNPs in salt solution because of the high density charge in the backbone of adsorbed ssDNA12. One way to induce AuNPs aggregation in the noncross-linking mechanism is the loss of steric stabilization for DNA-stabilized AuNPs by changing the DNA structure (e.g., by forming double-stranded DNA); another way is using enzymatic or oxidative cleavage to damage DNA on the nanoparticle surface as an AuNPs stabilizer13. Enzymatic cleavage, although accurate and highly sensitive, generally requires nucleases or proteases, increasing the experimental complexity and cost14. By contrast, DNA oxidative stress cleavage, which is a characteristic oxidative DNA damage induced by reactive oxygen species, such as hydroxyl radicals(·OH), alkoxyl and peroxyl radicals, and singlet oxygen, is a more convenient and cost-effective alternative strategy compared with enzymatic cleavage15, 16 . The Fenton reaction is a conventional generator of ·OH with important implications in health, disease, and environment, such as in the removal of organic pollutants in soils and waste water17. The ·OH produced by the Fenton reaction has been used for the oxidative damage of DNA and for cleaving DNA into mono- or oligonucleotide fragments18. The quantification relationship between the ·OH production and oxidative damage of ssDNA can be colorimetrically detected using unmodified AuNPs as indicators19. Therefore, designing new biosensing strategies based on variable ·OH production in the Fenton reaction by introducing a scavenger or accelerator is practical. We have previously demonstrated the label-free colorimetric detection of Cu2+ as the ·OH accelerator in the Fenton reaction20. In the current study, we propose the Fenton reaction-triggered colorimetric detection of phenols in water samples by using unmodified AuNPs as indicators for the following reasons. 1) Phenols are organic compounds. Being ·OH scavengers, phenols can react with ·OH to form carbon-centered radicals, and the formed organic intermediates may then further react with ·OH and oxygen, eventually mineralizing into CO2, H2O, and inorganic acids21. The ability of organic compounds to scavenge ·OH provides a quantitative basis for their biosensing. 2) Phenols, such as catechol, chlorogenic acid, malachite green etc., were reported to be degradable by the classical Fenton’s reagents which inspired us to develop a novel
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sensor for phenol detection based on the Fenton reaction22-25. In this study, we chose catechol and hydroquinone as the targets because of their toxicity and degradability in the Fenton reaction. The proposed approach eliminates the tedious modification of AuNPs and facilitates highly sensitive and selective detection through visualization with the naked eye and visible spectroscopy. 2. Experimental 2.1 Instruments and reagents. The absorption spectra were obtained using an ultraviolet–visible light (UV–Vis) spectrophotometer (UV-2700,Shimadzu, Japan) with a 1 cm quartz cell. An F-7000 fluorescence spectrophotometer (Hitachi, Japan) was used to measure plasmon resonance light scattering spectra by simultaneously scanning excitation and emission monochromators without wavelength difference. Transmission electron microscopy (TEM) was performed at 200 kV using an H-7650B microscope (Hitachi, Japan). Nanoparticle size was measured using Zetasizer Delsa Nano (Beckman Coulter, USA). Circular dichroism (CD) was conducted using a PiStar-180 spectropolarimeter (Applied Photophysics Ltd., UK) at from λ = 220 nm to 320 nm. Images of the agarose gel containing labeled DNA bands were obtained using a gel imaging system Doc XR (BioRad, USA). All reagents are of the highest available purity and at least of analytical grade. HAuCl4·4H2O, trisodium citrate, catechol, hydroquinone, o-cresol, o-methoxyphenol, dopamine, pentachlorophenol, butylated hydroxytoluene, p-methylhydroquinone, 3, 5-dinitrosalicylic acid, 4-phenylphenol, and H2O2 were purchased from Sinopharm Chemical Reagent Co., Ltd. (China). The ssDNA used is a commercially available fish sperm DNA from Sigma–Aldrich (St. Louis, MO, USA). Tris-HCl buffer (pH 7.4) was obtained by mixing Tris (0.1 mM) with HCl (0.1 mM). Deionized water of 18 MΩ cm was used in all experiments. 2.2 Preparation of AuNPs and Fenton-induced aggregation. AuNPs were synthesized through the citrate reduction of HAuCl4 as previously described26. In brief, 100 mL of 1.0 mM HAuCl4 solution was brought to a boil while stirring vigorously, Then, 10 mL of trisodium citrate solution (38.8 mM) was rapidly added, and the mixture was kept boiling for 30 min until a wine-red mixture was obtained. The mixture was filtered through a 0.22 µM membrane filter to remove the precipitate, and the filtrate was stored in a refrigerator at 4 °C for further use.
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For phenol detection, a Fenton reaction mixture containing 100 µL of 0.2 g L−1 ssDNA, 50 µL of 6 mM Fe2SO4, and an appropriate amount of phenols was incubated for 10 min at room temperature. Then, 100 µL of 3 mM H2O2 was added, and the mixture was again incubated for 10 min before thoroughly mixing by vortex. Afterward, 400 µL of AuNPs was added, and the mixture was incubated for another 10 min. The visible absorption spectra of the mixture were recorded after adding 100 µL of 40 mM Tris-HCl buffer (pH 7.4) containing 0.5 M NaCl and vortexing thoroughly. 2.3 Environmental samples. River water from Yuanmingyuan Park (Beijing, China), tap water from Qinghe (Beijing, China), and drinking water were collected to demonstrate the developed procedure. The water samples were filtered through a 0.22 µm membrane filter and adjusted to pH 7.4 before analysis. 3
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2.4 Agarose gel electrophoresis (AGE). A Fenton reaction mixture containing 100 µL of 0.2 g L−1 ssDNA, 50 µL of 6 mM Fe2SO4, and 100 µL of 50 µM catechol was incubated for 10 min at room temperature. Then, 100 µL of 3 mM H2O2 was added, and the mixture was again incubated for 10 min before thoroughly mixing by vortex. Then, 100 µL of 40 mM Tris-HCl (pH 7.4) containing 0.5 M NaCl was added, and the mixture was incubated at 37 °C for 30 min. Prior to electrophoresis, 10 µL of the mixture was mixed with a drop of 1×TAE buffer. The 0.8% (w/v) AGE was run at 150 V cm−1 for 30 min. The gel was stained with ethidium bromide nucleic acid (0.5 mg L−1) for 10 min for AGE imaging.
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3. Results and discussion 3.1 Principle of Fenton reaction-triggered colorimetric phenol detection Scheme 1a shows the biosensing scheme of Fenton reaction-triggered colorimetric phenol detection based on ssDNA and unmodified AuNPs. AuNPs can be stabilized in the presence of ssDNA because of electrostatic repulsion, which prevents the salt-induced aggregation of AuNPs in solution. However, the ·OH generated by the Fenton reaction can cleave the ssDNA on the AuNPs surface into mono- or oligonucleotide fragments, disrupting AuNP stability, whereas the phenolic compounds can be degraded or oxidized by ·OH through reduction–oxidation and free-radical reactions. Thus, the presence of phenols can effectively scavenge ·OH to avoid the cleavage of ssDNA, protecting AuNPs from salt-induced aggregation. The ability of phenols to scavenge ·OH provides a quantitative basis for phenol sensing. For example, in accordance with the classic mechanism of Fenton reaction and Hamilton catalytic cycle27, 28, Fe2+ ions can react with H2O2 to be oxidized to Fe3+ and ·OH, respectively. Fe3+ ions can form a 1:1 complex with catechol that decomposes the o-semiquinone radical and Fe2+ ions. The semiquinone radical is unstable and can be oxidized by other Fe3+ ions to generate 1, 2-benzoquinone. Meanwhile, the generated ·OH can attack catechol to degrade the organic compound. This redox cycle is illustrated in Scheme 1b, which shows that catechol can effectively scavenge ·OH to promote the reaction. Fig. 1 shows the typical UV–Vis spectra of AuNPs in the presence of Fenton’s reagent and catechol using unmodified 13 nm AuNPs as indicators. Initially, ssDNA can be adsorbed on the AuNPs surface to enhance the negative charge density, which causes enough electrostatic repulsion between ssDNA and AuNPs to resist aggregation at a fixed salt concentration, accompanied by a characteristic surface plasmon resonance absorption band at approximately 525 nm. After adding Fe2+ and H2O2, the ·OH generated by the Fenton reaction cleaves the ssDNA into different sequence fragments and even single bases29. The damaged ssDNA could not stabilize the AuNPs in salt solutions, leading to a broad UV–Vis absorption band (500–750 nm) along with a red-to-blue color change. When 5 µM catechol was introduced into the Fenton system, the absorption of AuNPs obviously decreased at 700 nm and sharply increased at 525 nm, causing another color change from blue to red. This result concurred with our speculation that catechol can scavenge ·OH. The control experiment shows that independent Fe2+or H2O2 had nearly no effect on AuNPs stability. Thus, the change in the agglomeration state of AuNPs associated with the color change was triggered by the addition of catechol.
