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Cite this: Chem. Commun., 2013, 49, 11200 Received 8th September 2013, Accepted 8th October 2013 DOI: 10.1039/c3cc46869k
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Target-stimulated metallic HgS nanostructures on a DNA-based polyion complex membrane for highly efficient impedimetric detection of dissolved hydrogen sulfide† Junyang Zhuang, Libing Fu, Wenqiang Lai, Dianping Tang* and Guonan Chen
www.rsc.org/chemcomm
Target-stimulated metallic HgS nanostructures formed on the DNAbased polyion complex (PIC) membrane were for the first time utilized as an efficient scheme for impedimetric detection of hydrogen sulfide (H2S) by coupling insoluble precipitation with sensitivity enhancement.
Hydrogen sulfide (H2S) is an extremely hazardous toxic compound.1 Recent investigations have demonstrated that H2S is the third endogenous gaseous signaling compound with cytoprotection properties,2 and its levels are altered in diseases ranging from Alzheimer’s disease and Down’s syndrome to diabetes and liver cirrhosis.3 Therefore, recent years have seen a steady increase in interest in understanding the physiological and pathological functions of H2S. Various methods and strategies have been developed for monitoring H2S, such as colorimetric methods, HPLC, GC, electrochemistry, and fluorescence.4 Among them, the electrochemical detection method is suitable for sensor miniaturization and automated detection because of simple instrumentation and easy signal quantification.5 Despite many advances in this field, there is still the quest for new schemes and strategies for improving the sensitivity of the assays. Electrochemical impedance spectroscopy (EIS) is an effective strategy to probe the interfacial properties of the modified electrode.6 The adsorption or desorption of insulating materials on conductive supports is anticipated to alter the interfacial electron-transfer features (capacitance and resistance) at the electrode surface. However, one of great challenges for the successful development of EIS-based sensors is to amplify the signal output. Significantly, many metal ions, e.g. PbII, CuII, HgII and AgI, can react with H2S to give the corresponding metal sulfides, and precipitate from solution upon exposure to H2S. The components of the resulting precipitate redissolve with selectivity. Hence, our aim in this work is to design a simple and feasible impedimetric detection method for toxic gases by virtue of the precipitation technique. Key Laboratory of Analysis and Detection of Food Safety (Ministry of Education & Fujian Province), Department of Chemistry, Fuzhou University, Fuzhou 350108, P.R. China. E-mail: [email protected]; Fax: +86 591 22866135; Tel: +86 591 22866125 † Electronic supplementary information (ESI) available: Experimental procedures and optimization of conditions. See DOI: 10.1039/c3cc46869k
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Chem. Commun., 2013, 49, 11200--11202
Scheme 1 Fabrication process of the impedimetric detection method for dissolved H2S based on precipitation of target-simulated metallic HgS nanostructures on a DNA-based polyion complex membrane.
Herein, we report the proof-of-concept of a novel and powerful impedimetric detection method for dissolved H2S by coupling signal amplification with the precipitation of target-stimulated metallic HgS nanostructures on the DNA-based polyion complex (PIC) membrane (Scheme 1, see ESI† for experimental details). Unlike the common polymer film, DNA self-assembled film on the electrode allows electron-transfer between solution and the electrode because of its relatively low density and ordered structure. Initially, the thiolated poly-T(25) oligonucleotide (5 0 -SH-TTTTT TTTTT TTTTT TTTTT TTTTT-3 0 ) is covalently conjugated onto the surface of the gold electrode by the classical Au–S bond, and then the resulting electrode is immersed in pH 7.4 tris-buffer (10 mM) solution containing 10 mM Hg2+. During this process, Hg2+ ions are intercalated into the polyion complex membrane based on the T–Hg2+–T coordination chemistry.7 Utilizing the high volatility of H2S in solution, the impedimetric assay is implemented using a headspace method that the H2S volatilizing from the test sample directly interacts with the as-prepared membrane on the top of the sample (Na2S is used as a H2S donor, see ESI† for experimental details). Upon introduction of target H2S, the reaction is carried out between the S2 ion and Hg2+ (Hg2+ + H2S - 2H+ + HgSk) to produce an insoluble HgS precipitate. The precipitate can coat on the electrode surface, and hinder the electron transfer of redox probes, e.g. Fe(CN)63/ Fe(CN)64, thereby resulting in the increase of the Faradaic impedance of the modified electrode. By monitoring the change in the resistance, we might quantitatively determine the concentration of target H2S in the sample. This journal is
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Fig. 1 SEM images of (a) the DNA-modified gold substrate, (b) probe ‘a’ after direct treatment with H2S, and (c) probe ‘a’ after incubation with Hg2+ and subsequent treatment with H2S.