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The change in the agglomeration state of AuNPs in the Fenton reaction system was verified by the TEM images. As shown in Figure S1, AuNPs were well-dispersed in the high salt concentration solution because of the protection of the adsorbed ssDNA. Upon the addition of Fenton’s reagent, AuNPs aggregated because of the oxidative cleavage of ssDNA by ·OH. In the presence of 10 µM catechol, the mono-dispersed AuNPs were unchanged because the cleavage of ssDNA was apparently inhibited by the scavenging ability of catechol on ·OH. A study on the aggregation of noble metal nanoparticles has reported enhanced plasmon resonance light scattering (PRLS) signals30. As shown in Fig. 2, the characteristic peak at 289 nm was the PRLS signal of the AuNPs solution. Initially, the PRLS signal was relatively weak because the AuNPs solution was well dispersed (bottom line). After the addition of Fenton’s reagent, the PRLS signals were significantly enhanced, indicating the change in agglomeration state of AuNPs from well dispersed to aggregated. In the presence of catechol, the PRLS signals gradually decreased with increasing catechol concentrations, which corresponded to the enhanced dispersion of AuNPs. The colorimetric probe exhibited distinct linear PRLS quenching at 289 nm toward catechol in the range of 0.5–5 µM with a correlation coefficient of 0.9820 and a linear regression equation of P = −477.2c + 5,844 (where c is the concentration of catechol in µM). Results indicated that the PRLS signals of the AuNPs presented here would also provide an alternative technique to quantify phenols for many applications. Dynamic light scattering was performed to identify the catechol-induced dispersion of AuNPs. As shown in Figure S2, the average particle size of ssDNA-stabilized AuNPs was 13 nm before the introduction of Fenton’s reagent. The Fenton reaction-triggered aggregation of AuNPs increased the particle size to 95 nm. However, the hydrodynamic diameter of AuNPs decreased with increasing catechol concentration, which corresponded to the catechol-dependent change in AuNPs agglomeration state. 3.2 Characterization of ssDNA by CD spectroscopy and AGE In the currently proposed bioassay, information on the base lengths of ssDNA was critical to understand the mechanism by which AuNPs agglomeration state changes. Thus, CD spectra and AGE data were obtained to provide experimental evidence of ssDNA cleavage from the Fenton reaction and introduction of phenols, as discussed below. Fig. 3 shows the CD spectra of the ssDNA helices influenced by ·OH. The CD spectra of ssDNA exhibited a positive absorption band at λ = 278 nm because of base stacking and a negative band at λ = 242 nm because of DNA helicity31. After the addition of Fenton’s reagent, a distinct decrease in the positive peak (i.e., blue shift) was observed, which could be related to the transition of ssDNA helicity and indicate the oxidative damage of Fenton’s reagent to ssDNA. By contrast, the addition of Fe2+ or H2O2 independently did not significantly change the negative and positive peaks of ssDNA, indicating a negligible cleavage effect when Fenton’s reagent was used. Despite the high oxidative capacity of Fenton’s reagent, a distinct increase in the amplitude of the positive absorption band at λ = 278 nm was observed and re-shifted to a longer wavelength when 5 µM catechol was added to the Fenton reaction system. This result indicated that catechol inhibited ssDNA cleavage by consuming the generated ·OH during degradation.
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AGE was also used to investigate the cleavage ability of ssDNA induced by ·OH and the scavenging ability of catechol for ·OH. As shown in Figure S3, Fe2+ was unable to cleave ssDNA (Lane 2), and the ssDNA band almost completely disappeared after the addition of Fenton’s reagent (Lane 3). This result indicated the high oxidative activity to cleave ssDNA and confirmed that ·OH-induced ssDNA cleavage occurred in the presence of both Fe2+ and H2O2. The addition of catechol inhibited ssDNA cleavage (Lane 4), indicating that catechol effectively scavenged ·OH to inhibit ssDNA cleavage. 3.3 Analytical performance of catechol and hydroquinone detection Fig. 4 demonstrate the sensitivity of the proposed colorimetric approach for catechol detection under optimal assay conditions (Figure S4). Calibration curves were obtained for catechol and hydroquinone detection by expressing the signal of each standard point as the ratio of A700/A525 response. The curves were plotted against the logarithm of catechol and hydroquinone concentrations through a four-parameter logistic model as follows:
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where [x] is the analyte concentration; A1 and A2 are the upper and lower asymptotes, respectively, (background signal) to the titration curve; [xo] is the analyte concentration at inflection; and p is the slope at the inflection point. The catechol or hydroquinone detection ranges were described to exhibit 20%–80% inhibition. The linear working ranges were 0.2–7.0 µM for catechol and 2.7–19 µM for hydroquinone (Figure S5). The detection limits calculated from the calibration curves were 0.11 µM for catechol and 1.6 µM for hydroquinone because the analyte concentration provided a 10% decrease in the blank signal. The proposed biosensing approach allowed the highly sensitive detection of phenols in aqueous solutions at the µM concentration level. However, the Fenton reaction has a wide substrate specificity; hence, the established colorimetric approach is not selective if the two phenols coexist in water samples just like the reported Laccase enzyme sensor for phenols4, 8. The proposed method is convenient for rapid and sensitive visual detection onsite without the need for instruments. Thus, a series of catechol solutions at different concentrations was prepared and assayed to demonstrate the usefulness of the colorimetric imaging for the naked-eye visualization of catechol. As shown in Fig. 4c, the minimal sensor response detectable by the naked eye was 5 µM catechol. The value represents the sensor response that provided a color change noticeable by the naked eye compared with that of the blank control. Compared with other visual detection methods, this sensor demonstrated a compared sensitivity for catechol detection1, 32. 3.4 Selectivity of the proposed colorimetric approach Although the mechanisms of organic matter degradation through the Fenton reaction are not yet fully understood, the optimal Fenton reaction conditions vary depending on the type of organic targets21, which would serve as a selective pressure to avoid interference from other organic matters. Under the optimal assay conditions currently established for catechol, we evaluated the
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A700/A525 response of ssDNA-regulated AuNPs to phenolic-related compounds, namely, resorcinol, o-cresol, o-methoxyphenol, dopamine, p-toluhydroquinone (THQ), pentachlorophenol (PCP), butylated hydroxytoluene (BHT), 3,5-dinitrosalicylic acid (DNS), and 4-phenylphenol. Fig. 5a shows the different A700/A525 responses of AuNPs to the selected compounds. Compared with 5 µM catechol and 10 µM hydroquinone, 50 µM of resorcinol, o-cresol, o-methoxyphenol, dopamine, PCP, and 4-phenylphenol demonstrated negligible responses to the ssDNA-regulated AuNPs in the established Fenton reaction system. The different responses of the selected compounds may be attributed to their different properties, such as acidity, reducing capability, and chelating property with metals, and steric effects. In particular, resorcin as an isomer of catechol and hydroquinonedid not significantly contribute to A700/A525even though a 10-fold higher concentration was used compared with the targets. This negligible effect can be attributed to the spatial instability of the semiquinone structure of resorcinol, which could not be oxidized into a quinone21. By contrast, THQ and BHT enhanced the dispersion-inducing effect on AuNPs. This result may partially be due to the attack of ·OH on a substituent rather than on the ring or to the number of sites available for attack21, 33. Moreover, a 10-fold higher concentration of DNS negatively affected the response of catechol and hydroquinone, which may be attributed to its stronger electron-withdrawing ability than catechol and hydroquinone, as well as to the electrophilic nature of ·OH. In summary, the interferences from the phenolic compounds depended on many factors, such as the strength of the C–H bond, the stability of the nascent organoradical, the number of equivalent H atoms or positions of attack, and steric effects21. Another interference study was performed on a series of mixtures of catechol and each of the individual interfering phenolic compounds (Fig. 5b). The response of the ssDNA-regulated AuNPs in the established Fenton reaction system to catechol in the absence and presence of the abovementioned phenolic compounds did not significantly differ. In particular, the same concentrations of BHT, THQ, and DNS with catechol, despite their negative effects on catechol detection, caused no significant effects on the UV–Vis spectra of A700/A525 values. This finding may be due to the partial selectivity of the proposed approach to catechol because the established Fenton reaction conditions were optimized using catechol as the target. In our fixed conditions, catechol had the most efficient scavenging ability for ·OH among the organic compounds studied. The similar conclusion can be found in previous reports28. Therefore, it is expected that the proposed Fenton reaction-triggered colorimetric detection for other phenols can be realized by optimizing the reaction conditions. To demonstrate the potential applications of the proposed colorimetric approach, we conducted selectivity trials and competition experiments to investigate the potential interferences from frequently encountered coexisting species in environmental samples. The absorption ratio of A700/A525 for competing species was recorded in the absence (as a blank control) and presence of catechol. As shown in Fig. 6, in the selectivity trials, 5 µM catechol significantly decreased the A700/A525 ratio, whereas 100 µM of competing species (including Mg2+, Zn2+, Fe3+, Mn2+, Cd2+, Cu2+, Co2+, lysine (Lys), alanine (Ala), valine (Val), glycine (Gly), threonine (Thr), and citric acid caused virtually no change inthe A700/A525 ratio. When 5 µM catechol was mixed with 100 µM of the abovementioned competing species, the A700/A525values remained unchanged compared with
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that obtained using only5 µM catechol. These results suggest that the developed biosensor for phenolic compounds enables the highly favorable and specific recognition of phenolic compounds comparable with synthetic receptors. 3.5 Application in real water sample analysis The colorimetric sensor was used to determine the presence of catechol in environmental water samples, namely, river water, tap water, and drinking water. The colorimetric sensor showed no significant change when the samples were added, indicating that catechol was not present in these samples. To demonstrate the potential application of this sensor on environmental samples, a spike-recovery test was performed after adding different concentrations of catechol. Table 1 shows the responses of the sensor to the spiked water samples. The results of the proposed method, along with the quantitative recoveries (95%–116%), are in good agreement with those of catechol added, indicating the promising functionality of the sensor in real samples. 4. Conclusions We described the use of ssDNA-regulated AuNPs as a colorimetric probe for the rapid and sensitive detection of phenols in water samples. Phenols were introduced as ·OH scavengers in the Fenton reaction, and ·OH production subsequently shared a quantifiable relationship to the oxidative damage of ssDNA on the AuNPs surface, which ultimately influenced the stability of AuNPs. Compared with other phenolic sensors, the proposed colorimetric approach can rapidly detect catechol and hydroquinone at the µM level with naked eye visualization and visible absorption spectroscopy. Moreover, using the unmodified AuNPs as indicators is convenient and cost effective because of the absence of elaborate, tedious, and expensive modification of AuNPs. The proposed detection procedure is practical for real-world applications because it eliminates the tedious sample pretreatment.
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Acknowledgements This research is supported by the Major Scientific Equipment Development Project of China (2012YQ030111) and China Postdoctoral Science Foundation (043208002) References [1] R. S. J. Alkasir, M. Ornatska, S. Andreescu, Colorimetric paper bioassay for the detection of phenolic compounds, Anal. Chem. 84 (22) (2012) 9729-9737. [2] S. Canofeni, S.D. Sario, J. Mela, R. Pilloton, Comparison of immobilization procedures for development of an electrochemical ppo-based biosensor for online monitoring of a depuration process, Anal. Lett. 27 (9) (1994) 1659-1669. [3] Commission of the European Communities, Urban Water Directive 91/271/EEC [4] X. H. Zhou, L. H. Liu, X. Bai, H. C. Shi, A reduced graphene oxide based biosensor for high-sensitive detection of phenols in water samples. Sensors and Actuators B: Chemical (181) (2013) 661-667. [5] K. M. Kalili, Andre de Villiers, Recent developments in the HPLC separation of phenolic compounds, J. Sep. Sci. 8 (34) (2011) 854-876.
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[22] R. Z. Chen, J. J. Pignatecllo, Role of Quinone intermediates as electron shuttles in Fenton and photoassisted Fenton oxidations of aromatic compounds, Environ. Sci. Technol. 8 (31) (1997) 2399-2406. [23] F. Chen, W. H. Ma, J. J. He, J. C. Zhao, Fenton degradation of malachite green catalyzed by aromatic additives, J. Phys. Chem. A 41 (106) (2002) 9485-9490. [24] J. Rodriguez, C. Parra, D. Contreras, J. Freer, J. Baesa, Dihydroxybenzenes: drive Fenton reactions, Water Sci. Technol. 5 (44) (2001) 251-256. [25] M. Strlic, T. Radovic, J. Kolar, B. Pihlar, Anti- and prooxidative properties of gallic acid in Fenton-type systems, J. Agric. Food Chem. 22 (50) (2002) 6313-6317. [26] K. G. Grabar, R. G. Freeman, M. B. Hommer, M. J. Natan, Preparation and characterization of Au colloid monolayers, Anal. Chem. 67 (4) (1995) 735-743. [27] G. A. Hamilton, J. P. Friedman, P. M. Campbell, The hydroxylation of anisole by hydrogen peroxide in the presence of catalytic amounts of ferric ion and catechol, scope, requirements, and kinetics studies, J. Amer. Chem. Soc. 20 (88) (1966) 5266-5268. [28] L. C. Friedrich, M. A. Mendes, V. O. Silva, C. L. P. S. Zanta, A. Machulek Jr. F. H. Quina, Mechanistic implications of zinc (II) ions on the degradation of phenol by the Fenton reaction, J. Braz. Chem. Soc.7 (23) (2012) 1372-1377. [29] M. K. Shigenaga, J. W. Park, K. C. Cundy, C. J. Gimeno, B. N. Ames, In vivo oxidative DNA damage: Measurement of 8-Hydroxy-2’-deoxyguanosine in DNA and urine by high-performance liquid chromatographywith electrochemical detection, Methods Enzymol. (186) (1990) 521-530. [30] J. Ling, Y. F. Li, C. Z. Huang, Visual sandwich immunoassay system on the basis of plasmon resonance scattering signals of silver nanoparticles, Anal. Chem. 4 (81) (2009) 1707-1714. [31] V. I. Ivanov, L. E. Minchenkova, A. K. Schyolkina, A. I. Poletayev, Different conformations of double-stranded nucleic acid in solution as revealed by circular dichroism, Biopolymers 1 (12) (1973) 89-110. [32] A. Hayat, J. Cunningham, G. Bulbul, S. Andreescu, Evaluation of the oxidase like activity of nanoceria and its application in colorimetric assays, Anal. Chim. Acta (885) (2015) 140-147. [33] D. L. Sedlak, A. W. Andren, Aqueous-Phase oxidation of polychlorinated biphenyls by hydroxyl radicals, Environ. Sci. Technol. 8 (25) (1991) 1419-1427.
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Dr. Li-pei Zhang received her Ph.D. degree in the analytical chemistry at Northeastern
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University, China, in October, 2012. After that, she joined Tsinghua University as a postdoc
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researcher. She is interested in the research field of nanomaterial and its application in
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environmental in recent years.
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Dr. Xiao-hong Zhou received her Ph.D. degree in Environmental engineering at Tsinghua
400
University, China, in July, 2007. She has been a full-time lecturer at the School of Environment,
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Tsinghua University since 2007. She focuses on the nanotechnology-based electrochemical
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sensors designed for the environmental monitoring in recent years.
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Prof. Han-Chang Shi received his Ph.D. degree in the environmental engineering in December,
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1990 at Tsinghua University, China. He has been a full-time professor at the School of
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Environment, Tsinghua University since 1998. His research interests include the principle and
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technology of water pollution control, water qualify analysis and biosensor technology.