To realize our design, one precondition for the successful development of the impedimetric assay was whether HgS nanostructures could be formed in the presence of target H2S. To demonstrate this issue, we used a scanning electron microscope (SEM) to investigate the PIC membrane-modified gold substrate before and after incubation with Hg2+ ions or H2S, respectively. Fig. 1a shows the SEM image of the PIC membrane-modified gold substrate. When the modified gold substrate was directly reacted with H2S in the absence of Hg2+ ions, no precipitation was observed on the surface (Fig. 1b). More inspiringly, upon addition of target H2S onto the Hg2+-treated DNA PIC membrane, an obvious particle-like topology could be achieved (Fig. 1c). The results revealed that the precipitation reaction could be implemented between H2S and the Hg2+. Another concern arises as to whether the formed HgS nanostructures could cause the change in the resistance of the modified electrode. To clarify this point, the DNA-modified electrode was used for detection of 200 nM H2S (as an example) by using the developed assay protocol. Fig. 2A shows the EIS of a variously modified electrode after each step. These EIS data were fitted to a Randles equivalent circuit (inset of Fig. 2A), which contains electrolyte resistance (Rs), the lipid bilayer capacitance (Cdl), charge transfer resistance (Ret) and Warburg element (Zw). The complex impedance can be presented as the sum of the real, Zre and imaginary, Zim, components that originate mainly from the resistance and capacitance of the cell. The two components of the scheme, Rs and Zw, represent bulk properties of the electrolyte solution and diffusion of the applied redox probe in solution, respectively. Thus, they are not affected by chemical transformations occurring at the electrode interface. The other two components of the circuit, Cdl and Ret, depend on the dielectric and insulating features at the electrode/ electrolyte interface. In EIS, the semicircle diameter of EIS equals the
Fig. 2 (A) Nyquist diagrams for (a) the bare gold electrode, (b) probe ‘a’ after modification with poly-T(25) DNA, (c) probe ‘b’ after reaction with Hg2+, and (d) probe ‘c’ after reaction with H2S (inset: equivalent circuit); and (B) Nyquist diagrams for (a) the DNA-modified gold electrode, (b) probe ‘a’ + 600 nM H2S, and (c) probe ‘b’ + Hg2+ + 600 nM H2S in 5 mM Fe(CN)64/3 + 0.1 M KCl with the range from 102 Hz to 105 Hz at an alternate voltage of 5 mV.