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Mr. Yun-peng Xing is a master degree candidate at Hebei University of Science and Technology.
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His current research interest field is the chemical sensor design and application in the
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environmental monitoring.
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Ms. Liu Lan-hua She received her M.S. degree in the analytical chemistry at Northeast
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University, China, in July, 2012. Now, she is a doctor at the School of Environment, Tsinghua
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University. She is interested in the research area of electrochemical sensor design and application
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in the environmental monitoring.
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Fig. 1 Absorption spectra of AuNPs in the presence ssDNA before and after treatment with Fenton's reagent and catechol. Colloidal gold: 2.4 nM; ssDNA: 20 mg L-1; Fe2+: 60 µM; H2O2: 0.3 mM; catechol: 5 µM; NaCl: 50 mM; pH 7.4. Fig. 2 PRLS spectra of the ssDNA-AuNPs in Fenton reaction system with addition of various amounts of catechol. Insert: Plot of the PRLS of AuNPs against the concentration of catechol. Colloidal gold: 2.4 nM; Ccatechol: 0, 0.5, 1.0 1.5, 2.0, 3.0, 4.0, 5.0, 10.0 µM; ssDNA: 20 mg L-1; Fe2+: 60 µM; H2O2: 0.3 mM; NaCl: 50 mM; pH 7.4. Fig. 3 CD spectra of ssDNA in the presence of Fenton's reagent and catechol. ssDNA: 20 mg L-1; Fe2+: 60 µM; H2O2: 0.3 mM; catechol: 20 µM; NaCl: 50 mM; pH 7.4. Fig. 4 Visible spectra of ssDNA-AuNPs in Fenton reaction system with addition of various amounts of catechol (a, in the order of curves 1-10: 0, 0.01, 0.05, 0.1,0.5, 1, 5, 10, 50, 100, 500 µM) and the absorption ratio A700/A525 vs. catechol concentration (b). The color display at the top from left to right indicates color changes corresponding to the concentration of catechol: 0, 0.1, 0.5, 1, 5, 10, 50 µM (c). Colloidal gold: 2.4 nM; ssDNA: 20 mg L-1; Fe2+: 60 µM; H2O2: 0.3 mM; NaCl: 50 mM; pH 7.4. Fig. 5 (A) The absorbance spectrum response of ssDNA-AuNPs in Fenton reaction system with addition of catechol (5 µM), hydroquinone (5 µM) and other structurally related compounds: resorcin, o-cresol, o-methoxyphenol, dopamine, PCP, THQ, BHT, DNS, 4-phenylphenol (all 50 µM). (B) in the presence of 5 µM catechol and coexisting species at the same concentrations as above-mentioned.
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Fig. 6 Variations of absorption ratio A700/A525 of ssDNA-AuNPs in Fenton reaction system for sensing 5 µM catechol and (A) in the presence of various species of cations: Mg2+, Zn2+, Fe3+, Mn2+, Cd2+, Cu2+, Ag+, Co2+, Lys, Ala, Val, Gly, Thr and Citric acid (100 µM); (B) in the presence of 5 µM catechol and coexisting species at the same concentrations as above-mentioned. Scheme 1 a) Colorimetric detection of phenols using unmodified gold nanoparticles and Fenton reaction; b) Mechanism of catechol oxidation because of ·OH generated by Fenton reaction.
Found Spiked (mmol Recovered (mmol L-1) -1 -1 (mmol L ) L )
Drink water
＜LOD
445
＜LOD
2.0
2.143±0.126
3.0
2.862±0.054
95.4±1.8
1.0
0.954±0.047
95.4±4.7
3.0
3.178±0.117
105.9±3.9
1.747±0.189
116.4±7.7
2.5
2.381±0.072
95.2±3.8
3.0
3.114±0.151
103.7±3.4
1.5
an
1.122±0.053
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Lake water
＜LOD
1.0
M
Tap water
Recovery (%)
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Table 1 Determination of catechol in three environment water samples (n=3, 95% confidence level).
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436 437 438 439 440 441 442 443 444
112.2±5.3
107.1±6.3
12
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448 449
13
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456 457
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S0925-4005(15)30665-1 http://dx.doi.org/doi:10.1016/j.snb.2015.11.083 SNB 19341
To appear in:
Sensors and Actuators B
Received date: Accepted date:
24-9-2015 18-11-2015
Please cite this article as: L.-p. Zhang, Y.-p. Xing, L.-h. Liu, X.-h. Zhou, H.-c. Shi, Fenton reaction-triggered colorimetric detection of phenols in water samples using unmodified gold nanoparticles, Sensors and Actuators B: Chemical (2015), http://dx.doi.org/10.1016/j.snb.2015.11.083 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.
Fenton reaction-triggered colorimetric detection of phenols in water samples using unmodified gold nanoparticles Li-pei Zhang, Yun-peng Xing, Lan-hua Liu, Xiao-hong Zhou*, Han-chang Shi*
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State Key Joint Laboratory of ESPC, School of Environment, Tsinghua University, Beijing 10084, China
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*Emails: [email protected] [email protected]
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Abstract This work demonstrates a rapid and sensitive colorimetric detection of phenols by using single-stranded DNA (ssDNA)-regulated gold nanoparticles (AuNPs) as indicators. AuNPs can be stabilized in the presence of ssDNA through electrostatic repulsion, which prevents the salt-induced aggregation of AuNPs in solution. However, hydroxyl radicals (·OH) generated by the Fenton reaction can cleave the ssDNA on the nanoparticle surface into mono- or oligonucleotide fragments, disrupting AuNPs stability. Phenolic compounds are known to be capable of being degraded or oxidized by ·OH produced by Fenton reaction. Thus, phenols can effectively scavenge ·OH to avoid ssDNA cleavage, protecting AuNPs from salt-induced aggregation. The ability of phenols to scavenge ·OH provides a quantitative basis for phenol sensing. In this study, catechol and hydroquinone were selected as analytes and detected using the proposed ssDNA–AuNPs colorimetric probe. The detection sensitivities of the colorimetric sensor are 0.2–7.0 µM for catechol and 2.7–19 µM for hydroquinone. The proposed bioassay eliminates tedious sample pretreatment and offers favorable sensitivity and selectivity for targets in the presence of other investigated metal ions and organic molecules. The detection limits are 0.11 µM for catechol and 1.6 µM for hydroquinone, with relative standard deviations of 3.7% for catechol and 4.8% for hydroquinone. The recovery rate of catechol in real water samples ranges from 95% to 116%, confirming the application potential of the method to measure phenols in real samples.