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ChemComm electron transfer resistance, Ret. This resistance controls the electron transfer kinetics of the redox-probe at the electrode interface. Its value varies when different substances are adsorbed onto the electrode surface. As seen from curve ‘a’ in Fig. 2A, a small Ret similar to a straight line was observed at the bare gold electrode. When thiolated DNA (curve ‘b’, Ret E 459 O) and Hg2+ (curve ‘c’, Ret E 812 O) were modified onto the electrode, however, the resistances increased thereupon. The reason might be the facts that (i) the negatively charged DNA repelled the ferricyanide with negative charge, and (ii) the formed T–Hg2+–T hindered the electron transfer. Significantly, the resistance largely increased (curve ‘d’, Ret E 1002 O) after the resultant electrode was reacted with H2S. Such an increase is basically attributed to the formation of insoluble HgS nanostructures on the electrode. However, an obscure question to be answered was whether the strong signal derived from the reaction between Hg2+ and H2S. To verify this issue, the DNA-modified electrode was used for the detection of 600 nM H2S in the presence and absence of Hg2+ using the same assay protocol, respectively (Fig. 2B). A resistance (Ret E 453 O) was observed at the DNA-modified electrode (curve ‘a’). When the electrode was directly incubated with 600 nM H2S, the resistance was almost the same as that of the DNA-modified electrode (Ret E 468 O, curve ‘b’). In contrast, the resistance largely increased in the presence of Hg2+ (Ret E 2123 O, curve ‘c’). The results further indicated that the increase in the resistance mainly originated from the reaction between H2S and Hg2+. Next, we optimized the conjugated time for T–Hg2+–T and the reaction time of H2S with Hg2+. Experimental results indicated that the optimal impedimetric signals were acquired at 15 min for T–Hg2+–T conjugation and 30 min for the formation of HgS, respectively (see Fig. S1 and S2† for detailed experimental results). Under the optimal conditions, the sensitivity and dynamic range was evaluated toward H2S standards with various concentrations based on the precipitation of target-stimulated metallic HgS nanostructures on the poly-T(25) DNA-modified gold electrode. Typical Nyquist plots of the poly-T-modified electrode before and after incubation with target H2S are shown in Fig. 3a. It is obvious that the Ret value shows a dependence upon the concentration of target H2S, and the Ret value increases with the increase of H2S concentration. As indicated in Fig. 3b, the increase in the resistance was proportional to H2S concentration ranging from 1.0 nM to 850 nM with a detection limit (LOD) of 0.35 nM at a signal to noise ratio of 3s (where s is the standard deviation of the blank, n = 15). Although the system has not yet been optimized for maximum efficiency, the LOD was obviously lower than those of fluorescent sensors,8a,b
Fig. 3 (a) Nyquist diagrams for the DNA-modified gold electrode toward H2S standards with various concentrations in 5 mM Fe(CN)64/3 + 0.1 M KCl. (b) Calibration plots (C vs. Ret) for H2S levels from 1.0 nM to 850 nM (error bars: SD, n = 3).
Chem. Commun., 2013, 49, 11200--11202
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Fig. 4 (a) The specificity of the developed impedimetric detection method against DTT, GSH, Cys, NO, CO, CO2, H2S and O2 (0.5 mM used in this case); and (b) comparison of the assay results for H2S spiked blank calf serum samples by using the developed impedimetric method with the standard values.
surface plasmon resonance-based sensors,8c colorimetric sensors,8d and near-infrared ratiometric fluorescent sensors.8e More importantly, the concentration of biological sulfides is usually in the range from 10 to 300 mM.9 Hence, the developed impedimetric assay protocol completely meets the requirement of H2S analysis in the biological sample. The specificity of the developed impedimetric detection method was also monitored by challenging the system with other biological thiols [e.g. dithiothreitol (DTT), glutathione (GSH) and cysteine (Cys), which are commonly found in the biological milieu and prove to interfere with the detection of physiological H2S] and gases [e.g. NO, CO, CO2 and O2, which usually coexist with H2S in biological milieu]. As shown in Fig. 4a, the stronger signal