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Keywords Gold nanoparticles, Fenton reaction, Phenol, Colorimetric sensor 1. Introduction Phenolic compounds are common industrial byproducts that are accumulated in the environment and the ecological food chain through consumption or uptake. Their presence in air, water, and food results in acute environmental problems and poses toxic risks to human health; for instance, chlorophenols and nitrophenols possess carcinogenic and immunosuppressive properties1, 2. As a result, both the European Union and the United States Environmental Protection Agency have included phenols in their lists of priority pollutants. The European Community set the maximum amount of phenols in wastewater at lower than 1 ppm3, 4. Various traditional analytical methods 1
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have been developed for phenolic compound detection; these methods include chromatography5, laboratory-based spectrophotometry6, immunochemical7 and enzyme-based electrochemical biosensing8. These conventional methods are accurate and have low detection limits but are time-consuming and usually require complicated sample pretreatment procedures. Therefore, developing simple, easy-to-operate, and cost-effective detection methods are critical to monitor phenolic compounds in actual conditions and evaluate the public risk of exposure to these compounds. Colorimetric biosensing using gold nanoparticles (AuNPs) as indicators have attracted considerable attention as a simple and rapid visual detection assay with a huge potential for field analysis9, 10. Previously reported colorimetric biosensors rely on AuNPs modification with specific ligands to realize the target-mediated cross-linking of AuNPs11. However, using unmodified AuNPs as indicators based on noncross-linking aggregation is convenient and cost effective because the elaborate and expensive synthesis of ligand-modified AuNPs is not required. For example, the randomly coiled single-stranded DNA (ssDNA) is selectively adsorbed onto the surface of AuNPs, leading to mono-dispersed AuNPs in salt solution because of the high density charge in the backbone of adsorbed ssDNA12. One way to induce AuNPs aggregation in the noncross-linking mechanism is the loss of steric stabilization for DNA-stabilized AuNPs by changing the DNA structure (e.g., by forming double-stranded DNA); another way is using enzymatic or oxidative cleavage to damage DNA on the nanoparticle surface as an AuNPs stabilizer13. Enzymatic cleavage, although accurate and highly sensitive, generally requires nucleases or proteases, increasing the experimental complexity and cost14. By contrast, DNA oxidative stress cleavage, which is a characteristic oxidative DNA damage induced by reactive oxygen species, such as hydroxyl radicals(·OH), alkoxyl and peroxyl radicals, and singlet oxygen, is a more convenient and cost-effective alternative strategy compared with enzymatic cleavage15, 16 . The Fenton reaction is a conventional generator of ·OH with important implications in health, disease, and environment, such as in the removal of organic pollutants in soils and waste water17. The ·OH produced by the Fenton reaction has been used for the oxidative damage of DNA and for cleaving DNA into mono- or oligonucleotide fragments18. The quantification relationship between the ·OH production and oxidative damage of ssDNA can be colorimetrically detected using unmodified AuNPs as indicators19. Therefore, designing new biosensing strategies based on variable ·OH production in the Fenton reaction by introducing a scavenger or accelerator is practical. We have previously demonstrated the label-free colorimetric detection of Cu2+ as the ·OH accelerator in the Fenton reaction20. In the current study, we propose the Fenton reaction-triggered colorimetric detection of phenols in water samples by using unmodified AuNPs as indicators for the following reasons. 1) Phenols are organic compounds. Being ·OH scavengers, phenols can react with ·OH to form carbon-centered radicals, and the formed organic intermediates may then further react with ·OH and oxygen, eventually mineralizing into CO2, H2O, and inorganic acids21. The ability of organic compounds to scavenge ·OH provides a quantitative basis for their biosensing. 2) Phenols, such as catechol, chlorogenic acid, malachite green etc., were reported to be degradable by the classical Fenton’s reagents which inspired us to develop a novel
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sensor for phenol detection based on the Fenton reaction22-25. In this study, we chose catechol and hydroquinone as the targets because of their toxicity and degradability in the Fenton reaction. The proposed approach eliminates the tedious modification of AuNPs and facilitates highly sensitive and selective detection through visualization with the naked eye and visible spectroscopy. 2. Experimental 2.1 Instruments and reagents. The absorption spectra were obtained using an ultraviolet–visible light (UV–Vis) spectrophotometer (UV-2700,Shimadzu, Japan) with a 1 cm quartz cell. An F-7000 fluorescence spectrophotometer (Hitachi, Japan) was used to measure plasmon resonance light scattering spectra by simultaneously scanning excitation and emission monochromators without wavelength difference. Transmission electron microscopy (TEM) was performed at 200 kV using an H-7650B microscope (Hitachi, Japan). Nanoparticle size was measured using Zetasizer Delsa Nano (Beckman Coulter, USA). Circular dichroism (CD) was conducted using a PiStar-180 spectropolarimeter (Applied Photophysics Ltd., UK) at from λ = 220 nm to 320 nm. Images of the agarose gel containing labeled DNA bands were obtained using a gel imaging system Doc XR (BioRad, USA). All reagents are of the highest available purity and at least of analytical grade. HAuCl4·4H2O, trisodium citrate, catechol, hydroquinone, o-cresol, o-methoxyphenol, dopamine, pentachlorophenol, butylated hydroxytoluene, p-methylhydroquinone, 3, 5-dinitrosalicylic acid, 4-phenylphenol, and H2O2 were purchased from Sinopharm Chemical Reagent Co., Ltd. (China). The ssDNA used is a commercially available fish sperm DNA from Sigma–Aldrich (St. Louis, MO, USA). Tris-HCl buffer (pH 7.4) was obtained by mixing Tris (0.1 mM) with HCl (0.1 mM). Deionized water of 18 MΩ cm was used in all experiments. 2.2 Preparation of AuNPs and Fenton-induced aggregation. AuNPs were synthesized through the citrate reduction of HAuCl4 as previously described26. In brief, 100 mL of 1.0 mM HAuCl4 solution was brought to a boil while stirring vigorously, Then, 10 mL of trisodium citrate solution (38.8 mM) was rapidly added, and the mixture was kept boiling for 30 min until a wine-red mixture was obtained. The mixture was filtered through a 0.22 µM membrane filter to remove the precipitate, and the filtrate was stored in a refrigerator at 4 °C for further use.
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For phenol detection, a Fenton reaction mixture containing 100 µL of 0.2 g L−1 ssDNA, 50 µL of 6 mM Fe2SO4, and an appropriate amount of phenols was incubated for 10 min at room temperature. Then, 100 µL of 3 mM H2O2 was added, and the mixture was again incubated for 10 min before thoroughly mixing by vortex. Afterward, 400 µL of AuNPs was added, and the mixture was incubated for another 10 min. The visible absorption spectra of the mixture were recorded after adding 100 µL of 40 mM Tris-HCl buffer (pH 7.4) containing 0.5 M NaCl and vortexing thoroughly. 2.3 Environmental samples. River water from Yuanmingyuan Park (Beijing, China), tap water from Qinghe (Beijing, China), and drinking water were collected to demonstrate the developed procedure. The water samples were filtered through a 0.22 µm membrane filter and adjusted to pH 7.4 before analysis. 3
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2.4 Agarose gel electrophoresis (AGE). A Fenton reaction mixture containing 100 µL of 0.2 g L−1 ssDNA, 50 µL of 6 mM Fe2SO4, and 100 µL of 50 µM catechol was incubated for 10 min at room temperature. Then, 100 µL of 3 mM H2O2 was added, and the mixture was again incubated for 10 min before thoroughly mixing by vortex. Then, 100 µL of 40 mM Tris-HCl (pH 7.4) containing 0.5 M NaCl was added, and the mixture was incubated at 37 °C for 30 min. Prior to electrophoresis, 10 µL of the mixture was mixed with a drop of 1×TAE buffer. The 0.8% (w/v) AGE was run at 150 V cm−1 for 30 min. The gel was stained with ethidium bromide nucleic acid (0.5 mg L−1) for 10 min for AGE imaging.
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3. Results and discussion 3.1 Principle of Fenton reaction-triggered colorimetric phenol detection Scheme 1a shows the biosensing scheme of Fenton reaction-triggered colorimetric phenol detection based on ssDNA and unmodified AuNPs. AuNPs can be stabilized in the presence of ssDNA because of electrostatic repulsion, which prevents the salt-induced aggregation of AuNPs in solution. However, the ·OH generated by the Fenton reaction can cleave the ssDNA on the AuNPs surface into mono- or oligonucleotide fragments, disrupting AuNP stability, whereas the phenolic compounds can be degraded or oxidized by ·OH through reduction–oxidation and free-radical reactions. Thus, the presence of phenols can effectively scavenge ·OH to avoid the cleavage of ssDNA, protecting AuNPs from salt-induced aggregation. The ability of phenols to scavenge ·OH provides a quantitative basis for phenol sensing. For example, in accordance with the classic mechanism of Fenton reaction and Hamilton catalytic cycle27, 28, Fe2+ ions can react with H2O2 to be oxidized to Fe3+ and ·OH, respectively. Fe3+ ions can form a 1:1 complex with catechol that decomposes the o-semiquinone radical and Fe2+ ions. The semiquinone radical is unstable and can be oxidized by other Fe3+ ions to generate 1, 2-benzoquinone. Meanwhile, the generated ·OH can attack catechol to degrade the organic compound. This redox cycle is illustrated in Scheme 1b, which shows that catechol can effectively scavenge ·OH to promote the reaction. Fig. 1 shows the typical UV–Vis spectra of AuNPs in the presence of Fenton’s reagent and catechol using unmodified 13 nm AuNPs as indicators. Initially, ssDNA can be adsorbed on the AuNPs surface to enhance the negative charge density, which causes enough electrostatic repulsion between ssDNA and AuNPs to resist aggregation at a fixed salt concentration, accompanied by a characteristic surface plasmon resonance absorption band at approximately 525 nm. After adding Fe2+ and H2O2, the ·OH generated by the Fenton reaction cleaves the ssDNA into different sequence fragments and even single bases29. The damaged ssDNA could not stabilize the AuNPs in salt solutions, leading to a broad UV–Vis absorption band (500–750 nm) along with a red-to-blue color change. When 5 µM catechol was introduced into the Fenton system, the absorption of AuNPs obviously decreased at 700 nm and sharply increased at 525 nm, causing another color change from blue to red. This result concurred with our speculation that catechol can scavenge ·OH. The control experiment shows that independent Fe2+or H2O2 had nearly no effect on AuNPs stability. Thus, the change in the agglomeration state of AuNPs associated with the color change was triggered by the addition of catechol.
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The change in the agglomeration state of AuNPs in the Fenton reaction system was verified by the TEM images. As shown in Figure S1, AuNPs were well-dispersed in the high salt concentration solution because of the protection of the adsorbed ssDNA. Upon the addition of Fenton’s reagent, AuNPs aggregated because of the oxidative cleavage of ssDNA by ·OH. In the presence of 10 µM catechol, the mono-dispersed AuNPs were unchanged because the cleavage of ssDNA was apparently inhibited by the scavenging ability of catechol on ·OH. A study on the aggregation of noble metal nanoparticles has reported enhanced plasmon resonance light scattering (PRLS) signals30. As shown in Fig. 2, the characteristic peak at 289 nm was the PRLS signal of the AuNPs solution. Initially, the PRLS signal was relatively weak because the AuNPs solution was well dispersed (bottom line). After the addition of Fenton’s reagent, the PRLS signals were significantly enhanced, indicating the change in agglomeration state of AuNPs from well dispersed to aggregated. In the presence of catechol, the PRLS signals gradually decreased with increasing catechol concentrations, which corresponded to the enhanced dispersion of AuNPs. The colorimetric probe exhibited distinct linear PRLS quenching at 289 nm toward catechol in the range of 0.5–5 µM with a correlation coefficient of 0.9820 and a linear regression equation of P = −477.2c + 5,844 (where c is the concentration of catechol in µM). Results indicated that the PRLS signals of the AuNPs presented here would also provide an alternative technique to quantify phenols for many applications. Dynamic light scattering was performed to identify the catechol-induced dispersion of AuNPs. As shown in Figure S2, the average particle size of ssDNA-stabilized AuNPs was 13 nm before the introduction of Fenton’s reagent. The Fenton reaction-triggered aggregation of AuNPs increased the particle size to 95 nm. However, the hydrodynamic diameter of AuNPs decreased with increasing catechol concentration, which corresponded to the catechol-dependent change in AuNPs agglomeration state. 3.2 Characterization of ssDNA by CD spectroscopy and AGE In the currently proposed bioassay, information on the base lengths of ssDNA was critical to understand the mechanism by which AuNPs agglomeration state changes. Thus, CD spectra and AGE data were obtained to provide experimental evidence of ssDNA cleavage from the Fenton reaction and introduction of phenols, as discussed below. Fig. 3 shows the CD spectra of the ssDNA helices influenced by ·OH. The CD spectra of ssDNA exhibited a positive absorption band at λ = 278 nm because of base stacking and a negative band at λ = 242 nm because of DNA helicity31. After the addition of Fenton’s reagent, a distinct decrease in the positive peak (i.e., blue shift) was observed, which could be related to the transition of ssDNA helicity and indicate the oxidative damage of Fenton’s reagent to ssDNA. By contrast, the addition of Fe2+ or H2O2 independently did not significantly change the negative and positive peaks of ssDNA, indicating a negligible cleavage effect when Fenton’s reagent was used. Despite the high oxidative capacity of Fenton’s reagent, a distinct increase in the amplitude of the positive absorption band at λ = 278 nm was observed and re-shifted to a longer wavelength when 5 µM catechol was added to the Fenton reaction system. This result indicated that catechol inhibited ssDNA cleavage by consuming the generated ·OH during degradation.
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AGE was also used to investigate the cleavage ability of ssDNA induced by ·OH and the scavenging ability of catechol for ·OH. As shown in Figure S3, Fe2+ was unable to cleave ssDNA (Lane 2), and the ssDNA band almost completely disappeared after the addition of Fenton’s reagent (Lane 3). This result indicated the high oxidative activity to cleave ssDNA and confirmed that ·OH-induced ssDNA cleavage occurred in the presence of both Fe2+ and H2O2. The addition of catechol inhibited ssDNA cleavage (Lane 4), indicating that catechol effectively scavenged ·OH to inhibit ssDNA cleavage. 3.3 Analytical performance of catechol and hydroquinone detection Fig. 4 demonstrate the sensitivity of the proposed colorimetric approach for catechol detection under optimal assay conditions (Figure S4). Calibration curves were obtained for catechol and hydroquinone detection by expressing the signal of each standard point as the ratio of A700/A525 response. The curves were plotted against the logarithm of catechol and hydroquinone concentrations through a four-parameter logistic model as follows:
219
y=
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where [x] is the analyte concentration; A1 and A2 are the upper and lower asymptotes, respectively, (background signal) to the titration curve; [xo] is the analyte concentration at inflection; and p is the slope at the inflection point. The catechol or hydroquinone detection ranges were described to exhibit 20%–80% inhibition. The linear working ranges were 0.2–7.0 µM for catechol and 2.7–19 µM for hydroquinone (Figure S5). The detection limits calculated from the calibration curves were 0.11 µM for catechol and 1.6 µM for hydroquinone because the analyte concentration provided a 10% decrease in the blank signal. The proposed biosensing approach allowed the highly sensitive detection of phenols in aqueous solutions at the µM concentration level. However, the Fenton reaction has a wide substrate specificity; hence, the established colorimetric approach is not selective if the two phenols coexist in water samples just like the reported Laccase enzyme sensor for phenols4, 8. The proposed method is convenient for rapid and sensitive visual detection onsite without the need for instruments. Thus, a series of catechol solutions at different concentrations was prepared and assayed to demonstrate the usefulness of the colorimetric imaging for the naked-eye visualization of catechol. As shown in Fig. 4c, the minimal sensor response detectable by the naked eye was 5 µM catechol. The value represents the sensor response that provided a color change noticeable by the naked eye compared with that of the blank control. Compared with other visual detection methods, this sensor demonstrated a compared sensitivity for catechol detection1, 32. 3.4 Selectivity of the proposed colorimetric approach Although the mechanisms of organic matter degradation through the Fenton reaction are not yet fully understood, the optimal Fenton reaction conditions vary depending on the type of organic targets21, which would serve as a selective pressure to avoid interference from other organic matters. Under the optimal assay conditions currently established for catechol, we evaluated the
p
+ A2 ,
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A700/A525 response of ssDNA-regulated AuNPs to phenolic-related compounds, namely, resorcinol, o-cresol, o-methoxyphenol, dopamine, p-toluhydroquinone (THQ), pentachlorophenol (PCP), butylated hydroxytoluene (BHT), 3,5-dinitrosalicylic acid (DNS), and 4-phenylphenol. Fig. 5a shows the different A700/A525 responses of AuNPs to the selected compounds. Compared with 5 µM catechol and 10 µM hydroquinone, 50 µM of resorcinol, o-cresol, o-methoxyphenol, dopamine, PCP, and 4-phenylphenol demonstrated negligible responses to the ssDNA-regulated AuNPs in the established Fenton reaction system. The different responses of the selected compounds may be attributed to their different properties, such as acidity, reducing capability, and chelating property with metals, and steric effects. In particular, resorcin as an isomer of catechol and hydroquinonedid not significantly contribute to A700/A525even though a 10-fold higher concentration was used compared with the targets. This negligible effect can be attributed to the spatial instability of the semiquinone structure of resorcinol, which could not be oxidized into a quinone21. By contrast, THQ and BHT enhanced the dispersion-inducing effect on AuNPs. This result may partially be due to the attack of ·OH on a substituent rather than on the ring or to the number of sites available for attack21, 33. Moreover, a 10-fold higher concentration of DNS negatively affected the response of catechol and hydroquinone, which may be attributed to its stronger electron-withdrawing