in the resistance was observed with target H2S than those of other components. The results clearly suggest that the DNA-modified electrode could be utilized as a sensitive and selective platform for the detection of the target molecule. The reproducibility of the DNA-modified electrode was also monitored by repeatedly assaying 3 H2S levels (high, middle and low), using identical batches of DNA-modified electrodes throughout (Table S1†). Experimental results indicated that the coefficients of variation (CVs) of the intra-assay were 8.8%, 5.8% and 4% for 5, 200 and 600 nM H2S (n = 5), respectively, whereas the CVs of the inter-assay with various batches were 10.3%, 9.6% and 6.7% for H2S towards the above-mentioned targets. The low CVs indicated the possibility of the DNA-modified electrode with batch-wise preparation. In addition, the modified electrodes exhibited satisfactory stability. In fact, as much as 90% of the initial resistance was preserved after storage of the sensors at 4 1C for 20 days. To further investigate the possibility of the newly developed technique to be applied for testing of real samples, 11 biological samples with various concentrations of H2S were studied. Initially, blank new born calf serum was diluted 10-fold with 10 mM trisHCl (pH 6.0, containing 50 mM NaCl), and then the Na2S donor with various concentrations is spiked into the serum sample. During this process, H2S is volatilized out of the serum under the meta-acid conditions. Following that, the as-prepared serum samples were assayed by using the poly-T-modified electrode, respectively. The results were compared with the standard values, which was performed via the use of a least-squares regression method (Fig. 4b). The regression line was fitted to y = 1.057x 16.203 (R2 = 0.9911, n = 33) where x stands for the H2S level
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Communication estimated with the impedimetric method and y stands for the reference values. The slope of the regression equation was close to the ideal of ‘1’. Hence, the methodology could be utilized for quantitative monitoring of target H2S in biological fluids. For the detection in the complex biological sample, H2S can be extracted by using the Ag power method in advance,10 and then tested using the developed scheme. In conclusion, we have demonstrated the ability of the polyT(25)-modified electrode for sensitive determination of H2S based on the precipitation of target-induced HgS nanostructures on the DNA-based PIC membrane for the first time. Compared with other H2S detection methods, the developed impedimetric assay protocol is simple and sensitive. Meanwhile, the gas–solid interface reaction of volatile H2S with the Hg2+ ions embedded into the DNA-based PIC membrane can produce an insoluble HgS precipitate, thereby resulting in the signal amplification. Importantly, this methodology does not require complex sample pretreatment, thus representing a versatile detection scheme. Support from the National Natural Science Foundation of China (21075019, 41176079), the ‘‘973’’ National Basic Research Program of China (2010CB732403), the Research Fund for the Doctoral Program of Higher Education of China (20103514120003), and the National Science Foundation of Fujian Province (2011J06003) is gratefully acknowledged.
Notes and references 1 W. Sun, J. Fan, C. Hu, J. Cao, H. Zhang, X. Xiong, J. Wang, S. Cui, S. Sun and X. Peng, Chem. Commun., 2013, 49, 3890. 2 N. Shibuya, S. Koike, M. Tanaka, M. Ishigami-Yuasa, Y. Kimura, Y. Ogasawara, K. Fukui, N. Nagahara and H. Kimura, Nat. Commun., 2013, 4, 1366. 3 M. Thorson, T. Majtan, J. Kraus and A. Barrios, Angew. Chem., Int. Ed., 2013, 52, 4641. 4 (a) A. Jarosz, T. Yep and B. Mutus, Anal. Chem., 2013, 85, 3638; (b) C. Yu, X. Li, F. Zeng, F. Zheng and S. Wu, Chem. Commun., 2013, 49, 403; (c) Q. Wan, Y. Song, Z. Li, X. Gao and H. Ma, Chem. Commun., 2013, 49, 502; (d) S. Pandey, K. Kim and K. Tang, TrAC, Trends Anal. Chem., 2012, 32, 87–99; (e) N. Lawrence, J. Davis, L. Jiang, T. Jones, S. Davies and R. Compton, Electroanalysis, 2000, 12, 1453–1460; ( f ) H. Peng, Y. Cheng, C. Dai, A. King, B. Predmore, D. Lefer and B. Wang, Angew. Chem., Int. Ed., 2011, 50, 9672; ( g) M. Wronski, Anal. Chem., 1971, 43, 606; (h) G. Mao, T. Wei, X. Wang, S. Huan, D. Lu, J. Zhang, X. Zhang, W. Tan, G. Shen and R. Yu, Anal. Chem., 2013, 85, 7875. 