ability than catechol and hydroquinone, as well as to the electrophilic nature of ·OH. In summary, the interferences from the phenolic compounds depended on many factors, such as the strength of the C–H bond, the stability of the nascent organoradical, the number of equivalent H atoms or positions of attack, and steric effects21. Another interference study was performed on a series of mixtures of catechol and each of the individual interfering phenolic compounds (Fig. 5b). The response of the ssDNA-regulated AuNPs in the established Fenton reaction system to catechol in the absence and presence of the abovementioned phenolic compounds did not significantly differ. In particular, the same concentrations of BHT, THQ, and DNS with catechol, despite their negative effects on catechol detection, caused no significant effects on the UV–Vis spectra of A700/A525 values. This finding may be due to the partial selectivity of the proposed approach to catechol because the established Fenton reaction conditions were optimized using catechol as the target. In our fixed conditions, catechol had the most efficient scavenging ability for ·OH among the organic compounds studied. The similar conclusion can be found in previous reports28. Therefore, it is expected that the proposed Fenton reaction-triggered colorimetric detection for other phenols can be realized by optimizing the reaction conditions. To demonstrate the potential applications of the proposed colorimetric approach, we conducted selectivity trials and competition experiments to investigate the potential interferences from frequently encountered coexisting species in environmental samples. The absorption ratio of A700/A525 for competing species was recorded in the absence (as a blank control) and presence of catechol. As shown in Fig. 6, in the selectivity trials, 5 µM catechol significantly decreased the A700/A525 ratio, whereas 100 µM of competing species (including Mg2+, Zn2+, Fe3+, Mn2+, Cd2+, Cu2+, Co2+, lysine (Lys), alanine (Ala), valine (Val), glycine (Gly), threonine (Thr), and citric acid caused virtually no change inthe A700/A525 ratio. When 5 µM catechol was mixed with 100 µM of the abovementioned competing species, the A700/A525values remained unchanged compared with
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that obtained using only5 µM catechol. These results suggest that the developed biosensor for phenolic compounds enables the highly favorable and specific recognition of phenolic compounds comparable with synthetic receptors. 3.5 Application in real water sample analysis The colorimetric sensor was used to determine the presence of catechol in environmental water samples, namely, river water, tap water, and drinking water. The colorimetric sensor showed no significant change when the samples were added, indicating that catechol was not present in these samples. To demonstrate the potential application of this sensor on environmental samples, a spike-recovery test was performed after adding different concentrations of catechol. Table 1 shows the responses of the sensor to the spiked water samples. The results of the proposed method, along with the quantitative recoveries (95%–116%), are in good agreement with those of catechol added, indicating the promising functionality of the sensor in real samples. 4. Conclusions We described the use of ssDNA-regulated AuNPs as a colorimetric probe for the rapid and sensitive detection of phenols in water samples. Phenols were introduced as ·OH scavengers in the Fenton reaction, and ·OH production subsequently shared a quantifiable relationship to the oxidative damage of ssDNA on the AuNPs surface, which ultimately influenced the stability of AuNPs. Compared with other phenolic sensors, the proposed colorimetric approach can rapidly detect catechol and hydroquinone at the µM level with naked eye visualization and visible absorption spectroscopy. Moreover, using the unmodified AuNPs as indicators is convenient and cost effective because of the absence of elaborate, tedious, and expensive modification of AuNPs. The proposed detection procedure is practical for real-world applications because it eliminates the tedious sample pretreatment.
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Acknowledgements This research is supported by the Major Scientific Equipment Development Project of China (2012YQ030111) and China Postdoctoral Science Foundation (043208002) References [1] R. S. J. Alkasir, M. Ornatska, S. Andreescu, Colorimetric paper bioassay for the detection of phenolic compounds, Anal. Chem. 84 (22) (2012) 9729-9737. [2] S. Canofeni, S.D. Sario, J. Mela, R. Pilloton, Comparison of immobilization procedures for development of an electrochemical ppo-based biosensor for online monitoring of a depuration process, Anal. Lett. 27 (9) (1994) 1659-1669. [3] Commission of the European Communities, Urban Water Directive 91/271/EEC [4] X. H. Zhou, L. H. Liu, X. Bai, H. C. Shi, A reduced graphene oxide based biosensor for high-sensitive detection of phenols in water samples. Sensors and Actuators B: Chemical (181) (2013) 661-667. [5] K. M. Kalili, Andre de Villiers, Recent developments in the HPLC separation of phenolic compounds, J. Sep. Sci. 8 (34) (2011) 854-876.
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[6] D.Puig, D. Barcelό, Determination of phenolic compounds in water and waste water, Trends Anal. Chem. 8 (15) (1996) 362-375. [7] E. P. Meulenberg, Phenolics: occurrence and immunochemical detection in environment and food, Molecules 14 (1) (2009), 439-473. [8] X. H. Zhou, X. R. Huang, L. H. Liu, X. Bai, H. C. Shi, Direct electron transfer reaction of laccase on a glassy carbon electrode modified with 1-aminopyrene functionalized reduced graphene oxide, RSC Advances, (3) (2013), 18036-18043. [9] W. A. Zhao, M. A. Brook, Y. F. Li, Design of gold nanoparticle-based colorimetric biosensing assays, ChemBioChem (9) (2008) 2363-2371. [10] Y. S. Kim, J. H. Kim, I. A. Kim, S. J. Lee, J. Jurny, M. B. Gu, A novel colorimetric aptasensor using gold nanoparticle for a highly sensitive and specific detection of oxytetracycline, Biosens. Bioelectron. 4 (26) (2010) 1644-1649. [11] M. E. Stewart, C. R. Anderton, L. B. Thompson, J. Maria, S. K. Gray, J. A. Rogers, R. G. Nuzzo, Nanostructured plasmonic sensors, Chem. Rev. 2 (108) (2008) 494-521. [12] Y. Jiang, H. Zhao, Y. Q. Lin, N. N. Zhu, Y. R. Ma, L. Q. Mao, Colorimetric detection of glucose in rat brain using gold nanoparticles, Angew. Chem. Int. Ed. 49 (28) (2010) 4800-4804. [13] N. L. Rosi, C. A. Mirkin, Nanostructures in biodiagnostics, Chem. Rev. 4 (105) (2005) 1547-1562. [14] J. H. Chen, X. M Zhou, L. W. Zeng, Enzyme-free strip biosensor for amplified detection of Pb2+ based on a catalytic DNA circuit, Chem. Comm. 49 (2013) 984-986. [15] R. Olinske, D. Gackowski, M. Foksinske, R. Rozalski, K. Roszkowski, P. Jaruga, Oxidative DNA damage: assessment of the role in carcinogenesis, atherosclerosis, and acquired immunodeficiency syndrome, Free Radical Biol. Med. 2 (33) (2002) 192-200. [16] A. M. Nowicka, A. Kowalczyk, F. Z Scholz, Z. Stojek, Changes in performance of DNA biosensor caused by hydroxyl radicals, Electroanalysis, 1 (23) (2001) 55-62. [17] R. F. F. Pontes, J. E.F. Moraes, Jr. A. Machulek, J. M. Pinto, A mechanistic kinetic model of phenol degradation by the Fenton process, Journal of Hazardous Materials, 1-3 (176) (2010) 402-413. [18] J. Borrás, G. Alzuet, M. González-Alvarea, J. L. Garcá-Giménez, B. Macías, M. Liu-González, Efficient DNA cleavage induced by copper (II) complexes of hydrolysis derivatives of 2,4,6-Triazine in the presence of reducing agents, Eur. J. Inorg. Chem. (6) (2007) 822-834. [19] Q. P. Shen, Z. Nie, M. L. Guo, C. J. Zhong, B. Lin, W. Li, S. Z. Yao, Simple and rapid colorimetric sensing of enzymatic cleavageand oxidative damage of single-stranded DNA with unmodifiedgold nanoparticles as indicator, Chem. Commun. (2009) 929–931. [20] L. P. Zhang, Y. P. Xing, C. Liu, X. H. Zhou, H. C. Shi, Label-free colorimetric detection of Cu2+ on the basis of Fenton reaction-assisted signal amplification with unmodified gold nanoparticles as indicator, Sensors and Actuators B: Chemical (215) (2015) 561-567. [21] J. J. Pignatello, E. Oliveros, A. Mackay, Advanced oxidation processes for organic contaminant destruction based on the Fenton reaction and related chemistry, Crit. Rev. Env. Sci. Techno. 36 (1) (2006) 1-84.