5 M. Xu, J. Zhuang, X. Chen, G. Chen and D. Tang, Chem. Commun., 2013, 49, 7304. 6 J. Tang, D. Tang, J. Zhu, H. Yang and G. Chen, Chem. Commun., 2012, 48, 2627. 7 (a) X. Liu, C. Qi, T. Ding, X. Cheng and D. Shangguan, Anal. Chem., 2009, 81, 3699–3704; (b) H. Torigoe, A. One and T. Kozasa, Chem.–Eur. J., 2010, 16, 13218–13225; (c) C. Kaul, M. Muller, M. Wagner, S. Schneider and T. Carell, Nat. Chem., 2011, 3, 794–800; (d) H. Mei, T. Ding, C. Qi, N. Zhang, X. Liu, T. Chang, J. Yan and D. Shangguan, Chem. Commun., 2013, 49, 164–166. 8 (a) X. Ou, C. Li, H. Chen, J. Mack, Z. Guo and Z. Shen, Chem. Commun., 2013, 49, 7510; (b) B. Wang, P. Li, F. Fu, J. Chen, Z. Qu and K. Han, Chem. Commun., 2013, 49, 5690; (c) R. Tabassum, S. Mishra and B. Gupta, Phys. Chem. Chem. Phys., 2013, 15, 11868; (d) L. Montoya, T. Pearce, R. Hansen, L. Zakharov and M. Pluth, J. Org. Chem., 2013, 78, 6550; (e) X. Wang, J. Sun, W. Zhang, X. Ma, J. Lv and B. Tang, Chem. Sci., 2013, 4, 2551. 9 Z. Xu, L. Xu, J. Zhou, Y. Xu, W. Zhu and X. Qian, Chem. Commun., 2012, 48, 10871. 10 M. Ishigami, K. Hiraki, K. Umemura, Y. Ogasawara, K. Ishii and H. Kimura, Antioxid. Redox Signaling, 2009, 11, 205.
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COMMUNICATION
Cite this: Chem. Commun., 2013, 49, 11200 Received 8th September 2013, Accepted 8th October 2013 DOI: 10.1039/c3cc46869k
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Target-stimulated metallic HgS nanostructures on a DNA-based polyion complex membrane for highly efficient impedimetric detection of dissolved hydrogen sulfide† Junyang Zhuang, Libing Fu, Wenqiang Lai, Dianping Tang* and Guonan Chen
www.rsc.org/chemcomm
Target-stimulated metallic HgS nanostructures formed on the DNAbased polyion complex (PIC) membrane were for the first time utilized as an efficient scheme for impedimetric detection of hydrogen sulfide (H2S) by coupling insoluble precipitation with sensitivity enhancement.
Hydrogen sulfide (H2S) is an extremely hazardous toxic compound.1 Recent investigations have demonstrated that H2S is the third endogenous gaseous signaling compound with cytoprotection properties,2 and its levels are altered in diseases ranging from Alzheimer’s disease and Down’s syndrome to diabetes and liver cirrhosis.3 Therefore, recent years have seen a steady increase in interest in understanding the physiological and pathological functions of H2S. Various methods and strategies have been developed for monitoring H2S, such as colorimetric methods, HPLC, GC, electrochemistry, and fluorescence.4 Among them, the electrochemical detection method is suitable for sensor miniaturization and automated detection because of simple instrumentation and easy signal quantification.5 Despite many advances in this field, there is still the quest for new schemes and strategies for improving the sensitivity of the assays. Electrochemical impedance spectroscopy (EIS) is an effective strategy to probe the interfacial properties of the modified electrode.6 The adsorption or desorption of insulating materials on conductive supports is anticipated to alter the interfacial electron-transfer features (capacitance and resistance) at the electrode surface. However, one of great challenges for the successful development of EIS-based sensors is to amplify the signal output. Significantly, many metal ions, e.g. PbII, CuII, HgII and AgI, can react with H2S to give the corresponding metal sulfides, and precipitate from solution upon exposure to H2S. The components of the resulting precipitate redissolve with selectivity. Hence, our aim in this work is to design a simple and feasible impedimetric detection method for toxic gases by virtue of the precipitation technique. Key Laboratory of Analysis and Detection of Food Safety (Ministry of Education & Fujian Province), Department of Chemistry, Fuzhou University, Fuzhou 350108, P.R. China. E-mail: [email protected]; Fax: +86 591 22866135; Tel: +86 591 22866125 † Electronic supplementary information (ESI) available: Experimental procedures and optimization of conditions. See DOI: 10.1039/c3cc46869k
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Chem. Commun., 2013, 49, 11200--11202
Scheme 1 Fabrication process of the impedimetric detection method for dissolved H2S based on precipitation of target-simulated metallic HgS nanostructures on a DNA-based polyion complex membrane.
Herein, we report the proof-of-concept of a novel and powerful impedimetric detection method for dissolved H2S by coupling signal amplification with the precipitation of target-stimulated metallic HgS nanostructures on the DNA-based polyion complex (PIC) membrane (Scheme 1, see ESI† for experimental details). Unlike the common polymer film, DNA self-assembled film on the electrode allows electron-transfer between solution and the electrode because of its relatively low density and ordered structure. Initially, the thiolated poly-T(25) oligonucleotide (5 0 -SH-TTTTT TTTTT TTTTT TTTTT TTTTT-3 0 ) is covalently conjugated onto the surface of the gold electrode by the classical Au–S bond, and then the resulting electrode is immersed in pH 7.4 tris-buffer (10 mM) solution containing 10 mM Hg2+. During this process, Hg2+ ions are intercalated into the polyion complex membrane based on the T–Hg2+–T coordination chemistry.7 Utilizing the high volatility of H2S in solution, the impedimetric assay is implemented using a headspace method that the H2S volatilizing from the test sample directly interacts with the as-prepared membrane on the top of the sample (Na2S is used as a H2S donor, see ESI† for experimental details). Upon introduction of target H2S, the reaction is carried out between the S2 ion and Hg2+ (Hg2+ + H2S - 2H+ + HgSk) to produce an insoluble HgS precipitate. The precipitate can coat on the electrode surface, and hinder the electron transfer of redox probes, e.g. Fe(CN)63/ Fe(CN)64, thereby resulting in the increase of the Faradaic impedance of the modified electrode. By monitoring the change in the resistance, we might quantitatively determine the concentration of target H2S in the sample. This journal is
c
The Royal Society of Chemistry 2013
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Communication
Published on 08 October 2013. Downloaded by Brown University on 22/10/2014 19:23:11.
Fig. 1 SEM images of (a) the DNA-modified gold substrate, (b) probe ‘a’ after direct treatment with H2S, and (c) probe ‘a’ after incubation with Hg2+ and subsequent treatment with H2S.
To realize our design, one precondition for the successful development of the impedimetric assay was whether HgS nanostructures could be formed in the presence of target H2S. To demonstrate this issue, we used a scanning electron microscope (SEM) to investigate the PIC membrane-modified gold substrate before and after incubation with Hg2+ ions or H2S, respectively. Fig. 1a shows the SEM image of the PIC membrane-modified gold substrate. When the modified gold substrate was directly reacted with H2S in the absence of Hg2+ ions, no precipitation was observed on the surface (Fig. 1b). More inspiringly, upon addition of target H2S onto the Hg2+-treated DNA PIC membrane, an obvious particle-like topology could be achieved (Fig. 1c). The results revealed that the precipitation reaction could be implemented between H2S and the Hg2+. Another concern arises as to whether the formed HgS nanostructures could cause the change in the resistance of the modified electrode. To clarify this point, the DNA-modified electrode was used for detection of 200 nM H2S (as an example) by using the developed assay protocol. Fig. 2A shows the EIS of a variously modified electrode after each step. These EIS data were fitted to a Randles equivalent circuit (inset of Fig. 2A), which contains electrolyte resistance (Rs), the lipid bilayer capacitance (Cdl), charge transfer resistance (Ret) and Warburg element (Zw). The complex impedance can be presented as the sum of the real, Zre and imaginary, Zim, components that originate mainly from the resistance and capacitance of the cell. The two components of the scheme, Rs and Zw, represent bulk properties of the electrolyte solution and diffusion of the applied redox probe in solution, respectively. Thus, they are not affected by chemical transformations occurring at the electrode interface. The other two components of the circuit, Cdl and Ret, depend on the dielectric and insulating features at the electrode/ electrolyte interface. In EIS, the semicircle diameter of EIS equals the
Fig. 2 (A) Nyquist diagrams for (a) the bare gold electrode, (b) probe ‘a’ after modification with poly-T(25) DNA, (c) probe ‘b’ after reaction with Hg2+, and (d) probe ‘c’ after reaction with H2S (inset: equivalent circuit); and (B) Nyquist diagrams for (a) the DNA-modified gold electrode, (b) probe ‘a’ + 600 nM H2S, and (c) probe ‘b’ + Hg2+ + 600 nM H2S in 5 mM Fe(CN)64/3 + 0.1 M KCl with the range from 102 Hz to 105 Hz at an alternate voltage of 5 mV.