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[22] R. Z. Chen, J. J. Pignatecllo, Role of Quinone intermediates as electron shuttles in Fenton and photoassisted Fenton oxidations of aromatic compounds, Environ. Sci. Technol. 8 (31) (1997) 2399-2406. [23] F. Chen, W. H. Ma, J. J. He, J. C. Zhao, Fenton degradation of malachite green catalyzed by aromatic additives, J. Phys. Chem. A 41 (106) (2002) 9485-9490. [24] J. Rodriguez, C. Parra, D. Contreras, J. Freer, J. Baesa, Dihydroxybenzenes: drive Fenton reactions, Water Sci. Technol. 5 (44) (2001) 251-256. [25] M. Strlic, T. Radovic, J. Kolar, B. Pihlar, Anti- and prooxidative properties of gallic acid in Fenton-type systems, J. Agric. Food Chem. 22 (50) (2002) 6313-6317. [26] K. G. Grabar, R. G. Freeman, M. B. Hommer, M. J. Natan, Preparation and characterization of Au colloid monolayers, Anal. Chem. 67 (4) (1995) 735-743. [27] G. A. Hamilton, J. P. Friedman, P. M. Campbell, The hydroxylation of anisole by hydrogen peroxide in the presence of catalytic amounts of ferric ion and catechol, scope, requirements, and kinetics studies, J. Amer. Chem. Soc. 20 (88) (1966) 5266-5268. [28] L. C. Friedrich, M. A. Mendes, V. O. Silva, C. L. P. S. Zanta, A. Machulek Jr. F. H. Quina, Mechanistic implications of zinc (II) ions on the degradation of phenol by the Fenton reaction, J. Braz. Chem. Soc.7 (23) (2012) 1372-1377. [29] M. K. Shigenaga, J. W. Park, K. C. Cundy, C. J. Gimeno, B. N. Ames, In vivo oxidative DNA damage: Measurement of 8-Hydroxy-2’-deoxyguanosine in DNA and urine by high-performance liquid chromatographywith electrochemical detection, Methods Enzymol. (186) (1990) 521-530. [30] J. Ling, Y. F. Li, C. Z. Huang, Visual sandwich immunoassay system on the basis of plasmon resonance scattering signals of silver nanoparticles, Anal. Chem. 4 (81) (2009) 1707-1714. [31] V. I. Ivanov, L. E. Minchenkova, A. K. Schyolkina, A. I. Poletayev, Different conformations of double-stranded nucleic acid in solution as revealed by circular dichroism, Biopolymers 1 (12) (1973) 89-110. [32] A. Hayat, J. Cunningham, G. Bulbul, S. Andreescu, Evaluation of the oxidase like activity of nanoceria and its application in colorimetric assays, Anal. Chim. Acta (885) (2015) 140-147. [33] D. L. Sedlak, A. W. Andren, Aqueous-Phase oxidation of polychlorinated biphenyls by hydroxyl radicals, Environ. Sci. Technol. 8 (25) (1991) 1419-1427.
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Dr. Li-pei Zhang received her Ph.D. degree in the analytical chemistry at Northeastern
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University, China, in October, 2012. After that, she joined Tsinghua University as a postdoc
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researcher. She is interested in the research field of nanomaterial and its application in
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environmental in recent years.
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Dr. Xiao-hong Zhou received her Ph.D. degree in Environmental engineering at Tsinghua
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University, China, in July, 2007. She has been a full-time lecturer at the School of Environment,
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Tsinghua University since 2007. She focuses on the nanotechnology-based electrochemical
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sensors designed for the environmental monitoring in recent years.
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Prof. Han-Chang Shi received his Ph.D. degree in the environmental engineering in December,
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1990 at Tsinghua University, China. He has been a full-time professor at the School of
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Environment, Tsinghua University since 1998. His research interests include the principle and
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technology of water pollution control, water qualify analysis and biosensor technology.
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Mr. Yun-peng Xing is a master degree candidate at Hebei University of Science and Technology.
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His current research interest field is the chemical sensor design and application in the
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environmental monitoring.
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Ms. Liu Lan-hua She received her M.S. degree in the analytical chemistry at Northeast
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University, China, in July, 2012. Now, she is a doctor at the School of Environment, Tsinghua
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University. She is interested in the research area of electrochemical sensor design and application
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in the environmental monitoring.
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Fig. 1 Absorption spectra of AuNPs in the presence ssDNA before and after treatment with Fenton's reagent and catechol. Colloidal gold: 2.4 nM; ssDNA: 20 mg L-1; Fe2+: 60 µM; H2O2: 0.3 mM; catechol: 5 µM; NaCl: 50 mM; pH 7.4. Fig. 2 PRLS spectra of the ssDNA-AuNPs in Fenton reaction system with addition of various amounts of catechol. Insert: Plot of the PRLS of AuNPs against the concentration of catechol. Colloidal gold: 2.4 nM; Ccatechol: 0, 0.5, 1.0 1.5, 2.0, 3.0, 4.0, 5.0, 10.0 µM; ssDNA: 20 mg L-1; Fe2+: 60 µM; H2O2: 0.3 mM; NaCl: 50 mM; pH 7.4. Fig. 3 CD spectra of ssDNA in the presence of Fenton's reagent and catechol. ssDNA: 20 mg L-1; Fe2+: 60 µM; H2O2: 0.3 mM; catechol: 20 µM; NaCl: 50 mM; pH 7.4. Fig. 4 Visible spectra of ssDNA-AuNPs in Fenton reaction system with addition of various amounts of catechol (a, in the order of curves 1-10: 0, 0.01, 0.05, 0.1,0.5, 1, 5, 10, 50, 100, 500 µM) and the absorption ratio A700/A525 vs. catechol concentration (b). The color display at the top from left to right indicates color changes corresponding to the concentration of catechol: 0, 0.1, 0.5, 1, 5, 10, 50 µM (c). Colloidal gold: 2.4 nM; ssDNA: 20 mg L-1; Fe2+: 60 µM; H2O2: 0.3 mM; NaCl: 50 mM; pH 7.4. Fig. 5 (A) The absorbance spectrum response of ssDNA-AuNPs in Fenton reaction system with addition of catechol (5 µM), hydroquinone (5 µM) and other structurally related compounds: resorcin, o-cresol, o-methoxyphenol, dopamine, PCP, THQ, BHT, DNS, 4-phenylphenol (all 50 µM). (B) in the presence of 5 µM catechol and coexisting species at the same concentrations as above-mentioned.
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Fig. 6 Variations of absorption ratio A700/A525 of ssDNA-AuNPs in Fenton reaction system for sensing 5 µM catechol and (A) in the presence of various species of cations: Mg2+, Zn2+, Fe3+, Mn2+, Cd2+, Cu2+, Ag+, Co2+, Lys, Ala, Val, Gly, Thr and Citric acid (100 µM); (B) in the presence of 5 µM catechol and coexisting species at the same concentrations as above-mentioned. Scheme 1 a) Colorimetric detection of phenols using unmodified gold nanoparticles and Fenton reaction; b) Mechanism of catechol oxidation because of ·OH generated by Fenton reaction.
Found Spiked (mmol Recovered (mmol L-1) -1 -1 (mmol L ) L )
Drink water
＜LOD
445
＜LOD
2.0
2.143±0.126
3.0
2.862±0.054
95.4±1.8
1.0
0.954±0.047
95.4±4.7
3.0
3.178±0.117
105.9±3.9
1.747±0.189
116.4±7.7
2.5
2.381±0.072
95.2±3.8
3.0
3.114±0.151
103.7±3.4
1.5
an
1.122±0.053
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Lake water
＜LOD
1.0
M
Tap water
Recovery (%)
us
Sample
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Table 1 Determination of catechol in three environment water samples (n=3, 95% confidence level).
d
436 437 438 439 440 441 442 443 444
112.2±5.3
107.1±6.3
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448 449
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450 451
452 453
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454 455
456 457
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458
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459 460
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