This journal is
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ChemComm electron transfer resistance, Ret. This resistance controls the electron transfer kinetics of the redox-probe at the electrode interface. Its value varies when different substances are adsorbed onto the electrode surface. As seen from curve ‘a’ in Fig. 2A, a small Ret similar to a straight line was observed at the bare gold electrode. When thiolated DNA (curve ‘b’, Ret E 459 O) and Hg2+ (curve ‘c’, Ret E 812 O) were modified onto the electrode, however, the resistances increased thereupon. The reason might be the facts that (i) the negatively charged DNA repelled the ferricyanide with negative charge, and (ii) the formed T–Hg2+–T hindered the electron transfer. Significantly, the resistance largely increased (curve ‘d’, Ret E 1002 O) after the resultant electrode was reacted with H2S. Such an increase is basically attributed to the formation of insoluble HgS nanostructures on the electrode. However, an obscure question to be answered was whether the strong signal derived from the reaction between Hg2+ and H2S. To verify this issue, the DNA-modified electrode was used for the detection of 600 nM H2S in the presence and absence of Hg2+ using the same assay protocol, respectively (Fig. 2B). A resistance (Ret E 453 O) was observed at the DNA-modified electrode (curve ‘a’). When the electrode was directly incubated with 600 nM H2S, the resistance was almost the same as that of the DNA-modified electrode (Ret E 468 O, curve ‘b’). In contrast, the resistance largely increased in the presence of Hg2+ (Ret E 2123 O, curve ‘c’). The results further indicated that the increase in the resistance mainly originated from the reaction between H2S and Hg2+. Next, we optimized the conjugated time for T–Hg2+–T and the reaction time of H2S with Hg2+. Experimental results indicated that the optimal impedimetric signals were acquired at 15 min for T–Hg2+–T conjugation and 30 min for the formation of HgS, respectively (see Fig. S1 and S2† for detailed experimental results). Under the optimal conditions, the sensitivity and dynamic range was evaluated toward H2S standards with various concentrations based on the precipitation of target-stimulated metallic HgS nanostructures on the poly-T(25) DNA-modified gold electrode. Typical Nyquist plots of the poly-T-modified electrode before and after incubation with target H2S are shown in Fig. 3a. It is obvious that the Ret value shows a dependence upon the concentration of target H2S, and the Ret value increases with the increase of H2S concentration. As indicated in Fig. 3b, the increase in the resistance was proportional to H2S concentration ranging from 1.0 nM to 850 nM with a detection limit (LOD) of 0.35 nM at a signal to noise ratio of 3s (where s is the standard deviation of the blank, n = 15). Although the system has not yet been optimized for maximum efficiency, the LOD was obviously lower than those of fluorescent sensors,8a,b
Fig. 3 (a) Nyquist diagrams for the DNA-modified gold electrode toward H2S standards with various concentrations in 5 mM Fe(CN)64/3 + 0.1 M KCl. (b) Calibration plots (C vs. Ret) for H2S levels from 1.0 nM to 850 nM (error bars: SD, n = 3).
Chem. Commun., 2013, 49, 11200--11202
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Fig. 4 (a) The specificity of the developed impedimetric detection method against DTT, GSH, Cys, NO, CO, CO2, H2S and O2 (0.5 mM used in this case); and (b) comparison of the assay results for H2S spiked blank calf serum samples by using the developed impedimetric method with the standard values.
surface plasmon resonance-based sensors,8c colorimetric sensors,8d and near-infrared ratiometric fluorescent sensors.8e More importantly, the concentration of biological sulfides is usually in the range from 10 to 300 mM.9 Hence, the developed impedimetric assay protocol completely meets the requirement of H2S analysis in the biological sample. The specificity of the developed impedimetric detection method was also monitored by challenging the system with other biological thiols [e.g. dithiothreitol (DTT), glutathione (GSH) and cysteine (Cys), which are commonly found in the biological milieu and prove to interfere with the detection of physiological H2S] and gases [e.g. NO, CO, CO2 and O2, which usually coexist with H2S in biological milieu]. As shown in Fig. 4a, the stronger signal in the resistance was observed with target H2S than those of other components. The results clearly suggest that the DNA-modified electrode could be utilized as a sensitive and selective platform for the detection of the target molecule. The reproducibility of the DNA-modified electrode was also monitored by repeatedly assaying 3 H2S levels (high, middle and low), using identical batches of DNA-modified electrodes throughout (Table S1†). Experimental results indicated that the coefficients of variation (CVs) of the intra-assay were 8.8%, 5.8% and 4% for 5, 200 and 600 nM H2S (n = 5), respectively, whereas the CVs of the inter-assay with various batches were 10.3%, 9.6% and 6.7% for H2S towards the above-mentioned targets. The low CVs indicated the possibility of the DNA-modified electrode with batch-wise preparation. In addition, the modified electrodes exhibited satisfactory stability. In fact, as much as 90% of the initial resistance was preserved after storage of the sensors at 4 1C for 20 days. To further investigate the possibility of the newly developed technique to be applied for testing of real samples, 11 biological samples with various concentrations of H2S were studied. Initially, blank new born calf serum was diluted 10-fold with 10 mM trisHCl (pH 6.0, containing 50 mM NaCl), and then the Na2S donor with various concentrations is spiked into the serum sample. During this process, H2S is volatilized out of the serum under the meta-acid conditions. Following that, the as-prepared serum samples were assayed by using the poly-T-modified electrode, respectively. The results were compared with the standard values, which was performed via the use of a least-squares regression method (Fig. 4b). The regression line was fitted to y = 1.057x 16.203 (R2 = 0.9911, n = 33) where x stands for the H2S level
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Chem. Commun., 2013, 49, 11200--11202
Communication estimated with the impedimetric method and y stands for the reference values. The slope of the regression equation was close to the ideal of ‘1’. Hence, the methodology could be utilized for quantitative monitoring of target H2S in biological fluids. For the detection in the complex biological sample, H2S can be extracted by using the Ag power method in advance,10 and then tested using the developed scheme. In conclusion, we have demonstrated the ability of the polyT(25)-modified electrode for sensitive determination of H2S based on the precipitation of target-induced HgS nanostructures on the DNA-based PIC membrane for the first time. Compared with other H2S detection methods, the developed impedimetric assay protocol is simple and sensitive. Meanwhile, the gas–solid interface reaction of volatile H2S with the Hg2+ ions embedded into the DNA-based PIC membrane can produce an insoluble HgS precipitate, thereby resulting in the signal amplification. Importantly, this methodology does not require complex sample pretreatment, thus representing a versatile detection scheme. Support from the National Natural Science Foundation of China (21075019, 41176079), the ‘‘973’’ National Basic Research Program of China (2010CB732403), the Research Fund for the Doctoral Program of Higher Education of China (20103514120003), and the National Science Foundation of Fujian Province (2011J06003) is gratefully acknowledged.
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